CHD4 mRNA expression was significantly higher in platinum-resistant samples in a subsequent clinical sample analysis, suggesting that CHD4 overexpression conferred platinum resistance to ovarian cancer cells, resulting in poor patient survival. In concordance with these findings, CHD4 knockdown enhanced the induction of apoptosis mediated by cisplatin in ovarian cancer cells TOV21G and increased cisplatin sensitivity in multiple ovarian cancer cells derived from different subtypes. However, CHD4 knockdown did not affect the expression of RAD51 or p21, the known targets of CHD4 in other cancer types that can modulate platinum sensitivity. Knockdown and overexpression assays revealed that CHD4 positively regulated the expression of multi-drug transporter MDR1 and its coding protein p-glycoprotein. In addition, a first-in-class CHD4/SMARCA5 inhibitor ED2-AD101 showed synergistic interactions with cisplatin. Our findings suggest that CHD4 mediates platinum sensitivity by modulating MDR1 expression in ovarian cancer. Further, CHD4 suppression has a potential to be a novel therapeutic strategy in combination with platinum agents.Platinum sensitivity is an important prognostic factor in patients with ovarian cancer. Chromodomain-helicase-DNA-binding protein 4 (CHD4) is a core member of the nucleosome remodeling and deacetylase complex, which functions as a chromatin remodeler. Emerging evidence indicates that CHD4 could be a potential therapeutic target for cancer therapy. The purpose of this study was to clarify the role of CHD4 in ovarian cancer and investigate its therapeutic potential focusing on platinum sensitivity. In an analysis of the Cancer Genome Atlas ovarian cancer dataset, CHD4 gene amplification was associated with worse overall survival.  More than half of the ovarian cancers are diagnosed in advanced stages, and present with carcinomatous peritonitis . The thePlatinum agents are the major DNA damaging agents that exert anticancer effects mainly by forming intrastrand and interstrand DNA crosslinks ,8. AlthoChromodomain-helicase-DNA-binding protein 4 (CHD4) is a component of the nucleosome remodeling and deacetylase (NuRD) complex. As the name suggests, NuRD complex acts as a chromatin remodeler and CHD4 is considered to play a pivotal role by conferring ATPase activity on the complex –14. The The purpose of this study was to elucidate the potential of CHD4 as a new therapeutic target for the treatment of ovarian cancer, with a particular focus on its interaction with platinum agents.TOV21G was obtained from the American Type Culture Collection. KURAMOCHI was obtained from the Japanese Cancer Research Resources Bank. JHOS2, and JHOC5 were obtained from RIKEN. A2780 and A2780cis were obtained from Sigma-Aldrich. OSE2 and OSE4 were kindly provided by Dr. Makoto Nitta  Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: PartlyReviewer #3: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: I Don't KnowReviewer #3: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: NoReviewer #3: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: In the manuscript, “CHD4 regulates platinum sensitivity through MDR1 expression in ovarian cancer: A potential role of CHD4 inhibition as a combination therapy with platinum agents”, the authors have characterized the role of CHD4 in sensitizing ovarian cancer cells towards platinum. The authors found that CHD4 expression is increased in platinum resistant clinical samples and knockdown of CHD4 enhanced cisplatin induced apoptosis. Furthermore, they found that CHD4 regulates platinum sensitivity via MDR1 expression. The findings are interesting and demonstrate great therapeutic potential in terms of combination therapy, however, the authors should address following concerns:1) Have the authors compared CHD4 expression in platinum sensitive and platinum resistant ovarian cancer cell lines?2) Supplementary Figure 3A: Knockdown of CHD4 is not convincing based on the immunoblotting for CHD4 and loading control. The authors should include a better representative blot along with densitometry analysis.3) Figure 4F: Can the authors explain why there are two CDH4 bands in the empty vector lane?4) Have the authors determined if cisplatin+CDH4 knockdown affect cell cycle progression?5) Does treatment with cisplatin enhance the expression of MDR1 in ovarian cancer cells? Furthermore, does combination of CHD4 knockdown and cisplatin treatment affect MDR1 expression?6) Can the authors speculate how CDH4 regulates MDR1 expression? What are the upstream regulators of MDR1? These points should be addressed in the discussion.7) This study will greatly benefit by a phenotype rescue experiment. The authors can overexpress MDR1 in CHD4 knockdown cells and investigate if this changes cell viability or cisplatin induced apoptosis.Reviewer #2: The goal of this study is to determine how overexpression of CHD4 contributes to platinum resistance in ovarian cancer.The authors have genetically knocked down CHD4 expression in TOV21G cells and overexpressed CHD4 in the same cell line and determined cisplatin sensitivity using cell viability assays. It is not clear on what basis this cell line was chosen. Lack of a western blot showing the levels of CHD4 in the cell lines used is another major weakness. Were there any OV cell lines without CHD4 expression that the authors could have chosen to use for their overexpression studies.Q-PCR analysis of what the authors define as cisplatin sensitive vs resistant clinical samples, the authors show that the resistant cells express higher levels of CHD4. Since the # of patient derived resistant samples is so few and the error bars overlap with the sensitive samples- the significance is not very clear. It is also not clear why the authors have not performed western blots for markers in the sensitive and resistant samples or by IHC for CHD4 and/or MDR1to claim CHD4 expression positively impacts the resistance to platinum treatment.They also show synergy between SMARCA5/CHD4 dual inhibitor and cisplatin. Since ED2-AD101 is a novel drug, the author should show the cytotoxicity in the normal cells vs OC cells with positive control like 5FU with SRB or MTT and clonogenic assay and the same has to be performed in CHD4 knockdown cells with cisplatin also. Cisplatin sensitivity should be performed over a concentration range 0, 0.5, 1, 2,5,10 uM for better validation.In fig 4, the authors show increased levels of both cleaved and non-cleaved PARP at 5uM of cisplatin and which makes it difficult to interpret these results. However, the authors revalidated the same in FACS analysis using a very high dose of cisplatin and claimed in CHD4 kd cells induces the cell death but author has not explained why there was an sudden increase in 5 to 20uM cisplatin concentration.Although, the authors show in fig 4a, CHD4 kd with cisplatin induces phospho yH2AX, a marker for DNA damage and in ,Fig 4c CHD4 kd induces RAD51expression, a DNA repair factor, authors has to explain how cisplatin/ED2-AD101 inhibitor synergy will work on apoptosis?Finally they have determined that MDR1 downregulation in CHD4 KD cells and overexpressed in cells with enhanced expression of CHD4.Over all the study does not robustly add anything to the literature on the role of CHD4 due to the lack of well accepted methods to determine cell viability such as colony forming assays. It was very difficult to determine the IC50 values since the #s are not mentioned in the text. Lack of p value in Q-PCR based siRNA results made it difficult to assess the significance. In general the clarity of the figures also was not good. The manuscript is not acceptable for publication in PLOSONE with the limited amount of data presented in its current form.Reviewer #3: Platinum drugs are the most effective in the ovarian cancer treatment. Platinum resistance is a critical issue to be solved. It is important to study the mechanism about platinum sensitivity or platinum resistance.　The authors first found that CHD4 mRNA expression was significantly higher in the platinum resistant samples than in the platinum sensitive samples in ovarian cancer tissues. Then, the authors investigated whether CHD4 is associated with platinum sensitivity using ovarian cancer cell lines in vitro. Suppression of CHD4 expression increased the platinum sensitivity by decreasing MDR1 expression in ovarian cancer cells.The present study clearly showed that CDH4 is associated with platinum sensitivity in ovarian cancer cells.Major comments1. Generally, the platinum sensitivity is much higher in high grade serous carcinoma than in clear cell carcinoma. According to Figure 2, there seems to be no difference in the platinum sensitivity between clear cell carcinoma cells (TOV21G and JHOC5) and high grade serous carcinoma cells (KURAMOCHI and JHOS2). Furthermore, the effect of CHD4 knockdown seems to be high in clear cell carcinoma cells compared with that in high grade serous carcinoma cells. Reviewer is wondering whether these cell-lines used in this study are suitable for evaluating platinum sensitivity in ovarian cancer cells.2. Figure 3 only showed the data from clear cell carcinoma cell lines (TOV21G and JHOC5). The data from high grade serous carcinoma cell lines (KURAMOCHI and JHOS2) should be provided or described in the result section.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  25 Mar 2021We are extremely thankful to the editor and reviewers for reviewing our manuscript and providing insightful comments. We have revised the manuscript based on all these comments, which have helped us improve our manuscript greatly. The revisions have been indicated as tracked changes in the revised manuscript.　We would like to inform the editor and reviewers that English proofreading was performed before resubmitting the manuscript, resulting in minor grammatical corrections as well as the revision in response to the reviewer’s comments. Below, we have provided point-by-point responses to each of the reviewers’ comments. Should there be any further concerns regarding our manuscript, we will gladly clarify. Reviewer #1: In the manuscript, “CHD4 regulates platinum sensitivity through MDR1 expression in ovarian cancer: A potential role of CHD4 inhibition as a combination therapy with platinum agents”, the authors have characterized the role of CHD4 in sensitizing ovarian cancer cells towards platinum. The authors found that CHD4 expression is increased in platinum resistant clinical samples and knockdown of CHD4 enhanced cisplatin induced apoptosis. Furthermore, they found that CHD4 regulates platinum sensitivity via MDR1 expression. The findings are interesting and demonstrate great therapeutic potential in terms of combination therapy, however, the authors should address following concerns:1) Have the authors compared CHD4 expression in platinum sensitive and platinum resistant ovarian cancer cell lines?　Thank you for the valuable comment. To address your concern, we additionally compared CHD4 expression by western blotting in variable ovarian cancer cell lines including A2780 and A2780cis, a pair of parental cells and their derivatives that experimentally acquired platinum resistance. The result was demonstrated in S2 Fig, and additionally described in page 16, line 223-229. CHD4 expression did not differ across the cell lines. As there are plenty of mechanisms involved in platinum sensitivity, we considered that this result does not directly contradict our findings. We appreciate your suggestion.2) Supplementary Figure 3A: Knockdown of CHD4 is not convincing based on the immunoblotting for CHD4 and loading control. The authors should include a better representative blot along with densitometry analysis.Thank you for the valuable suggestion. We have replaced the bands with a better representative blot with the results of densitometry .3) Figure 4F: Can the authors explain why there are two CDH4 bands in the empty vector lane?Thank you for the valuable comment. The CHD4 antibody used in this study frequently detects two bands close to each other. In CHD4 knockdown, the expression of the lower band always decreased as shown in S5 Fig and S9 Fig. Hence, it is fair to conclude that the lower band represents CHD4. As the expression of the lower band obviously increases by the transfection of the CHD4 overexpression vector, we considered that overexpression of CHD4 was successfully introduced. 4) Have the authors determined if cisplatin+CDH4 knockdown affect cell cycle progression?Thank you for the valuable suggestion on cell cycle assessment. We performed this analysis additionally, and the result has been displayed in S8 Fig. The result has also been described in the results section of the revised manuscript. Briefly, cisplatin treatment alone facilitated S phase arrest in TOV21G. CHD4 suppression also increased the population of the cells at S phase. Mild increase of G2/M was also observed. A combination of CHD4 knockdown and cisplatin administration does not seem to interact with each other in the perspective of cell cycle regulation. 5) Does treatment with cisplatin enhance the expression of MDR1 in ovarian cancer cells? Furthermore, does combination of CHD4 knockdown and cisplatin treatment affect MDR1 expression?Thank you for the valuable comment. We examined the MDR1/p-glycoprotein expression by western blotting. It is shown in a figure 1 of the additional information for the reviewers. The expression of p-glycoprotein was not significantly affected by cisplatin treatment, CHD4 knockdown, or the combination. However, we could only get very faint bands for the p-glycoprotein once, after the replacement with a new one of a different lot.6) Can the authors speculate how CDH4 regulates MDR1 expression? What are the upstream regulators of MDR1? These points should be addressed in the discussion.Thank you for the valuable comment. Accordingly, we have addressed the points mentioned in this comment in the discussion section of the revised manuscript, with some relevant references.  7) This study will greatly benefit by a phenotype rescue experiment. The authors can overexpress MDR1 in CHD4 knockdown cells and investigate if this changes cell viability or cisplatin induced apoptosis.We appreciate your suggestion. We transfected MDR1 overexpression vector by lipofection in TOV21G. Quantitative PCR analysis revealed the increase of MDR1 mRNA quickly disappeared within 72 hours and p-glycoprotein expression does not increase. These results have been demonstrated in a figure 2 of the additional information for the reviewers. We concluded that it is not possible to address the reviewer’s comment with transient vector transfection. As our laboratory currently does not have permission to handle viral vectors, we could not proceed to create a stable overexpression system. Reviewer #2: The goal of this study is to determine how overexpression of CHD4 contributes to platinum resistance in ovarian cancer.The authors have genetically knocked down CHD4 expression in TOV21G cells and overexpressed CHD4 in the same cell line and determined cisplatin sensitivity using cell viability assays. It is not clear on what basis this cell line was chosen. Lack of a western blot showing the levels of CHD4 in the cell lines used is another major weakness. Were there any OV cell lines without CHD4 expression that the authors could have chosen to use for their overexpression studies.Thank you for pointing out an important issue. We agree that it is important to select the cell lines based on the CHD4 expression level. We have added a supplementary figure  to demonstrate the expression of CHD4 in several ovarian cancer cells lines including the cells used in this study. There was no clear difference in CHD4 expression across the cell lines.From the perspective of translational research, we believe it is also important to select the cells that retain the characteristics unique to each histologic subtype as ovarian cancer is quite heterogenous. As there was no clear difference in CHD4 expression in ovarian cancer cells, we decided to mainly use TOV21G, JHOC5, and KURAMOCHI, JHOS2 that are reported to appropriately reflect the phenotype of clear cell carcinoma and high-grade serous carcinoma, respectively.　We have added several comments on this point in the revised manuscript. ReferencesDomcke S, Sinha R, Levine DA, Sander C, Schultz N. Evaluating cell lines as tumour models by comparison of genomic profiles. Nat Commun. 2013;4: 2126. Beaufort CM, Helmijr JC, Piskorz AM, Hoogstraat M, Ruigrok-Ritstier K, Besselink N, et al. Ovarian cancer cell line panel (OCCP): clinical importance of in vitro morphological subtypes. PLoS One. 2014;9: e103988. Anglesio MS, Wiegand KC, Melnyk N, Chow C, Salamanca C, Prentice LM, et al. Type-specific cell line models for type-specific ovarian cancer research. PLoS One. 2013;8: e72162. Q-PCR analysis of what the authors define as cisplatin sensitive vs resistant clinical samples, the authors show that the resistant cells express higher levels of CHD4. Since the # of patient derived resistant samples is so few and the error bars overlap with the sensitive samples- the significance is not very clear. It is also not clear why the authors have not performed western blots for markers in the sensitive and resistant samples or by IHC for CHD4 and/or MDR1to claim CHD4 expression positively impacts the resistance to platinum treatment.Thank you for the valuable comment. As mentioned in the manuscript, we considered a specimen to be a platinum resistant sample only if the remaining tumor in the patient clearly showed progressive or non-regressive behavior toward subsequent chemotherapy. As the majority of the samples were collected in the primary debulking surgery and up to 80 % of ovarian cancer is initially sensitive to platinum-based chemotherapy, there were only 6 samples left in the resistant group. The error bars in the figure represent standard deviation; thus, overlapping of two error bars does not deny the presence of a statistically significant difference. IHC was additionally performed. The results are demonstrated in a supplementary figure  and described in the revised manuscript .Although statistically not significantly different, the resistant samples were all positive for CHD4 immunostaining and all of the CHD4 negative samples were included in the platinum sensitive group. We believe that the results support our findings. As to western blotting, the amount of stored frozen samples was substantially not enough to extract protein and subject to western blotting. We appreciate your comments on our clinical data set.They also show synergy between SMARCA5/CHD4 dual inhibitor and cisplatin. Since ED2-AD101 is a novel drug, the author should show the cytotoxicity in the normal cells vs OC cells with positive control like 5FU with SRB or MTT and clonogenic assay and the same has to be performed in CHD4 knockdown cells with cisplatin also. Cisplatin sensitivity should be performed over a concentration range 0, 0.5, 1, 2,5,10 uM for better validation.Thank you for the valuable comment. Based on your comment, we additionally performed clonogenic assays with ED2-AD101 in multiple cell lines including non-cancerous ovarian surface epithelial cells OSE2 and OSE4, and the results are summarized in a figure 3 of the additional information for the reviewers. The response to ED2-AD101 varies depending on each cell line and suppression of colony formation by ED2-AD101 was not specific to ovarian cancer cells. As CHD4 knockdown alone did not drastically suppress cancer cell proliferation as shown in the supplementary figure , the results obtained were unsurprising.In contrast to ovarian cancer cell lines, cisplatin sensitivity was not significantly increased by CHD4 knockdown in either OSE2 or OSE4 . The result indicates that an increase in the platinum sensitivity by CHD4 inhibition could be cancer-cell specific.We additionally performed the cisplatin sensitivity assay at the indicated concentrations. CHD4 knockdown significantly increased platinum sensitivity in TOV21G, JHOC5, and KURAMOCHI. The result is illustrated in S3 Fig and described in the revised manuscript .In fig 4, the authors show increased levels of both cleaved and non-cleaved PARP at 5uM of cisplatin and which makes it difficult to interpret these results. However, the authors revalidated the same in FACS analysis using a very high dose of cisplatin and claimed in CHD4 kd cells induces the cell death but author has not explained why there was an sudden increase in 5 to 20uM cisplatin concentration.Thank you for the insightful comment. As we found that PARP cleavage was not an appropriate marker for the assessment of apoptosis in this experimental setting, we preliminarily performed an apoptosis assay by flow cytometry with 5 �M of cisplatin and observed little increase in Annexin-V positive fraction regardless of CHD4 knockdown. The preliminary data is shown in a figure 4 of the additional information for the reviewers. Thus, we determined to increase the cisplatin concentration to 20 �M and successfully detected the increase in annexin V positive cell fraction by cisplatin. As DNA double-strand breaks do not always result in apoptosis, we considered that it is plausible to see the increase of Annexin V positive cells only at higher concentrations of cisplatin. Taken together, we conclude that CHD4 knockdown enhances cisplatin-mediated DNA-double strand breaks and facilitates apoptotic changes under a certain degree of DNA double strand accumulation.Although we have not shown the flow cytometry result with 5�M of cisplatin in our manuscript, as this is preliminary data, we are happy to add it as a supplementary figure if the reviewer recommends that it should be included.Although, the authors show in fig 4a, CHD4 kd with cisplatin induces phospho yH2AX, a marker for DNA damage and in ,Fig 4c CHD4 kd induces RAD51expression, a DNA repair factor, authors has to explain how cisplatin/ED2-AD101 inhibitor synergy will work on apoptosis?Thank you for the valuable suggestion. The experiment was repeated three times independently; however, a consistent trend was not observed. We also quantified the density of RAD51 and did not find a significant difference in this as well. This result has been depicted in a supplementary figure  and described in the revised manuscript.  Finally they have determined that MDR1 downregulation in CHD4 KD cells and overexpressed in cells with enhanced expression of CHD4.Over all the study does not robustly add anything to the literature on the role of CHD4 due to the lack of well accepted methods to determine cell viability such as colony forming assays. It was very difficult to determine the IC50 values since the #s are not mentioned in the text. Lack of p value in Q-PCR based siRNA results made it difficult to assess the significance. In general the clarity of the figures also was not good. The manuscript is not acceptable for publication in PLOSONE with the limited amount of data presented in its current form.Thank you for the valuable comments. We apologize that some fundamentally important information were missing in the original manuscript. In the revised manuscript, a supplementary table (S table) has been added to show the actual IC50 for each dose-response curve. The p-value in the quantitative PCR has also been added in the figure legend .Further, the results of clonogenic assays for TOV21G and JHOC5 have also been added in a supplementary figure  in addition to cell viability assay.Regarding the clarity of the figures, we confirmed whether the resolution of the original data submitted by us complied with the requirements instructed by PLOS ONE. We also found that the resolution of the PDF file was much lower than the original data. Therefore, kindly enquire with the editor regarding the provision of original files submitted, if necessary.Reviewer #3: Platinum drugs are the most effective in the ovarian cancer treatment. Platinum resistance is a critical issue to be solved. It is important to study the mechanism about platinum sensitivity or platinum resistance.　The authors first found that CHD4 mRNA expression was significantly higher in the platinum resistant samples than in the platinum sensitive samples in ovarian cancer tissues. Then, the authors investigated whether CHD4 is associated with platinum sensitivity using ovarian cancer cell lines in vitro. Suppression of CHD4 expression increased the platinum sensitivity by decreasing MDR1 expression in ovarian cancer cells.The present study clearly showed that CDH4 is associated with platinum sensitivity in ovarian cancer cells.Major comments1. Generally, the platinum sensitivity is much higher in high grade serous carcinoma than in clear cell carcinoma. According to Figure 2, there seems to be no difference in the platinum sensitivity between clear cell carcinoma cells (TOV21G and JHOC5) and high grade serous carcinoma cells (KURAMOCHI and JHOS2). Furthermore, the effect of CHD4 knockdown seems to be high in clear cell carcinoma cells compared with that in high grade serous carcinoma cells. Reviewer is wondering whether these cell-lines used in this study are suitable for evaluating platinum sensitivity in ovarian cancer cells.We appreciate your comment about cell lines. As the phenotype of ovarian cancer is quite heterogenous according to each histologic subtype, it is important to select the cell lines which retain their original biomolecular phenotype. Several studies have pointed out that many of the ovarian cancer cell lines commercially available lost their original features after a certain period of time. Some cell lines have obviously different characteristics compared to the cell lines they were derived from. As for platinum sensitivity, we consider short-term in vitro assays do not always reflect the actual response in the clinical setting. Thus, we prioritized using the cell lines that well represent the features of high-grade serous carcinoma or clear cell carcinoma. We added descriptions of the cell lines in the revised manuscript.  2. Figure 3 only showed the data from clear cell carcinoma cell lines (TOV21G and JHOC5). The data from high grade serous carcinoma cell lines (KURAMOCHI and JHOS2) should be provided or described in the result section.Thank you for your recommendation. We added the results of KURAMOCHI and JHOS2 in Fig 3 with some comments in the result section of the revised manuscript.  6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.AttachmentResponse to reviewers.docxSubmitted filename: Click here for additional data file. 20 Apr 2021CHD4 regulates platinum sensitivity through MDR1 expression in ovarian cancer: A potential role of CHD4 inhibition as a combination therapy with platinum agentsPONE-D-20-33496R1Dear Dr. Tokunaga,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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For more information, please contact Kind regards,Carlos Telleria, PhDAcademic EditorPLOS ONEAdditional Editor Comments :The authors have responded mostly in its majority to the comments of the reviewers and is now found acceptable for publication. Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: (No Response)Reviewer #3: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #3: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #3: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #3: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #3: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: 1. The authors have determined CHD4 expression in platinum sensitive and platinum resistant ovarian cancer cell lines and claimed that CHD4 expression remains consistent in all the cell lines tested . According to the authors, the lower band corresponds to CHD4 protein. However, the band is very faint, and the loading control is inconsistent to draw any conclusions. In fact, if the loading control is normalized, the cisplatin resistant A2780cis cells might demonstrate lower CHD4 expression compared to the parental cell line.2. The authors have performed clonogenic assay to determine the effect of CHD4 knockdown on cisplatin sensitivity. The results from this assay should be quantified in terms of number of colonies. However, the authors have stained the colonies in 72 hr which is a short time period to form visible colonies for quantification. Is there a reason, the authors have used a shorter time point?Reviewer #3: The manuscript has been appropriately revised according to the reviewer's comment, and is now acceptable for publication.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article (If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1: NoReviewer #3: No 1 Jun 2021PONE-D-20-33496R1 CHD4 regulates platinum sensitivity through MDR1 expression in ovarian cancer: A potential role of CHD4 inhibition as a combination therapy with platinum agents Dear Dr. Tokunaga:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofProfessor Carlos Telleria Academic EditorPLOS ONE


T cell exhaustion plays critical roles in tumor immune evasion. Novel strategies to suppress immune evasion are in urgent need. We aimed to identify potential compounds to target T cell exhaustion and increase response to immune checkpoint inhibitors (ICIs). Differentially expressed genes (DEGs) were identified between tumors with different immune evasion potential by comparing the transcriptome data. DEGs were then analyzed in the Connectivity Map (CMap) platform to identify potential compounds to increase response to ICIs. Gene set enrichment analysis, LDH release assay, Chromatin immunoprecipitation (ChIP), and Co-IP were performed to explore the potential mechanisms in vitro. Patients derived organoids and humanized xenograft mouse model were utilized to validate the finding ex vivo and in vivo. We identified 25 potential compounds that may play critical roles in regulating tumor immune evasion. We further pinpointed a specific compound, dexamethasone, which shows potent anti-tumor effect in multiple cancer cell lines when cocultured with T cells. Dexamethasone can suppress T cell exhaustion by decreasing the activity of two immune checkpoints simultaneously, including PD-L1 and IDO1. Functional study shows dexamethasone can increase the sensitivity of ICIs in coculture system, 3D organoid model and humanized mouse model. Mechanism study shows dexamethasone mediated transcriptional suppression of PD-L1 and IDO1 depends on the nuclear translocation of GR/STAT3 complex. These findings demonstrate dexamethasone can suppress immune evasion by inducing GR/STAT3 mediated downregulation of PD-L1 and IDO1 pathways. T cell immunity against cancers is regulated by co-stimulatory signals and co-suppressive signals. The co-suppressive signals are called immune checkpoints, which weaken T cell functions and play critical roles in tumor immune escape . The immCancer Cell Line Encyclopedia (CCLE) is an open database consisting of genomic, transcriptomic and methylation data of over 1000 cancer cell lines covering 24 types of cancer . The CanIn the current study, we carried out cross-database analysis on multiple immune-related molecules and potential targeted therapies. A total of 25 potential compounds were identified, which may play critical roles in regulating tumor immune evasion. Among them, dexamethasone shows potent anti-tumor effect in multiple cancer cell lines when cocultured with T cells. Dexamethasone can decrease T cell exhaustion by suppressing the activity of two immune checkpoints simultaneously, including PD-L1 and IDO1. Functional study shows dexamethasone can increase the sensitivity of ICIs in coculture system, 3D organoid model and humanized mouse model. Mechanism study shows dexamethasone mediated transcriptional suppression of PD-L1 and IDO1 depends on the nuclear translocation of GR/STAT3 complex. These findings suggest dexamethasone as a potential regimen to increase sensitivity of ICIs.https://portals.broadinstitute.org/ccle) and TCGA . The mRNA levels of 10 critical regulators of tumor immune evasion were manually curated, including BTLA, VISTA, CD160, PD-L1, CTLA4, IDO1, LAG3, LGALS9, TNFRSF14, and VTCN1. Then we performed correlation analysis between these regulators. Those with correlation coefficients larger than 0.3 or less than −0.3 were presented by heatmap using “pheatmap” package in R. The CMap database (RRID:SCR_016204) was used for matching chemicals based on 150 differential expressed genes between PD-L1(+)/IDO1(+) vs PD-L1(−) /IDO1(−) GC or CRC cancer cells.Transcriptomic data was obtained from CCLE  and Pembrolizumab  were purchased from SELLECK. Other chemicals and antibodies were listed in Supplementary Table Cell lines were obtained from American Type Culture Collection, Japanese Collection of Research Bioresources Cell Bank and several other cell banks  for 30 min, and then, minced for enzymatic digestion by 1.5 mg/mL collagenase IV  for 1 h at 37 °C. Cells were washed twice by advanced DMEM/F12, and filtered by 40 µm cell strainer . After centrifuged for 5 min at 1000 rpm, cells were seeded into Matrigel  and overlayed with human IntestiCult™ organoid growth medium  containing 10 µM Y-27632, 1% Primocin and 1% penicillin–streptomycin. TrypLE™ Express Enzyme  was used to digest and resuspended cancer cells for organoid passages.GC tissues were obtained from Department of Surgery, Ruijin Hospital of Shanghai Jiaotong University School of Medicine. No chemotherapy, radiotherapy, or immunotherapy was received pre-surgery. This study was approved by the Research Ethics Committee of Shanghai Ruijin hospital. The written informed consents were signed by all patients. A 1 cm6) were washed with PBS, and harvested with immunoprecipitation lysis buffer . Antibodies to STAT3, GR, and magnetic beads  were added to lysates and incubated for 1 h at 4 °C. After washing three times with immunoprecipitation lysis buffer, the precipitates were analyzed with antibody against STAT3 or GR. The working dilutions of antibodies were stated in Supplementary Table After incubation with 50 nM dexamethasone for 48 h, cancer cells (2 × 106) were cross-linked with formaldehyde at a final concentration of 1% for 10 min at room temperature, then stopped by glycine. Chromatin was sonicated in the lysis buffer to 100–300 bp. One-tenth of the total lysate was removed to use as an input DNA control. Supernatant fraction was pre-cleared with 50% slurry of Salmon Sperm DNA/Protein A/G agarose for 1 h at 4 °C on a rocker. Immunoprecipitation was performed with anti-STAT3 and control rabbit IgG  was used. Briefly, cancer cells  was used for library construction. Illumina HiSeq Xten  was used for RNA-seq. Gene counts were normalized to transcripts per million (TPM). Gene Set Enrichment Analysis (GSEA) was performed for pathway analysis by “clusterProfiler” package in R . The top 20 pathways were displayed by bubble diagram utilizing “ggplot2” package in R .The lactate dehydrogenase (LDH) releasing was evaluated as cell damage by LDH Assay Kit-WST CK12 . Injury rate (%) of cancer cells was calculated as: /.Cell Counting Kit-8 kit was purchased from DOJINDO . Cancer cells (5000) or PBMCs  were placed on 96-well plates (100 μl/well). The experiments were performed as previously described . After cThe kynurenine was analyzed by ultra-performance liquid chromatography mass spectrometry (UPLC/MS). The cell medium of 300 μl was mixed with 600 μl acetonitrile for 10 min rotation, and centrifuged at 12000 rpm for 10 min. A 1 μl supernatant was injected into a 2.1 × 100 mm column of ACQUITY UPLC BEH Amide. The mobile phase contained 0.1% formic acid (solvent A) and acetonitrile (solvent B) at a flow rate of 0.3 ml/min at 40 °C. Chromatography was performed as following: 5% A, held for 1 min; 1–3 min, increased to 50% A; 3–5 min, held at 50% A; 5–5.1 min, decreased to 5% A; 5.1–7 min, held at 5% A. Mass spectrometry was performed in the conditions of source temperature 400 °C, capillary voltage 1.5 kV, cone voltage 10 V. The Cone Gas Flow was 10 L/Hr, and the Desolvation Gas Flow was 800 L/Hr. High-purity helium and nitrogen were used for the collision gas. The Masslynx mass spectrometry data acquisition software  was used for extracting m/z, retention time and ion intensity. All spectra were aligned and normalized to the total peak intensity.Peripheral blood was got from a healthy donor who has signed informed consent. Peripheral blood mononuclear cells (PBMCs) were separated using FICOLL PAQUE PLUS . Non-adherent cells were harvested and suspended in AIM-V medium supplemented with 1000 U/ml IFN-γ on day 1. Then, human CD3 NALE HIT3a (100 ng/ml) and human recombinant IL-2 (300 U/ml) were added at day 2 and day 3. The cell culture medium was half replaced by AIM-V medium containing 10% FBS, IL-2 (200 U/ml), IL-7 (25 ng/mL), and IL-15 (10 ng/mL) every 2-day.n = 5 per group) according to literature and our previous studies. Investigators were blinded to the group allocation. In this study, animal experiment was performed on mouse model with reconstituted immune system [7) via tail veins at 1st and 14th day. One day after PBMCs infusion, 100 µL mixture of SGC-7901 or SW1990 (5 × 106) and Matrigel was inoculated subcutaneously. Dexamethasone  was injected intraperitoneal every three days, and pembrolizumab (5 mg/kg) was injected via tail vein every week. The same experiments of SGC-7901 were also performed in non-immune reconstituted NOD/SCID model, which was not injected human PBMCs to humanize the immune system.Female NOD/SCID mice (4-week-old) were used . The animal experiments were approved by the Research Ethics Committee of Shanghai Jiaotong University School of Medicine. Simple randomization was applied to allocate the mice into different groups  staining and immunohistochemistry. Cancer organoids were embedded in Optimum Cutting Temperature Compound (OCT), and cut into 6-µm thick for hematoxylin–eosin (HE) and protein expression analysis . The wort test was carried out in GraphPad Prism 6.0  unless otherwise specified. The variance is similar between the groups that are being statistically compared. Correlation diagrams were plotted by hierarchical clustering by “corrplot” package in R software. Gene ontology (GO) analysis was performed using online tool Metascape . Alpha was set to 0.05 in the power analysis. P value less than 0.05 was considered significant. RNA-seq data of cancer cells treated by dexamethasone was uploaded to the GEO repository (GSE134195).All results are expressed as mean ± 95% CI from three repeats. Pearson correlation analysis of PD-L1 and IDO1 was performed. The student’s P < 0.05), and were 0.224 and 0.354 in CRC   and 0.783 (P < 0.001), respectively  Fig. . We furtely Fig. .Fig. 1Pon = 13, CRC: n = 19), PD-L1(−)/IDO1(+) , PD-L1(+)/IDO1(−)  and PD-L1(+)/IDO1(+) . We collected the top 150 upregulated genes in PD-L1(+)/IDO1(+) tumors compared to PD-L1(−)/IDO1(−) tumors, and applied them for CMap analysis. A series of connective compounds mimicking the phenotypes were identified. According to the connectivity scores, we selected compounds negatively regulating PD-L1 and IDO1 in GC and CRC (connectivity score <−90), and identified top 25 compounds as potential inhibitors for PD-L1 and IDO1  and CRC (correlation coefficient is 0.354), we further analyzed the transcriptomic data of 38 GC cell lines and 61 CRC cell lines in CCLE database. We set median value of PD-L1 or IDO1 as cutoff value, and stratified them into four groups: PD-L1(−)/IDO1(−) . We pretreated cancer cells and PBMCs with a gradient concentration of dexamethasone and identified the most effective concentration as 50 nM  and “resistant cell lines” (RKO and MV3) according to their response to dexamethasone. NCIH-747 is an intermediate responsive cell line to dexamethasone. Then we performed RNA-seq on these cell lines treated with dexamethasone and identified differentially activated and inactivated pathways Fig. . The resPD-L1 (ch9:5449027-5449342) and IDO1 (ch8:39770147-39770462) via UCSC ChIP database (https://genome.ucsc.edu/index.html)  showed higher expression of phosphorylated STAT3 and glucocorticoid receptor (GR) than resistant cell lines (RKO and MV3) Fig. . We founml) Fig. , and furml) Fig. .We successfully constructed organoids from the above GC tissue with high activity of PD-L1 and IDO1 pathway. Organoids expressed epithelial marker CK19, stomach specific marker H+/K+ ATPase, but not muscular marker SMA Fig. . Dexamet7) at 1st day and 14th day via tail veins  were inoculated subcutaneously. Dexamethasone was given intraperitoneally from the 3rd day, and repeated every 3 days (Fig. To verify the efficacy of dexamethasone on enhancing efficacy of ICIs in vivo, we constructed humanized mouse model Fig. . After tins Fig. . One dayays Fig. . At the ays Fig. . Dexametays Fig. . CD8+ ceays Fig. . To illuays Fig. –G. Synerays Fig. –C. Theseays Fig. .Fig. 6PDThe therapeutic efficacy of immune checkpoint inhibitors has drawn great attentions in the field of cancer research . HoweverDrug repositioning is an emerging field. There are several publicly accessible databases in this area . CMap isPD-L1 and IDO1 are regulated by JAK/STAT, MAPK, and PI3K/AKT pathways –44, whicThe strengths of this study are: the usage of multiple cancer cell lines, the incorporation of bioinformatic screening and in vitro study and the validation in the ex vivo and in vivo models. This study also has limitations, including the validation of the efficacy of dexamethasone in patients receiving ICIs is lacking and the mechanism of the positive correlation between PD-L1 and IDO1 remains uncharacterized.In conclusion, we identified dexamethasone as a potent suppressor of tumor immune evasion, which can target PD-L1 and IDO1 simultaneously. Dexamethasone can increase the sensitivity of ICIs in vitro, ex vivo and in vivo by triggering nuclear translocation of GR/STAT3 and transcriptional suppression of PD-L1 and IDO1. This study provides preclinical evidence of dexamethasone as a potential treatment to increase response to ICIs.Supplementary figuresSupplementary Table S1Supplementary Table S2Supplementary Table S3


S,24S)-24-methyl-22,25-epoxyfurost-5-ene-3β,20β-diol (16), 3β,11-dihydroxy-24-methylene-9,11-secocholestan-5-en-9-one (23), (24S)-ergostane-3β,5α,6β,25 tetraol (146), sinulerectadione (227), sinulerectol C (229), and cladieunicellin I (277) exhibited stronger cytotoxicity than their respective positive control and that their mechanism of action has not yet been further investigated.The octocoral family Alcyoniidae represents a rich source of bioactive substances with intriguing and unique structural features. This review aims to provide an updated overview of the compounds isolated from Alcyoniidae and displaying potential cytotoxic activity. In order to allow a better comparison among the bioactive compounds, we focused on molecules evaluated in vitro by using the 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, by far the most widely used method to analyze cell proliferation and viability. Specifically, we surveyed the last thirty years of research, finding 153 papers reporting on 344 compounds with proven cytotoxicity. The data were organized in tables to provide a ranking of the most active compounds, to be exploited for the selection of the most promising candidates for further screening and pre-clinical evaluation as anti-cancer agents. Specifically, we found that (22 Marine environments, and especially coral reefs, are among the richest and most complex Earth ecosystems in terms of biodiversity . Althoug132) is an allelopathic molecule usually found in Sarcophyton and used by this soft coral genus in the competition for space with scleractinian corals -3-methyldodec-2-enamide, 5α,7αH-eduesm-11(13)en-4α-ol, 24-methylenecholesterol, and 1β-hydroxy-α-cyperone were isolated from Sinularia sp. by Sheu et al. -3-methyldodec-2-enamide (228) exhibited cytotoxicity toward CCRF-CEM and MOLT-4 cells with IC50 values of 6.3 ± 1.5 and 9.7 ± 3.6 μM, respectively. Moreover, sinulerectol C (229) showed cytotoxicity toward the K-562 cell line with an IC50 value of 9.2 ± 3.3 μM. The metabolites displayed comparable and even higher cytotoxicity than the positive control 5-fluorouracil. Four new isoprenoids, including norcembranoids (sinulerectols A and B), a cembranoid (sinulerectol C), and a degraded cembranoid (sinulerectadione), along with three known isoprenoids  and a known nitrogen-containing compound, were isolated from an extract of g et al. . The act205), a known active compound isolated from S. flexibilis, against two human gastric cancer cell lines, AGS and NCI-N87. Sinularin at the concentrations of 3, 6, 12, and 18 μM showed a dose-dependent effect on these two cell lines. The effectively reduced cell proliferation of sinularin on AGS cells is 52%  and on NCI-N87 cells it is 53.5% .Wu et al.  investigS. leptoclados by Ngoc et al. [230) was found to be active against HepG2 (Hepatocellular carcinoma) and SW480 (colon adenocarcinoma) cells with IC50 values of 21.13 ± 0.70 and 28.65 ± 1.53 µM, respectively. Moreover, ergost-5-en-3β,7β-diol (231) resulted cytotoxic against HL-60 (acute leukemia) and SW48 cell lines with IC50 values of 20.53 ± 2.26 µM and 26.61 ± 1.59 µM, respectively. Moreover, 3β,7β-dihydroxyergosta-5,24(28)-diene (232) showed cytotoxic activity on all tested cell lines with IC50 values ranging from 13.45 ± 1.81 to 29.01 ± 3.21 μM.Fifteen steroids, including two new compounds, leptosteroid and 5,6β-epoxygorgosterol, were derived from c et al. . LeptostSinularia brassica [233), J (234), and K (235), exhibited activity toward the four cell lines with IC50 values ranging from 8.7 ± 1.4 to 39.8 ± 7.7 μM. Moreover, also sinubrasolide A (236) was found to display activity against the above cancer cells with IC50 of 29.9 ± 3.0 μM, 12.1 ± 1.1 μM, 8.7 ± 1.4 μM, and 18.7 ± 2.5 μM, respectively.brassica  containeSinularia brassica [237) exhibited higher cytotoxicity against all three tested cell lines with IC50 values of 27.12 ± 1.69, 24.64 ± 1.28, and 20.51 ± 2.72 μM, respectively, compared to that of the positive controls, camptothecin on A-459 (IC50 of 12.65 ± 1.01 μM), etoposide on HeLa (IC50 of 27.99 ± 2.01 μM), and PANC-1 (IC50 of 1.17 ± 0.42 μM). Cytotoxicity was also observed for sinubrassione (238) against PANC-1 and A-549 cells with IC50 values of 15.24 ± 0.38 and 39.36 ± 0.72 μM. Moreover, ergosta-1α,3β,5α,6β,11α-pentaol (239) and pregnedioside A (240) showed variable cytotoxic activity toward the three cancer cell lines with IC50 values from 16.59 ± 1.77 to 41.76 ± 1.76 μM, while ergosta-1β,3β,5α,6β-tetraol (241) displayed activity against PANC-1 with IC50 value of 15.39 ± 1.09 μM.Six ergostane-type steroids, including one new compound (sinubrassione), and two pregnene-type steroid glycosides, including the new sinubrassioside, were isolated from methanol extracts of brassica . The natSinularia terspilli. Ergost-24(28)-ene--triol, (242), ergost-24(28)-ene--pentol, (243), and ergost-24(28)-ene--tetra-acetyl-5-ol, (244) with IC50 values of 0.004, 0.002, and 0.025 µM, respectively for human acute leukemia HL-60 cells and 0.005, 0.003, and 0.04 µM for the human chronic myelogenous leukemia K-562 cell line compared to taxol, which displayed IC50 values of 0.0005 and 0.0023 µM against HL60 and K562, respectively. The other metabolites were less active with IC50 values ranging from 0.2 to 0.4 µM. The structures of the cytotoxic compounds 217–244 derived from the genus Sinularia are shown in Mohammed et al.  isolated245), a sesquiterpenoid isolated by Huong et al. [Sinularia erecta, exhibited selective cytotoxicity against the A-549 cell line with IC50 of 14.79 ± 0.91 μM, relative to that of the positive control, camptothecin, with an IC50 value of 11.42 ± 0.13 μM.3β,5α-dihydroxyeudesma-4(15),11-diene , human T-lymphoid (MOLT-4), human erythroleukemia (K-562), and human colon carcinoma (HT-29), were evaluated and sinubrasones B (247) and C (248) were found to show cytotoxicity against all cell lines , while sinubrasone A (246) and D (249) exhibited only cytotoxic activity toward the four cancer cell lines.A chemical investigation  of Sinul250), extracted from Sinularia compacta by Wang et al. [50 values of 10.1 and 14.7 μM, respectively.A known compound, 5-episinuleptolide , a known active compound isolated from S. flexibilis, exhibited IC50 values of 17.5 ± 6.7 μM and 43.2 ± 8.1 μM against HepG2 and Hep3B, respectively [Sinularin (ectively .Sinularia nanolobata [251) was found to be cytotoxic against P-388, A-549, and HT-29 cancer cell lines with ED50 values of 1.0, 1.2, and 0.5 μg/mL, respectively.A novel tetranorditerpenoid (sinubatin A), a new norditerpenoid (sinubatin B), and a known diterpenoid (gibberosin J), were isolated from nolobata . Only gi252), isolated from Sinularia cf. molesta, was found to be cytotoxic against HeLa and HCT-116 cell lines with IC50 values of 5.26 and 8.37 μM, respectively [A new cembranolide, molestin E , ximaosteroid F (254), and (20S)-20-hydroxycholest-1-ene-3,16-dione (255) were found to show selective cytotoxicity against the HL-60 cancer cell line with IC50 values of 1.79, 4.03, and 0.69 μM, respectively.Two steroids, ximaosteroid E and ximaosteroid F, along with two known compounds, were extracted from i et al. . XimaostSinularia flexibilis by Wu et al. [197), 11-episinulariolide acetate (202) and 14-deoxycrassin (44) were found to inhibit the proliferation of P-388 (murine leukemia), K-562 (human erythromyeloblastoid leukemia), and HT-29 (human colon carcinoma) cell lines with 11-episinulariolide acetate (202) exhibiting the most potent cytotoxicity with IC50 values ranging from 6.9 μM to 12.2 μM. However, the new compounds were nontoxic toward these cancer cell lines .Five new cembranoid-related diterpenoids, flexibilisins D and E, secoflexibilisolides A and B, and flexibilisolide H, along with nine known compounds, were isolated from u et al. . The knoSinularia sp., sinuketal, showed a cytotoxic toward MDA-MB-231 and U2OS cancer cell lines . Moreover, the known cembranoid 5-episinuleptolide (250) exhibited cytotoxicity against HeLa and HCT-116 with IC50 values of 11.6 and 33.3 μM, respectively.A sesquiterpenoid isolated by Qin et al.  from Sin256, 257) were isolated from the soft coral Sinularia sp. The metabolites exhibited anti-proliferation effect against five human cancer cell lines, including MDA-MB-436, A-549, Hep3B, HT-29, and H157 with IC50 values ranging from 10.14 to 41.71 µM [Two new highly oxygenated ergostane-type sterols (41.71 µM .S. flexibilis. The compounds were tested against A-549 (lung adenocarcinoma), HT-29 (colonic carcinoma), SNU-398 (hepatocellular carcinoma), and Capan-1 (pancreatic carcinoma) human cancer cell lines. 5-dehydrosinulariolide (258) was found to exhibit cytotoxicity against all the above four cell lines with IC50 values of 27.4, 22.7, 8.9, and 9.4 µM, respectively, while 11-episinulariolide acetate (202) and sinulariolide (165) showed cytotoxicity against HT-29, SNU-398, and Capan-1 cells with IC50 values ranging from 24.9 to 33.6 µM.Wu et al.  isolatedSinularia erecta by Liu et al. [259) were found to exhibit activity against the proliferation of A-549, HT-29, SNU-398, and Capan-1 cancer cell lines with IC50 values of 40.55 ± 7.51, 32.83 ± 14.81, 15.57 ± 5.26, and 23.51 ± 10.46 μM, respectively.Two novel steroidal derivatives, erectsterates A and B, were isolated from u et al. . ErecsteSinularia polydactyla. Among the tested molecules, 7β-acetoxy-24-methyl-cholesta-5,24(28)-dien-3β,19-diol (260) and 7β-acetoxy-cholest-5-en-3β,19-diol (261) showed cytotoxic activity against HeLa  and MCF-7  cancer cell lines as compared to the control cisplatin . However, the compounds also showed cytotoxicity toward normal cells (BJ) with IC50 values of 14.8 ± 5.8 and 14.5 ± 3.9 μM, respectively.Tammam et al.  isolatedSinularia variabilis by Pour et al. [50 values of 31.44 ± 1.17 and 25.67 ± 1.05 μM, respectively. The structures of the cytotoxic compounds 245–261 derived from the genus Sinularia are shown in A new steroid, 16,17-epoxy-23-methylergostane, isolated from r et al. , was fouCladiella has proven to be a rich source of cytotoxic eunicellin-based diterpenoids. In 2005, Ahmed et al. [Cladiella australis four new eunicellin-based diterpenoids, australins A–D. Australin B (262) was found to be cytotoxic against MDA-MB-231 (human breast adenocarcinoma), MCF-7 (human breast carcinoma), and HepG2 DMEM-12 (human liver carcinoma) cell lines , while australin D (263) showed only activity toward MDA-MB-231 (ED50 24.4 µg/mL).The genus d et al.  extracteS)-3β-acetoxyergost-5-en-21-oic acid, 5′-O-acetylthymidine, 3′-O-acetylthymidine, and p-vinylbenzyl alcohol, along with a known steroid, (24S)-3β-hydroxyergost-5-en-21-oic acid (264), from the EtOAc extract of C. australis. These compounds were tested against HepG2, Hep3B, MCF-7, and MDA-MB-231 cancer cell lines and the results showed that the known steroid (264) was the most potent toward the cancer cells with IC50 values ranging from 2.2 to 14.5 µg/mL.Ahmed et al.  also isoCladiella hirsuta and extracted eight new eunicellin-based diterpenoids, hirsutalins A–H. The cytotoxicity of metabolites against a panel of cancer cell lines including human liver carcinoma (HepG2 and Hep3B), human breast carcinoma (MDA-MB-231 and MCF-7), human lung carcinoma (A-549), and human oral cancer cells (Ca9-22) was investigated. The results showed that hirsutalin A (265) had a cytotoxicity toward Hep3B, A-549, and Ca9-22 cell lines with IC50 values of 29, 28, and 35 μM, respectively. Moreover, hirsutalin E (266) showed to cytotoxicity  toward the proliferation of Hep3B, MDA-MB-231, MCF-7, A-549, and Ca9-22 cells, respectively, and cytotoxicity against the HepG2  cell line. Finally, hirsutalin F (267) exhibited cytotoxicity toward HepG2, Hep3B, and MCF-7 cell lines with IC50 values of 29, 29, and 32 μM, respectively. Chen et al.  investigCladiella sp. Cladielloide B (268) showed cytotoxicity toward CCRF-CEM (human T-cell acute lymphoblastic leukemia) and DLD-1  cells with IC50 value of 4.7 and 10.2 μM, respectively. The same authors [Cladiella sp. Cladieunicellin B (269) were found to be cytotoxic toward DLD-1 cancer cells with a IC50 value of 2.0 μM and cladieunicellin E (270) showed cytotoxicity against the HL-60 leukemia cell line with a IC50 value of 2.7 μM. Chen et al. [C. hirsuta. The cytotoxicity of these products against the proliferation of a limited panel of cancer cell lines, including human liver (HepG2 and Hep G3B), breast (MDA-MB-23), and gingival (Ca9-22) carcinoma cells, was evaluated. Hirsusterol A (271) was the most potent one toward HepG2, Hep G3B, MDA-MB-23, and Ca9-22 cancer cell lines with IC50 values of 16.9, 9.4, 8.2, 18.4, 17.8, and 16.1 μM, respectively. Chen et al.  isolated authors  also ison et al.  also isoR,2R,3R,6S,7S,9R,10R,14R)-3-butanoyloxycladiell-11(17)-en-6,7-diol from Cladiella krempfi. The cytotoxicity of diterpenoids toward lung adenocarcinoma (A-549 and H1299), breast carcinoma (BT-483), liver carcinoma (HepG2), oral cancer (SAS), and human lung bronchial (BEAS-2B) cell lines was evaluated. Only the known metabolites resulted cytotoxic: litophynol B (272) showed activity against the proliferation of H1299 and BT-483 cancer cells  and -3-butanoyloxycladiell-11(17)-en-6,7-diol (273) exhibited cytotoxicity toward A549, BT483, and SAS cancer cell lines . Furthermore, this study also highlighted that these two compounds were non-cytotoxic toward the normal cell BEAS2B.Tai et al.  isolatedC. krempfi, krempfielins E–I, and seven known compounds. Krempfielin I (274), litophynol B (272), 6-acetoxy litophynin E (275), and litophynin F (276) displayed cytotoxicity toward A-549, BT-483, H1299, HepG2, and SAS cancer cells with 6-acetoxy litophynin E (275) being the most potent one . Unfortunately, except for krempfielin I (274), these compounds were also cytotoxic for the human normal cell line BEAS-2B.Tai et al.  also iso277), a new eunicellin diterpenoid isolated from Cladiella sp. by Cheng et al. [50 value of 1.59 μM, being more effective than the positive control doxorubicin (IC50 of 10.98 μM).Cladieunicellin I  resulted in cytotoxic towards Molt-4 (human acute T lymphoblastic leukemia) cells with a IC50 value of 14.42 μM.Cladieunicellin K and cladieunicellin L were extracted from h et al.  and cladCladiella sp. Cladieunicellins M (279), O (280), and Q (281) showed a cytotoxicity toward Molt-4 cancer cells with IC50 values of 16.43, 14.17, and 15.55 μM, respectively. Chen et al. [282) from Cladiella sp. The natural product was cytotoxic toward K-562, Molt-4, CCRF-CEM, and DLD-1 cancer cells with IC50 values of 10.9, 6.6, 4.3, 13.4 μg/mL, respectively.Chen et al.  isolatedn et al.  also iso283), isolated from C. hirsuta by Huang et al. [50 values of 13.8 and 36.3 μg/mL, respectively, which compared to those of the positive control 5-fluorouracil  showed cytotoxicity. Unlike the drug, however, hirsutalin R (283) was inactive on HT-29 (human colon adenocarcinoma) and A-549 (human lung adenocarcinoma) cell lines.Hirsutalin R , B (285), and two new α-tocopherylhydroquinone glycosides, cladophenol glycosides A (286) and B (287) were isolated from C. hirsuta by Chao et al. [50 values ranging from 10.2 to 39.3 μM. However, hirsutosteroside A (284) was inactive against the growth of P388 and HT-29 cells and hirsutosteroside B (285) against the proliferation of A-549 cells.Two new pregnane glycosides, hirsutosterosides A , a new eunicellin-type diterpenoid isolated from Cladiella tuberculosa by Peng et al. [50 values of 6.04, 6.80, and 6.90 μM, respectively.Cladieunicellin S  and sclerophytin B (290) were found to exhibit cytotoxicity toward the leukemia K-562 cells . Moreover, cladieunicellin V (291) displayed cytotoxicity toward the leukemia MOLT-4 cells (IC50 of 18.8 μg/mL). The structures of the cytotoxic compounds 262–291 derived from the genus Cladiella are shown in Cladieunicellin U, along with a new natural eunicellin, cladieunicellin V, and two known analogues, sclerophytins A and B, were obtained from n et al. . CladieuKlyxum simplex and isolated nine eunicellin-based diterpenoids, simplexins A–I, and studied cytotoxicity against human medulloblastoma (Daoy), human breast carcinoma (MCF-7), human cervical epithelioid (HeLa), and human laryngeal (Hep 2) carcinoma cells. The results showed that simplexin E (294) exhibited cytotoxicity (in comparison to the positive control doxorubicin) toward the four tested cancer cell lines  and simplexins A (292) and D (293) displayed cytotoxicity toward Daoy and MCF-7 cancer cell lines . Moreover, simplexin A (292) displayed cytotoxicity against Hep2 cancer cells with IC50 values of 12.10 μg/mL, while simplexins B, C, F, and I were not cytotoxic toward the above cancer cells.Wu et al.  investigK. simplex by Chen et al. [295) and H (296) were cytotoxic toward HepG2 and Hep3B (human hepatocellular carcinoma), MDA-MB-231 and MCF-7 (human breast carcinoma), A-549 , and Ca9-22  cell lines with IC50 values ranging from 1.8 to 6.9 μg/mL. The results revealed that the α,β-unsaturated ketone in klysimpplexin B (295) might be able to enhance the cytotoxicity.Eight eunicellin-based diterpenoids, klysimplexins A–H, were isolated from the soft coral n et al. . KlysimpK. simplex. The results show that klysimplexins Q (297) and T (298) exhibited cytotoxicity toward HepG2, Hep3B, MDA-MB-231, MCF-7, A-549, and Ca9-22 cell lines with IC50 values ranging from 26.4 to 53.2 μM.Chen et al.  also extK. simplex. Simplexin R (300) showed activity against the proliferation of human chronic myelogenous leukemia (K-562), human T-cell acute lymphoblastic leukemia (CCRF-CEM), human breast carcinoma (T-47D), and human acute lymphoblastic leukemia (MOLT-4) cell lines with ED50 values of 7.2 ± 2.4, 2.7 ± 0.1, 13.5 ± 2.8, and 3.8 ± 0.5 µg/mL, respectively. Moreover, simplexins P (299), S (301), and A (292) exhibited to cytotoxicity against CCRF-CEM and MOLT-4 cancer cells with ED50 values ranging from 12.0 ± 1.6 to 30.3 ± 3.4 µg/mL.Wu et al.  isolatedKlyxum molle. Klymollin M (302) was found to inhibit the proliferation of K-562, Molt-4, and T-47D cancer cell lines with ED50 values 7.97 ± 2.55, 4.35 ± 0.63, and 8.58 ± 1.72 μM, respectively.Lin et al.  isolatedK. molle [290) showed activity against the proliferation of CCRF-CEM, K-562, Molt-4, and T-47D cells , and klymollin W (303) exhibited cytotoxicity toward CCRF-CEM, Molt 4, and T47D cancer cell lines with ED50 values of 9.6, 8.5, and 19.9 μg/mL, respectively.Five new eunicellin-based diterpenoids, klymollins T–X, along with two known compounds were isolated from K. molle . Among tKlyxum flaccidum [304) showed selective cytotoxicity toward the growth of A-549  cells with ED50 7.7 μg/mL, while klyflaccisteroid E (307) showed the strongest cytotoxicity toward the HT-29 (human colon adenocarcinoma) cell line (ED50 6.9 μg/mL). Klyflaccisteroids C (305) and E (307) displayed the strongest cytotoxic activity against A-549 and P-388 (murin lymphocytic leukemia) cells with ED50 values of 6.1 and 3.7 μg/mL, respectively. Also, the known steroid 9,11-secosteroid 3β,11-dihydroxy-9,11-secogorgost-5-en-9-one (308) exhibited cytotoxicity against HT29 (IC50 of 13.9 μg/mL), A-549 (IC50 of 12.5 μg/mL), and P-388 (IC50 of 7.1 μg/mL) cell lines, whereas klyflaccisteroid F (309) was selectively cytotoxic against A-549 cancer cells with a IC50 value of 14.5 μg/mL. Moreover, only klyflaccisteroid C (305) e D (306) were active against the K-562 (chronic myelogenous leukemia) cell line with ED50 of 17.3 and 12.9 μg/mL, respectively.Six new steroids, klyflaccisteroids A–E and the new 9,11-secogorgosteroid klyflaccisteroid F, along with two known steroids and a known eunicellin-based diterpenoid, were extracted from laccidum . Klyflac310) and klyxumollin D (311), isolated from K. molle exhibited cytotoxicity against the CCRF-CEM cancer cell line, with ED50 values of 17.6 and 9.9 μg/mL, respectively [Two new eunicellin-derived diterpenoid, klymollin Z (ectively .K. flaccidum [313) was found to be cytotoxic toward the growth of HT-29, P-388, and K-562 cancer cells with IC50 values of 15.1, 14.8, and 12.7 μM, respectively. Moreover, Klyfaccisteroid H (312) showed selective inhibition toward P-388 cells .Another chemical investigation on laccidum  led to tK. flaccidum. Klyflaccicembranol B (314), F (316), and H (317) showed cytotoxicity against A-549 human cancer cell lines  in comparison to the positive control Fluorouracil with a IC50 value of 110 μM. Klyflaccicembranol B (314), D (315), and H (317) exhibited cytotoxicity toward the growth of K-562 human cancer cells . Moreover, klyflaccicembranol H (317) and I (318) were found to be active against P-388 murine cancer line cells with IC50 values of 34.6 and 25.9, respectively. Ahmed et al.  discoverK. flaccidum led to the isolation of new steroids, klyflaccisteroids K–M. Klyflaccisteroid K (319) possessed to cytotoxicity (in comparison to the positive control Doxorubicin) toward HT-29, K-562, A-549, and DLD-1 cancer cell lines , while klyflaccisteroid M (320) exhibited cytotoxicity against HT-29 and P388 cells, with IC50 values of 36.7 and 31.8 μM, respectively.Chemical investigation  on K. flK. flaccidum, by Tseng et al. [321) was found to inhibit the growth of human lung adenocarcinoma (A-549), human colorectal adenocarcinoma (DLD-1), and mouse lymphocytic leukemia (P-388D1) cells with IC50 values of 9.7 ± 1.2, 6.0 ± 0.4, and 7.2 ± 1.8 µg/mL, respectively. Moreover, 14-O-acetylsarcophytol B (322) showed cytotoxicity against the above cancer cells , whereas 7-epipavidolide D (323) displayed cytotoxicity toward the tested cells and flaccidodioxide (324) was found to be active only against P-388D1 cells. The structures of the cytotoxic compounds 292–324 derived from the genus Klyxum are shown in Two new capnosane-based diterpenoids, flaccidenol A and 7-epipavidolide D, two new cembranoids, flaccidodioxide and flaccidodiol, and three known compounds were characterized from g et al. . Flaccid325) was isolated from the soft coral Alcyonium patagonicum and was found to be cytotoxic against the P-388 cell line with an IC50 value of 1 µg/mL [A new sterol, 24-methylenecholest-4-ene-3β,6β-diol  displayed cytotoxicity against the P-388 cancer cell line with a IC50 of 7.8 μg/mL.Seo et al.  extracte327) and B (328), isolated from Alcyonium aureum, were found to be cytotoxicity against P-388 (murine leukemia), A-549 (human lung carcinoma), MEL-28 (human melanoma), and HT-29 (human colon carcinoma) cell lines with IC50 values of 1 µg/mL for almost all cancer cells [Xenicane-type diterpenoid, zahavin A  showed cytotoxicity toward the Hep-2 (human larynx carcinoma) cell line , while alcyopterosins A (329), C (330), and H (332) were found to be cytotoxic toward the HT-29 (human colon carcinoma) cell line with a IC50 value of 10 μg/mL.Fifteen illudalane sesquiterpenoids, alcyopterosins A–O were isolated from o et al. . Alcyopt333), was isolated from Bellonella albiflora [A new diterpene, (Z)-sarcodictyin A -3-oxocholesta-1,4,24-trien-26-oate (334) and methyl (22E)-3-oxo-24-norcholesta-1,4,22-trien-26-oate (335) displayed activity against MDA-MB-231 (breast adenocarcinoma), A-549 (lung carcinoma), and HT-29 (colon adenocarcinoma) cell lines with GI50 values ranging from 15.3 to 22.4 μM. Moreover, methyl -22-acetoxy-3-oxocholesta-1,4,24-trien-26-oate (336) exhibited cytotoxicity toward MDA-MB-231 (GI50 of 18.4 μM), while (20S)-20-hydroxyergosta-1,4,24(28)-trien-3-one (337) against A-549 cells (GI50 of 23.4 μM).Seven new steroids were isolated from the Antarctic species o et al. . Methyl Paraminabea acronocephala. The cytotoxicity of metabolites against HepG2, Hep3B, MDA-MB-231, MCF-7, and A-549 cancer cells was evaluated. The results showed that paraminabeolide A (338) and minabeolide-1 (340) were selectively cytotoxic toward the HepG2 cancer cell lines with IC50 values of 8.0 and 5.2 μM, respectively. Moreover, paraminabeolide B (339) was cytotoxic toward MDA-MB-231 and MCF-7 cancer cells with IC50 values of 19.3 and 14.9 μM, respectively [Six new withanolides, paraminabeolides A–F, along with five known compounds, minabeolides-1, -2, -4, -5, and -8, were isolated from ectively .Paraminabea sp. and reported one new compound (341) and five known cholic acid type keto-steroids possessing enone or dienone A-rings and desmosterol. All compounds resulted cytotoxic against NBT-T2  cells with IC50 values of 6.7, 3.1, 17, 5.6, 3.2, 2.1, and 12 μg/mL, respectively.Uddin et al.  investigParaminabea acronocephala. Paraminabic acid C (342) showed cytotoxicity toward Hep3B, MDA-MB-231, MCF-7, and A-549 cancer cell lines, with IC50 values ranging from 2.05 to 2.83 μg/mL [Three new steroidal carboxylic acids, paraminabic acids A–C, were isolated from 83 μg/mL .Protodendron repens. The cytotoxicity of the natural products against human cancer cell lines NSLC A-549 (lung), HT-29 (colon), and MDA-MB-231 (breast) were evaluated and the result showed that both compounds had a cytotoxic activity: protoxenicin A (343) was cytotoxic against the three tested cells  with GI50 values of 2.1, 0.6, and 1.1 μM, respectively, while protoxenicin B (344) exhibited cytotoxic toward HT-29 (GI50: 1.7 μM) and cytotoxicity against MDA-MB-231 and NLSC A-549 cells with GI50 values of 6.3 and 6.1 μM, respectively. The structures of the cytotoxic compounds 325–344 derived from other genera of Alcyoniidae are shown in Two new xenicanes, named protoxenicins A and B, were isolated by Urda et al.  from an For a better reading of the data, data reported in the previous section was organized in three tables  compiled207), sinulariaoid A (215), sinularin (205), durumolide C (211), and cladieunicellin I (277), which displayed IC50 values of the same order of magnitude of doxorubicin against several cell lines . In comparison to vincristine, lobophytolin D (49) proved to be a very promising compound with a similar cytotoxicity against HT-29 cells and an even higher toxicity against Capan-1 and A-549 cells . In comparison to etoposide, (24S)-ergostane-3β,5α,6β,25 tetraol (146) was shown to be the most interesting compound, since it was more cytotoxic than the drug against HepG2 cells. Moreover, acutumosterol A (147) and sarcoaldosterol B (148) showed IC50 values comparable to etoposide toward MCF-7 and A-549 cells. In comparison to 5-fluorouracil, glaucumolides A (116) and B (117), sinulerectol C (229), sinulerectadione (227), sinubrasolides H (233), J (234), K (235), and A (236) were found to be promising compounds, with a stronger activity than the control against HL-60, CCRF-CEM and K-562 cells. In comparison to mitoxantrone, four compounds were more cytotoxic against several cell lines , namely -3,7,11,15-cembratetraen-17,2-olide (13), lobophytosterol (15), -24-methyl-22,25-epoxyfurost-5-ene-3β,20β-diol (16), and 3β,11-dihydroxy-24-methylene-9,11-secocholestan-5-en-9-one (23). Noteworthily, the experiments carried out using the anticancer drugs paclitaxel, cisplatin, and vinblastine as positive controls highlighted that no molecules from corals are so far capable to display a comparable cytotoxic activity. This suggests that further tests employing these controls are needed to better verify the cytotoxicity of coral-derived natural products and to identify possible differences in the mechanism of action. The structures of the anticancer drugs used as positive control for the MTT assay in the considered studies are shown in Considering doxorubicin as positive control, the most active compounds discovered in the last thirty years are 5-episinuleptolide acetate (70) with 2790.7 mg per kg of coral (dry weight), 13-acetoxysarcocrassocolide (42) with 2033 mg per kg of coral (wet weight), 11-episinulariolide acetate (202) with 1757.1 mg per kg of coral (dry weight), and lobohedleolide (4) with 1090.9 mg per kg of coral. For the other compounds, the very low yields (more than 1 ton to isolate 1 g of active compound) represent a significant obstacle for the preclinical development, and a synthetic replacement would be required.Considering the isolation yields, the best results were obtained in the extraction of emblide  represent the largest class of natural products in the biosphere, and their variety of functions in mediating antagonistic and beneficial interactions among organisms is still largely unexplored . TerpeneSarcophyton with durumolide C (211) (5-membered), and in Sinularia with manaarelonide A (6-membered) and sinuladiterpene (7-membered); (3) furanocembranoids characterized by a 14-membered carbocyclic nucleus, as well as a furan heterocycle, as observed in Sinularia with pukalide; (4) biscembranoids showing a 14-6-14 membered tricyclic backbone of tetraterpenoids, as in lobophytone A found in Lobophytum pauciflorum. (5) special cembranes that in turn include several subtypes. Our review clearly shows that among the cembranoids, the best activity at the MTT assay is obtained by molecules owing an odd membered lactone ring (five and seven) such as in the case of deoxycrassin (44), flexilarin D (196), 11-dehydrosynulariolide (197), but also simple cembranes bearing various oxidation display a potent cytotoxic action, such as in sinulerectol C (229).Among the diterpenoids, cembranoids were the most represented. This name indicates the structure of an isopropyl- and three methyl-substituted 14-membered rings originated by the cyclization of a geranylgeraniol derived precursor between carbons 1 and 14 . StructuSteroids resulted in the second most represented group in terms of compounds active against cancer cell lines, with a total of 72 compounds. The use of steroids for cancer treatment is clinically well established, and the pharmacology of several active compounds has been described. Steroids mostly work by interacting with specific nuclear hormone receptors that are known to drive cell growth, proliferation, and metastasis for specific tumors . In contrast, glucocorticoids work through glucocorticoid receptors, which are not considered oncogenes, to perform a variety of functions, including arresting growth or inducing apoptosis in lymphocytes; for these reasons they are widely used in the treatment of all lymphatic cancers . On the According to a recent review of Ermolenko et al. , about 2304) and -24-methyl-22,25-epoxyfurost-5-ene-3 (16). Interestingly, several active molecules showed the methyl/ethyl substitution typical of phytosterol, as in sarcoaldosterol B (148) and acutumosterol A (147). Another interesting modification displayed in the coral cytotoxic steroids was the change of the position of the unsaturation in the steroid ring, something commonly observed in algae. On the other hand, the loss of the methyl substituents, which is equally common in the algae, was not observed. Since corals are not capable of providing de novo synthesis of cholesterol and its derivatives, these structures are obtained from phytosterols  through de-alkylation and successive oxidation reactions. Signs of the oxidative pathway are clearly observable in various cytotoxic structures, such as the addition of an 11α-hydroxyl group in acutumosterol A (147) and sarcoaldosterol B (148), a 17α hydroxylation in klyfaccisteroid A (304), the oxidation in the lateral chain, even if this does not come with extensive dealkylation, the presence of a conjugated Δ4-3-keto and of an additional unsaturation at Δ1 as in sinubrasolide A (236), K (235) and in sinubrasones .A variety of structural variations from the classical steroidal architecture was found in both the tetracyclic ring and the alkyl chain, such as in the case of klyffacisteroid A -3,7,11,15-cembratetraen-17,2-olide (13) increased the sub-G1 phase population and induced apoptosis in HT-29 cells through reactive oxygen species generation. Moreover, -3,7,11,15-cembratetraen-17,2-olide (13) induced apoptosis through the modulation of Wnt/β-catenin and the TGF-β pathways in human colon cancer cells SNU-C5 [Hong et al.  showed ts SNU-C5 .15) was found to induce apoptosis, in fact chromatin condensation in apoptotic bodies was observed [Lobophytosterol (observed .205) induced apoptosis and suppressed the migration capacity of A2058 cancer cells [205) inhibited cell migration capacity and induced apoptosis through mitochondrial dysfunction and inactivation of the pI3K/AKT/mTOR pathway in human gastric carcinoma cell lines. Moreover, sinularin (205) activated ATM/Chk2 DNA damage pathway, induced G2/M phase arrest, decreased mitochondial memebrane potential and caused apoptosis in HepG2 (human hepatocellular carcinoma) cell line [205) induced oxidative stress mediated G2/M arrest and apoptosis in Ca9-22 cancer cells [205) induced G2/M arrest and induced apoptosis as well as activation of MAPKs and repression of PI3K/AKT pathways, which are dependent on ROS generation, in human renal cancer (786-O) cells. Recently, Ko et al. [205) on human hepatocellular cancer (SK-HEP-1) cells and found that wound healing, cell migration, and potential colony formation were attenuated as well as DNA fragmentation and apoptosis were induced and mitochondrial and intracellular reactive oxygen species (ROS) levels were significantly increased following sinularin treatment, which also decreased the mitochondrial membrane potential and caused cytoskeleton disruption [Sinularin  was investigated by Huang et al. [2+ as well as decreased the mitochondrial membrane potential and induced apoptosis through the Hsp90 chaperone machinery inhibition in HL-60 (human promyelocytic leukemia) cells.5-Episinuleptolide acetate  induced apoptosis but its selective toxicity toward HepG2/ADM cells was not related to P-glycoproteins [Sinulariaoid A -24-methyl-22,25-epoxyfurost-5-ene-3β,20β-diol (16), 3β,11-dihydroxy-24-methylene-9,11-secocholestan-5-en-9-one (23), (24S)-ergostane-3β,5α,6β,25 tetraol (146), sinulerectadione (227), sinulerectol C (229), and cladieunicellin I (277) exhibited stronger cytotoxicity than their respective positive control, but they have not yet been submitted to further investigations devoted to the mechanism of action elucidation.The last thirty years of research on cytotoxic compounds from the family Alcyoniidae were reviewed and data were organized to highlight the most promising natural products for further preclinical development. Our work highlighted that 344 distinct secondary metabolites, mainly from the class of cembranoids and steroids, display a cytotoxic activity proven by in vitro assays and deserve attention for further development. Specifically, while for other bioactive compounds mechanistic studies have been undertaken, we found that  looks still small when compared to the vastness of biodiversity available and the possibilities offered by coral farming.


The development of the medical applications for substances or materials that contact cells is important. Hence, it is necessary to elucidate how substances that surround cells affect gene expression during incubation. In the current study, we compared the gene expression profiles of cell lines that were in contact with collagen–glycosaminoglycan mesh and control cells. Principal component analysis-based unsupervised feature extraction was applied to identify genes with altered expression during incubation in the treated cell lines but not in the controls. The identified genes were enriched in various biological terms. Our method also outperformed a conventional methodology, namely, gene selection based on linear regression with time course. RegulatThe recently proposed principal component analysis (PCA)-based unsupervised feature extraction (FE)  has the Gene expression profiles were downloaded from the Gene Expression Omnibus (GEO) database (GEO ID: GSE6432). The dataset in GSE6432_series_matrix.txt.gz is available in the Series Matrix File(s) section. It consists of 32 gene expression profiles of the IMR90 cell lines, and the relevant details are provided in ith probe at the jth sample. Before applying singular value decomposition (SVD), they were standardized asGene expression profiles are formatted as matrices After applying SVD, we obtained the left-hand singular value vector jth sample. We used the lm function in R [p-values were corrected using the Benjamini–Hochberg criterion [p-values less than 0.05, whereas p-values less than 0.05. This result is appropriate because the simple cell division process may not be associated with any time development other than cell senescence [In order to see which hours in  association in R , and theriterion . v3j fornescence , which mp-values to the probes asx, and p-value less than 0.01 were selected for the treated cell lines.Probes are selected by assuming that jth sample. Subsequently, the adjusted p-values that were less than 0.01 were selected. The number of probes selected for treated cell lines was 813, and no probes were selected for the control cell lines.As an alternative method to PCA-based unsupervised FE, we utilized linear regression-based FE. Linear regression is applied to hours in  associatThe IDs of the selected probes were converted to gene symbol using the ID converter in DAVID . Then, tAs mentioned in the Materials and Methods Section, genes associated with the 318 probes for the treated cell lines (contact with collagen–glycosaminoglycan mesh) were uploaded to Enrichr (no probes were selected for control cell lines using this method). The full list of probes, genes, and enrichment analysis is provided in the The top ranked term in the GO biological process (BP)  is “regu“Focal adhesion” is the top ranked term in “GO Cellular Component 2021”  and the Other than these three categories, there are some additional categories that support the suitability of our analysis. For example, “ARCHS4 Cell-lines” lists IMR90, which is the cell line used in this study, as the top ranked cell line .Moreover, although it is not the top ranked term, “FETAL LUNG”, from which IMR90 cell lines were derived, is ranked within the top 10 ranked terms in “ARCHS4 Tissues” .Although we provide only a few examples, our results suggest that our analysis was robust.Although we successfully applied our methodology to the dataset, one might wonder whether more conventional methods can achieve similar performance. Since this dataset was generated using archaic technology, namely, microarray, more modernized methodologies adapted to high-throughput sequencing technology  in For KEGG, not only are the generally adjusted p-values generally larger  than those in p-values attributed to IMR90 in p-values . These finding suggest the inferiority of linear regression as compared to PCA-based unsupervised FE.For “ARCHS4 Cell-lines” and “ARCHS4 Tissue”, the results are similar. In p-value is Although “FETAL LUNG” is fourth ranked in Finally, we attempted to conduct a time-series analysis, which is more widely used than linear regression for time course data. To this end, we used the fsMTS  package The limitation of our methodology is that because of its unsupervised nature, when it fails to select biologically reasonable genes, there are no ways to improve it, although it occasionally worked effectively in the present study.In the current study, we applied PCA-based unsupervised FE to gene expression profiles for IMR90 cell lines incubated in collagen–glycosaminoglycan mesh. Whereas no genes whose expressions vary over time were detected in control cell lines, the expression profiles of several genes were altered during the cell division process. These genes are associated with several enriched biological terms. One conventional method, linear regression, was employed for comparison. Although it could select several hundred genes whose expressions vary over time, their enrichment was inferior to that seen using PCA-based unsupervised FE. Thus, not only can PCA-based unsupervised FE achieve a good performance, but it can also outperform a conventional method. We demonstrated that collagen–glycosaminoglycan is an effective medium that could be used for cell culture.


Ononis diffusa and Ononis variegata, were screened against colorectal cancer cell lines. The cytotoxic activity of the crude extracts was tested on a panel of colon cancer cell models including cetuximab-sensitive , intrinsic (HT-29 and HCT-116), and acquired  cetuximab-resistant cell lines. Ononis diffusa showed remarkable cytotoxic activity, especially on the cetuximab-resistant cell lines. The active extract composition was determined by NMR analysis. Given its complexity, a partial purification was then carried out. The fractions obtained were again tested for their biological activity and their metabolite content was determined by 1D and 2D NMR analysis. The study led to the identification of a fraction enriched in oxylipins that showed a 92% growth inhibition of the HT-29 cell line at a concentration of 50 µg/mL.In the search of new natural products to be explored as possible anticancer drugs, two plant species, namely  Natural products play a crucial role in the discovery of anticancer compounds even today, when new technologies are available to easily obtain a wide range of drug candidates. The success of natural products is inherent to their structure: they possess a well-defined three-dimensional scaffold with functional groups precisely oriented in space. Furthermore, they are characterized by an enormous structural and functional diversity ,2. TheseIn recent years, the need for anticancer compounds has become particularly pressing and, in this context, colorectal cancer is definitely under the spotlight. This cancer is indeed one of the leading causes of cancer-related death worldwide and one of the most frequently diagnosed malignant diseases in Europe . AlthougIn the search for compounds with anticancer activity in plants of Mediterranean region, we recently screened a set of plants of the Fabaceae family with a metabolomics-based approach . This stOnonis diffusa Ten. and Ononis variegata L. Ononis is a large genus of perennial herbs and shrubs, including food, foraging and medicinal plants [Ononis species have been studied, and bioactive compounds including flavonoids, isoflavonoids, alkylresorcinols, coumarin derivatives, and terpenes have been reported from them [O. diffusa and O. variegata.Herewith, we undertook the study of two further Fabaceae plants which were not analyzed in our previous study: l plants . Severalrom them ,15,16,17Ononis diffusa showed a marked cytotoxic effect in all the cell lines and especially in those exhibiting drug resistance. Thus, O. diffusa crude extract was further tested on cetuximab-sensitive (GEO and SW48) and on acquired cetuximab-resistant (GEO-CR and SW48-CR) human colon cancer cell lines. GEO-CR and SW48-CR represent a preclinical model for the study of acquired resistance to cetuximab. The potential cytotoxicity of the extracts was evaluated through MTT assays. In the attempt to identify the metabolites responsible for the activity, NMR profiles of the extracts were obtained. Furthermore, a partial purification, always paired with the NMR analysis, and biological tests led to the identification of a mixture of oxylipins as putative bioactive compounds responsible for the antiproliferative properties of the crude extract.Extracts from the two species which are the objects of this study were first screened for their antiproliferative activity on colorectal cancer cell lines  characterized by different genetic profiles. Specifically, the Caco-2 cell line is sensitive to cetuximab as it has no genetic defects associated with anti-EGFR therapy resistance. Meanwhile, the HT-29 and HCT-116 cell lines are intrinsically resistant to cetuximab since they harbor BRAF V600E and KRAS/PIK3CA mutations, respectively . Ononis O. variegata showed no inhibition of cell proliferation at any of the tested concentrations, O. diffusa showed a significant inhibition against all of the cell lines, beginning at a concentration of 50 µg/mL (Ononis diffusa extract exhibited a more pronounced effect on HT-29 cell line.Plant extracts were obtained with a mixture of methanol and water (1:1). The potential antiproliferative activity of the plant crude extracts was evaluated on a panel of colon cancer cell lines . While 50 µg/mL . Ononis O. diffusa extract on the three colon cancer cell lines, including the intrinsically cetuximab-resistant ones, this extract was further explored for its activity against both cetuximab-sensitive (GEO and SW48) and secondary cetuximab-resistant cancer cell lines (GEO-CR and SW48-CR). The extract showed a remarkable inhibition of the proliferation of the acquired resistant cell lines  and the solution thereby obtained was analyzed by NMR.NMR spectra were obtained for the extracts of the two Ononis species. The plant material was extracted with a mixture of methanol-O. variegata showed no activity in the biological tests, a comparison of the profiles of the two species helped us narrow down the set of the possible bioactive compounds. It was clear, indeed, from the comparison of the 1H-NMR spectra . The columns were eluted with methanol first, and then with water. The methanolic fractions were joined together due to their very similar TLC profiles. The fraction (OdM) thereby obtained was analyzed by NMR and the 1H-NMR spectrum, along with the HSCQ experiment, clearly showed that the aim was achieved  and 6.97 . The latter signal showed cross peaks with two oxygenated aromatic carbons at δC 147.8 and 146.1, both of which correlated with a third aromatic proton at δH 6.76  and with a singlet methylene at δH 5.87 (δC 101.2), indicating the presence of a 3,4-dioxymethylenephenyl group bound to the amide nitrogen. In addition, in the CIGAR experiment, the amidic proton correlated with a further aromatic carbon at δC 157.9, which in turn displayed cross-peaks with a proton at δH 8.01 , as an indicator of a second 1,2,4-tribubstituted aromatic system. This proton correlated in the long-range experiment with the amide carbonyl and with a quaternary aromatic carbon at δC 118.8, which in turn correlated with other two aromatic protons resonating at δH 7.07  and 7.22 , showing cross-peaks with an oxygenated aromatic carbon at δC 162.2. This carbon correlated with an anomeric proton at δH 4.98, therefore suggesting the presence of a sugar moiety, putatively identified, on the basis of an HSQC-TOCSY experiment, as glucose. These data were in agreement with the presence of a glucopyranosyl-2-hydroxy-N-benzamide. However, the isolation and complete structural elucidation of this compound will be needed to confirm the hypothesized structure.However, the spectrum was still rich in signals, especially in the aromatic region. The HSQC experiment  and the periment , with a J = 2.0 Hz) at δH 6.20 and 6.39. These protons were correlated in the HSQC experiment to the carbons at δC 93.1 and 98.4, respectively. Furthermore, the former proton also showed long-range correlations with the two carbons resonating at δC 161.2 and 165.0. This second carbon was also correlated to the δH 6.39, which showed further long-range correlations with the carbons at δC 156.9 and 104.0. It was not possible to unambiguously identify these metabolites, due to the lack of further correlations, but the NMR data here described prompted us to hypothesize that the flavonoids were all characterized by a hydroxy function bound to the C-3 [More signals were detected in the aromatic region. Signals belonging to the A-ring of flavonoids were observed as two COSY-correlating doublets .O. diffusa is a source of potential anticancer compounds acting on drug-resistant colon cancer cell lines. The aim of the present work was not only to identify cytotoxic extracts, but particularly sources of compounds able to overcome anti-EGFR therapy resistance in mCRC.The present study demonstrates that EGFR is a very important target in cancer therapy , being aO. variegata  and identified by Prof. Assunta Esposito. Voucher specimens were deposited at the Herbarium of DiSTABiF of Università degli Studi della Campania “Luigi Vanvitelli.” Leaf samples were collected and immediately frozen in liquid N2 to avoid unwanted enzymatic reactions, and stored at −80 °C before the freeze-drying process. Lyophilized samples were powdered in liquid nitrogen and stored at −20 °C.Plant samples belonging to the two species, 2O  containing 0.1% w/w trimethylsilylpropionic-2,2,3,3-4d acid sodium salt  and CD3OD  (1:1). The mixture was vortexed at room temperature for 1 min, ultrasonicated  for 40 min, and centrifuged  at 13,000 rpm for 10 min. A volume of 0.65 mL was transferred to a 5 mm NMR tube and analyzed by NMR [An aliquot (50 mg) of freeze-dried and powdered plant material extracted with 1.5 mL of phosphate buffer  in Dd by NMR .1H and 75.45 MHz for 13C. CD3OD was used as the internal lock. 1D and 2D NMR spectra were acquired using Varian standard pulse sequences and as previously described [NMR spectra were recorded at 25 °C on a Varian Mercury Plus 300 Fourier transform NMR operating at 300.03 MHz for O. diffusa were powdered and underwent three cycles of an ultrasound-assisted extraction with a MeOH:H2O (1:1) solution (1.8 L), obtaining a crude extract (16 g), which was dissolved in H2O and separated by liquid–liquid extraction using EtOAc as an extracting solvent, obtaining an ethyl acetate fraction  and a water fraction. The aqueous fraction was chromatographed on Amberlite XAD-4 and XAD-7 with water and then with methanol. The two alcoholic eluates were joined together to give the OdM fraction (1.7 g). Aliquots were used for bioassays and NMR analyses.Dried leaves (60 g) of 2 at 37 °C, and routinely screened for the presence of mycoplasma .Human HCT-116, HT-29, Caco-2, and SW48 colorectal cancer cell lines were obtained from the American Type Culture Collection (ATCC) . The human GEO colon cancer cell was a gift of Dr. N. Normanno . The HCT-116 and HT-29 cancer cells were cultured in RPMI 1640 medium , and supplemented with 10% FBS, 2 mM L-glutamine, 50 U/mL penicillin, and 100 µg/mL streptomycin . Caco-2 cell line was cultured in DMEM medium , and supplemented with 10% FBS, 2 mM L-glutamine, 1% non-essential amino acid, 50 U/mL penicillin, and 100 µg/mL streptomycin . The GEO and GEO-CR clones were grown on DMEM medium supplemented with 20% FBS, 1% penicillin/streptomycin . The SW48 and SW48-CR cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. All cell lines were maintained in a humified atmosphere of 95% air and 5% COCell proliferation was measured with 3--2,5-diphenyltetrazolium bromide (MTT) assay . Brieflyt test (p < 0.001) was used to determine the statistical significance of the experimental results.Bioassays were carried out in six replicates. Statistical analyses were performed using Excel 2010 . A Student’s