Conversely, O2 levels in most mammalian tissues range from 1–6% (physioxia). Such hyperoxic conditions in cell culture can alter reactive oxygen species (ROS) production, metabolism, mitochondrial networks, and response to drugs and hormones. The aim of this study was to investigate the transcriptional response to different O2 levels and determine whether it is similar across cell lines, or cell line-specific. Using RNA-seq, we performed differential gene expression and functional enrichment analyses in four human cancer cell lines, LNCaP, Huh-7, PC-3, and SH-SY5Y cultured at either 5% or 18% O2 for 14 days. We found that O2 levels affected transcript abundance of thousands of genes, with the affected genes having little overlap between cell lines. Functional enrichment analysis also revealed different processes and pathways being affected by O2 in each cell line. Interestingly, most of the top differentially expressed genes are involved in cancer biology, which highlights the importance of O2 levels in cancer cell research. Further, we observed several hypoxia-inducible factor (HIF) targets, HIF-2α targets particularly, upregulated at 5% O2, consistent with a role for HIFs in physioxia. O2 levels also differentially induced the transcription of mitochondria-encoded genes in most cell lines. Finally, by comparing our transcriptomic data from LNCaP and PC-3 with datasets from the Prostate Cancer Transcriptome Atlas, a correlation between genes upregulated at 5% O2 in LNCaP cells and the in vivo prostate cancer transcriptome was found. We conclude that the transcriptional response to O2 over the range from 5–18% is robust and highly cell-type specific. This latter finding indicates that the effects of O2 levels are difficult to predict and thus highlights the importance of regulating O2 in cell culture.Standard cell culture is routinely performed at supraphysiological oxygen levels (~18% O At sea level, atmospheric O2 levels are ~21%, and headspace O2 in conventional incubators thus equilibrates to ~18% O2 due to high humidity and the addition of 5% CO2. Despite being referred to as ‘normoxia’, 18% O2 is substantially hyperoxic relative to the cellular microenvironment in vivo. Increasing evidence indicates that the hyperoxic conditions of cell culture affect multiple biological processes, including reactive oxygen species (ROS) production [2 levels can compromise the ability of cell culture models to recapitulate in vivo disease pathophysiology  versus physioxia (5% O2). While physioxia is a range  rather than a set value, we chose 5% O2 for this study as we have accumulated data from other studies using this value [2 entry into media. We have previously shown that culture at 5% O2 in the incubator headspace maintains pericellular O2 in media at above 3.7% O2 under conditions used here [Cancer cell lines are widely used in basic research to study cancer pathophysiology; however, they are routinely cultured in 18% Ohysioxia % O2. Whiis value ,9, whichsed here .2. The cell lines used in this project were LNCaP (prostate adenocarcinoma), Huh-7 (hepatocellular carcinoma), PC-3 (prostate adenocarcinoma), and SH-SY5Y (neuroblastoma). We chose these cell lines because we have previously measured the effects of O2 on a wide range of cellular activities in them, including energy metabolism, mitochondrial dynamics, ROS production, and response to drugs such as resveratrol [2 levels in the range from 5% to 18% O2, and whether the effects of O2 were similar amongst cell lines, or cell-line specific. We found that the effects of O2 on the transcriptomes of these cell lines were substantial and largely cell-line specific. Nonetheless, we identified interesting patterns in gene expression among these cell lines, such as a differential but strong induction of genes encoded by mitochondrial DNA (mtDNA) and upregulation of HIF-1/2 targets in physioxia.We used RNA-seq and bioinformatic approaches to analyze differential gene expression of four human cancer cell lines cultured for 14 days at either 5% or 18% Overatrol . Inclusiveratrol  and ischveratrol  and Huh-veratrol  and fatt2 (~18% O2) to allow for acclimatization to Plasmax  every 3 or 4 days, when cells reached ~80% confluence. Media was refreshed every 24 h and cells were routinely monitored for mycoplasma contamination. Cell culture at either 5% O2 or 18% O2 was performed for 14 days. Cells were seeded at a density of 2 × 106 cells/plate prior to RNA extraction.LNCaP, SH-SY5Y, Huh-7, and PC-3 cell lines were purchased from ATCC . Cell passages between 15–19 were used throughout this study. Upon thawing, cells were cultured in 10-cm plates with Plasmax  supplemented with 2.5% FBS and 1% penicillin/streptomycin  for a week in a humidified 5% COTotal RNA was extracted using the RNeasy Plus Mini Kit  according to the manufacturer’s instructions. RNA integrity was assessed using 1.5% agarose gel electrophoresis, while RNA concentration and purity were evaluated as A260/280 ratio using a Thermo Fisher Scientific Nanodrop spectrophotometer. RNA samples were snap frozen in liquid nitrogen and stored at −80 °C until being sent to Novogene  for sequencing and analysis.Homo sapiens reference genome (GRCh38) using Hisat2 v2.0.5 [p-value < 0.05 and |log2FC|≥ 1 were set as threshold for significantly differential expression, as done previously [Quality check (QC), library preparation, sequencing, and differential expression analysis were performed by Novogene. Paired-end at 150 bp (PE150) high throughput Illumina sequencing was performed at a sequencing depth of 40 million reads per sample. Reads were aligned to the 2 v2.0.5 . Gene ex2 v2.0.5  through eviously .p-value (padj) < 0.1 and a FPKM ≥ 1 in at least one of the experimental groups in order to produce a concise list of enrichment terms which reflect the most strongly affected genes. Functional enrichment analysis was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) [p-value and Benjamini adjusted p-value (padj) of 0.05 were applied for identifying the most statistically significant enriched annotation terms.Prior to functional enrichment analysis, the list of differentially expressed genes (DEGs) was further reduced to genes with a Benjamini adjusted  (DAVID) . Enriche2 and 18% O2 in both LNCaP and PC-3 were independently cross-referenced with the PCTA dataset to find overlapping genes. We then analyzed the correlation between the change in expression levels (log2FC) in our samples and the PCTA dataset (mean log2FC from the 2115 samples) by performing a Pearson correlation test with the software GraphPad Prism 8.Data from the Prostate Cancer Transcriptome Atlas (PCTA) was downloaded from , contain2 in each cell line. In general, more differentially expressed genes (DEGs) showed higher expression at 5% O2 than at 18% in all cell lines. In addition, there was substantial variation between the four cell lines in their sensitivities to O2  were exclusively affected in this cell line. This indicates that O2 effects on gene expression are highly specific to a given cell line. This in turn makes it difficult to predict how the non-physiological O2 levels of standard cell culture are affecting cell biology in general terms.A remarkable result was the extremely limited overlap between cell lines in terms of the identities of the DEGs B. Only fgulation . Differe2 level in the four cell lines (padj < 0.05), which was found to be enriched at 18% O2. In Huh-7 cells, pathways such as extracellular matrix (ECM) organization  and drug metabolism by the cytochrome P450 (CYP450) enzymes  were strongly enriched at 5% O2, while oxidative phosphorylation  and oxidative stress-induced senescence  were enriched at 18% O2. Interestingly, in contrast to Huh-7 cells, both PC-3 and SH-SY5Y showed enrichment of annotation terms related to mitochondrial respiration and oxidative phosphorylation at 5% O2 (see padj < 0.05) and neurogenesis  were among the processes enriched at 18% O2 in SH-SY5Y cells. The full lists of functional annotation terms enriched by O2 level in all cell lines are available in Functional enrichment analysis revealed that different biological processes and pathways were enriched by Oll lines . For exa% O2 see . Signalip-value, we found that most of the genes highly affected by O2 are implicated in cancer cell biology, including several with roles in cancer cell proliferation, tumor progression, metastasis, invasion, and chemosensitivity to anticancer therapy. A selection of these genes is shown in 2 conditions and their corresponding log2FC values are shown in By sorting the DEGs according to their adjusted 2, of which six are subunits of respiratory complexes I, IV, and V, while the rest are mitochondrial tRNAs. Interestingly, all of these were upregulated at 18% O2. Eleven mtDNA-encoded genes were affected by O2 in PC-3 cells and 10 in SH-SY5Y, however, all were upregulated at 5% O2, in striking contrast with the observation in Huh-7 cells. Again, these DEGs encoded subunits of the respiratory chain and tRNAs. In contrast, only two mtDNA-encoded genes were affected in LNCaP. These results suggest that O2 levels in cell culture affect the expression of mtDNA-encoded genes, but in a highly cell-type specific manner.Oxygen induced differential expression of mtDNA-encoded genes in most cell lines . In Huh-2 is not hypoxic, and we have previously shown that, under the conditions used here, pericellular O2 levels do not fall below 3.7%, even during extensive static incubation periods in culture [2 levels  [2 in all cell lines, a selection of which is shown in VEGFA and ADM, were enriched. Similarly, genes that encode enzymes involved in the metabolic reprograming of cells towards a glycolytic phenotype were upregulated in Huh-7 cells grown at 5% O2. These genes include the glucose transporter SLC2A3 (GLUT3), the glycolytic enzyme ENO2 (enolase), and the gluconeogenic enzyme PCK1 (phosphoenolpyruvate carboxykinase 1). A lower number of HIF-1/2 targets were detected in PC-3 and SH-SY5Y cells, consistent with our initial observation that these two cell lines were less sensitive to O2 than Huh-7 and LNCaP cells.Five percent O culture . A handfysioxia) ,29,30. H2 in our cells were regulated by HIF-1α, HIF-2α, or both. To do this, we compared our DEG datasets with the dataset from the study by Downes et al. who sequenced the transcriptional outputs of stabilized forms of HIF-1α and HIF-2α [2 in our cell lines  with the expression data from the Prostate Cancer Transcriptome Atlas (PCTA), which contains a dataset of expression levels of 18,390 genes from 2,115 human prostate tumor samples [2 and at 18% O2 from both cell lines with the PCTA dataset. Upon obtaining matched gene lists from our data and the PCTA dataset, we performed a correlation analysis of the change in gene expression levels (log2FC) in both datasets. In LNCaP cells, gene expression patterns from the DEGs upregulated at 5% O2 showed a highly statistically significant (p < 0.0001) albeit weak (r = 0.1837) correlation with the PCTA expression data  with the PCTA expression data .To determine if physiological O samples . We indeion data A. On theion data B. Finall2 tension in the standard cell culture environment (18% O2) impacts the cancer cell transcriptome compared to a more representative in vivo environment (5% O2). We were particularly interested in the extent to which any effects of O2 were shared amongst cell lines versus cell-line specific. Our results indicate broad transcriptional effects of O2 between 5% and 18% that are highly cell-type specific. Even in LNCaP and PC-3, both prostate cancer cell lines, the overlap in O2-dependent DEGs was only ~5%. These results are consistent with a previous study showing little overlap in the proteome of three diffuse large B-cell lymphoma cell lines cultured in the same two O2 conditions [2 levels affects a variety of pathways, including the p53 pathway and mitogen-activated protein kinase (MAPK) pathways . Given 2 level. For example, prostate cancer commonly metastasizes to bone, forming primarily osteoblastic lesions. TGF-β and bone morphogenetic proteins (BMPs), released by prostate cancer cells, induce osteoblast differentiation, which in turn releases growth factors that stimulate the proliferation of prostate cancer cells [padj < 0.05) in LNCaP cells at 18% O2. Signaling by BMP (R-HSA-201451) and osteoblast differentiation (GO:0001649) were also enriched (p < 0.05). Another interesting observation was the upregulation of the androgen receptor (AR gene) in PC-3 cells grown at 18% O2 , in accordance with a previous observation that CYP1A1, CYP1A2, and CYP2E1, along with a number of phase II enzymes, were upregulated in HepG2 cells cultured in physioxia, compared with cells at atmospheric O2 [2 tensions leads to altered hepatic metabolism of drugs and toxins, which in turn results in altered biological responses to these compounds when tested in vitro. Indeed, DiProspero et al. showed that the toxicity and potency of acetaminophen, cyclophosphamide, and aflatoxin B1 were dependent on O2 tension in HepG2 cells cultured at ~18% O2, 8% O2, and 3% O2 [2 tension should be considered as an important factor in the design of experiments aimed to study the effects and mechanisms of bioactive molecules in vitro.One of the main functions of hepatic cells is detoxification of xenobiotics through phase I (CYP450) and phase II enzymes. In Huh-7 cells, drug metabolism by the CYP450 (hsa00982) was among the annotation terms enriched at 5% Oheric O2 . Differend 3% O2 . Notably2 in LNCaP and Huh-7 cells. Although traditionally known as transcription factors that mediate the response to hypoxia, some studies have shown HIF-1/2 expression and activity in the physiological O2 range [2) is provided in [2 were observed in PC-3 and SH-SY5Y cells supports the notion that these cells are less sensitive to O2 than LNCaP and Huh-7. Moreover, because cells were grown at either 5% O2 or 18% O2 for 14 days, the induction of HIF targets shown in this study suggests a role for HIF activity in physioxia, rather that it being a mere consequence of an acute reduction in O2 levels while performing the experiments. Similar observations have been made in other studies [2 in our cell lines  was one of the Reactome pathways enriched at 18% O2 in Huh-7 cells  was observed in three of the four cell lines , although the directionality of the effects were inconsistent. While mitochondrial terms were enriched at 18% O numbers . Moreoveheric O2 . No dire2 levels is cancer cell stemness [2 levels in our experimental groups on the expression of Oct4/Nanog/Sox2 related genes. While the expression of Oct4/Nanog/Sox2 was not affected by O2 levels in most of the cell lines used here—except SH-SY5Y where POU5F1 (Oct4) was upregulated at 5% O2—we did observe differential expression of their gene targets at 5% and 18% O2  shows a highly significant correlation (p < 0.0001) with the PCTA gene expression data, whereas the DEGs upregulated at 18% O2 has no significant correlation with the PCTA data. The overall weak correlation degree (Pearson r = 0.1837) of DEGs upregulated in physioxia and PCTA data could be explained by the fact that LNCaP is derived from a metastatic tumor from a single patient whereas the PCTA is a collection of data from a total of 2,115 patients. This shows the high heterogeneity of cancers within different patients. No significant correlation with the PCTA data was found from the transcriptomic profiles of PC-3 cells at either O2 level, which supports the notion that PC-3 cells are not as sensitive to O2 tension (between 5–18% O2) as LNCaP and Huh-7 cells. Additional research should be conducted using multi-omics approaches to investigate how culturing cells in physioxia produces more similar genomic, transcriptomic, proteomic, and epigenomic profiles to the ones observed in tissues in vivo.Finally, we analyzed the correlation between the transcriptomic data from the two prostate cancer cell lines used here and in vivo prostate tumor transcriptional profiles, by using the dataset from the PCTA ,91. In L2 levels on the epigenome, proteome, metabolome, and lipidome of different cell types is certainly warranted. In addition, data obtained from functional enrichment analysis needs to be interpreted carefully, as their significance in terms of cell behavior is not always clear. As an example, “regulation of apoptotic process” (GO:0042981) is one of the biological processes (GO) found to be enriched in PC-3 cells at 18% O2, with 11 DEGs associated with this process being upregulated at this O2 tension. Out of these eleven genes, five have an antiapoptotic role, five are proapoptotic, and one has dual roles in the regulation of apoptosis. Thus, the functional effect of 18% O2 in the regulation of apoptosis in PC-3 cells on apoptosis cannot be predicted by our bioinformatic analysis alone, and an experiment challenging PC-3 cells to apoptotic stimuli at both 5% O2 and 18% O2 would be necessary. Nonetheless, our results provide insight into the cellular processes and pathways that may be affected by O2 in these cancer cells, which can direct subsequent research questions.This study has some limitations. We recognize that transcriptional effects do not necessarily reflect changes in the cell proteome or phenotype, and this may be due to complex epigenetic, post-transcriptional, and post-translational mechanisms governing gene expression and protein function ,93. Inve2 levels in cell culture significantly alter the global transcriptomes of cancer cell lines in highly cell-line specific ways. This makes it difficult to establish general rules regarding how non-physiological O2 levels might affect experiments. Our results, together with the increasing amount of functional data regarding the effects of physioxia versus standard cell culture hyperoxia [2 levels in their experiments. This would certainly be expected to increase the likelihood that results will translate to in vivo.In conclusion, our results show that supraphysiological Oyperoxia , should


DRS) for each cell by integrating drug-response genes (DRGs) and gene expression in scRNA-seq data. Then, scDR was validated through internal and external transcriptomics data from bulk RNA-seq and scRNA-seq of cell lines or patient tissues. In addition, scDR could be used to predict prognoses for BLCA, PAAD, and STAD tumor samples. Next, comparison with the existing method using 53,502 cells from 198 cancer cell lines showed the higher accuracy of scDR. Finally, we identified an intrinsic resistant cell subgroup in melanoma, and explored the possible mechanisms, such as cell cycle activation, by applying scDR to time series scRNA-seq data of dabrafenib treatment. Altogether, scDR was a credible method for drug response prediction at single-cell resolution, and helpful in drug resistant mechanism exploration.Heterogeneity exists inter- and intratumorally, which might lead to different drug responses. Therefore, it is extremely important to clarify the drug response at single-cell resolution. Here, we propose a precise single-cell drug response (scDR) prediction method for single-cell RNA sequencing (scRNA-seq) data. We calculated a drug-response score ( Drug resistance, mainly caused by tumor heterogeneity (TH), is one of the huge challenges for effective treatment for cancers. The complex genetic contexts of tumor cells, exhibiting inter- and intra-TH, account for different drug responses. Targeted therapy is an optimal therapeutic management of tumors. Some anti-cancer drugs have been developed and applied in clinical cancer therapies by targeting genes such as EGFR, KRAS, etc. . HoweverIn contrast to tissue samples, cell lines, from the original tumor, are still considered to be the main vector for exploring molecular and pharmacological characteristics because of their properties of being relatively easy to culture, observe, and carrying rich genetic information about drug response . SeveralWith the development of single-cell RNA sequencing (scRNA-seq) technology, gene expression profile enables resolution at the single-cell level . Over thhttps://sites.broadinstitute.org/ccle, accessed on 17 September 2021)  in each test. For each drug, we performed 100 permutations and obtained 900 tests to verify the accuracy of scDR. We evaluated the difference in the predicted DRSs between resistant and sensitive cell lines corresponding to one drug using one-sided Wilcoxon test. Similarly, the significant difference  was regarded as an accurate prediction and calculated accuracy in all 481 drugs. In addition, we verified scDR with external datasets, including the bulk RNA-seq of cell lines  and tissue samples , as well as the scRNA-seq of cell lines . Similarly, the significance of DRSs difference between resistant and sensitive samples was used to evaluate the accuracy of scDR. Finally, scDR was also compared with the existing method, Beyondcell [Firstly, we used scDR to predict yondcell , using tyondcell . WPCI isDRSs could serve as prognostic marker or not, we performed survival analysis for STAD, PAAD, and BLCA . In addition, we downloaded the expression and clinical data of SKCM in TCGA to perform survival analysis for upregulated genes in d0-R cluster in the Application Cohort. Survival analysis was performed using the survfit function in the R survival package (v3.4-0); Kaplan–Meier (KM) survival curves were plotted using the ggsurvplot function in the R package survminer (v0.4.9).To explore whether We performed pseudotime analysis for melanoma cells to determine the process of evolution under dabrafenib treatment. Pseudotime analysis was carried out using the Monocle toolkit . SubsequTo validate that the d0-R cluster in the Application Cohort was the intrinsic resistant cell subgroup, we performed single sample gene set enrichment analysis (ssGSEA) for d0-R and d0-S cells. Tumor microenvironment (TME)-associated signatures, which were cancer-malignant and drug resistance-related, were downloaded from the previous collection in IOBR . ssGSEA https://metascape.org, accessed on 2 November 2021) [p-value Cutoff = 0.05, Min Overlap = 3.To detect the key potential mechanisms associated with resistance-related markers, gene set enrichment analysis for the Kyoto Encyclopedia of Genes and Genomes (KEGG)  and Geneer 2021) . The parDRSs and ssGSEA scores between different groups. A p-value < 0.05 was considered statistically significant.One-sided Wilcoxon tests were used to compare the DRSs)  for different drugs (DRS) for each cell using the scRNA-seq data of the DRGs. Then, we validated the accuracy and the stability of scDR using three data cohorts, including the bulk RNA-seq of cell lines and patient tissues, as well as the scRNA-seq of cell lines. In addition, scDR was applied to time series scRNA-seq data of melanoma cell lines during dabrafenib treatment. We identified an intrinsic resistant cell subgroup in the untreated cell lines, probably maintained by the upregulation of cell cycle genes such as RAN and TUBA1B.The scDR framework involves three major steps: (1) identifying resistant and sensitive cell lines; (2) predicting drug-response genes (DRGs); and (3) calculating drug-response scores  of 481 drugs in CTRP. We evaluated the difference in the predicted DRSs between the resistant and sensitive cell lines corresponding to each drug using one-sided Wilcoxon test. The significant difference  was regarded as an accurate prediction. The results showed that the accuracy was 100% (481 of 481) in the whole Discovery Cohort  higher than those of the sensitive cell lines for 479 of 481 (99.58%) drugs  and tissues , as well as scRNA-seq data of cell lines . The Validation Cohort I contained the gene expression profiles and the matched drug response data of 424 cell lines. We calculated the %) drugs . Especiall lines B. The peDRSs of three datasets. Higher DRSs were observed in the resistant samples for all three drug-cancer pairs  were observed in all three cancers  in TCGA . The patients were assigned to two groups based on the different drug responses: resistant  and sensitive (patients showing “Complete Response”). We calculated the er pairs C–E. Thes cancers F–H. OverDRS of each cell for 481 drugs. There was no true information about the drug response at single-cell level; therefore, we calculated the mean DRSs values (mDRS) of cells in each cell line and performed a comparison between resistant and sensitive cell lines. Thus, the differences in mDRS showed that, for 460 of 481 (95.63%) drugs, the resistant cell lines had significantly  higher mDRS values than the sensitive cell lines  higher than that of Beyondcell  . We used the function “FindCluster” in the R package Seurat to perform clustering analysis. These cells were grouped into five clusters at the resolution of 0.12, which were named d0-R, d0-S, C1, C2, and C3 A. The ceroceeded C. This ied (DRSs D. Howeve 0.0001) . The higo 3 days E. We obstimeline F, and thtimeline . These rp-value < 0.05) higher ssGSEA scores  prognosis-related including RAN and TUBA1B , such as the “cell cycle” and “apoptosis” pathways [To further demonstrate that d0-R was an intrinsic resistant cluster, we performed ssGSEA for TME-associated signatures, which were collected as cancer-malignant and drug resistance-related signatures in a previous study . In 51 TA scores A, such a in d0-R B, of whid TUBA1B . In addipathways ,35,36 (FDRSs of drug-resistant cells was higher than that of sensitive cells (For tumor precision therapy, to accurately distinguish resistant cells from sensitive cells is necessary. Although amounts of scRNA-seq data arise to push on the exploration of tumor heterogeneity, a computational method of predicting drug response and identifying resistant cells for scRNA-seq data is still lacking. Here, we present scDR, a precise single-cell drug response prediction method for scRNA-seq data. From bulk RNA-seq data, drug response signatures were obtained and then transferred to predict drug responses in scRNA-seq. scDR could accurately predict drug responses for both bulk RNA-seq and scRNA-seq data, as well as for both cell lines and clinical patient tissues. In addition, compared with the existing method, scDR achieved higher accuracy. Finally, we showed a case that scDR could aid in intrinsic resistant cell identification and dissection of biological mechanisms. Thus, scDR could help accurate resistant cells identification and the determination of biological mechanisms. Similarly, we also used scDR to predict the drug response in an additional scRNA-seq data for breast cancer cells (GSE131135, detailed in ve cells . Thus, uThe effectiveness of scDR has been validated in drug response prediction; however, it still has potential for improvement. First, drug response signature was simply obtained by differential expression analysis. Other signature identification method might lead to improvements, such as scDEAL . Second,


In our study, our aim was to examine the cytotoxic and radio-sensitizing effect of the alkaloid piperine, a major pungent of black pepper, on two different human epithelial tumor cell lines in vitro. The growth of the human cell lines T98G (glioblastoma) and FaDu  was examined under the influence of piperine in different concentrations. In addition, after combined treatment with ionizing radiation, long-term survival was investigated with a colony formation assay. The proliferation was analyzed using the BrdU-assay, while the DNA repair capacity was examined via the γH2AX assay. Piperine reduced the growth of both cell lines in a concentration-dependent manner as well as a time-dependent one. After combined treatment with piperine and ionizing radiation, an inhibition of clonogenic survival could be proven. A reduced proliferation capacity and an additive effect on DNA damage 24 h after irradiation are possible causal mechanisms, which were also demonstrated for both cell lines. Based on the results presented in this study, piperine was shown to have cytotoxic antitumor activity and a radio-sensitizing effect in micromolar concentrations in the human tumor cells that were tested. Based on these results piperine represents a potential therapeutic option in radio-oncological treatment.  Owing to the increasing incidence of malignant tumor diseases, there is a great medical need for modern treatment options. An important strategy in today’s radiation oncology is the combination of radiation therapy with cytostatic or cytotoxic substances. In addition to the development of new therapeutic approaches, the search for active ingredients in nature still offers great potential. It was already possible in the past to obtain active ingredients from plants, such as taxanes, which are still used today in oncology, e.g., in combined radio-chemotherapy. Piperine is one such substance that has become the focus of interest in recent years because of its anti-tumor effects.Piperaceae family, which can be isolated from the fruits or roots of black pepper (P. nigrum) and long pepper (P. longum) [Piper nigrum has long played an important role in traditional phytomedicine and is mainly used for digestive and respiratory problems [Cip1 and p27Kip1 could be observed [Piperine (1-piperoylpiperidine) is an alkaloid from plants of the  longum) . Piperinproblems . Its effproblems . Recent observed ,6. In adobserved  and an iobserved .Hence, various in vitro analyses have already been able to show a large number of anti-tumorigenic starting points in different established cell lines for the active substance piperine, so that a combination with ionizing radiation appears promising. The aim of this work was therefore to investigate the combined effect of the alkaloid piperine and ionizing radiation on the human tumor cell lines T98G (glioblastoma) and FaDu . The cell lines examined are representative of in vivo tumor entities which, despite modern therapy options, are associated with a poor prognosis for the affected patients. An established treatment option for both tumor entities is radiation therapy. Additive or supra-additive effects of the active ingredient piperine could lead to a further reduction in the tumor burden and a reduction in radiation doses in order to minimize side effects for the patient.The growth curves without the influence of piperine showed an almost typical course. The population doubling time of T98G cells was 26.3 h. A population doubling time of 27.7 h in the exponential growth phase was determined for the FaDu cells. Dose- and time-dependent growth delays were demonstrated for both cell lines under the influence of piperine .50 value was 133 µM after a six-day incubation period. The growth-inhibiting effect was evident earlier in the FaDu cells than in the T98G cell line. The inhibition of growth was already clearly recognizable after a 48-h incubation from a piperine concentration of 50–75 µM and was statistically significant for the 75 µM concentration. After six days of incubation with piperine, the IC50 value was 74 µM. Thus, the FaDu cells were more growth sensitive to piperine than the T98G cells.Three days after treatment with piperine, the T98G cells showed growth inhibition from a concentration of 150 µM, which was even statistically significant after the addition of 200 µM. Five days after the addition of piperine, an inhibitory effect on growth could also be demonstrated for lower piperine concentrations of 25 μM, 50 μM, 75 μM, and 100 μM. The IC2 value and serves as a measure of the radiation sensitivity of a cell line. An SF2 value of 0.66 was determined for the T98G cells; 0.75 was calculated for the FaDu cells. The solvent DMSO had no effect on the clonogenic survival of either cell line. The influence of the active ingredient piperine was already evident in the morphology of the cell colonies.In the cells treated without the drug, an increasing radiation dose resulted in a decrease in survival in both cell lines . The proMicroscopically, the T98G cells lost their fibroblast-like shape and formed significantly more compact colonies A. The ceProliferation was assessed using the BrdU assay. Proliferation-inhibiting effects were found in both cell lines examined after irradiation alone .ANOVA analysis revealed significant dose- and concentration-dependent significant changes for all proliferations at all radiation doses, compared with untreated corresponding controls. In the T98G cell line, the proliferation of the untreated control (100%) was statistically significantly reduced to 71% after a radiation dose of 6 Gy A. In theUsing the γ-H2AX test, it was possible to analyze the number of DNA double-strand breaks and, hence, their repair capacity in T98G and FaDu cells after ionizing radiation or/and under the influence of piperine. In both cell lines, a small number of DNA double-strand breaks was already noticeable in the control batches, which were neither irradiated nor treated with the active substance. On average, 1–2 foci per cell nucleus could be counted in the control samples . After irradiation with 6 Gy alone, there was a significant increase in the number of DNA double-strand breaks in both tumor cell lines compared with the non-irradiated control. Already, 0.5 h after radiation exposure, an average of 28 DSB per cell nucleus could be determined in both the T98G and FaDu cells, with a maximum of 30 DSB/cell nucleus being counted. Significant differences between the individual batches within the same cell line could, therefore, not be observed. Piperine without ionizing radiation led to a concentration-dependent increase in the number of DNA double-strand breaks. The T98G cells under the influence of the 200 µM active substance were more sensitive than the FaDu cells (approx. 6 DSB/cell nucleus) at both fixation times with an average of approx. 11–12 DSB. Even with 100 µM piperine, a significant increase in DNA double-strand breaks could be seen in both cell lines. Under the combination of radiation and piperine, a maximum of 30 DNA-DSB/cell nucleus were counted. The DNA damage after irradiation with 6 Gy was so pronounced that, with this maximum number of foci counted, no additional foci as differences between the batches could be detected even after the addition of piperine. After 24 h, the number of residual foci of piperine-induced DNA-DSB was unchanged compared with 0.5 h. Thus, no repair of this genotoxic damage caused by piperine took place. However, notes for an additive effect after combined treatment with irradiation and piperine could be observed. At this time point, the repair of the DNA-DSB is already complete. The number of residual foci of the irradiated cells was significantly higher under the influence of piperine than without the active substance. Significant results could be observed for both cell lines after treatment with 100 µm and 200 µM piperine. Without piperine, in irradiated cells nine DSB/cell could be counted in the T98G cell line. In contrast, after the addition of 200 μM piperine, in irradiated cells 19 DSB/cell were detected. Similar results were obtained for irradiated cells of the FaDu cell line: 6 DSB/cell were counted without the influence of the active substance, whereas 13 DSB/cell were visible after treatment with 200 µM piperine. In both cell lines, the repair capacity of radiation-induced DNA-DSB was reduced under the influence of piperine; additive effects were observed.The aim of this study was to investigate the combined effect of the alkaloid piperine and ionizing radiation on the human tumor cell lines T98G (glioblastoma) and FaDu . Based on the available data, dose- and time-dependent growth retardations under the influence of piperine were demonstrated for both cell lines, although the cell line FaDu was slightly more sensitive. Piperine-mediated inhibition of cell growth was already described for other human cell types or, for example, for different breast cancer cells , prostatTo assess the influence of piperine on the cellular radiation response, the clonogenic survival of cells was determined using clinically relevant doses from 2 Gy to 8 Gy. The cell survival curves obtained from the results of the colony-forming test allow conclusions to be drawn about the radiation sensitivity of the cells. In the cells treated without piperine, increasing the radiation dose resulted in a decrease in survival in both cell lines. A dose–response relationship with regard to treatment with ionizing radiation could, therefore, be demonstrated for both the T98G and the FaDu cells. After adding the drug piperine, synergistic effects from the combination of radiation and piperine could be observed on the basis of the cell survival curves. For both cell lines, a radiation-sensitizing effect could be demonstrated by piperine in higher concentrations. The FaDu cell line showed these effects from a lower piperine concentration than the T98G cell line and was, therefore, again more sensitive. Ionizing radiation or other toxic agents can lead to the loss of the reproductive integrity of the tumor cells and thereby cause clonogenic cell death, which is multifactorial. Investigations regarding a radio-sensitizing effect by piperine have not yet been published for the two investigated cell lines T98G and FaDu. However, an increased radiation effect was observed for the breast carcinoma cell line MDA-MB-468 . In thesCip1 were described as possible causal mechanisms [Cip1 and p27Kip1. Hence, the G1 arrest induced by piperine represents a possible cause for the inhibition of proliferation measured in this work. The combined treatment with ionizing radiation and the active ingredient piperine had a synergistic effect on both cell lines with regard to proliferation inhibition. This also corresponds to the observations of two other working groups. In the human colon carcinoma cell line HT-29, for example, after the addition of 25 µg/mL piperine and after irradiation with only 1.25 Gy, the relative proliferation was reduced by almost 60% compared with the untreated control [By examining cell proliferation using the BrdU assay, the causal mechanisms—for example, of the results observed using growth curves and colony formation tests—could be further classified. Irradiation alone, as well as piperine from a concentration of 50 µM, resulted in an inhibition of proliferation. A combination effect of piperine and ionizing radiation was detectable for both cell lines. Proliferation-inhibiting effects through ionizing radiation have already been described for both the cell lines that were examined, whereby a dose dependency was also shown here; both the FaDu and the T98G cells can be classified as relatively radiation-resistant cell lines ,21. A prchanisms . Similarchanisms . These i control . SynergiCip1. This cell cycle arrest was associated with increased DNA damage and increased apoptosis. Twenty-four hours after irradiation, the repair of the DNA-DSB is already complete and the number of residual foci can be detected to make a statement for the repair capacity of the cells. Notes for an additive effect after combined treatment with irradiation and piperine could be observed at this time point. Under the influence of the two drug concentrations examined, significantly more residual DNA double-strand breaks were found 24 h after irradiation compared with the irradiated control. This effect increased with increasing drug concentration. Induced DNA double-strand breaks represent the most important radiobiological effect of ionizing radiation. The influence of piperine on the number of DNA-DSB and their repair capacity was investigated using the γ-H2AX assay. Under the sole influence of piperine, an increased number of DNA double-strand breaks could be determined both in the T98G and in the FaDu cells. The genotoxic effects of the alkaloid piperine appear to be manifold. Investigations of the human melanoma cell line SK MEL 28 revealed dose- and time-dependent increased levels of reactive oxygen species after the addition of piperine. In particular, in the S phase, these led to strand breaks during DNA replication and hence to increased recruitment of γ-H2AX . FurtherBoth examined cell lines in our study are characterized by a mutation in the p53 tumor suppressor  ,22, are In conclusion, this study demonstrates the antiproliferative and radio-sensitizing effects of piperine. The drug causes a significant increase in the radiation response in the cells. This finding gives crucial evidence of the high potential of piperine for use in combined radio-chemotherapy in the future. Next investigations are following to get deeper insights into the mechanism of the radio-sensitizing effect of piperine.Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Lonza Group Ltd. . Phosphate buffered saline (PBS) was obtained from PAN BioTech  and fetal bovine serum (FBS) from Biochrom AG . Penicillin/streptomycin (100 U/mL/100 µg/mL) and piperine (>97%) were purchased from Sigma-Aldrich GmbH . 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), non-essential amino acids (NEA), and sodium pyruvate were purchased from ThermoFisher Scientific GmbH . Trypsin/EDTA (0.05%/0.25%) was obtained from PAA Laboratories GmbH . Piperine was dissolved in dimethylsulfoxide  to obtain a 150 mM stock solution. For experiments, piperine stock solution was diluted with DMEM to produce the final concentrations.2 in DMEM, supplemented with 10% FBS, and 1% penicillin/streptomycin. In addition, 1% NEA and sodium pyruvate and 2% HEPES were added to the medium of the FaDu cells. Both cell lines were passaged twice weekly to ensure exponential growth.T98G and FaDu cells were purchased from American Type Culture Collection . The cell lines were cultivated at 37 °C, 5% COThe cells were irradiated in cell culture flasks or in multi-well plates at room temperature using a Versa HD linear accelerator from Elekta Ltd. . The irradiation was carried out with a nominal photon energy of 6 MV and a dose rate of 6.6 ± 0.6 Gy/min with doses of 2 Gy, 4 Gy, 6 Gy, or 8 Gy. A control sample (0 Gy) was transported to the radiation room to ensure comparable environmental factors.4 cells per well, for each experimental approach. Twenty-four hours after seeding, piperine was added in concentrations of 25 μM, 50 μM, 75 μM, 100 μM, 150 μM, or 200 μM respectively. A 1:100 dilution of the piperine stock solution was prepared for the concentrations of 25–75 µM, and a 1:10 dilution for the concentrations of 100–200 µM. In addition, the solvent was checked by adding DMSO (0.1%). On the day the piperine was added, the cell count of the cells seeded on the previous day was determined. In the following six days, the cell counts for all drug concentrations as well as the DMSO control and the untreated (native) growth control were then determined in triplicates. Growth curves were created on the mean of three independent experiments.The cells were seeded in a 24-well plate in triplicates, with a seeding density of 1 × 103 cells/T25 cell culture flask. The active substance was added 24 h after cell seeding . In addition, a control without active substance and a solvent control with DMSO were carried out. After a further 24 h, the cells were irradiated using a linear accelerator with doses of 0 Gy (control), 2 Gy, 4 Gy, 6 Gy, and 8 Gy. Seven days after the start of the experiment, the medium and the active substance dissolved in it were removed and a new active substance-free complete medium was added. After a further five days of incubation, the cells were fixed and stained. For this purpose, the medium was discarded and the cells were then fixed with 5 mL ethanol (70%). The cell colonies were stained with 1% crystal violet solution. First, the size and cell number of the colonies were assessed under the phase contrast microscope. Only colonies of at least 50 cells in size were counted. The counting then took place with the help of the light plate.The colony formation test was carried out with a cell density of 2 × 103 cells were seeded into each well. After 24 h, the piperine was added in different concentrations . After an incubation of a further 24 h, BrdU was added (20 μL/well). Two hours later, the cells were irradiated using a linear accelerator . BrdU incorporation was measured another 24 h after irradiation. The further procedure was based on the information provided by the cell proliferation kit . The absorption was then measured at a wavelength of 450 nm on the spectral photometer.Proliferation was assessed using the BrdU assay. A 96-well plate was used to carry out the experiment. A quadruple determination was carried out for each batch. The 2 × 10Chambered coverslips with four chambers  were used for the assay. The test was carried out in duplicate for each experiment. Twenty-four hours after cell seeding, piperine was added (100 μM or 200 μM). After a further 24 h incubation time, irradiation was carried out using a linear accelerator with 6 Gy. The cells were fixed 30 min or 24 h after irradiation. The maximum of DNA double-strand breaks can be seen about 30–90 min after the damage event, which is why the first fixation point in this work was chosen after 0.5 h. The 24 h point in time was also examined, since the repair of radiation-induced DNA-DSB is then completed and conclusions can be drawn about the repair capacity. The detached slides were then fixed and stained in accordance with the protocol already described [t-test. p ≤ 0.05 was considered as the statistically significant difference. To compare a variable under different conditions (γH2AX assay), ANOVA analysis followed by a Bonferroni post test with SigmaPlot  was utilized. There was no adjustment for multiple testing, since all experiments were carried out individually, and were, therefore, considered independently of one another.Calculations were performed on the mean of at least three independent experiments. After examination on normal distribution, statistical analyses were carried out using Student’s


Advanced systemic mastocytosis (AdvSM) is a rare malignant disease with a poor prognosis due to the drug resistance of neoplastic mast cells. We found that drugs targeting the cell cycle regulators CDK4 and CDK6 profoundly suppress the growth and survival of neoplastic mast cells. Furthermore, these drugs can overcome resistance against KIT D816V-targeting drugs, including midostaurin, in neoplastic mast cells. Finally, the CDK4/CDK6 inhibitors applied induced apoptosis in CD34+/CD38− stem cells in AdvSM. Based on these results, we believe that CDK4/CDK6 inhibition may be a new and interesting therapeutic approach with curative potential for AdvSM. Whether combinations of KIT D816-targeting drugs and CDK4/CDK6 inhibitors can induce long-term remission in patients with AdvSM remains to be determined in clinical trials.KIT D816V. However, despite their disease-modifying potential, KIT D816V-targeting drugs, including midostaurin and avapritinib, may not produce long-term remissions in all patients. Cyclin-dependent kinase (CDK) 4 and CDK6 are promising targets in oncology. We found that shRNA-mediated knockdown of CDK4 and CDK6 results in growth arrest in the KIT D816V+ MC line HMC-1.2. The CDK4/CDK6 inhibitors palbociclib, ribociclib, and abemaciclib suppressed the proliferation in primary neoplastic MC as well as in all HMC-1 and ROSA cell subclones that were examined. Abemaciclib was also found to block growth in the drug-resistant MC line MCPV-1, whereas no effects were seen with palbociclib and ribociclib. Anti-proliferative drug effects on MC were accompanied by cell cycle arrest. Furthermore, CDK4/CDK6 inhibitors were found to synergize with the KIT-targeting drugs midostaurin, avapritinib, and nintedanib in inducing growth inhibition and apoptosis in neoplastic MCs. Finally, we found that CDK4/CDK6 inhibitors induce apoptosis in CD34+/CD38− stem cells in AdvSM. Together, CDK4/CDK6 inhibition is a potent approach to suppress the growth of neoplastic cells in AdvSM. Whether CDK4/CDK6 inhibitors can improve clinical outcomes in patients with AdvSM remains to be determined in clinical trials.In most patients with advanced systemic mastocytosis (AdvSM), neoplastic mast cells (MC) express  KIT is detected in neoplastic MC and is considered to act as a driver of disease evolution and MC expansion [Systemic mastocytosis (SM) is a rare disease characterized by the accumulation of neoplastic mast cells (MC) in one or more organs, such as the bone marrow (BM), the skin, the spleen, and the gastrointestinal tract ,2,3,4. Sxpansion ,10,11.KIT D816V-negative AHN or MCL, have been reported [Several pharmacologic approaches have been established to counteract the expansion of neoplastic MC in AdvSM, including cytostatic drugs, interferon-alpha, cladribine, poly-chemotherapy, allogeneic hematopoietic stem cell transplantation (HSCT), and KIT-targeting drugs ,19,20,21reported ,28,29.KIT [All in all, single agent KIT-targeting treatment may not be sufficient to suppress all cells and sub-clones in a complex disease such as AdvSM. Therefore, research focuses on identifying drug combinations that are able to induce more durable remissions in AdvSM. Indeed, a number of potential combination partners for midostaurin or other KIT D816V-targeting TKIs have been examined ,30,31,32KIT ,36,37,38Cyclin-dependent protein kinases (CDKs) have various functions in normal and malignant cells. CDK4 and CDK6 regulate the activity of the retinoblastoma protein (RB1) and are required for G1 phase progression and G1/S transition of the cell cycle ,41,42,43In the current study, we explored the expression and role of CDK4/CDK6 in neoplastic MC and evaluated the anti-neoplastic effects of CDK4/CDK6-targeting drugs alone or in combination with KIT D816V-targeting TKI.Reagents used in this study are described in the n = 6), ASM (n = 2), SM-AHN (n = 9), and MCL (n = 4), according to WHO criteria (n = 27), SSM (n = 1), ASM (n = 5), SM-AHN (n = 11), and MCL (n = 6)  see . The stuKIT WT, ROSAKIT D816V) [The following human MCL-like cell lines were employed in this study: HMC-1.1 and HMC-1.2 , two ROST D816V) ,57, and T D816V) . A detaiCDK4, CDK6, cyclin D1, cyclin D2 and RB1 in BM samples obtained from patients with SM and in the MCL-related cell lines, RNA-isolation and a quantitative polymerase chain reaction (qPCR) were performed as reported in [ABL1 was used as reference gene. A detailed description of the technique is provided in the To determine the expression of orted in  using spExpression of CDK4, CDK6, cyclin D1, cyclin D2, and RB1 protein was analyzed in MC cell lines by western blotting as described ,59. To s3H-thymidine uptake was measured as previously described [3H-thymidine uptake was measured. All experiments were performed in triplicates. A detailed description of the technique is provided in the Primary neoplastic cells and cell lines were incubated in a control medium or various concentrations of CDK4/CDK6 inhibitors  at 37 °C for 48 h. Thereafter, escribed ,55. In aFor flow cytometric determination of apoptosis, cell lines were cultured in a control medium or in a medium supplemented with a CDK4/CDK6 inhibitor (0.5–10 μM), midostaurin (0.1–0.5 µM) or avapritinib (0.3–0.7 µM), or a combination of two drugs (‘CDK4/CDK6 inhibitor + midostaurin’ or ‘CDK4/CDK6 inhibitor + avapritinib’) at 37 °C for 48 or 72 h and subjected to combined Annexin V/4′,6-diamidino-2-phenylindole (DAPI) staining to determine apoptosis by flow cytometry as described ,55. In aFor analysis of cell cycle progression, cell lines (HMC-1 and ROSA cells) were kept in the presence or absence of various concentrations of palbociclib, ribociclib, or abemaciclib for 24 h. Thereafter, propidium iodide was added and cell cycle distribution was analyzed as described previously . These fn = 3) were incubated in a control medium or in the presence of palbociclib, ribociclib, or abemaciclib (0.01–10 μM each) at 37 °C for 30 min. Then, histamine release was determined as described [Dextran-enriched blood basophils obtained from healthy individuals (escribed . A detaiHMC-1.1 and HMC-1.2 cells were transfected simultaneously with short hairpin RNA (shRNA) constructs directed against CDK4  and CDK6 (induced shRNA labeled with GFP) or with control shRNAs, as previously described  values using Calcusyn software [t test. The results were considered to be significantly different when p was <0.05.To determine the level of significance of differences seen in growth inhibition, cell cycle progression, and apoptosis studies, analysis of variance testing (ANOVA) with post-testing using the Dunnett test was applied. Results were considered to be significantly different when software . A CI ofsoftware ,63. A syn = 27; SSM: n = 1; ASM: n = 5; SM-AHN: n = 11; MCL: n = 6). However, mRNA expression levels varied from patient to patient without a clear correlation to a subtype of SM  in neoplastic MC. As evidenced by qPCR, transcripts of CDK4, CDK6, cyclin D1, cyclin D2, and RB1 were detectable in all BM samples analyzed   E. Growth1–10 µM) . We next1–10 µM) . Indeed,50-values ranged between 5 and 200 nM for palbociclib, 38 and 362 nM for ribociclib, and 5 and 160 nM for abemaciclib , no loss of sensitivity against CDK4/CDK6 inhibitors was observed. Of note, in two patients with relapsed MCL following midostaurin treatment . In one patient who had received avapritinib, resulting in a partial response (patient #14.2), neoplastic cells also remained sensitive to palbociclib  and abemaciclib . These observations suggest that CDK4/CDK6 inhibitors may be suitable drugs for cytoreductive therapy in all patients with AdvSM, regardless of the subtype of disease, the presence of KIT D816V, or previous treatment with KIT-targeting drugs. In addition to malignant cell expansion, symptoms caused by mediator release upon MC activation often represent a problem in patients with SM. We were therefore interested to investigate whether CDK4/CKD6 inhibitors are also able to block anti-IgE induced histamine release. In these experiments, high concentrations of palbociclib (10 µM) suppressed anti-IgE-mediated histamine release in basophils  may lead to different expression patterns of CDKs and cyclins. However, which oncogenes may contribute to the overexpression of CDKs and cyclins in neoplastic MCs remains unknown.CDK4/CDK6 is a well-established target in applied oncology ,40,41 anCDK4/CDK6 have been described to play an essential role in cell cycle progression, and thus in cell proliferation in normal and neoplastic cells ,40,41. HKIT D816V. Interestingly, total RB1 expression was also decreased in HMC-1 cells after exposure to CDK4/CDK6 inhibitors. In this regard, our data confirm previous observations in other cell types [Within the last decade, the concept of targeting CDK4/CDK6 with suitable drugs has been well established in oncology ,41,42,43ll types ,65,66. SKIT D816V at low IC50-values. However, in MCPV-1 cells expressing RAS G12V, Large T, and hTert, palbociclib and ribociclib failed to produce growth inhibition and a relative resistance against abemaciclib was observed. This is somehow intriguing, as RAS G12V has not been associated with resistance against CDK4/CDK6 inhibitors in the context of breast cancer or other malignancies [RAS G12V-mutated cells against chemotherapeutic agents in pancreatic cancer [RAS G12V mediates resistance against CDK4/CDK6 inhibitors exclusively in the cellular background of neoplastic MC. On the other hand, resistance may also be related to the particular expression pattern of cell-cycle-related kinases in MCPV-1 cells. Indeed, we found that MCPV-1 cells overexpress cyclin D1 but fail to express cyclin D2, contrasting to the other MCL-like cell lines analyzed. Abnormal expression of cyclins D1 and D2 in cancer cells has been discussed as being associated with resistance against CDK4/CDK6 inhibitors [In our study, all three CDK4/CDK6 inhibitors were found to induce growth inhibition, cell-cycle arrest, and, to a lesser degree, apoptosis in HMC-1 and ROSA cells expressing or lacking gnancies . On the c cancer . One exphibitors ,72,73. Hhibitors .KIT D816V+ ASM and MCL. The effects of CDK4/CDK6 inhibitors were independent of the presence or absence of an AHN and of the nature of the AHN. These observations suggest that CDK4/CDK6 inhibitors may be suitable drugs for cytoreductive therapy in a majority of patients with AdvSM. Of note, anti-proliferative effects were also confirmed in samples obtained from relapsed cases after midostaurin-treatment and from one patient who underwent treatment with avapritinib. This was predictable to a certain degree, as midostaurin/avapritinib and CDK4/CDK6 inhibitors have different cellular targets and a different mode of action, and no cross-resistances were expected. CDK4/CDK6 inhibitors may, therefore, be interesting drug candidates that may be developed for use as salvage therapy in relapsed drug-resistant AdvSM.We next asked whether CDK4/CDK6-targeting drugs would also be able to inhibit the proliferation of primary neoplastic cells and if—by way of analogy to our cell line models—resistance would occur in a subset of samples. In these experiments, all three drugs were found to induce growth inhibition in the nanomolar range in neoplastic cells in all subtypes of SM, including In the recent past, it has been postulated that LSCs, which reside in a CD34+/CD38− cell compartment in AdvSM, may survive conventional or targeted therapy and may, therefore, give rise to disease relapse . To achiIt has been previously observed that patients with AdvSM often fail to achieve long-lasting remissions after treatment with a single drug, such as midostaurin or avapritinib ,28. TherThe implication of CDK4/CDK6 inhibitors—as single drugs or in combination with KIT-targeting TKI—in treatment concepts for AdvSM may have several benefits from a clinical point of view. Palbociclib, ribociclib, and abemaciclib are all approved and therefore available for application in patients. Furthermore, all three drugs are generally well-tolerated when used as single agents. Therefore, these drugs might be used in patients who are resistant or intolerant against midostaurin. The combination “midostaurin + CDK4/CDK6 inhibitor” may result in a more powerful inhibition of neoplastic MC-proliferation than both agents when applied alone. In the case of midostaurin-pretreated ASM/MCL, the combinations “avapritinib + CDK4/CDK6 inhibitor” or “nintedanib + CDK4/CDK6 inhibitor” may be preferable, as these inhibitors show higher affinity to D816V-mutated KIT than midostaurin. Last, two-drug combinations involving a KIT-targeting TKI and a CDK4/CDK6 inhibitor would allow the application of each drug at a lower dose and therefore avoid specific side effects. However, whether these drug combinations would provoke unexpected side effects remains to be carefully determined in upcoming trials.Together, our study shows that CDK4 and CDK6 are promising targets in neoplastic MC in AdvSM. The FDA-approved CDK4/CDK6 inhibitors palbociclib, ribociclib, and abemaciclib exert anti-proliferative and pro-apoptotic effects in most MCL cell lines and primary neoplastic MCs and CD34+/CD38− LSC isolated from patients with AdvSM and synergize with KIT-targeting TKIs in producing growth arrest. The value of such drug combinations needs to be evaluated within the frame of upcoming clinical trials.


Colorectal Cancer (CRC) ranks third in terms of incidence and second in terms of mortality and prevalence worldwide. In relation to chemotherapy treatment, the most used drug is 5-fluorouracil (5-FU); however, the use of this drug generates various toxic effects at the systemic level. For this reason, new therapeutic strategies are currently being sought that can be used as neoadjuvant or adjuvant treatments. Recent research has shown that natural compounds, such as genistein, have chemotherapeutic and anticancer effects, but the mechanisms of action of genistein and its molecular targets in human colon cells have not been fully elucidated. The results reported in relation to non-malignant cell lines are also unclear, which does not allow evidence of the selectivity that this compound may have. Therefore, in this work, genistein was evaluated in vitro in both cancer cell lines SW480 and SW620 and in the non-malignant cell line HaCaT. The results obtained show that genistein has selectivity for the SW480 and SW620 cell lines. In addition, it inhibits cell viability and has an antiproliferative effect in a dose-dependent manner. Increased production of reactive oxygen species (ROS) was also found, suggesting an association with the cell death process through various mechanisms. Finally, the encapsulation strategy that was proposed made it possible to demonstrate that bacterial nanocellulose (BNC) is capable of protecting genistein from the acidic conditions of gastric fluid and also allows the release of the compound in the colonic fluid. This would allow genistein to act locally in the mucosa of the colon where the first stages of CRC occur. According to the Global Cancer Observatory (GCO), in 2020, colorectal cancer (CRC) ranked as the type of cancer with the fourth highest incidence worldwide (approximately 19.5%). In women, an incidence rate of 23.4% is estimated and in men a rate of 16.2%. This type of cancer represents the third most common cause of death and is the second most prevalent for men and women of all ages worldwide [In the clinical field, most cases of CRC are diagnosed as a terminal chronic condition or in a metastatic state due to the non-obvious development of the disease and symptoms during its initial phase. Additionally, the inevitable drug tolerance and side effects associated with chemotherapy make it more difficult to treat CRC effectively. Consequently, any candidate component with potential application for the treatment of metastatic CRC should be explored in depth . In gene5-FU is an analog of uracil with a fluorine atom at the C-5 position instead of hydrogen, allowing it to rapidly enter the cell using the same facilitated transport mechanism as uracil. The mechanism of 5-FU cytotoxicity has been attributed to the misincorporation of fluorinated nucleotides into RNA and DNA and the inhibition of the nucleotide-synthesizing enzyme thymidylate synthase (TS) . This drRecent research has shown that various natural compounds have chemotherapeutic and anticancer effects; these investigations focus on the relationship of these effects with the specific biological targets associated with cancer on which these compounds act . Among tGenistein is a biologically active flavonoid found in high amounts in soybeans. Many studies have described a relationship between a soy-rich diet and cancer prevention, further demonstrating the pharmacological effects of genistein, including antiestrogenic action, antioxidant action, inhibition of angiogenesis, and anticancer activity against breast and ovarian cancer tumor cells. Therefore, it is considered as a promising chemopreventive agent in the treatment of cancer ,11. In tIn CRC, several in vitro studies have shown that genistein exhibits growth-inhibitory activity and promotes apoptosis in a dose-dependent manner. Genistein causes cell cycle arrest in colon cancer cell lines HCT-116 and SW480, mainly participating in cell cycle regulation and apoptosis . In otheThe following information demonstrates the ability of genistein to inhibit the proliferation and migration of tumor cells due to the inhibition of the activity of several molecular targets, making it a promising natural compound for the chemoprevention and treatment of CRC. However, the effect of this compound on non-malignant cell lines that show the selectivity of this compound has not yet been reported. By definition, an ideal compound should have a relatively high toxic concentration but a very low active concentration. Under this premise, the compound would affect cancer cells but should not affect non-malignant ones ,19,20. TAdditionally, in this research, the encapsulation of genistein is proposed as a strategy to improve pharmacokinetic activity, achieving a better local effect at the target site of this compound when administered orally. This is because, despite the beneficial properties of genistein, the use of this compound in vivo is limited due to its low water solubility, rapid biotransformation to inactive metabolites, poor accumulation in target tissues and cells, and low concentration in the blood after oral administration ,22.To evaluate the maximum desorption capacity of genistein when it is encapsulated in BNC, a release of the compound was performed in simulated physiological fluids of the stomach, small intestine, and colon. In the release profiles found in 2 = 0.968 and R2 = 0.888, respectively. In physiological stomach fluid, a gradual release of genistein is observed with a release rate of 0.001 min−1, and for the small intestine, a release rate of 0.021 min−1 was obtained, which stabilizes after 60 min. This is due to the protection generated by bacterial nanocellulose to the compound. On the other hand, the experimental data of the release in colonic fluid fit with R2 = 0.927 to a pseudo-second-order model. The release of genistein in this medium was much greater and in a controlled and prolonged manner.For release into the stomach and intestine fluid, the experimental data had a better fit to a pseudo-first-order kinetic model with R50) was determined by dose–response curves and the selectivity index by the formula IS = IC50 Control cells (HaCat)/IC50 Tumor cells (SW480 and SW620). A selectivity index greater than 1 indicates that the treatment is more cytotoxic to tumor cells than to control cells. The results show that genistein has selectivity in the SW480 and SW620 cell lines at 24 h and 48 h of treatment against the non-malignant HaCat cell line. Based on the IC50 value of free genistein, cytotoxicity assays of empty BNC capsules and BNC/GEN were performed. In 50 value of genistein was used. To evaluate ROS production in SW480 and SW620 cell lines, the ICTM kit (Chemicon Cat. N°APT110) with PI/F-dUTP makes it possible to differentiate, at the top of each graph, the population where a cell death process takes place. For the SW480 cell line, higher percentages of cell death were observed compared to the control . For the SW620 cell line, higher percentages of cell death were also observed compared to the control .The apoptotic capacity of genistein on cell lines SW480 and SW620 can be seen in To determine the intracellular proteins that participate in the apoptosis process of the SW480 and SW620 cell lines, Caspase 3, p53, Cytochrome c, BCL2, and cleaved PARP proteins were analyzed. In the SW620 cell line, after 24 h of treatment with genistein, a significant increase in Caspase 3 and cleaved PARP proteins was observed, and after 48 h of treatment, a significant increase in Caspase 3, p53, Cytochrome c, and cleaved PARP proteins was observed. The antiapoptotic protein BCL2 was not detected at 24 h and did not show significant differences compared to the control at 48 h of treatment . In the Cell supernatants were analyzed after 24 and 48 h incubation, with and without genistein. In the SW480 cell line, there were no significant changes in the cytokines tested (results not shown).On the other hand, in the SW620 cell line, several cytokines showed a significant increase both at 24 h and 48 h after genistein treatment . At 24 hThis work was carried out in the Laboratory of Medical and Experimental Mycology CIB-UdeA-UPB-UDES located in the Corporación para Investigaciones Biológicas, Medellín, Colombia.A commercial genistein CAS No. 446-72-0 , with 98% purity, was used for the analyses.Komagataeibacter medellinensis NBRC 3288, which was isolated in the Central Retail of Medellín and was identified in the Universidad Pontificia Bolivariana [w/v), peptone at 0.5% (w/v), yeast at 0.5% (w/v), disodium phosphate at 0.267% (w/v), and citric acid to adjust the pH up to 3.5. After fermentation for 7 days, the BNC membranes were removed and purified in a 5% (w/v) KOH solution to remove biomass and debris. Finally, the NCB was processed in a monobloc blender and passed to a MKCA6-3 ultrafine friction mill , with a total of 27 passes for the individualization of the nanofibers. The BNC was sterilized for later use.Bacterial nanocellulose (BNC) was used as encapsulating agent, which was obtained through the fermentation of the bacterium ivariana . Nanocel3/h, an air pressure of 6 bars, and an air inlet temperature of 150 °C.The encapsulation of genistein in BNC was carried out using the spray-drying technique in a . First, a solution of 0.1% BNC (1 mg/mL) and 5.52 mg of genistein were prepared. Then, the solution was dried with a feed flow rate of 5 mL/min, an air flow of 35 mThe morphology and size of the capsules obtained were analyzed by scanning electron microscopy (SEM) using JEOL JSM 6490 LV equipment  in a high vacuum with a secondary electron detector to obtain high-resolution SEI images at an acceleration voltage of 20 kV. The capsules were placed on a carbon ribbon and coated with a thin layer of gold. The size of the capsules was measured using the free distribution software: Image J 1.49, adjusting each capsule to an ellipse. A total of 100 capsules were measured to find the size distribution.For genistein release profiles, the membrane dialysis method with a size of 12–14 kDa was used  in simulMycoplasma spp. by PCR, using specific primers [For the biological tests, the human colon adenocarcinoma cell line SW480, its metastatic derivative SW620, and the non-malignant cell line HaCaT were used. These cell lines were obtained from the European Collection of Authenticated Cell Cultures  and cultured in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% horse serum  heat-inactivated (60 °C), Penicillin/Streptomycin 1%  and Non-Essential Amino Acids 1% (Sigma-Aldrich). For the experiments, the serum concentration was reduced to 3% and the medium was supplemented with 5 mg/mL transferrin, 5 ng/mL selenium, and 10 mg/mL insulin  . Before  primers .2. They were then incubated for 24 h to allow their adherence, and subsequently, they were treated with different concentrations of genistein (3.7–740 µM), as well as with the vehicle used (Dimethyl sulfoxide DMSO at 1%) as growth control. After each treatment for 24 and 48 h, cells were fixed with trichloroacetic acid  for one hour at 4 °C. Cellular proteins were determined by staining with 0.4% SRB (Sigma-Aldrich) for 30 min at room temperature. Subsequently, 5 washes with 1% acetic acid were performed. For these latter procedures, the plate was allowed to dry at room temperature. SRB-bound proteins were solubilized with 10 mM Tris-base and the reading was performed by absorbance at 490 nm in a microplate reader .Cell viability after genistein treatments was evaluated by the Sulforhodamine B (SRB) assay. A colorimetric assay consisted of staining the cellular protein content of adherent cells. Cell lines were seeded in 96-well plates at a density of 20,000 cells per well at 37 °C with 5% CO2. The cells were incubated for 24 h to allow their adherence and were subsequently treated with the vehicle as growth control (1% DMSO) and with 5 different concentrations of genistein based on the IC50 found in the cell viability assay . The evaluation times were 0, 2, 4, 6, and 8 days. Every 48 h, the culture medium was changed with the respective treatments [The antiproliferative effect of genistein was also evaluated with SRB. Cell lines were seeded in 96-well plates with a density of 2500 cells per well at 37 °C with 5% COeatments . After c2. Cells were incubated for 24 h to allow their adherence and were subsequently treated with the vehicle as growth control, with the positive control (ferrous sulfate) and the IC50 of genistein corresponding to each cell line and evaluation time. The treatments were evaluated for 24 h and 48 h. After this time, washings were carried out with DMEM medium, DCFH-DA staining, and cell lysis and reading in the varioskan equipment at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Fluorescence intensity was normalized from the total protein concentration measured for each treatment. Triplicate evaluations were performed for this assay.For the determination of reactive oxygen species, a quantitative analysis was carried out in the Varioskan Lux reader  by measuring fluorescence in a 96-well plate using fluorescein 2′, 7′ Dichlorodihydrofluorescein Diacetate (DCFH-DA)  that allows ROS detection. In this procedure, cells were incubated in 6-well plates at a density of 250,000 cells per well for the SW480 cell line and 350,000 cells per well for the SW620 cell line at 37 °C with 5% COTM kit  allowing a staining method that marks DNA breaks. In this procedure, cells were incubated in T75 flasks at a density of 1.1 million cells per flask for the SW480 cell line and 1.6 million cells for the SW620 cell line at 37 °C with 5% CO2. Cells were incubated for 24 h to allow their adherence and were subsequently treated with genistein (IC50 of each cell line) for 24 h and 48 h. After this time, cells underwent cell fixation, the addition of staining solution, incubation, and subsequent reading in the flow cytometer , according to the kit instructions.The detection of DNA fragmentation related to cell death processes was detected by flow cytometry using the APO-DIRECT2. Cells were incubated for 24 h to allow their adherence. Subsequently, they were treated with the vehicle as growth control (DMSO at the highest concentration used with the compound in the test) and with the concentrations of genistein corresponding to the IC50 of 24 h and 48 h, evaluating these same times. For the immunological marker measurement assay, the supernatant was collected at each of the evaluated times and taken to the equipment for measurement following the protocol indicated by the manufacturer; the kit used in this assay was the Th1/Th2/Th9/Th17 Cytokine 18-Plex Human ProcartaPlex™ Panel. For the apoptosis assay, a lysis buffer was used to release intracellular proteins and was subsequently taken to the equipment for measurement following the protocol indicated by the manufacturer. The kit used in this assay was Apoptosis 6-Plex Human ProcartaPlex™ Panel . Triplicate evaluations were performed for these assays. The samples were normalized in protein concentration for analysis.For these evaluations, the Magpix equipment was used , which allows a qualitative and quantitative analysis of proteins. Cell lines were seeded in 6-well plates at a density of 250,000 cells per well for the SW480 cell line and 350,000 cells per well for the SW620 cell line, incubating at 37 °C with 5% COp < 0.05 was considered significant.In the cell viability assays, non-linear regression was applied to find the IC50. The results were expressed as the mean ± standard deviation (SD). For the antiproliferation, apoptosis, ROS, and expression of immunological markers assays, a two-way ANOVA was applied followed by the Sidak test after verifying the assumption of normality of the data using the Kolmogorov–Smirnov test. Statistical analyses and graphs were performed using the GraphPad Prism Version 8 program. In all cases, a value of Komagataeibacter [Many studies show that genistein has great potential as a chemopreventive agent for different types of cancer ,15,30,31eibacter . In the eibacter . As an eeibacter ,37. BNC eibacter , and wheeibacter ,37. As oeibacter .The release profiles obtained in this research indicate that the BNC/GEN capsules present a release of the compound that depends on the pH of the solution. This behavior is characteristic of NCB-encapsulated and oven-dried compounds . The low50 of each cell line after treatment with genistein, it was possible to determine a selectivity of 3.17 for the SW480 cell line at 24 h of evaluation and 1.75 at 48 h of evaluation. For the SW620 cell line, at 24 h the selectivity was 1.34 and at 48 h it was 1.47 against the non-malignant HaCaT cell line. Additionally, to date, the evaluation of the effect of this type of compound on a tumor cell line and its metastatic derivative, both from the same patient, had not been reported in the same study. Based on these IS and IC50s, an antiproliferative effect was observed over the time points tested in a dose-dependent manner for both the SW480 cell line and the SW620 cell line. This inhibition of cell growth is associated with the cumulative dose of genistein that was given every 48 h for up to eight days.Although genistein has been described as an agent with anticancer properties in several types of cancer, including CRC, there are many mechanisms that need to be described in relation to its biological activity . The meaOne of the mechanisms by which a compound can inhibit cell growth and proliferation is by inducing apoptosis in malignant cells. In cancer cells, the mechanism of programmed cell death is reduced, demonstrating an imbalance in the proteins involved in the apoptosis process, both proapoptotic and antiapoptotic ,45. In tROS are highly reactive molecules and their generation in cells occurs in a balanced manner. They have various functions in normal physiological regulation, such as cell-cycle progression, proliferation, differentiation, and cell death. Additionally, they play an important role in the activation of various cell signaling pathways, and their high levels can lead to damage to proteins, nucleic acids, lipids, membranes, and organelles, leading cells to a process of apoptosis . Higher In the SW480 cell line, no levels of these proteins were detected at any of the evaluation times after treatment with genistein. However, a population in the process of cell death was observed in the flow cytometric assay. This test allows us to identify ruptures presented in the process of DNA fragmentation, a process that not only occurs in apoptosis but also in other mechanisms of cell death, including necroptosis, which can also be evaluated and identified with Propidium Iodide (PI) and presents a signaling pathway independent of the caspase pathway and the proteins involved in the apoptosis process . TherefoIn CRC, cytokines play a crucial role in the development of this type of cancer. However, the available data are insufficient to describe the changes in the cytokine profile during the development of CRC, as well as the mechanisms that lead to changes in the cytokine levels in this type of cancer . In the IL-13 has effector functions, including allergic inflammation, tissue remodeling, and fibrosis. It is a structurally and functionally similar cytokine to IL-4, and the components of its receptors are similar. They regulate the immune response and are involved in several neoplasms. Both cytokines are important in the Th2 response. Several studies have shown discrepancies in the antiproliferative effect of these cytokines in various cell lines and CRC clinical studies . These iIL-6 can show both pro- and antineoplastic activity  and is rIt is important to highlight that the effects exhibited by these cytokines will depend on the tumor microenvironment in which they are found. Perhaps, for this reason, there is no clear immunomodulatory effect or profile due to the fact that they are in vitro tests with single cell lines, an effect that could, perhaps, be presented in a tumor microenvironment to evaluate different processes in relation to the evolution of tumors.Finally, one of the great problems of chemotherapeutic agents is the resistance that occurs against them. For this and the other reasons previously described, it is of great importance to develop a cancer therapy based on natural products such as genistein. In this sense, the efficiency of drugs used in chemotherapy depends on the mechanisms to exert their action. One of these mechanisms is apoptosis, in which the deregulation of pro- or antiapoptotic genes in tumor cells has been related to increased resistance to chemotherapy . ApoptosIn conclusion, genistein has a chemopreventive effect on the colorectal adenocarcinoma cell line SW480 and its metastatic derivative SW620 through cytotoxicity and antiproliferation evaluations. In both cell lines, there is evidence of the expression of immunological markers that plays an important role in the carcinogenic process and metastasis of CRC and that have been described in other studies with antitumor properties in this type of cancer. By exerting a dual role in this process, depending on the tumor microenvironment, they can have various effects on cell lines, in this case, an increase in expression. The production of ROS in both cell lines may be associated with the cell death process through different mechanisms. Thus, due to the lack of response in the evaluated proteins, it is hypothesized that a necroptotic process mediated by ROS production could occur in the SW480 cell line, while in the SW620 cell line, it is suggested that genistein induces the formation of ROS with the consequent activation of intrinsic apoptosis mediated by caspases and p53. Although genistein has great potential as a chemotherapeutic agent, when administered orally, its bioavailability and, therefore, its effect on the target site, in this case the colon, are decreased. Therefore, new strategies such as encapsulation should be used to improve its effectiveness. BNC encapsulation improved the release profile of genistein, protecting it from gastric pH and allowing its release in the colon.


Alternaria tenuissima AUMC14342 ethyl acetate extract on HeLa , SKOV-3 (ovarian cancer), and MCF-7 (breast adenocarcinoma) cell lines. The extract showed potent effect on MCF-7 cells with an IC50 value of 55.53 μg/mL. Cell cycle distribution analysis of treated MCF-7 cells revealed a cell cycle arrest at the S phase with a significant increase in the cell population (25.53%). When compared to control cells, no significant signs of necrotic or apoptotic cell death were observed. LC-MS/MS analysis of Alternaria tenuissima extract afforded the identification of 20 secondary metabolites, including 7-dehydrobrefeldin A, which exhibited the highest interaction score  in molecular docking analysis against human aromatase. Regarding ADME pharmacokinetics and drug-likeness properties, 7-dehydrobrefeldin A, 4’-epialtenuene, and atransfusarin had good GIT absorption and water solubility without any violation of drug-likeness rules. These findings support the anticancer activity of bioactive metabolites derived from endophytic fungi and provide drug scaffolds and substitute sources for the future development of safe chemotherapy.Breast, cervical, and ovarian cancers are among the most serious cancers and the main causes of mortality in females worldwide, necessitating urgent efforts to find newer sources of safe anticancer drugs. The present study aimed to evaluate the anticancer potency of mycoendophytic  Human health is at constant risk due to the occurrence of different types of non-communicable chronic diseases . Among tThe massive replicative potential and loss of balance between cell proliferation and apoptosis are attributes that increase the failure of damaged cells to be eliminated via apoptosis ,6. Many It is well known that estrogens are required for the growth of breast cancer in both premenopausal and postmenopausal women who have an elevated risk of occurrence . Thus, dRecently, natural products from endophytic fungi have been identified as an untapped valuable source of many new biologically active chemical analogs against infectious diseases, cancer, and a variety of other diseases ,16,17. EAlternaria tenuissima is a species of the Alternaria genus that belongs to the Dematiaceae family of fungi that inhabit various plant species and have a wide distribution in the environment − fit to isocoumarin derivatives altenuene , alternariol, 3-O-demethylaltenuisol, and alternariol-9-methyl ether. In addition, (S)-alternariphent A, alternapyran, 4-methoxy-6-methyl-5-(3-oxobutyl)-2H-pyran-2-one, and resveratrodehyde C were also detected at tR 12.43, 12.84, 15.04, and 30.02, respectively.The negative mode results  revealedThe compounds’ docking results revealed an affinity range of −8.0156 to −4.7618 kcal/mol. The top-scoring compounds were 4’-epialtenuene (4’), 7-dehydrobrefeldin A (9), and atransfusarin (19), which had a stronger affinity to the human placental aromatase cytochrome P450 (CYP19A1) active site relative to the complexed inhibitor ligand 4-androstene-3-17-dione .Molecular docking analysis revealed that 7-dehydrobrefeldin A (9) showed the highest interaction score with human aromatase  as compared to 4-androstene-3-17-dione, standard inhibitor . 7-dehydrobrefeldin A interacts through hydrogen bond formation with the Met 374 amino acid residue in the catalytic cleft of aromatase in addition to several hydrophobic interactions with the active site .Moreover, the compound 4’-Epialtenuene showed a pose score of −7.2929 kcal/mol with an RMSD value of 1.30 Å and was found to bind with the receptor active site through the formation of hydrogen bonds with Leu 477 and Met 374 amino acid residues as H-donors as well as through a pi-H bond with VAL 370 amino acid .Furthermore, atransfusarin (19) interacts with Met 374 amino acid residue as an H-donor and with VAL 370 amino acid through a pi-H bond  with a pThe compounds that showed the highest affinity to the human aromatase cytochrome P450 active site were screened for their drug-likeness and ADME pharmacokinetics using the website servers. All tested compounds had good GIT absorption and water solubility without any violation of drug-likeness rules. In addition, the compounds had promising bioavailability scores with a value of 0.55. 4’-epialtenuene and dehydrobrefeldin A showed promising lead-likeness and synthetic accessibility .Alternaria spp. of various chemical classes and biological activities have been identified [Alternaria extracts and secondary metabolites have been shown to have a wide range of biological activities and functions, including cytotoxic, antimicrobial, and herbicide effects. The promising bioactivities have attracted the interest of pharmaceutical researchers in the discovery of novel natural therapeutics [Fungi are a valuable source of bioefficient natural products that can be employed to fabricate new analogs for cancer treatment . Severalentified . Alternaapeutics ,34.50 values of the A. tenuissima EtOAc extract on the viability of HeLa, SKOV-3, and MCF-7 cancer cell lines determined by SRB assay were 67.76, 74.60, and 55.53 μg/mL, respectively. Cell cycle distribution analysis of the treated MCF-7 cell line showed a cell cycle arrest at the S phase with a significant increase in the cell population (25.53%), while no significant signs of necrotic or apoptotic cell death were observed. It is noteworthy that the previous studies of A. tenuissima revealed the isolation of toxic metabolites such as alternariol and alternariol monomethyl ether which possessed strong cytotoxicity against KB and KBv200 tumor cell lines [Alternaria species, exhibited a broad spectrum of biological activity, such as antineoplastic, antiviral, and antibiotic effects [A. tenuissima TER995 was employed as a promising source for the submerged fermentation-based production of paclitaxel (taxol), the most valuable anticancer drug [In the present study, the ICll lines  and L517ll lines . Interesll lines . Further effects ,50,51. Acer drug .A. tenuissima extract in positive ion mode revealed several compounds, including N-acetyltyramine and solanapyrone P, which were previously isolated from A. tenuissima SY-P-07 and SP-07 [A. carthami [Alternaria sp. [Alternaria sp. NH-F6 [A. alternata MT-47 [A. tenuissima [A. solani [A. tenuis [Alternaria sp. HG1 [A. atrans MP-7 [Alternaria sp. and A. tenuis [A. tenuis [A. tenuissima [Alternaria sp. PfuH1 [Alternaria sp. SCSIO41014 [Alternaria sp. ZG22 [A. phragmospora [Alternaria sp. R6 [Analyzing the nd SP-07 ,54. Morecarthami , 5-butylcarthami , altertearia sp. , 2--alt sp. HG1 , atransfans MP-7 , altenue. tenuis ,64, alte. tenuis , alternanuissima , 3-O-demp. PfuH1 , (S)-altSIO41014 , alternasp. ZG22 , 4-methogmospora , and resa sp. R6 .A. tenuissima has a potent impact on MCF-7 cells, with an IC50 value of 55.53 μg/mL. In order to evaluate the possible affinity of identified compounds and understand their binding possibility as inhibitors for the aromatase enzyme as a selective target for breast cancer treatment, a molecular docking analysis was conducted [The cytotoxicity assay revealed that onducted ,33. Aromonducted ,70. Aromonducted . Both stonducted . The genonducted . The aroonducted ,74 and sAlternaria tenuissima AUMC14342 showed anticancer potential against HeLa , SKOV-3 (ovarian cancer), and MCF-7 (breast adenocarcinoma) cell lines in an SRB assay used to assess cancer cell viability. Cell cycle distribution analysis of treated MCF-7 cells revealed cell cycle arrest at the S phase with a significantly increased cell population. Bioactive secondary metabolites in the EtOAc extract were characterized using LC-MS/MS, and their molecular docking analysis against human placental aromatase exhibited a promising affinity to the aromatase active site. Moreover, ADME pharmacokinetics and drug-likeness properties of 7-dehydrobrefeldin A, 4’-epialtenuene, and atransfusarin revealed good GIT absorption and water solubility. The current research will enrich and enhance the bioprospecting of anticancer natural metabolites from endophytic fungi to achieve a sustainable supplement for safe chemotherapy.The ethyl acetate extract from endophytic