TP53 mutated ultra high-risk neuroblastoma (NB) cell lines, SK-N-DZ, Kelly, SK-N-AS, SK-N-FI, and BE(2)-C. Following treatment, a dose-dependent accumulation in either S- and/or G2/M-phase was observed. This coincided with a heterogeneous increase of cell cycle checkpoint proteins, i.e., phos-ATM, phos-CHK1, phos-CHK2, Wee1, p21Cip1/Waf1, and p27Kip among the cell lines. Combination treatment with doxo and a small-molecule inhibitor of ATM showed a delay in regrowth in SK-N-DZ, of CHK1 in BE(2)-C, of Wee1 in SK-N-FI and BE(2)-C, and of p21 in Kelly and BE(2)-C. Further investigation revealed, in all tested cell lines, a subset of cells arrested in mitosis, indicating independence on the intra-S- and/or G2/M-checkpoints. Taken together, we mapped distinct cell cycle checkpoints in ultra high-risk NB cell lines and identified checkpoint dependent and independent druggable targets.Most chemotherapeutics target DNA integrity and thereby trigger tumour cell death through activation of DNA damage responses that are tightly coupled to the cell cycle. Disturbances in cell cycle regulation can therefore lead to treatment resistance. Here, a comprehensive analysis of cell cycle checkpoint activation following doxorubicin (doxo) treatment was performed using flow cytometry, immunofluorescence and live-cell imaging in a panel of  Neuroblastoma (NB) is the most common extracranial solid tumor in early childhood. It is a heterogenous neoplasia with clinical presentation ranging from spontaneous regression of metastatic disease to a rapidly progressive course . Fifty pSmall-molecule inhibitors of anaplastic lymphoma kinase (ALK), antibodies against the NB antigen GD2 and radiolabelled somatostatin analogues ,7 represCip1/Waf1 (p21), which in turn inhibit the catalytic activity of cyclin-dependent kinases [DNA damage leads to the activation of cell cycle checkpoint surveillance mechanisms causing cells to either arrest in order to ensure DNA repair  or to un kinases ,14. Ther kinases . Further kinases .TP53 mutations (mut) at diagnosis are rare, occurring in <2% of cases independent of tumour stage [TP53 mut, are identified in 49% of NB following relapse, indicating a mechanistic relevance in the development of therapy resistance [TP53 mut NB.While checkpoint proteins maintain genomic intregitry, dysregulation might be associated with therapy resistance . Among cur stage . Insteadsistance . In addisistance . TherefoTP53 mut NB cell lines, SK-N-DZ, Kelly, SK-N-AS, SK-N-FI, and BE(2)-C, to study cell cycle checkpoints and regrowth capacity following doxo treatment. The aim is to increase the understanding of conditions leading to treatment resistance and relapse, and to identify targetable vulnerabilities. Here, we utilized a panel of five ultra high-risk We demonstrate heterogeneity in cell cycle distribution among the tested cell lines following doxo treatment. Furthermore, a fraction of cells showed activation of checkpoint proteins and sensitivity to chemical inhibition of these proteins. However, a subset of cells were able to progress though the S-phase and arrest in mitosis, indicating independence of the intra-S- and G2/M-checkpoints. These results demonstrate heterogeneity in cell cycle regulation with the presence of both checkpoint-dependent and independent subpopulations of tumour cells in all tested cell lines. Identification of CT escape mechanisms does not only increase the knowledge on chemoresistance–but helps to identify specific vulnerabilities that can be targeted in conjunction with conventional therapy.TP53 mut cell lines, we measured DNA fragmentation (sub-G1) after 48 h of treatment with doxo serially diluted at 0.01 µM, 0.1 µM and 1 µM, using flow cytometry. Importantly, the highest dose tested, 1 µM, corresponds to clinically relevant plasma peak level in patients [The cytotoxic effect of doxo has been shown to be highly varying among p53 inactive NB cell lines, with IC50 values ranging from 0.06 to 0.2 µM . To invepatients ,26.p < 0.01 and from 0.5 to 7.4%, p < 0.05, respectively). However, SK-N-FI and BE(2)-C showed no change in sub-G1 fractions after any of the tested concentrations. Thus, the data indicated a high resistance to doxo-induced apoptosis, except in SK-N-DZ, Kelly, and SK-N-AS cell lines, where full resistance was not observed.As shown in Since doxo-induced DNA damage is suggested to trigger cell cycle arrest, we investigated cell cycle distribution following doxo exposure using flow cytometry. Forty-eight hours following treatment with the lowest tested dose (0.01 µM doxo), all cell lines maintained the bulk of cells in G0/G1 (46–66%), similar to the results after mock-treatment (51–66%).p < 0.0001), with a decreased G0/G1 population  Treatment with the intermediate dosing (0.1 µM doxo), resulted in accumulation of G2/M-phases in all cell lines as compared to mock treatment  presented, similar to 0.1 µM, decreased levels of diploid cells (G0/G1) in all cell lines. Notably, all cell lines, except BE(2)-C, presented S-phase accumulation, suggesting an intra-S-phase arrest. Especially two cell lines, Kelly and SK-N-AS, showed a high preference for S-phase accumulation reaching levels of approximately 70% compared to 30% when mock-treated To determine the long-term effect of doxo treatment, regrowth assays were conducted. Regrowth (confluency) was mapped during a period of 44 days following a pulse treatment of mock or doxo at 0.01 µM, 0.1 µM or 1 µM, for 48 h. Two cell lines, SK-N-DZ and Kelly, showed detachment from the plates once reaching high confluency following mock treatment, most likely due to crowding effects. Following the lowest tested dose (0.01 µM doxo) regrowth followed the same growth pattern as with mock treatment. Moreover, for all cell lines, except BE(2)-C, a dose dependency with a lag period of approximately 20 days following 0.1 µM doxo, and 24 days following 1 µM doxo was observed before an increase in confluency (regrowth). However, BE(2)-C cells showed regrowth at day 6 following 0.1 µM and at day 14 following 1 µM doxo, indicating a less prominent lag period compared to the other four cell lines TP53 mut cell line in their cell cycle response upon doxo treatment. This is especially seen after 1 µM doxo treatment, where SK-N-AS and Kelly accumulated in S-phase, SK-N-DZ and SK-N-FI in both S- and G2/M-phase and BE(2)-C in G2/M-phase. Furthermore, all cell lines showed regrowth capacity following all tested concentrations of doxo, albeit with different kinetics indicating the presence of resistant cells.The combined data illustrate heterogeneity among the Kip1 in single-cells using immunofluorescence. This was done to correlate doxo-induced alterations in the cell cycle distribution with intra-S- and/or G2/M-checkpoint activation.Next, the activation of checkpoint proteins was investigated following doxo treatment by determining the expression of ATM-phospho S1981 (pATM), CHK1-phospho S296 (pCHK1), CHK2-phospho T68 (pCHK2), Wee1, p21, and p27Kip1 positive cells following treatment Forty-eight hours following 1 µM doxo treatment a robust increase in the number of pATM positive cells was observed for all cell lines, except SK-N-AS. The fraction of positive cells increased for pCHK1 in Kelly and BE(2)-C, and for pCHK2, a downstream target of ATM, for all cell lines, except SK-N-AS Kip1 were generally low, the exception was pATM which was strongly increased for all cell lines, except SK-N-AS.These results illustrate that the doxo-induced frequencies of cells positive for cell cycle checkpoint proteins, pCHK1, pCHK2, Wee1, p21 and p27An increase in the number of positive cells for one or several cell cycle checkpoint proteins was shown after doxo treatment for all NB cell lines, indicating their role in doxo-induced cell cycle arrest.Kip1.To validate the functional role of cell cycle checkpoint proteins we employed pharmacological inhibitors. Small molecular inhibitors of ATMi (KU 60019), CHK1i (PF-477736), CHK1/2i (AZD 7762), Wee1i (Adavosertib), and p21i (UC2288) were tested in order to assess the effect of the inhibitors on NB viability and growth. A small molecular inhibitor was not commercially available for p27The ability of each drug was tested as a single-agent, using a 4-log dose–response cell viability assay with a 72-h time point. Inhibition of ATM or p21 did not give a decrease in cell viability post-treatment for any of the cell lines. However, all tested NB cell lines were sensitive to single-agent inhibitions of CHK1, CHK1/2, or Wee1, compared to mock treatment. Kelly and SK-N-AS showed the most sensitivity, followed by SK-N-DZ, SK-N-FI, and BE(2)-C Current developmental strategies for inhibitors of cell cycle checkpoint proteins have been focused on their chemosensitizing properties. Here, a sub-sequential assessment of the ability of the molecular inhibitors to potentiate the CT doxo in the panel of NB cell lines was made. Each inhibitor was combined at a concentration of 10 µM for ATMi and p21i or the corresponding IC50 value with 1 µM doxo and cellular growth (confluency) was measured for 44 days.p < 0.05–0.0001). Kelly showed delay in growth following combination treatment with doxo and p21i (p < 0.05) and BE(2)-C showed delay in growth following combination treatment with doxo and either CHK1i (p < 0.05–0.0001), Wee1i (p < 0.05–0.0001), or p21i (p < 0.05–0.0001). SK-N-FI showed an initial delay in growth  following combination treatment with doxo and Wee1i however, this was not observed from experimental day 8 and later. None of the combination treatments showed a delay in growth compared with a single doxo treatment in the SK-N-AS cell line A significant delay in growth was observed in the SK-N-DZ cell line after combination treatment with doxo and ATMi compared to single doxo treatment  was observed in all cell lines, except for Kelly. SK-N-FI and BE(2)-C displayed the highest fraction of cells in mitotic arrest following doxo treatment reaching 20% and 15% PH3 positive cells respectively A significant reduction of EdU positive cells was observed in all tested cell lines after exposure to 1 µM doxo, as expected from doxo-induced cell cycle arrest. The most dramatic reductions of EdU positive cells, indicating abundant arrest, were noted for Kelly and SK-N-FI . Despite statistically significant reductions, SK-N-DZ, SK-N-AS, and BE(2)-C, maintained relatively high levels, ranging 64–81%, of EdU positive cells following doxo treatment A small, but significant, increase in PH3+/EdU+ cells (between 4.7–9.3%) was detected in SK-N-DZ, SK-N-AS, SK-N-FI, and BE(2)-C, but not in Kelly, which showed a reduction to 0.8% PH3+/EdU+ cells Furthermore, all tested cell lines showed an increase in PH3+/EdU- cells Considering the longstanding clinical experience of enhanced anti-tumour effect from repeated CT we investigated cell cycle behaviour after repeated doxo treatment, were 1 µM doxo was added for 48 h followed by medium change and a second dose of 1 µM doxo in combination with EdU for another 48 h.In all tested cell lines, after adding doxo (1 + 1 µM), resilient cells could be shown to mostly reside in cellular arrest, as demonstrated by either increased expression of PH3 and/or failure to incorporate EdU Furthermore, a small fraction of PH3+/EdU+ cells (0.8–3.5%) were detected in all cell lines, except Kelly These data illustrate the presence of cells in mitotic arrest (PH3 positive) following both single and repeated doxo treatment in all tested cell lines. Moreover, the subset of cells arrested in mitosis can be further subdivided depending on their cell cycle behavior, indicated by cells either with or without progression through an S-phase prior to the arrest.To investigate the consequence of replicating cells during treatment, we pulse labelled with EdU during the single 1 µM doxo treatment and examined regrowth capacity (colonies). Three cell lines, Kelly, SK-N-FI, and BE(2)-C, were selected as they displayed a range of cell cycle behaviours following doxo treatment, with Kelly preferentially accumulating in S-phase and having a low fraction of EdU positive cells (20%), whereas SK-N-FI showed accumulation in both S- and G2/M-phase and had a low fraction of EdU positive cells (27%) and BE(2)-C showed accumulation in G2/M-phase and had a higher fraction of EdU positive cells (64%) p < 0.05, N = 3, Student’s t-test) but not compared to SK-N-FI . The consequence of these EdU positive cells remains to be investigated.In the Kelly cell line, EdU positive cells persisted in 2/45 newly formed colonies, in SK-N-FI EdU positive cells persisted in 12/36 newly formed colonies and in BE(2)-C EdU positive cells persisted in 37/40 newly formed colonies. Besides observing EdU positive cells in colonies, there was also a fraction of EdU positive cells not directly adjacent to colonies Taken together, the results indicate heterogeneity among the NB cell lines regarding the fraction of regrowth colonies with descendants from tumor cells completing replication during the doxo challenge.Based on our findings, and due to having the highest fraction of EdU positive colonies, the BE(2)-C cell line was selected for additional regrowth tracing using EdU in combination with repeated (1 + 1 µM) doxo treatment. BE(2)-C cells were pulse labelled with EdU during the second dose of doxo and colonies were investigated for the presence of EdU positive cells. However, no EdU positive cells were detected in 63/63 identified colonies. This suggests that cells responsible for regrowth following repeated doxo treatments have not progressed through an S-phase, at least during the second doxo dose. p < 0.001), similar to that found following a single doxo treatment. The fraction of pCHK1 or pCHK2 positive cells was reduced, reaching levels similar to the mock treatment. However, there was a persistent increase in the fraction of cells positive for Wee1 , and p21 , supporting the notion of sustained checkpoint activation. Furthermore, there was also an increase in the fraction of p27Kip1 expressing cells  indicating that a subset of cells was arrested for an extended period of time.In fact, investigating the fraction of BE(2)-C cells positive for cell cycle checkpoint proteins following repeated doxo These data illustrate that cell progression through the S-phase during single doxo treatment is able to regrow. However, this is not the case when doxo is given repeatedly. Instead, cells that are most likely already arrested following the first doxo treatment, are capable of regrowth.The challenge of ultra high-risk and relapse tumours remains a major hurdle in the successful treatment of NB. Cell cycle checkpoint activation following therapy has been linked to tumour cell survival and although exploiting these checkpoints has been a successful treatment strategy in several adult tumours , the potTP53 mut NB cell lines. The flow cytometry data showed, for all cell lines, high, but not full, resistance to doxo induced apoptosis Here, the cell cycle checkpoint activation following treatments with doxo was investigated in a panel of five in vitro ultra high-risk MYCN amplification contributes to driving proliferation but it also transcriptionally upregulates CHK1 [MYCN amplification, and two of these upregulated CHK1 following doxo treatment, whereas only BE(2)-C showed a reduction in growth following combination treatment with CHK1i. Moreover, BE(2)-C cells also responded with delay in growth following combination treatment with Wee1i and p21i, in agreement with an active G2/M-checkpoint. On the other hand, SK-N-DZ, which following doxo treatment displayed the highest fraction of pATM expressing cells (89%) among the tested cell lines, showed a delay in growth following combination treatment with ATMi, indicating its sensitivity. Synthetic lethality has been suggested between inhibitors of CHK1 and ATM, thus this combination might be an alternative in MYCN-amplified NB cells [Among common genetic alterations in NB, tes CHK1 . Three oNB cells .Moreover, the SK-N-AS cell line, which harbours the loss of one 11q copy rendering the cell line with impaired functional ATM and CHK1 due to haploinsufficiency , showed Moreover, when using a single-cell approach we were able to detect heterogeneity in the endogenous expression of cell cycle checkpoint proteins among the different NB cell lines. Both Kelly and SK-N-FI displayed a high fraction of p21 expressing cells  in the mock-treated samples, both also showed increased S-phase accumulation and few EdU positive cells  following doxo treatment. This cell cycle behaviour might be correlated to a p53 independent expression of p21 since endogenous expression of p21 has been linked to cell cycle behaviour with high expression leading to a senescence fate . FurtherOur results, even though they illustrate a heterogeneic response among NB cell lines summarized in Kip1 in the BE(2)-C cell line following repeated doxo treatment, indicating maintained arrest and/or quiescence.Furthermore, co-labeling with PH3 and EdU revealed that a subset of cells in mitotic arrest had progressed through the S-phase during the single doxo treatment. These cells seem to have bypassed the intra-S- and the G2/M-checkpoint, a phenomenon known as checkpoint adaptation . AlthougTP53 mutations are more frequently occurring. Furthermore, these in vitro findings may have an impact on the development of new treatment strategies through that doxo today is commonly included in first-line and salvage therapy protocols. The presence of a subset of resistant cells that are able to actively replicate during the treatment adds complexity when it comes to sensitizing tumour cells to DNA damaging drugs. One alternative treatment comes from a study where a CHK1/2 inhibitor revealed strong synergy with the antimetabolite gemcitabine, which targets tumour cell progression in S-phase . It is a2 with high humidity.BE(2)-C and Kelly were obtained from ATCC . SK-N-AS, SK-N-FI and SK-N-DZ were a kind gift from professor Per Kogner at Karolinska Institutet. STR analysis  confirmed the authenticity of the cell lines (data not shown). Basic characteristics of the NB cell lines are described in Doxorubicin (doxo)  was diluted in 1X PBS and administered to the culture medium. Doxo was added for 48 h at concentrations of 0.01 µM, 0.1 µM or 1 µM, to cells in logarithmic growth. For the repeated (1 + 1 µM) doxo treatment 1 µM doxo was added for 48 h followed by medium change and a second dose of 1 µM doxo for another 48 h. For mock treatment, 1X PBS was added to the culture medium.® . The data were analysed and quantified by using NovoExpressTM software . Each condition was run in triplicate. Statistics were performed on the proportion of cells within a specific gate.All five NB cell lines, in logarithmic growth, were treated with mock (1X PBS), or 0.01 µM, 0.1 µM, 1 µM doxo. Forty-eight hours post-treatment the cells were fixated with 70% EtOH before incubation with Hoechst 33343  according to the manufacturer’s protocol and run on a FACS Novocyte3 in 1X PBS overnight at +4 °C. Following washing, cells were incubated with 2’ab diluted in TNB buffer, for 2 h at room temperature. The cells were washed and mounted with Prolong Gold antifade with DAPI  to visualize the nuclei. Stained cells were analysed using a Metafer® Slide Scanning Platform . A list of the antibodies and kits used is presented in Following fixation for 15 min in ice-cold freshly prepared 4% paraformaldehyde , cells were incubated in TNB Buffer: 0.5g of blocking reagent  to 100 mL TBS buffer (Tris/NaCl pH 7.4) for 30 min at room temperature. Cells were then incubated with the 1’ab diluted in 0.3% Triton X-100, 0.1% NaN® AQueous One Solution Assay  was added per 100 μL media. The plates were then incubated for 3 h and analysed in the microplate reader FLUOstar Omega . The absorbance at 490 nm and 690 nm was measured to assess the cell viability post-treatment. Each treatment was completed in triplicates. DMSO was used as a negative control.Cells, in logarithmic growth, were treated with a titration series of 10 to 0.01 μM ATM inhibitor (ATMi) , CHK1 inhibitor (CHK1i) , CHK1/2 inhibitor (CHK1/2i) , Wee1 (Wee1i) , or p21 (p21i)  diluted in dimethyl sulfoxide (DMSO). After 72 h a 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was performed where 20 μL CellTiter 96® Cell Analysis System  every 48 h for 44 days. Fresh medium was added every 4 days. The proliferation was determined by measuring the cell confluency. Each treatment was done in five replicates and the data are presented as mean ± Standard Error of the Mean (SEM).Each cell line, in logarithmic growth, was treated at a concentration of 10 µM for ATMi and p21i or the corresponding IC50 value for each drug. The appropriate drugs either as a single agent ATMi, CHK1i, CHK1/2i, Wee1i, p21i, or as a combination with 1 µM doxo were added for 48 h followed by medium change. An amount of 1 μM doxo, 1X PBS (mock), or DMSO (mock) was used as negative controls. The plates were scanned by an IncuCyte S3 LiveThe thymidine analogue 5-Ethynyl-2′-deoxyuridine (EdU)  was added at a concentration of 10 µM 48 h before fixation. EdU labelling was performed according to the manufacturer’s protocol.® Slide Scanning Platform. Colony formation, with a threshold of >10 cells in proximity set for the definition of a colony, was assayed by ocular inspections.Kelly, SK-N-FI, and BE(2)-C cells, in logarithmic growth, were treated with 1 µM doxo in combination with 10 µM EdU for 48 h followed by medium change. Cells were allowed to grow, with fresh medium added every 4 days, until colonies were visible. The cells were fixed for 15 min in ice-cold freshly prepared 4% paraformaldehyde, EdU labelling was performed according to the manufacturer’s protocol. Cell density was visualised by DAPI staining and the number of EdU positive cells was deciphered as cells per scanned image (10× image) using a Metafert-test and two-way analysis of variance (ANOVA). Dunnett or Bonferroni post-hoc test was applied to adjust for multiple testing as indicated in the text. The software GraphPad Prism 7.03  was used for testing the normal distribution and generation of graphs. The immunofluorescence data were analysed in RStudio  and the percentage of positive cells followed by the mean ± Standard Deviation (SD) were calculated for each combination.Data were analysed using Student’s


The development of reliable predictive models for individual cancer cell lines to identify an optimal cancer drug is a crucial step to accelerate personalized medicine, but vast differences in cancer cell lines and drug characteristics make it quite challenging to develop predictive models that result in high predictive power and explain the similarity of cell lines or drugs. Our study proposes a novel network-based methodology that breaks the problem into smaller, more interpretable problems to improve the predictive power of anti-cancer drug responses in cell lines. For the drug-sensitivity study, we used the GDSC database for 915 cell lines and 200 drugs. The theory of optimal mass transport was first used to separately cluster cell lines and drugs, using gene-expression profiles and extensive cheminformatic drug features, represented in a form of data networks. To predict cell-line specific drug responses, random forest regression modeling was separately performed for each cell-line drug cluster pair. Post-modeling biological analysis was further performed to identify potential biological correlates associated with drug responses. The network-based clustering method resulted in 30 distinct cell-line drug cluster pairs. Predictive modeling on each cell-line-drug cluster outperformed alternative computational methods in predicting drug responses. We found that among the four drugs top-ranked with respect to prediction performance, three targeted the PI3K/mTOR signaling pathway. Predictive modeling on clustered subsets of cell lines and drugs improved the prediction accuracy of cell-line specific drug responses. Post-modeling analysis identified plausible biological processes associated with drug responses. Importantly, the genomic and transcriptomic profiles of all cancer cell lines employed in GDSC were extensively characterized as a part of the COSMIC cell line project . These resources have the potential to link anti-cancer drug sensitivity to detailed genomic information and facilitate the discovery of relevant molecular biomarkers when coupled with powerful analytical tools to cope with the high-dimensionality and complexity of these datasets.Recent significant advances in investigating drug sensitivity have been driven by advances in high-throughput technologies that can generate large amounts of biological data at low cost. Pioneers of such datasets include the NCI-60 database , GenomicA variety of approaches have been proposed for investigating drug sensitivity in cancer cell lines. One of the first models was developed by Staunton et al., which employed a weighted voting classification model for anti-cancer drug sensitivity based on NCI-60 gene-expression data . Recent http://www.hprd.org, accessed on 9 December 2021). [In the present study, we demonstrate that cell-line and drug clustering prior to machine learning modeling can significantly improve the accuracy of cell-line drug-sensitivity prediction. We first represented genomic profiles of cell lines and chemical features of drugs in the form of separate feature networks. Several network-based papers for drug-sensitivity prediction have been previously published. For instance, Wang et al. proposed a heterogeneous network model of cell lines, drugs, and targets ,18. Zhanr 2021). . This rer 2021). ,24,25. Tr 2021). ,26. It wIn summary, the heterogeneity of pan-cancer cell lines and structurally diverse drugs in large-scale pharmacogenomic databases makes prediction of drug sensitivity challenging. In this work, we propose a novel computation method for clustering cell lines and drugs in an unsupervised way, followed by a supervised prediction-modeling of drug responses. Our results demonstrate that modeling on homogeneous data significantly improves the prediction accuracy. Moreover, clustering increases the focus for understanding potential biomarkers and mechanisms of drug sensitivity.Hierarchical clustering of the cell lines resulted in six clusters with the highest average silhouette score. The numbers of cell lines in each cluster were 149, 113, 130, 174, 208, and 141, respectively, labeled clusters 1 through 6. 2) values were computed for the predicted and observed log(IC50) values. 2 of the predicted and observed log(IC50) values in the 30 paired clusters of cell lines and drugs.For each of the 30 paired clusters (six clusters for cell lines and five clusters for drugs), random forest regression models were trained and validated, using 635 genes and 165 cheminformatic features. A three-fold cross-validation approach was employed, such that in each cross validation, 2/3 of the data were used for training, and 1/3 of the data were used for validation of the model. After performing the three-fold cross validation in each paired cluster, correlation (R) and coefficient of determination  obtained via random forest on the whole dataset  using a three-fold cross validation scheme. Further, R and R2 in the best and worst paired clusters with respect to prediction accuracies were (R = 0.96 and R2 = 0.93) and (R = 0.79 and R2 = 0.62), respectively  values for all the 30 clusters and then calculated R and R2 . For comectively . The celectively . In addiAfter applying the modeling pipeline, we investigated the prediction accuracy for individual cell lines and drugs. t-test, differentially expressed genes were further identified. For example, we investigated a paired cluster: cell-line cluster 4 and drug cluster 1, which is one of the highest performing cluster pairs. Initially, the top 200 genes were selected based on the importance score in random forest modeling, and 70 out of the 200 genes met a Bonferroni corrected p-value < 0.05. For these 70 genes, gene ontology enrichment analysis was performed using MetaCore software to discover significant biological correlates. −20. The hypergeometric distribution was used to compute unadjusted p-values. For further insight, a protein–protein interaction (PPI) network with direct connections among the set of 70 gene products was constructed as shown in To identify significant genes, we employed a two-step approach: (1) the importance score for each gene was derived based on its contribution to the random forest accuracy  and (2) In this study, we developed a network-based method for predicting the drug sensitivity of pan-cancer cell lines in the GDSC database. Several studies have proposed network-based methods for drug response prediction on single omics data ,21, wherBCL6 gene, was initially discovered as an oncogene in B-cell lymphomas, driving a malignant phenotype via the repression of DNA damage and proliferation checkpoints [BCL6 has also been implicated in an expanding spectrum of solid and hematologic tumors, including leukemia, breast cancer, and NSCLC [BCL6 expression has been implicated in the modulation of apoptotic responses of malignant cells to chemotherapeutic reagents, suggesting the development of BCL6 inhibitors as a potential therapeutic option [In the application of machine-learning techniques to biology, interpretability is very important. Clustered cell lines and drugs, and the resulting random forest models in individual paired clusters, can be deeply interrogated to gain further insights into the determinants of cell-line drug effectiveness. We have demonstrated that post-modeling analysis using bioinformatics techniques enables the identification of plausible biological correlates. For example, we investigated the pair of cell-line cluster 4 and drug cluster 1 see A. Cell-lckpoints . BCL6 hand NSCLC . Additioc option .A limitation of this study is that using IC50 as the measure of drug sensitivity may be biased, due to different growth rates of cancer cells growing in culture . MoreoveWe used the anti-cancer drug-response data from the GDSC database. GDSC is a publicly available large-scale pharmacogenomic database that includes drug-screening data for more than a thousand human pan-cancer cell lines. The dataset consists of 265 compounds, including cytotoxic chemotherapeutics as well as targeted therapeutics. GDSC drug responses are given as log-transformed IC50 values  and the area under the curve (AUC) for a fitted model. We used log(IC50) as the degree of drug responses. Genomic mRNA expression profiles (Affymetrix Human Genome U219 Array) of the cell lines within GDSC were obtained from the CCLP database. A protein–protein interaction (PPI) network was obtained from the HPRD database. Cell lines with missing data for more than 80% of the drugs were removed, leaving 915 cell lines .http://oncokb.org/, accessed on 9 December 2021) that consists of 1019 genes. Among those 1019 genes, 796 genes were common to both CCLP and HPRD. In the HPRD network with those 796 genes, the largest connected network component consisted of 635 genes, which we focused on in this study  and downloaded the SMILES (Simplified Molecular Input Line Entry Specification) string of 241 drugs, for which the PubChem ID was provided in the GDSC database. Two-hundred drugs had response values for more than half of the cell lines, resulting in 183,000 cell-line drug pairs . The descriptors included functional groups, fragment counts, and estimated chemical properties as well as simple atomic descriptors. The overview of data analysis is shown in The CCLP and HPRD datasets had 8483 genes in common. Even though our method is applicable to large scale data, in this study, we wanted to focus on genes of known relevance in cancer. Thus, we used a smaller set of genes from the OncoKB (Precision Oncology Knowledge Base) database (gej) . By normIf we let this stochastic signal-passing process proceed from an initial state based on gene expressions, in repeated steps according to these probabilities , it can be shown that the system reaches a stationary distribution, implying that the system is invariant under a right multiplication by e., πp=π . SolvingWe initially extracted molecular descriptors of the 200 drugs from Dragon software. The following descriptors were removed: descriptors that are constant or near constant and descriptors with missing values, yielding 1500 features. We further removed many highly correlated features via unsupervised clustering, using the Spearman’s correlation between the features. We selected a representative feature from each cluster, which had the highest average correlation to all other intra-cluster features. This further reduced the number of features to 500. We then constructed a network of these cheminformatic features via the graphical LASSO, which suppressed unimportant feature connections to build a sparse network (see the W1 Wasserstein distance (EMD) metric  metric . OMT is The resultant pair-wise Wasserstein distance matrix was input to a hierarchical agglomerative clustering method, resulting in a set of clusters of cell lines or drugs. To find the optimal number of clusters, the silhouette score was used . The silmtry parameter was set to p/3, where p is the number of features [Predictive modeling of drug responses was conducted in each cell-line drug cluster, employing random forest regression on the associated gene-expression profiles and cheminformatic features. We chose the number of decision trees to be 100. For all other parameters, the default settings were used: the minimum number of samples in the terminal nodes was set to 5, and the features . Figure features , which ifeatures . We asseThis study proposed a novel network-based clustering method based on OMT theory for drug response prediction. Clustering was performed for cell lines and drugs using gene-expression profiles and cheminformatic drug features, respectively, represented in the form of data networks. Random forest modeling was then performed for each cell-line drug cluster pair. Prediction modeling on clustered homogeneous data is likely to improve the prediction accuracy for drug sensitivity, as well as enhance the biological interpretability compared to modeling using all the data together. We plan to apply the proposed approach to several biomedical problems with multi-modal data, including genomics and medical imaging.


In vivo and in vitro gene functional studies showed that LPCAT1 contributed to the pathogenesis of gefitinib resistance in LUAD, where an LPCAT1-EGFR positive feedback loop formed and then regulated its downstream signaling molecules of the EGFR/PI3K/AKT signaling pathway. The results provided novel insights into the acquired resistance mechanism of EGFR TKI from the perspective of phospholipid metabolism. These findings suggest LPCAT1 may serve as a potential therapeutic target for patients with EGFR TKI-resistant NSCLC.Currently, the mechanisms of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) resistance have been a focus of clinical research. Despite that most of the mechanisms of acquired EGFR TKI resistance have been revealed, about 30% of non-small-cell lung cancer (NSCLC) cases have not been fully elucidated, especially for lung adenocarcinoma (LUAD). Recently, LPCAT1, an important enzyme of phospholipid metabolism, has been found to bridge the gap between the oncogene and metabolic reprogramming. In NSCLC, LPCAT1 has been shown to participate in progression and metastasis. However, little is known about the role of LPCAT1 in acquired EGFR TKI resistance. In this study, elevated LPCAT1 expressions were observed in an EGFR TKI-resistant cell line (PC-9R) relative to a corresponding EGFR TKI-sensitive cell line (PC-9).  Lung cancer is the leading cause of cancer mortality in China, with 714,699 deaths from the disease recorded in 2020 Besides the classical EGFR-T790M mutation and transformation to a small-cell lung cancer (SCLC) phenotype, aberrant activations of bypass signaling pathways responsible for EGFR TKI resistance have been identified, such as c-MET amplification and abnormal activation of the PI3K/AKT pathway As is well known, metabolic reprogramming is becoming a hotspot in the field of cancer research. Cancer cells can alter their capability to metabolize carbohydrates, lipids, and proteins in an effort to strive for cell proliferation and survival, including lung cancer cells in vitro and in vivo.In the present report, three cell lines  representing normal human embryonic lung cells, gefitinib-sensitive cells harboring an EGFR exon 19 deletion mutation, and gefitinib-resistant cells, respectively, were selected. Gefitinib was chosen as the research agent due to the wide usage of EGFR TKI in China. We first explored the baseline level of LPCAT1 among three cell lines, and our data revealed that LPCAT1 expression was significantly higher in PC-9R cells than in PC-9 or MRC-5 cells. Then, LPCAT1 knockout was performed and found to be strongly associated with restoring the sensitivity to gefitinib in the PC-9R cell line. Finally, the PI3K/AKT pathway was confirmed to positively participate in the development of LPCAT1-mediated gefitinib resistance both PC-9 and PC-9R cell lines were purchased from Beinabiology Co., Ltd. , and the MRC-5 cell line was purchased from Procell Life Science & Technology Co., Ltd. . Five-week-old male BALB/c nude mice were obtained from Hunan SJA Laboratory Animal Co., Ltd. . Gefitinib (CAS: 184475-35-2) and the inhibitor of PI3K/AKT pathway, LY294002 (CAS: 154447-36-6), were purchased from Shanghai Yuanye Bio-Technology Co., Ltd.  and MedChemExpress , respectively.2 and 95% atmosphere, 37 °C). The medium was changed every three days and cells were passaged with 0.25% trypsin for digestion when the cell density reached 80% to 90% confluence.PC-9, PC-9R, and MRC-5 cell lines were cultured in Roswell Park Memorial Institute 1640 medium  containing 10% fetal bovine serum and 1% double antibiotics  and then maintained in standard conditions  according to the instruction manual. PC-9 and PC-9R cell lines stably expressing LPCAT1-specific short hairpin RNA (shRNA) or scrambled control-shRNA were constructed using a lentiviral shRNA technique from GeneChem Company . The LPCAT1 shRNA was designed based on the small interfering RNA (siRNA) sequence (GenBank identification no.: 79888).The primer used to obtain the overexpression sequence of LPCAT1 is as follows: F: 5'-CCCTGTGACCATGACGATGT-3', R: 5'-TGAATGCACCAGGTTTGAAGGT-3'. Meanwhile, the primer used for the overexpression negative control (NC) of LPCAT1 is as follows: F: 5'-UUCUCCGAACGUGUCACGUTT-3'; R: 5'-ACGUGACACGUUCGGAGAATT-3.Separately, the employed LPCAT1 shRNA sequences are as follows: (1) F: 5'-GGACAG AUACUCAGAAAGATT-3', R: 5'-UCUUUCUGAGUAUCUGUCCTT-3'; (2) 5'-CCAUUG ACCAAGAGGAGAATT-3', R: 5'-UUCUCCUCUUGGUCAAUGGTT-3'; and (3) F: 5'-CUGAGGAGGAGAAGAGGAATT-3', R: 5'-UUCCUCUUCUCCUCCUCAGTT-3'. Finally, the sequence of carboxyfluorescein (FAM)-labeled NC siRNA is as follows: F: 5'-UUCUCCGAACGUGUCACGUTT-3'; R: 5'-ACGUGACACGUUCGGAGAATT-3'. All primer pairs were synthesized by Beyotime .4 cells/well, and then treated with different concentrations of gefitinib  for 48 hours. The cells were subsequently incubated with a 10% solution of CCK-8 at 37 °C for two hours. Finally, the absorbance at 450 nm was measured using a multifunctional enzyme-linked analyzer . Each experiment was repeated at least three times.A cell counting kit 8 (CCK-8) assay was conducted using a CCK-8 kit from KeyGEN Biotechnology . The PC-9 and PC-9R cell lines were plated in 96-well plates at 1.0 × 106 cells from various groups were collected, washed with phosphate-buffered saline (PBS) and suspended in 500 μL of binding buffer. Then, the cells were stained with an annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit  at room temperature for 10 minutes according to the manufacturer's instructions. Cell apoptosis was analyzed by flow cytometry.A total of 1 × 10LPCAT1 gene. Total RNA was extracted using the Ultrapure RNA kit ; then, reverse transcription was conducted using the HiScript® II Q RT SuperMix for qPCR . qRT-PCR was performed with 2× SYBR Green PCR Master Mix  on a real-time fluorescent quantitative PCR meter . The β-actin gene was used as an internal reference gene, and the data were analyzed using the 2-ΔΔct method. PCR primers were ordered from Beyotime . All experiments were conducted according to the manufacturer's protocols. Experiments were performed at least in triplicate.Quantitative real-time transcription-PCR (qRT-PCR) was used to detect the messenger RNA (mRNA) expression of the The PCR primer sequences used are as follows: LPCAT1 (F) 5'-CCCTGTGACCATGACGATGT-3, (R) 5'-TGAATGCACCAGGTTTGAAGGT-3' and β-actin (F) 5'-TGGCACCCAGCACAATGAA-3', (R) 5'-CTAAGTCATAGTCCGCCTAGAAGCA-3'.The cells collected from various groups were lysed in radioimmunoprecipitation assay lysis buffer . Subsequently, the cells were centrifuged for 10 minutes at 12,000 rpm. Then, the supernatants were taken, and total protein was determined via bicinchoninic acid assay (CoWin Biosciences). An equivalent amount of protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes . Then, the membranes were blocked with 5% bovine serum albumin (Solarbio) and subsequently probed using respective primary antibodies overnight at 4 °C. Membranes were then incubated with the secondary antibody conjugated with horseradish peroxidase  for two hours at room temperature. Finally, the target proteins were measured by enhanced chemiluminescence  and Chemi Doc™ XRS+ System (Bio-Rad Laboratories). Glyceraldehyde 3-phosphate dehydrogenase (GADPH)  served as the internal control. The information of primary antibodies were listed as followed: anti-LPCAT1 , anti-EGFR , anti-p-EGFR, anti-PI3K , p-AKT (S473) , t-AKT, and horseradish peroxidase (HRP) conjugated anti-rabbit secondary antibody .6 cells in 200 μL of serum-free PBS, then subcutaneously inoculating them into the right rear flanks of five-week-old female BALB/c nude mice. Gefitinib was prepared by dissolving a 250-mg clinical-grade tablet in 150 μL of sterile water of 0.05% Tween-80 . When the volume of the transplantation tumor reached 100 mm3 , the mice were treated with different drugs as indicated below (n = 3 per group). Xenograft tumor volumes were measured every three days when they were palpable.PC-9R cells were prepared by suspending 1 × 10Mice were treated with (1) gefitinib (10 mg/kg) once daily via oral gavage, (2) LY294002 (2.5 mg/kg) every three days via intraperitoneal injection, (3) gefitinib and LY294002 co-treatment at the same doses, or (4) an equal volume of diluents as control therapy. The mice were humanely euthanized using conventional cervical dislocation after 12 days of treatment. Tumors were excised and dissected for further pathological evaluation. Hematoxylin and eosin staining was performed to evaluate the pathological and morphological changes in the xenograft tumor tissues. Ethical approval of this study was obtained from the Ethics Committee of the Jiujiang University Affiliated Hospital.p < 0.05.The data are presented as mean ± standard deviation values. Intergroup differences between two groups were assessed by the Student's t-test. Differences among multiple groups were assessed by one-way analysis of variance. The comparisons between the groups after the analysis of variance test were confirmed with the Student-Newman-Keuls method. In the present study, the experiments conducted were performed independently at least in triplicate. Significant differences were defined as when Figure 1, the mRNA expression of LPCAT1 in the PC-9R cell line was significantly higher than that in the PC-9 and MRC-5 cell lines. The PC-9 cell line displayed an enhanced LPCAT1 mRNA level relative to that of the MRC-5 cell line . Similarly, the ranking order of LPCAT1 protein levels in descending order of expression was as follows: PC-9R, PC-9, and MRC-5 cell lines .Normal human embryonic lung cells (MRC-5 cells) were used as the control cell line. As shown in Figures  and 2D, the IC50 values of gefitinib in the PC-9 and PC-9R cell lines were 12,653 nM (12.653 μM) and 53,480 nM (53.48 μM), respectively, which further verified the resistance of PC-9R cells to gefitinib relative to that of PC-9 cells. Considering experimental efficiency, we chose the concentrations of 12.653 μM and 53.48 μM as the optimal ones for the following experiments.To explore the sensitivity to gefitinib in PC-9 and PC-9R cell lines, we conducted a cell viability analysis by CCK-8 method. As evidenced in Figure 3, the levels of LPCAT1 mRNA and protein were robustly elevated in groups treated with the LPCAT1 overexpression vector compared to in groups treated with the empty vector and NC among either PC-9 or PC-9R cell lines by qRT-PCR and western blotting . Furthermore, we designed three siRNAs to interfere with LPCAT1 expression in PC-9 and PC-9R cell lines and, as shown in Figure 3, either the PC-9 or PC-9R cell line in siRNAs groups exhibited lower levels of LPCAT1 mRNA and protein than did the empty vector and NC groups. These findings substantiated that the transfection was successful.In this study, we first transfected PC-9 and PC-9R cell lines with the LPCAT1 overexpression vector and empty vector, respectively. As presented in Figures ) (and hereafter refer to this as the “interfere” group).Owing to the highest interference efficiency being present in the PC-9R cell line, we chose siRNA2-LPCAT1 for the subsequent experiments , which strongly indicated that LPCAT1 knockdown restored gefitinib sensitivity and inhibited cell proliferation in the PC-9R cell line.As illustrated in Figures ). However, the interference of LPCAT1 siRNA failed to induce cell apoptosis in PC-9R cell lines. These results suggest that other mechanisms  are involved in the proliferative inhibition of PC-9R cells transfected with LPCAT1 siRNA.In the PC-9 cell line, the cell apoptosis rate was significantly higher in the gefitinib/interfere group compared to in the other three groups  protein expression was observed. As depicted in Figure , compared to in the gefitinib group, gefitinib and LY294002 co-treatment significantly decreased the levels of PI3K and p-AKT proteins. In comparison with gefitinib monotherapy, LPCAT1 overexpression distinctly elevated the expression levels of EGFR, p-EGFR, PI3K, and p-AKT proteins. Furthermore, the addition of LY294002 obviously decreased the levels of EGFR, p-EGFR, PI3K, and p-AKT proteins compared to the group of LPCAT1 overexpression plus gefitinib treatment among PC-9R cells. Similarly, there was no significant change in t-AKT expression. These findings above indicated that LPCAT1 was the important upstream molecule that regulated the EGFR/PI3K/AKT signaling pathway in the PC-9R cell line.As shown in As the preceding results showed that LPCAT1-mediated gefitinib resistance of PC-9R cells is closely associated with the EGFR/PI3K/AKT pathway, we next verified this result by establishing a tumor-bearing mouse model.Figures 7, compared to in the control group, the volumes of xenograft tumors were slightly decreased in the groups of gefitinib and interfere NC plus gefitinib . However, in comparison with gefitinib monotherapy, the treatment of gefitinib plus interfere group markedly diminished the volume of xenograft tumors . Similarly, compared with interfere NC plus gefitinib, the addition of LY294002 substantially triggered tumor shrinkage . In particular, the volume of the xenograft tumor dramatically shrunk in the combination group of interfere, LY294002, and gefitinib compared to that in the other five groups .As described in Figure 8, compared with the control group, gefitinib treatment led to slight cell necrosis and smaller cell nuclei . In comparison with in the gefitinib group, cells in the groups of either interfere plus gefitinib or interfere NC, LY294002 plus gefitinib presented tumor cells with markedly smaller nuclei, greater cell necrosis, and larger gaps between cells Figures B, 8D-8E. Of note, the combination treatment of interfere, LY294002, and gefitinib resulted in massive tumor-cell necrosis, nuclei pyknosis, and lower intratumoral vascularity compared to the other five groups .Furthermore, hematoxylin and eosin staining was conducted to elucidate the pathological and morphological changes in the xenograft tumor tissues. As illustrated in EGFR TKI, especially gefitinib, has widely been used in advanced NSCLC patients with EGFR-sensitive mutations and has dramatically improved the clinical outcome of NSCLC. However, nearly all NSCLC patients will eventually progress to an EGFR TKI-resistant status. The mechanism of EGFR TKI acquired resistance is very complex, including both T790M-positive and T790M-negative resistance, each of which account for about 50% of cases In recent years, metabolic reprogramming, including lipid metabolisms, has been confirmed to contribute to tumor development and metastasis and drug resistance in vivoIn normal lung tissue, LPCAT1 has been found to be highly expressed and to regulate surfactant lipid generation in vitro revealed that LPCAT1 expression was markedly higher in the PC-9 cell line (EGFR 19 del E746_A750) than in the A459 cell line  and H460 cell line (EGFR wild-type) A study in vivo study was performed to observe the effect of LPCAT1 knockout in PC-9R cell tumor-bearing mice. We found that, in the xenograft model, LPCAT1 silencing resulted in tumor shrinkage and restored gefitinib sensitivity in the PC-9R cell line. Further in vivo experiments demonstrated that LY294002 substantially circumvented resistance to gefitinib and promoted gefitinib-induced shrinkage of xenograft tumors on the basis of LPCAT1 knockdown. Of coincidence, the study by Zhou et al. reported that LY294002 exerted the activity of sensitizing EGFR wild-type NSCLC cell lines to erlotinib chemotherapy According to the foregoing literature, the LPCAT1-EGFR positive feedback loop contributes to the tumorigenesis of glioma cells In summary, our study reported that enhanced LPCAT1 was observed in the EGFR TKI-resistant cell line compared to the corresponding EGFR TKI-sensitive cell line. A functional investigation found that LPCAT1 contributed to the pathogenesis of gefitinib resistance in LUAD, the mechanism of which may be at least partly attributed to positive regulation of EGFR/PI3K/AKT signaling cascade. The present study also provided a novel insight into the mechanism underlying acquired resistance to EGFR TKIs from the perspective of phospholipid metabolism. Since high LPCAT1 expression exists in a variety of cancers, LPCAT1 may serve as a potential target of targeted therapy.Of course, there are shortcomings in this study. One is that only a few single cell types were studied; the other is that the association of LPCAT1 with major resistant mutations  was not explored. These questions will need to be further explored in our future research.


Daphne from the Thymelaeaceae family, featuring a 5/7/6-tricyclic ring system and usually with an orthoester group. So far, a total of 135 diterpenoids has been isolated from the species of the genus Daphne, which could be further classified into three main types according to the substitution pattern of ring A and oxygen-containing functions at ring B. A variety of studies have demonstrated that these compounds exert a wide range of bioactivities both in vitro and in vivo including anticancer, anti-inflammatory, anti-HIV, antifertility, neurotrophic, and cholesterol-lowering effects, which is reviewed herein. Meanwhile, the fascinating structure–activity relationship is also concluded in this review in the hope of providing an easy access to available information for the synthesis and optimization of efficient drugs.There are abundant natural diterpenoids in the plants of the genus  Daphne Linn., with its ca. 95 species, is the most diverse genus in the Thymelaeaceae family [Daphne have been applied for a long history in traditional treatments for aches, rheumatism, inflammation, and abortion in Asia, Africa, and Europe [Daphne mezereum L. and other Daphne species in 1970. Subsequently, an increasing number of diterpenoids have been discovered from Daphne species.The genus e family . Some ofd Europe . Yet, noDaphne, and the genus Daphne itself also acts as an important role in the discovery of phytochemical and bioactive properties of diterpenoids. The archetype of one class, daphnetoxin, was first isolated from D. mezereum, which was named after the genus Daphne. Then, daphnetoxin and its analogues have been collectively known as the daphnetoxin class. Similarly, genkwanine A from D. genkwa is the archetypical diterpenoid of genkwanines.The diterpenoids are believed to be representative components of the genus Daphne genus also contribute to the pharmacological study of diterpenoids and have been demonstrated to possess a variety of important biological activities including anticancer, anti-inflammatory, anti-HIV, antifertility, neurotrophic, and cholesterol-lowering effects [The diterpenoids from the  effects , and somDaphne. The occurrence and distribution of these diterpenoids are also discussed thoroughly, including the source species of every diterpenoid listed in a chronological order of discovery and the parts of plants which they were isolated from. Besides, detailed information on every class of diterpenoids is provided in this review. When the adequate information is given, the structure–activity relationship (SAR) is discussed.The current review provides a comprehensive coverage of all natural diterpenoids in the genus Daphne in total. Most of the natural diterpenoids occurring in the genus Daphne belong to the daphnane type featuring a 5/7/6-tricyclic ring system with polyhydroxyl groups at C-3, C-4, C-5, C-9, C-13, C-14, or C-20 and an orthoester function located at ring C, which could be further categorized into four major classes:1–83), sharing the characteristic of an epoxy ring at C-6 and C-7;6-epoxy daphnane diterpenoids (84–95), possessing a saturated ring A and a 6,7-dihydroxyl group in the ring B;genkwanines (96–97), which could be viewed as 5-deoxy-6,7-double bond daphnetoxin derivatives;resiniferonoids (98–107), featuring a saturated ring A and a macrocyclic bridge connecting C-1 in the ring A and the end of the aliphatic orthoester group  and daphnetoxins (55–83) according to the possession of an oxygen group at C-12 in the ring C [For the 6-epoxy daphnane diterpenoids, they could be further subdivided into 12-hydroxydaphnetoxins (xins 55–8 accordin108–123). As for compounds 108–112, the skeleton resembles the genkwanine one but possesses a 3-ketone group, a C-12 oxygenated substituents, and an additional 1,2-double bond, which makes it not accord with the definition of genkwanine. What attracts more attention is the unique 4,7 or 4,6-ether structure of neogenkwanine A-I and daphneodorin H (113–123), which is believed to never be found in any other daphnane diterpenoid [Nonetheless, it is worth noticing that the existing classification system could not apply to some daphnane diterpenoids (124–133) in Daphne species, instead of a caged 9,13,14-orthoester, distinguish themselves with a cyclopropane ring D, which is regarded to be closely related to the daphnane one [134 and 135) were also reported to be isolated from Daphne genkwa, featuring a characteristic 5/11/3-membered ring system.Besides daphnane diterpenoids, tigliane diterpenoids  and daphnetoxins (55–83), based on the oxygenated substituent at C-12 in the ring C.The diterpenoids from this class share the common features of a 6α,7α-epoxy and 4β,5β-dihydroxy in the seven-membered ring B. The 6-epoxy daphnane diterpenoids could be further divided into two classes, 12-hydroxydaphnetoxins , 3-deoxy derivative (54), and the ones with a 5,20-acetonide (27 and 28). The archetypal compound of 12-hydoxydaphnanetoxins is 12-hydroxydaphnetoxin (39), which was first found as a degradation product in Lasiosiphon Burchellii Meisn. [Compared to daphnetoxins, 12-hydroxydaphnetoxins have an additional oxygen group at C-12. There are more 12-hydroxydaphnanetoxins than daphnetoxins existing in the species from the ives 43–4, 3-deoxyDaphne. Compounds of this class were mainly isolated from D. genkwa, D. tangutica, and D. acutiloba and these diterpenoids were abundant in the flower buds of D. genkwa, stems, roots, and especially bark of the plants . The archetype of this class is daphnetoxin (14), which was separated from the bark of D. mezereum for the first time [D. papyracea [D. giraldii [D. tangutica [D. acutiloba [There are less compounds from the genus rst time  and lateapyracea , D. giragiraldii , D. tangangutica , and D. cutiloba  in chron84). Among these genkwanines, only genkwanine L (95) possesses a ketone function at C-3 and an oxygen-containing function at C-12  of D. genkwa [Up to now, all compounds of this class were reported to occur in . genkwa ,57,66,73E. resinifera, showing significant transient receptor potential vanilloid 1 (TRVP1) activating activity and strong irritant effect [The resiniferonoids are a group of 5-deoxy-6,7-double bond daphnetoxin derivatives, in which the A/B ring system possesses the pattern of phorbol. One of the most representative resiniferonoids, resiniferatoxin (RTX), was isolated from the dried latex of t effect ,5, and cEuphorbia genus in the Euphorbiaceae [96 and 97) obtained from the Daphne species [Resiniferonoids have a quite narrow distribution, mostly limited to several rbiaceae ,5. Two n species  are prac98–107) have an obvious feature of bridging between the C-1′ at the end of 9,13,14-orthoester group and C-1 in the ring A with a C-C bond. Meanwhile, the ring A of 1-alkyldaphnanes is usually saturated. The 1-alkyldaphnanes also has two subdivisions, 3-hydroxy and 3-acyloxy classes, depending on the oxidation degree of C-3 , which was obtained from Gnidia subcordata originally [Daphne plants. The 1-alkyldaphnanes (98–107) tend to exist mainly in the buds of D. genkwa [D. odora [Daphne genera, there appeared to be more 1-alkyldaphnane diterpenoids in the leaves and roots of the plants from the Stellera [Wikstroemia genera [The archetype of this category is gnidimacrin  remain uncategorized into four classes mentioned above, which could be divided into two major types. The skeleton of daphgenkin A–C (109–111) and yuanhuaoate B (112) resembles the one of genkwanines  feature a 4,7-epoxy-bridged structure  in ring B, which is very rare in daphnane diterpenoids, yet there is no appropriate category for these diterpenoids. These diterpenoids (108–123) were obtained from the buds of D. genkwa with only the exception of daphneodorin H (113) isolated from the leaves and branches of D. odora , there are structural variations of a 7-ketone function and a 5,6-double bond in the ring B in the others  was isolated from commercial “mezeron” bark  in 1970 and identified as a major toxic component possessing both a similar skeleton and similar sites of functions as phorbol, which was a known toxic principle in Croton species [24) with the same skeleton as daphnetoxin (56) was identified as another toxic principle of D. mezereum [However, the poisonous principles of any  species ,96 at thmezereum .Daphne genus possess a wide range of pharmacological activities including anticancer, anti-inflammatory, anti-HIV, cholesterol-lowering, neurotrophic, antifertility, skin irritant, nematicidal, and pesticidal activities. Among these biological activities, the anticancer ones have received the most attention since there remains a need for both efficient and safe anticancer drugs with novel structures.With a considerable number of modern pharmacological and chemical studies, it was demonstrated that diterpenoids from the Daphne species exhibit potent anticancer activities against various types of cancers both in vitro and in vivo [50 values ranging from 10−6–98.46 μM , yuanhuatine (47), and yuanhuaoate B (112) share the similar structure with the only exception of an unsaturated bond in the ring A, and yuanhuatine (47) among them is the least active anticancer compound [111) and yuanhuaoate B (112), it appears that the change from the 1,10- to 1,2-double bond enhances the anticancer activity [It is generally accepted that the 1,2-double bond could be a possible alkylating functionality in a diterpenoid and thus the reduction of it might make it a less active compound . Genkwadcompound ,14. The O-benzoyl-3,5-hydroxy-6,7-epoxy-resiniferonol-9,13,14-orthobenzoate, which is a 3-hydroxyl derivative of genkwadaphnin (15), showed a promising cytotoxicity against seven carcinoma cell lines, much better than genkwadaphnin (15) itself, indicating the possibility that the hydronation of a 3-ketone favors a bioactivity [84) with a 3-hydroxyl was basically inactive against A549 cells, while its 3-acylated analogues (85–87) all possessed certain cytotoxicity with the IC50 values in the range of 0.79–8.70 μM [87) was the most potent one with a 3-benzoate group at the IC50 values of 0.79–8.00 μM [In the ring A, the variation of the oxygen-containing function at C-3 influences the anticancer activity. 12-activity . However–8.70 μM , indicat–8.00 μM  suggesti30), yuanhuadine (31), yuanhuafine (32), and yuanhuapine (46) generally showed stronger cytotoxicity against A549 cell line than genkwanine A-L (84–95) possessing a 6α,7α-dihydroxyl in the same assay [100) possessed a stronger activity in seven cell lines than genkwadane C (107) [The 6α,7α-epoxy is a characteristic structure in 6-epoxy and 1-alkyldaphnane diterpenoids, and it was reported to help enhance the anticancer activity. Yuanhuacine (me assay . Additio C (107) , suggest30) showed no inhibitory activity against P-388 in vivo, while its 20-palmitate derivative gnidilatidin 20-palmitate (19) exhibited a strong inhibitory activity at dosages of 0.5–2.0 mg/kg/d, and the 20-plamitate derivative of gnidilatin was also observed to be more active than gnidilatin itself against P-388 [The 20-acyloxy group in the ring B might have an important role in affecting the anticancer activity in diterpenoids. One type of 20-acylocy groups that could favor the bioactivity is a 20-palmitate ester. Yuanhuacine (st P-388 . This fa68) with a 20-benzoate was demonstrated to inhibit various cell lines more significantly, especially HL-60 leukemia cells, A375-S2 melanoma cells, and HT-1080 fibrosarcoma cells, than orthobenzoate 2 (71) [91) possessing a 20-benzoate exhibit much stronger cytotoxicity against P388 and A549 (IC50: 13.0 and 1.60 μM) than its analogues [Genkwanine M (e 2 (71) . Similarnalogues , indicat93–94) with a 9,13,14-orthoester were more cytotoxic (IC50: 4.20–42.0 μM) than genkwanine F–G  in P-388 and A549 cells [The caged 9,13,14-orthoester is another characteristic structure in the daphnane-type diterpenoids in general. Genkwanines J–K (49 cells .92) showed potent inhibition against P388 and A549 (IC50: 4.2 and 25.0 μM), while genkwanine F (84) were moderately active with the IC50 values of 39.0–24.0 μM [92) compared to genkwanine F (84) in different cell lines [Interestingly, for a more statured skeleton, for instance, genkwanine, the caged 9,13,14-orthoester seems to reversely have a negative impact on anticancer activity. Genkwanine J (–24.0 μM . More st30) and yuanhuadine (31) showed promising inhibition against HepG2 cell line with the IC50 values in the range of 5.56 to 17.06 μM, while yuanhuafine (32) showed more limited activity at the IC50 value of 42.37 μM [Evidence showed that the presence of a long-chain fatty could enhance the inhibitory activity against carcinoma cells. Yuanhuacine (42.37 μM .31) was inactive with the introduction of 2′,3′- and 4′,5′-double bonds [33) with one more unsaturated bond in the long-chain substituent in orthoester group presents weaker bioactivity (IC50: 809.4 nM) than yuanhuadine (31) and isoyuanhuadine  in SK-BR-3 [Whether this long chain at the end of the 9,13,14 orthoester is statured or not could influence the antineoplastic activity as well. Gnidilatin was a potent antileukemia compound against P-388 in vivo; meanwhile, yuanhuacine  and yuanhuadine (31) possessing a 2′E,4′E-double bond could both inhibit SK-BR-3 proliferation more significantly (IC50: 172.6 and 61.6 nM) than their conformational isomers, respectively (IC50: 217.1 and 83.7 nM) [As for the unsaturated long-chain at C-1′, the stereochemistry matters. Yuanhuacine  and yuan24), a major toxic principle of D. mezereum, showed significant inhibitory activity in vivo against P-388 and L-1210 leukemia cell lines at dosage of 50 μg/kg in mice [16), gnididin (18), and gniditrin (20) with the closely related skeleton isolated from Gnidia lamprantha Gilg were also proved to be substantial antileukemia agents. Huratoxin (59) and simplexin (60) exhibited similar inhibitory activities against L1210 and K562 in vitro, but less active than an esterified derivative subtoxin [39) bearing a hydroxyl at C-12 in the ring C showed no antileukemia activity and esterification of it could establish the bioactivity [15), which is in essence the benzoyl derivative of 12-hydroxydaphnin (39), was found to exert both in vitro and in vivo antileukemia activity against P-388 cells [The absence of a 12-acyl group generally causes the reduction of anticancer bioactivity. Mezerein ( in mice . Gnidicisubtoxin . By compactivity . Genkwad30) showed stronger cytotoxicity against HL-60 and K562 cells (IC50: 26.81 and 16.08 μM) than yuanhuadine . Genkwadaphnin (15), with a 12-benzoate function, was also more active in antileukemia than yuanhuafine (32), as the latter showed limited inhibitory activity in HL-60 and K562 cell lines [47) was cytotoxic against human and chronic myeloid promyelocytic leukemia cell lines at the IC50 values of 17.72 and 17.54 μM [46) has not been reported to possess antileukemia property. Yuanhuacine (30) and isoyuanhuacine (21) showed moderate cytotoxicity against SK-BR-3 (IC50: 172.6 and 217.1 nM), while and yuanhuadine (31) and isoyuanhuadine (22) were more active (IC50: 61.6 and 83.7 nM) [The type of ester group at C-12 may also have an impact on the antileukemia activity. Yuanhuacine  is one of the most potent antileukemia agents both in vitro (IC50 in the range of 0.16–0.28 nM) to HL-60, K562, and CCRF-CEM cells [Daphne species [It is noteworthy that gnidimacrin  and pimelotide C (106), it seems that the one with α-CH3 (106) exhibits more significant antineoplastic bioactivities against a variety of cell lines including HeLa, MCF-7, HepG2, HCT116, A549, A375-S2, U937, HL-60, and K562, which may suggest that the stereochemistry of substituents is related to the anticancer activity as well [Interestingly, for pimelotide A ( as well .15) could oppose the protein and DNA synthesis of P-388 cells to exert both in vitro and in vivo antileukemia activities [15, IC50: 11.8 μM) and yuanhuacine  were determined to suppress Bcl-2 and Bcl-XL in a dose-dependent manner to induce apoptosis in human myelocytic HL-60 cells [It is also revealed that genkwadaphnin  showed a significant antiproliferative effect against K562 cell lines at the IC50 of 1.2 nM by activating protein kinase C (PKC) and arresting the cell cycle at G1 phase [Gnidimacrin (G1 phase .31), yuanhuadine (63), yuanhualine (64), and yuanhuagine (33) exhibited stronger antiproliferative activity against A549 cells  than the positive control ellipticine without displaying cytotoxicity against the human normal lung epithelial cell line MRC-5, with especially yuanhuacine  presenting the most potent bioactivity [30) could also be anticancer bioactive against H1993 human non-small cell lung cancer (NSCLC) cells both in vitro and in vivo by modulation of the AMPK/mTOR signaling pathway [Several 12-hydrodaphnetoxins including yuanhuadine ( pathway .64), yuanhuadine (63), and yuanhuagine (33) showed notable inhibitory effects in drug-resistant cell lines including gemcitabine-resistant A549, gefitinib- and erlotinib-resistant H292. Further research indicated that these diterpenoids were able to arrest cell cycle in the G0/G1 and G2/M phase in A549 cells by upregulating the expression of cyclin dependent kinase inhibitors P21 and P53 as well as downregulating cell-cycle regulators, for example, c-Myc and cyclin B1/cell division cycle 2 (CDC2) complex and suppress Akt/STAT/Src signaling pathway [64) was observed to have synergistic effects with certain chemotherapeutic agents  in the treatment of A549 cell line [Yuanhualine ( pathway . Additioell line .15) on hepatocellular carcinoma (HCC) cells both in vitro and in vivo with Hep3B and PLC/PRF/5 cell lines and BALB/c nude mice, respectively, the results showed that genkwadaphnin (15) suppressed growth and invasion of HCC cells both in vitro and in vivo by blocking DHCR24-mediated cholesterol biosynthesis and lipid rafts formation [30) and genkwadaphnin (15) were hepatotoxic on normal human liver cells HL-7702 in a dose- and time-dependent manner; meanwhile, the change of cell morphology and increased AST and ALT were observed as well [Wu et al. evaluated the effects of genkwadaphnin (ormation . Evidenc as well .30) was found to be an active inhibitor in both MCF-7 and MDA-MB-231 cell lines, and the preliminary mechanism of strong cytotoxicity of yuanhuacine (30) against MCF-7 was investigated further by using Western blot and flow cytometry analysis; the results suggested that yuanhuacine (30) induced apoptosis via the regulation of Bcl-2, Bax, and cleavage of PARP expression in MCF-7 cells [47) was also observed to inhibit the growth of estrogen receptor alpha (ERα)-positive cells MCF-7 (IC50: 0.62 μM) significantly compared to tamoxifen (IC50: 14.43 μM) through mitochondrial dysfunction and apoptosis in ERα-positive breast cancer cells MCF-7 caused by ERα-downregulation [Yuanhuacine , Yuanhuatine (47), and genkwanine M (68) isolated from the buds of D. genkwa displayed pronounced inhibitory bioactivity against the human melanoma cell line A375-S2 at the IC50 levels of 8.72, 9.31, and 3.62 μM, respectively [30) and yuanhuadine (31) were obtained from the dichloromethane fraction and showed potent cytotoxicity to melanoma B16 as well as A2058 cell lines, while genkwanine C (86) and genkwanine VIII (118) were selectively active in the A2058 cells [30), yuanhuadine (63), genkwadaphnin (15), genkwanine A (84), genkwanine F (89), genkwanine H (91), and daphneresiniferin A (96) and B (97) could inhibit the α-MSH-induced melanin production in B16 melanoma cells remarkably with the IC50 in the range of 0.57–9.0 μM in comparison with the positive control arbutin (IC50: 140 μM) and kojic acid (IC50: 39 μM), especially yuanhuadine  with the striking inhibitory activity [97) showed obvious cytotoxicity against B16 cells at the IC50 level of 6.6 μM [15) was also investigated to exert apoptosis-triggering effect in squamous cell carcinoma (SCC) cells in a JNK-dependent manner [Yuanhuacine  cell and cytotoxic T lymphocyte (CTL) [Besides, the antiproliferative activity of the ethyl acetate and aqueous extract from the leaves of 10 cells ; furtherte (CTL) , hopefulD. mucronata was evaluated to possess anticancer property both in vitro and in vivo and its mechanism, similar to the natural anticancer drug Taxol, was probably related to the downregulation of human tumor necrosis factor alpha receptors (TNF-αR) [54), was isolated from D. mucronata afterwards and showed a strong antiproliferation effect on WEHI-164 by mediating the progress of DNA synthesis [The alcohol–water extract of the aerial parts of (TNF-αR) ; the proynthesis .15) was revealed when it was found that it enhanced the p21 expression and simultaneously suppressed the c-Myc expression in a PRDM1-denpendent manner to arrest the cell-cycle progression in the human colon cancer SW620 cell line [The mechanism of anticancer effect of genkwadaphnin (ell line .30) and yuanhuadine (31) were revealed to exhibit more significant inhibitory effects on the proliferation of the COLO250 (IC50: 2–3 μM) than HT-29 cell line (IC50: 13–23 μM) [30) inhibited the HCT116 cell line by upregulating p21 expression and transcription via a p53 protein independent cascade [108), along with yuanhuacine (30) and yuanhuadine (31) obtained from the petroleum ether extract from D. genkwa, showed definite cytotoxic effects on both SW620 and RKO cell lines with the respective IC50 value of 3.0 and 6.5 μM; then, the results of further research revealed that daphgenkin A (108) inhibited SW620 cell proliferation by stalling the cell cycle at G0/G1 phase, causing cell death by apoptosis as well as inducing cell cycle arrest via regulating the PI3K/Akt/mTOR signaling pathway [Yuanhuacine (3–23 μM) , suggest cascade . Daphgen30), yuanhuadine (31), yuanhuatine (47), genkwanine F (89), genkwanine N (69), and wikstroemia factor M1 (72) obtained from D. genkwa were moderately cytotoxic with IC50 levels in the range of 25.61 to 27.32 μM against the human gastric carcinoma MGC-803 cell line [30, IC50: 17 μM) and yuanhuadine  were detected to oppose the proliferation of human gastric adenocarcinoma AGS cell lines, along with genkwanine VIII  [Yuanhuacine (ell line . Further: 12 μM) .30) and yuanhuajine (7) presented more obvious inhibitory activity against DNA topoisomerase I (DNA topo I) at the IC50 levels of 40.0 and 38.3 μM than a known topo I inhibitor hydroxycamptothecin , and further study with the prepared derivatives suggested that less electron-withdrawing groups at C-5, C-12, and C-20 facilitated the combination between the compounds and DNA topo I [Yuanhuacine (A topo I .30) was also reported to be cytotoxic against two bladder cancer cell lines UMUC3 (IC50: 1.89 μM) and T24T (IC50: 1.83 μM), and it functioned in T24T cells by inducing a G2/M phase arrest significantly via modulation of Sp1 protein expression [Yuanhuacine (pression .50), yuanhuacine (30), and yuanhuatine (47) with the IC50 levels of 11.62–12.35 μM [54) showed a stronger cytotoxicity (IC50: 1 μM) [The human lymphoma cell line U937 has been reported to be moderately suppressed by daphnane-type diterpene ester-7  .D. altaica was also found to significantly suppress the proliferation of esophageal squamous carcinoma Eca-109 cell line at the IC50 level of 10.6 μM and in a dose-dependent manner [D. altaica was reported to function by inducing apoptosis and cell cycle arrest in the S phase in the Eca-109 cell line via the PPARγ-mediated pathway [The chloroform extract from t manner , and the pathway . HoweverDaphne species were demonstrated to possess an anti-HIV activity even stronger than some anti-HIV agents such as 3′-azido-3′-deoxythymidine (AZT), and structure-activity relationship in anti-HIV bioactivity is quite similar to that in the anticancer one (Both daphnane- and tigliane-type diterpenoids from ncer one .11–12), presented stronger anti-HIV activity than daphneodorins F–H  without one (EC50 > 25 nM) [35) and its isomer, isoyuanhuadine (22), showed promising anti-HIV activity (EC50 < 0.9 nM), while yuanhuaoate C (53) without a 9,13,14-orthoester merely exhibited moderated activity [The presence of a 9,13,14-orthoester in the ring C is also favorable for anti-HIV activity as diterpenoids with an orthoester, for instance, daphneodorins D–E  from D. aurantiaca showed definite anti-HIV-1 activity against C8166 cell line with EC50 value of 0.282 nM and SI value of 65177.305, while phorbol 13-monoacetate (130) possessing a 12-hydroxyl showed limited activity, which suggests an acyl group at C-12 might favor anti-HIV-1 bioactivity [The substituent at C-12 in the ring C acts as an important role in anti-HIV activity as well. A tigliane diterpenoid 12-activity .102–103) presented more potent activity in inhibiting HIV-1 replication in MT4 cell line  than daphneodorin C (EC50: 2.9 nM) suggesting that a 20-benzoyloxy reduces the anti-HIV activity [Daphneodorins A–B (activity .129) is known as a potent anti-HIV agent [98), a HIV-latency-reversing compound that is strikingly 2500-fold more potent that prostratin (129), functioned by regulating various signaling pathways including the MAPK, PI3K-Akt, JAK-Stat, TNF, and NF-κB ones [Prostratin , genkwanine N , genkwadaphnin , kirkinine , excoecariatoxin , and 14′-ethyltetrahydrohuratoxin  were obtained from D. acutiloba, all of which exhibited strong anti-HIV-1 bioactivity, especially genkwanine N  [Acutilobins A–G , gnidicin (16), gniditrin (20), and excoecariatoxin (58) were determined to be the principles of anti-HIV bioactivity according to the HPLC-based profiling; meanwhile, a more detailed study showed that daphnetoxin (56) selectively interfered with the expression of two key cell-surface factors CXCR4 and CCR5 for HIV-1 entry [Based on the discovery of the antiretroviral activity of the dichloromethane extract of -1 entry .77) inhibited not only HIV-1 but HIV-2 strains in a concentration-dependent manner with high SI values and low cytotoxicity, and its mechanism might be related to the inhibition of HIV-1 reverse transcription and integrase nuclear translocation [It is worth noting that wikstroelide M (location .15), gniditrin (20), and yuanhuatine (47) showed significant inhibitory effect against LSP-induced nitric oxide (NO) production in RAW 264.7 macrophages with the IC50 values of 0.03–0.07 μM, especially 12-O-benzoylphorbol-13-nonanoate (124) and 12-O-benzoylphorbol-13-octanoate (85) with the IC50 values of 0.01 μM could be potential therapeutic agents for inflammation [132, IC50: 7.2 μM), a tigliane-type diterpenoid derived from D. genkwa, and orthobenzoate 2  showed stronger inhibitory activity than aminoguanidine (IC50: 17.4 μM) on NO production in RAW 264.7 cells [Genkwadaphnin , the mechanism includes the activation of PKD1/NF-κB signaling to induce CD44 expression in a time- and concentration-dependent manner and thus promoting the migration of K562 cells, resulting in an innate immune response [15) was also observed to restore exhausted LCMV-specific CD4+ and CD8+ T cells by downregulating negative regulatory molecule Tim-3 [As for another inflammatory inhibitor genkwadaphnin (response . Genkwadle Tim-3 .128), genkwadaphnin (15), isoyuanhuacine (21), yuanhuacine (30), yuanhuaoate C (53), yuanhuagine (33), isoyuanhuadine (22), and yuanhuadine (31) in LPS-induced RAW264.7 cells by downregulating the overexpression of IL-6, IL-1β, and TNF-α as well as decreasing NO production as the results of principal component analysis (PCA) and hierarchical cluster analysis (HCA) indicated, among these, prostratin Q (128) also showed bioactivity on VEGF, MMP-3, and ICAM, which has the potential to be developed as a novel drug for rheumatoid arthritis treatment [It is reported that diterpenes showed anti-inflammatory efficacy sorted from highest to lowest as follows: prostratin Q (nhuadine  in LPS-i20) and daphnetoxin (56) extracted from D. giraldii were found to present potent cholesterol-lowering activity in vitro at the EC50 levels of 0.59 and 4.3 μM, respectively, by up-regulating the low-density lipoprotein receptor (LDLR) level and consequently promoting LDLR expression [20) had a lower EC50 for activating LDLR-promoter than that of daphnetoxin (56), which might indicate that the acyl group at C-12 might improve the cholesterol-lowering bioactivity as well.Gniditrin (pression . Gniditr30) and genkwanine N (69) significantly enhanced the function of the orphan nuclear receptor Nurr1 at a concentration of 0.3 μM and inhibited LPS-induced neuroinflammation in vitro as well as improved behavioral deficits in a hydroxydopamine (6-OHDA) -induced rat model of Parkinson’s disease [Yuanhuacine ( disease .133), a tigliane diterpene from the EtOAc extract of D. holosericea, was discovered to be a moderate acetylcholinesterase (AChE) inhibitor at a concentration of 100 μM, which suggested its potential usage in the mediation of Alzheimer’s disease [Dapholosericin A ( disease .134) and B (135), two lathyrane-type diterpenes isolated from the chloroform extract from D. genkwa, were demonstrated to inhibit LPS-induced NO production in microglial BV-2 cells moderately (IC50: 43.08–46.77 μM) with nearly non-cytotoxicity, while yuanhuapine (46), yuanhuatine (47), genkwanine M (68), genkwanine N (69), yuanhuafine (32), and genkwadaphnin (15) showed much stronger anti-neuroinflammatory effect with the IC50 value below 0.44 μM, affecting cell viability slightly (17.5%–32.5% cell death at 10 μM) [Genkwalathins A (t 10 μM) .D. genkwa are also used as a traditional Chinese herbal remedy for abortion. Both yuanhuacine (30) and yuanhuadine (31) have already been used clinically as labor-induced drugs at the per capita dose of 70–80 and 60 μg, respectively [47) [26) [The flowers of ectively . Yuanhuaely [47)  and tang47) [26)  also sho47) [26) , and the47) [26) .59), mezerein (24), simplexin (60), and pimelea factor P2 (99), are highly skin irritant and toxic, thus considered as toxic principles in Daphne species [Many naturally occurring daphnane and tigliane diterpenoids, including huratoxin  was isolated from the seeds of D. mezereum and verified to be skin irritant just as daphnetoxin (56) [55), daphnetoxin (56), and 12-hydroxydaphnetoxin (39) obtained from D. giraldii were discovered to exert skin irritant activity [59), genkwadaphnin (15), prohuratoxin (77), and 1,2α-dihydrodaphnetoxin (73) were isolated from D. feddei, all of which have been determined to be skin irritant, and Huratoxin (59) exhibited the highest irritant activity among these four compounds [Mezerein (xin (56) . Daphnegactivity . Huratoxompounds .D. odora was nematicidal to Aphelenchoides besseyi; then, further study showed that odoracin (25) had nematicidal activity towards A. besseyi was 100% (5 ppm) after 5 days [56) was reported to be a major poisonous component in D. mezereum [25), along with odoratrin (45), gniditrin (20), 12-O-(E)-cinnamoyl-9,13,14-ortho--decadienylidyne-5β,12β-dihydroxyresiniferonol-6α,7α-oxide (41) and 12-O-(E)-cinnamoyl-9,13,14-ortho--decatrienylidyne-5β,12β-dihydroxyresiniferonol-6α,7α-oxide (40), was found to have ornithine decarboxylase (ODC)-inducing activity in mouse skin, which correlated with a tumor-promoting effect; these compounds were simultaneously determined to be piscicidal [It has been discovered that the benzene extract from the roots of r 5 days . Daphnetmezereum . Odoraciscicidal . Daphne species, daphnetoxin (56), was isolated as a toxic principle [Daphne after decades. These diterpenoids could be classified as daphnane-, tigliane-, and lathyrane-types according to the oxygen-containing functions and substitution pattern, and daphnane-type could be subdivided into 6-epoxy, genkwanine, 1-alkyldaphnane, and resiniferonoid. Within more and more novel diterpenoids isolated from Daphne genera, some of them could not fall into any classification. For example, genkwanine L (95) with a 3-ketone and 12-acetyl group does not completely satisfy the definition of genkwanines. As for compound 108–123, one or more suitable classifications have not been established yet.Back to 1970, the very first diterpenoid from rinciple . A totalDaphne embraces more than ninety species distributed in Asia, Africa, and Europe [Daphne species and diterpenoids have been reported to occur mainly in D. genkwa. For D. mucronata, only two isolated diterpenoids but one of those, gnidilatimonoein (54), showed potent anticancer activity [Daphne. The distributions of every diterpenoid in different parts of plants are available in the hope that it would be a guide for isolation and identification of diterpenoids.Although the genus of d Europe , the oriactivity ,122,123,Daphne species have been assayed and found to possess pharmacological activities which might be closely related to diterpenoids. The methanol extract of D. malyana showed significant antimicrobial potential [D. linearifolia, of which the stem bark was used to treat inflammation and rheumatism such as some Daphne species mentioned above, showed affinity towards a promising anticancer target Hsp90 [D. cneorum extract exhibits potent antimicrobial and antioxidant activities, which makes it a possible source of new agents [D. alpina, its antioxidant and antimicrobial bioactivities have been investigated [20) has been isolated from this species [Daphne species.Furthermore, some otential  and the et Hsp90 . The D. w agents . For D. stigated ,128 and  species . These sFor the structure–activity relationship (SAR) summarized in this review, despite the fact that some of the SAR remains unclear and needs to be further investigated, it showed that the pharmacological effect, to a large extent, depends on the substituents at C-3, C-12, C-20, and C-1′ and the saturation of the ring A in these molecules. The SAR study is believed to provide very useful information for the optimization and synthesis of diterpenoids and lead to the development of novel agents; however, the SAR of diterpenoids has not been completely clarified.Daphne were covered. Many of the Daphne species are bioactive and thus have been used as traditional treatments for various diseases. Both the source species and parts from which diterpenoids were isolated have been provided as detailed reference information. The natural diterpenoids from Daphne species present interesting structures with complicated stereochemistry, which are closely related to their abundant bioactivities. The biological activities and the structure–activity relationship of certain classes of diterpenoids were reviewed in the hope of providing an easier way for researchers to understand the general situation of phytochemical and pharmacological properties in diterpenoids from the genus Daphne.In this review, a total of 135 natural diterpenoids in the past decades from the plants of the genus