Epigenetic Regulation Underlying Bee Venom–Induced Negative Anastasis in Cancer Cells but Positive Anastasis in Normal Cells

Epigenetic Regulation Underlying Bee Venom–Induced Negative Anastasis in Cancer Cells but Positive Anastasis in Normal Cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Epigenetic Regulation Underlying Bee Venom–Induced Negative Anastasis in Cancer Cells but Positive Anastasis in Normal Cells Sinan TETİKOĞLU, Selcen ÇELİK UZUNER This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8465237/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Anastasis—defined as cellular recovery from the brink of apoptosis—has been previously characterized in ethanol-induced apoptosis models, yet its regulatory mechanisms and relevance in normal cells remain insufficiently understood. This study investigates the epigenetic regulation underlying divergent anastatic responses in normal breast cells (MCF10A) and breast cancer cells (MDA-MB-231) following bee venom and cisplatin. In our previous work, bee venom induced selective recovery (positive anastasis) in MCF10A cells while promoting persistent cell death in MDA-MB-231 cells. This work explores whether epigenetic mechanisms contribute to this selective response using an anastasis scoring approach to quantify recovery after exposure to bee venom or cisplatin. We found that cisplatin triggered persistent cell death in both cell types, whereas bee venom elicited robust anastatic recovery exclusively in normal cells. Gene expression analyses targeting key epigenetic regulators—responsible for reading, writing, and erasing DNA and histone modifications—revealed distinct transcriptional alterations associated with positive anastasis, suggesting that epigenetic reprogramming may facilitate the loss of cellular memory of bee venom cytotoxicity. These findings provide the first evidence that positive anastasis in normal cells is shaped by specific epigenetic mechanisms, highlighting a potential therapeutic advantage in cancer treatment strategies that preserve normal tissue integrity while limiting cancer cell survival. Biological sciences/Cancer Biological sciences/Cell biology Biological sciences/Molecular biology anastasis epigenetics cancer bee venom cisplatin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Introduction Anastasis is described as “a return” from induced cell death. For the first time, this has been defined by a return of ethanol-induced apoptosis, and some genes are related to its regulation (H. M. Tang et al. 2017 ; H. M. Tang et al. 2022 ). Anastasis is considered a responsive strategy for drug resistance in cancer cells (Mohammed et al. 2022 ). However, the anastatic response of normal cells to cytotoxic agents has not been elucidated. In our previous study, we revealed that anastasis can occur selectively in normal breast cells after exposure to bee venom. In contrast, it induced persistent cell death in metastatic breast cancer cells (Tetikoglu et al. 2025 ). Another group of cells exhibited tenacious cell death after treatment with cisplatin, a commonly used chemotherapeutic agent in cancer therapies. Selective anastasis for normal cells only can be defined as “positive anastasis,” indicating an advantageous cellular regulation in cancer therapies that does not affect normal cell survival (Tetikoglu et al. 2025 ). The accumulated data on anastasis research suggest the need for a detailed investigation of positive anastasis, focusing on its differential regulation in normal cells versus cancer cells. Epigenetic regulation of positive anastasis is one of the important candidates that needs to be studied, and there is a lack of research on the role of epigenetic (re)programming during positive anastasis, even in regular anastasis (as described initially). This study aimed to elucidate the expression profiles of key genes involved in epigenetic mechanisms, including the reading, erasing, and writing of epigenetic modifications on DNA and histones. This study employed a method called “anastasis scoring” to define anastasis in each cell after exposure to bee venom or cisplatin. Cisplatin treatment showed persistent cell death in both breast cancer (MDA-MB-231) and normal breast (MCF10A) cells. Therefore, the results suggest that bee venom-mediated positive anastasis is regulated by epigenetic reprogramming, particularly through the deregulation of genes involved in epigenetic regulation, and by distinct patterns of DNA methylation. The findings of this study indicate that cellular memory for bee venom cytotoxicity has been lost, leading to anastasis-mediated cell survival in normal cells but not in cancer cells. Materials and Methods 1. Cell culture, bee venom/cisplatin treatment, and anastasis experiment design MDA-MB-231 (metastatic breast cancer) and MCF10A (normal breast) cell cultures (ATCC) were cultured in RPMI (Wisent, Cat. No. 350-000 RL) and DMEM (Capricorn, Cat. No. HPA) media, respectively. Media were prepared containing 1% penicillin-streptomycin antibiotic and 10% or 20% fetal bovine serum (FBS) for MCF10A and MDA-MB-231 cells, respectively. Media were sterilized by passing through a 0.22 µm pore size filter. Cells in sterile media were incubated in a 37°C incubator with 5% CO 2 . Confluent cells were treated with 8 µg/ml or 12 µg/ml of bee venom or cisplatin for 24h (Tetikoglu et al. 2025 ). Control cells were left untreated. After 24h, bee venom/cisplatin was removed, and cells were washed with 1xPBS. Cells were then cultured in fresh media without bee venom or cisplatin for an extra 24h and 72h to evaluate the anastatic response of the cells. Sequential incubations were classified into 3 groups: i) first 24h with media including bee venom /cisplatin (+ 24h), ii) 24h with clean media after 24h-treatments (-24h), and iii) 72h with clean media after 24h-treatments (-72h). A batch of cells was used for extended incubations for anastasis (-24h and − 72h), and the rest was examined by trypan blue assay, Annexin V-PI assay, cellular morphology assessment, mitochondrial membrane potential, and senescence profiles for each incubation as detailed below. 2. Cell viability by trypan blue staining Trypan blue is a negatively charged dye used to determine cell viability. Since the membrane structure is intact in living cells, the dye cannot enter them, whereas dead cells absorb the dye and appear blue under the microscope. Healthy and cancerous cells were treated separately with bee venom or cisplatin for 24 hours and then washed once with 1x PBS (phosphate-buffered saline) (Wisent, 311-010-CL). Cells removed with trypsin were collected by centrifugation at 230 rpm. The supernatant was removed, and the cells were resuspended in the medium. 10 µL of cell suspension was mixed with 10 µL of 0.4% trypan blue (Biological Industries, B 103-102-1B) (1:1 ratio) and incubated for approximately 10 minutes at room temperature. The cell and dye mixture was loaded onto the coverslip of the device at 10 µL per well. Cell viability was determined using the Countess FL II automated cell counter (Thermo Fisher). Standard errors of the mean (S.E. +/- standard error of the mean) were calculated using SPSS software. All experiments were performed in at least three independent replicates, and each sample was measured at least three times within a replicate to test the reliability of the device. 3. Cell viability by MTT assay The MTT experiment was designed in 3 of 96-well plates for each MDA-MB-231 and MCF10A cells and bee venom or cisplatin (for + 24h, -24h, and − 72h incubations). 5000 cells per well were cultured in 96-well plates in triplicate and incubated at 37 ᵒ C with 5% CO 2 humidification, and treated with bee venom or cisplatin as mentioned above. Control wells were treated with 0.9% NaCl, the solvent for bee venom, as a negative control. After 24h incubation, the media was removed, and fresh media (190µL) and 10 µL of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, 5mg/mL) (Invitrogen, Cat.No: M6494) was added to each well and incubated at 37°C and 5% CO2 for 4 hours (Meantime, the other 2 plates were washed, fresh media were added, and then left for anastatic incubations, -24h and − 72h). After incubation with MTT, the media with MTT was removed, 100 µL of DMSO (Sigma-Aldrich, Cat.No: D4540) was then added to each well, and incubated on a shaker for 30 minutes. After incubation, color development was observed, and the absorbance at 570 nm was measured using a microplate reader. Cell viabilities were then calculated from absorbance values by proportioning untreated control wells (taken as 100% viable). Cell viabilities were then arcsine-transformed for use in the UNIANOVA test. The arcsine transformation is a statistical technique used to stabilize the variance of proportional data. Bar graphs presented data. The y-axis represents cell viability (arcsine-transformed), and the X-axis represents the dose of the treatment agent in microgram/mL. 4. Apoptosis and necrosis determination by flow cytometry A million cells were separated for Annexin-V and PI staining. Annexin V (FITC)-PI staining protocol (BD, Cat. No. 556547) was applied to separate apoptotic and necrotic cell populations by flow cytometry. Briefly, the protocol includes (i) washing the cells once with cold 1xPBS, (ii) suspending the cells with 1x binding buffer (100 µl), (iii) adding Annexin V (FITC) (5 µl) and PI (5 µl), and finally, (iv) making the volume up to 500 µl with 1x binding buffer. Cells were stained with Annexin V (FITC), and PI was analyzed by flow cytometry at one hour. This analysis was performed according to the untreated control cells by grouping the cells in the FSC-SSC (forward scatter-side scatter) graph, dividing them into four regions (live – necrotic – early apoptotic – late apoptotic), and determining the cell percentages in these regions (UL, upper left: necrotic cells, UR; upper right: cells in late apoptosis, LL, lower left: live cells, LR, lower right: early apoptotic cells) (Koç et al. 2018 ). The accuracy and reliability of the assay were evaluated by measuring cellular autofluorescence in unstained cells (data are provided in the supplementary material Tables S1-S4 ). Autofluorescence was detected in all assays using flow cytometry (BD Accuri). 5. Cellular morphology assessment by flow cytometry In order to quantitatively measure the morphological changes in the cells, a batch of cells after each incubation and dose was washed with PBS, and read by flow cytometry (BD Accuri). FSC-SSC (forward scatter-side scatter) plots were obtained for each sample to morphologically define cells of interest based on cell size and intracellular granularities, respectively. 6. Mitochondrial membrane potential (MMP) by flow cytometry For MMP analysis, after bee venom and cisplatin treatments, cells were centrifuged, and a batch of cells was incubated in cell media containing 400 nM MitoTracker Red dye (Invitrogen, Cat. No M7512) at 37°C for 45 minutes. At the end of incubation, the media in the cells was removed by centrifugation and washed once with 1xPBS. Finally, the cells were analyzed by flow cytometry (BD Accuri) in fresh PBS. The amount of mitochondrial membrane potential was measured in the FL-3 channel, where red staining was detected, as before (Celik-Uzuner 2020 ). 7. Senescence profile by flow cytometry Another 1 million cells were separated for senescence using CellEvent™ Senescence Flow Cytometry Assay Kit (Invitrogen, C10841). The protocol includes the following steps: i) treated cells were washed with PBS and collected with trypsin. Cells were resuspended in 1X PBS at 500,000-1,000,000 cells per 100 µL. Then, 100 µL of the cell suspension was taken into flow cytometry tubes. Cells were pelleted by centrifugation and resuspended in 100 µL of Fixation Solution. The suspension was incubated for 10 min at room temperature in the dark. At the end of the period, cells were washed with PBS containing 1% BSA to remove the fixation solution. Cells were resuspended in 100 µL of working solution and then incubated for 1–2 hours at 37°C incubator without CO 2 , protected from light. After incubation, the working solution was removed, and cells were washed with PBS containing 1% BSA. Cells were resuspended in 1% BSA in PBS buffer and then read on a BD Accuri device with a 488 nm laser and a 530 nm/30 filter. The mean fluorescence intensity of fluorescence (by FL1-H channel-detected FITC signal) was obtained. 8. Anastasis scoring We have developed, for the first time, an extensive method, called “anastasis scoring,” to determine the tendency of cells towards anastasis. The reasoning for the necessity to develop a comprehensive method for anastasis is based on 1) there is a range of cell death types, 2) it is hard to conclude cell death with only one cellular parameter, and 3) there is also a wide range of methods for detecting cell death. Anastasis is considered a return from apoptosis so far. Tang et al. have reported a method for tracking anastasis by live cell imaging (H. L. Tang et al. 2015 ). However, apoptosis is not the only type of cell death, and cells have been shown to recover from different cell death mechanisms, such as ferroptosis (Cao et al. 2024 ) and necroptosis (Gong et al. 2017 ), suggesting that a range of cellular and molecular parameters should be considered. “Anastasis scoring” method, therefore, includes scoring cells according to the different parameters as follows: 1) total cell death by trypan blue assay, 2) cell death by MTT assay, 3) apoptosis rate, 4) necrosis rate, 5) cellular morphology, including cell size and cell granularity, 6) mitochondrial membrane potential, 7) senescence, and 8) expression profiles of BAX , DFFB and BCL2 genes. The cells were scored as none (score 0, p > 0.05), less (score 1, p ≤ 0.05), more (score 2, p ≤ 0.01), high (score 3, p ≤ 0.001), or the highest (score 4, p ≤ 0.0001) for each parameter, with a comparison of treated cells and untreated cells. Expected significance (decrease or increase) is given in Table 1 . In our case, we compared “12µg/ml treated cells for 72h anastatic incubation (-72h)” with “0µg/ml untreated cells for − 72h”. Table 1 Parameters and significance for anastasis scoring Cellular Event Considered significance Scoring for each Cell Death (Trypan Blue) Increased cell death none (score 0, p > 0.05), less (score 1, p ≤ 0.05), moderate (score 2, p ≤ 0.01), high (score 3, p ≤ 0.001), the highest (score 4, p ≤ 0.0001) Cell Death (MTT) Increased cell death Apoptosis Increased apoptosis Necrosis Increased necrosis Cell size (FSC) Increase or decrease in FSC Cell granularity (SSC) Increase or decrease in SSC Mitochondrial membrane potential Increase or decrease in MMP Senescence Increased senescence BAX gene expression Increased expression DFFB gene expression Increased expression BCL2 gene expression Decreased expression 9. RNA isolation and cDNA conversion After bee venom/cisplatin, total cellular RNA was isolated using an RNA isolation kit (BioBasic, BS88003) specifically developed for cultured cells. The steps followed in this kit were as follows: A maximum of 10 7 cells were collected from the flask. Lysis buffer was added, and centrifuged. After centrifugation, the pellet was transferred to an RNA tube. Ethanol was added to the RNA tube and transferred to the RNA column, and after centrifugation, GT and NT solutions were added, respectively, to precipitate the RNA. The purity and concentration of RNAs were measured in the NanoDrop device. The total RNA obtained from the samples was first converted into complementary DNA (cDNA) using a kit from A.B.T. Laboratory Industry (Cat. No: C03-01-05). For cDNA synthesis, 100 ng of isolated total RNA, 5X Reaction Buffer, Reverse Transcriptase (200 U/µl), RNase Inhibitor (10 U/µl), Random Hexamers (50 µM), and 10 mM dNTP Mix were added to the PCR tube. The reaction volume was completed to 20 µl with RNase-Free Water. The tube was kept on ice until the reverse transcriptase reaction took place. The tube was placed in the thermal cycler, run by a cycle of 25°C 10 min, 37°C 120 min, 85°C 5 min, respectively, and kept at 4°C. 10. Primers and Quantitative PCR We performed QPCR for a list of genes involved in epigenetic regulation pathways. The genes were TET1, IGF2, H19, EZH1, EP300, BAX, BCL-2, HOXB13, DNMT1, DNMT3A, TP53, HDAC1, KDM6A, KMT2A, DFFB, RBBP4 , and LHX4 . The Primer3 program was used for QPCR primer design (Untergasser et al. 2012 ; Koressaar and Remm 2007). Primers used are given in Table S5 . The expression levels of target genes from cDNA samples were analyzed by QPCR reaction. Real-time PCR reactions were performed using A.B.T.™ 2X qPCR EVA-Green MasterMix (with ROX) (Atlas Biotechnology, Cat.No. Q02-02-05). The reaction workflow consisted of the following steps: (1) initial denaturation, (2) denaturation, (3) primer annealing, (4) extension, and (5) melting curve analysis. For each qPCR reaction, forward and reverse primers (10 µM), template DNA (cDNA), and RNase-free water were added to PCR tubes. The reaction volume was adjusted to 20 µl. Reagents were kept on ice during preparation, gently mixed, and briefly centrifuged. Reaction tubes were placed into a thermal cycler and amplified under the following cycling conditions: 95°C for 300 seconds (initial denaturation), followed by cycles of 95°C for 10–30 seconds (denaturation), 55–68°C for 10–60 seconds (annealing), and melting curve analysis performed from 65–95°C with 2–5 seconds per step, applied sequentially according to the required number of cycles. The amplification curves and threshold cycle (Ct) values were analyzed using the qPCR instrument software. Changes in gene expression were determined using the ΔΔCt (delta–delta Ct) method by calculating fold-change values. The ΔΔCt method (also known as the comparative Ct method) is widely used for the analysis of qPCR data, particularly for comparing relative gene expression levels between experimental and control samples (Livak and Schmittgen 2001 ). The calculation steps are as follows: 1) Ct (Cycle threshold): The PCR cycle number at which the fluorescence signal exceeds the background level for each gene. 2) ΔCt calculation: The difference between the Ct value of the target gene and that of the reference (housekeeping) gene in each sample. In this study, ACTB was used as the reference gene. 3) ΔΔCt calculation: The difference between the ΔCt value of the treated sample and that of the control group. In this study, the control group consisted of untreated cells (neither bee venom nor cisplatin exposure). 4) Relative gene expression (Fold change): The fold-change value indicates how many times the expression of a gene is increased or decreased compared with the control group. 11. The STRING database for protein interactions involved in epigenetics To confirm the relationship between the proteins encoded by the genes analyzed using QPCR, the gene list was uploaded to the STRING database (version 12.0; https://string-db.org ) to explore potential protein–protein interactions (PPIs) and predict associated signaling pathways ( Fig. 1 ) . The analysis was performed using the “multiple proteins” option, restricting the organism to Homo sapiens . The minimum required interaction score was set to 0.7 (high confidence) to ensure biological relevance. Both known and predicted interactions (from curated databases, experiments, gene co-expression, and text mining) were included in the network. The generated PPI network was visualized within STRING, and clusters of functionally related proteins were identified using the Markov Cluster Algorithm (MCL) with an inflation parameter of 3. Pathway enrichment analyses were conducted using the KEGG (Kanehisa et al. 2025 ) and Reactome databases (Fabregat et al. 2018 ) integrated in STRING (Szklarczyk et al. 2023 ), enabling the identification of up- and down-regulated biological pathways related to the experimental conditions. Functional annotations with the highest enrichment scores (FDR < 0.05) were considered significant. Figure 1 confirms the PPI of the genes TET1, IGF2, H19, EZH1, EP300, BAX, BCL-2, HOXB13, DNMT1, DNMT3A, TP53, HDAC1, KDM6A, KMT2A, DFFB, RBBP4 , and LHX4 (by the String database). The PPI enrichment p -value was detected as 3.15×10 − 14 , confirming high significance. The pathway of these genes is related to transcriptional misregulation of cancer (KEGG pathway, hsa05202) (Kanehisa et al. 2025 ), and epigenetic regulation of gene expression (by Reactome database HSA-212165, and Gene ontology). The enrichment approaches confirmed the interaction of these genes in epigenetics, and therefore, the expression profiles of these genes were analyzed using QPCR. 12. Statistical analyses The cell percentages obtained from Annexin-V/PI /PI results, mitochondrial membrane potential (as an arbitrary unit of total fluorescence amount), FSC-SSC profiles of cells, trypan blue-assisted cell viability percentages detected by automated cell counter, and the level of senescence were analyzed using the SPSS program UNIANOVA. The levels of significance used were p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), and p ≤ 0.0001 (****). Post-Hoc test was applied for pairwise comparisons. Percentage values ​​were first arcsine transformed before tests. All experiments were performed in triplicate. Results The general cell death profiles of each cell line after bee venom and cisplatin were analyzed by conventional trypan blue staining, as mentioned in the methods. Bee venom induced continuing cell death in MDA-MB-231 cells but anastasis in MCF10A cells at 72h of anastatic incubation ( Fig. 2 ) . Cell viability significantly increased at 72h in MCF10A cells ( p = 0.000) after bee venom, while the same significance was detected in increased cell death in MDA-MB231 cells. However, cisplatin treatment resulted in persistent cell death in both cells ( p = 0.000) ( Fig. 2 ) . The results confirmed our previous findings (Tetikoglu et al. 2025 ). To detail the cell death, we performed Annexin-V and PI dual-staining by flow cytometry. We then performed MTT cytotoxicity assay for + 24h, -24h, and − 72h in each cell after bee venom and cisplatin. The results demonstrate the concentration- and time-dependent effects of bee venom and cisplatin on the viability of MCF10A ( p < 0.0001) and MDA-MB-231 cells ( p < 0.0001) ( Fig. 3 A, top) . Both agents induced cytotoxicity in both cell lines, with viability decreasing as the dose and incubation time increased. Cisplatin reduced the viability of both MCF10A cells ( p < 0.0001) and MDA-MB-231 cells ( p < 0.0001) ( Fig. 3 A, bottom) . Regarding anastasis, the viability of MCF10A cells increased after the maximum bee venom dose (12µg/mL) at -72h anastatic incubation ( p < 0.0001) compared to the viability after the same dose at + 24h. The viability percentages are also given in Fig. 3 B, and importantly, bee venom showed a degree of selective cytotoxicity, reducing the viability of the MDA-MB-231 cancer cells but maintaining viability in MCF10A cells under the same conditions, and significant anastasis in MCF10A cells at -72h ( Fig. 3 B ) . This suggests that while cisplatin is more toxic with no specificity, and bee venom may possess a therapeutic advantage due to its preferential toxicity towards the aggressive cancer cell line over the normal epithelial line, which is in an anastatic trend. Bee venom (12 µg/mL) induced necrosis after 24h treatment (+ 24), followed by a significantly increased apoptosis ( p < 0.01) in extended 24h (-24) for anastasis in MDA-MB-231 cells (Fig. 4 A). Similarly, at the end of 24 hours with bee venom treatment (+ 24) at a dose of 8 µg/mL, the apoptosis rate increased significantly compared to 24 hours without bee venom (-24) ( p < 0.01) and 72 hours without bee venom (-72) ( p < 0.05) (Fig. 4 A). However, at -72h of anastasis incubation, apoptosis decreased in treated cells with 12 µg/mL. Although MCF10A cells tended to undergo necrotic cell death after bee venom for 24h, bee venom did not significantly induce necrosis or apoptosis in comparisons between different doses within each incubation period and in comparisons with different incubation times at the same dose, according to the UNIANOVA test (Fig. 4 B). Cisplatin treatment provoked significant apoptosis in MDA-MB-231 cells. In general, the apoptosis rate in untreated cells was significantly lower compared to all cisplatin doses. However, there was no significant difference between the doses (Fig. 4 C). Similarly, the apoptotic cell population was higher after cisplatin in MCF10A compared to their untreated counterparts. But, there was no difference in the rate of apoptotic cells between the doses at each incubation (+ 24h, -24h, and − 72h) (Fig. 4 D). Regarding cellular morphology, bee venom decreased cell size (FSC) in MDA-MB-231 cells at + 24h, -24h, and − 72h, but increased granularity at -24h and − 48h ( Fig. 5 A ) . A similar pattern was observed in MCF10A cells, but cell size was not significantly changed at + 24h ( Fig. 5 B ) . In contrast, cisplatin generally induced a decrease in cell size in MDA-MB-231 ( Fig. 5 C ) and MCF10A cells ( Fig. 5 D ) . Granularity (SSC) significantly increased only at + 24 after cisplatin in MCF10A ( Fig. 5 D ). Statistical analyses confirmed that neither FSC nor SSC changed significantly in MDA-MB-231 or MCF10A cells after bee venom treatment. However, cisplatin induced a significant decrease in FSC, but not in SSC, in both cell lines ( p < 0.001). Cellular senescence was only induced in MDA-MB-231 cells after 8µg/ml bee venom for − 24h, compared to untreated cells ( p < 0.05) and to the higher dose 12µg/ml ( p < 0.05) ( Fig. 6 A ) . Inconsistent results were obtained for senescence in other groups ( Fig. 6 B-D ) . Bee venom did not change the mitochondrial membrane potential ( Fig. 7 A-B ) . However, the MMP profile was inclined to reduce after bee venom in both cells ( Fig. 7 C-D ) . To evaluate anastasis in the cells, anastasis scoring was used as detailed in the methods. According to the scoring, MCF10A cells treated with bee venom were the most anastatic group, followed by MCF10A cells treated with cisplatin, MDA-MB-231 cells treated with bee venom, and MDA-MB-231 cells treated with cisplatin ( Table 2 ) . Table 2 Cellular anastasis score of each cell after bee venom or cisplatin (incubation, -72h and concentration, 12µg/ml, statistically compared with untreated control cells at -72h). The lower the score, the higher the anastasis Cellular Event MCF10A MDA-MB-231 Bee Venom Cisplatin Bee Venom Cisplatin Cell Death (Trypan Blue) 0 4 4 4 Cell Death (MTT) 4 4 4 4 Apoptosis 0 0 0 1 Necrosis 0 0 0 0 Cell size (FSC) 0 4 0 4 Cell granularity (SSC) 0 0 0 0 Mitochondrial membrane potential 0 4 0 3 Senescence 0 0 0 0 BAX gene expression 0 0 0 0 DFFB gene expression 0 0 0 3 BCL2 gene expression 0 0 4 1 Cellular Anastasis Score 4 15 12 20 Scoring; 0; none, 1; less, 2; moderate, 3; high, 4; the highest Following cellular parameters, gene expression profiles including anti-apoptotic (BCL-2) and anti-apoptotic genes (BAX, DFFB) were analyzed. Figure 8 A-D presents the changes in gene expression for each experimental group based on the calculated ΔΔCt values. All values ​​obtained in Fig. 8 are presented as heat maps in Fig. 9 . Genes with fold-change values ​​of 2 and above were considered to have significantly increased expression. In contrast, genes with fold-change values ​​of 0.5 and below were considered to have significantly decreased expression. Therefore, among all the findings in Fig. 9 , the most significant data are selected and presented in Fig. 10 . Discussion This study investigated the epigenetic regulation in selective anastasis in normal breast cells compared to breast cancer cells after bee venom. Normal cells (MCF10A) responded to bee venom by reversing cell death; however, breast cancer cells (MDA-MB-231) showed persistent cell death. On the other hand, cisplatin, a common chemotherapeutic drug, resulted in irreversible cell death in both cells, suggesting its non-selective effect on cancer cells. In recent years, the term anastasis has been primarily used to describe the recovery of cells from apoptosis. The pioneering study by Tang et al. (H. L. Tang et al. 2012 ) demonstrated that cells displaying apoptotic morphology can revert to a proliferative state once the death stimulus is removed, and this process has since been confirmed by several subsequent studies (H. M. Tang et al. 2022 ; Sun et al. 2017 ; H. M. Tang et al. 2017 ). However, whether similar reversal phenomena occur in other cell death modalities such as necrosis, necroptosis, or ferroptosis remains a matter of debate. In classical necrosis, the irreversible loss of plasma membrane integrity precludes any anastasis-like recovery. Nevertheless, in necroptosis, it has been shown that some cells can survive despite activation of RIPK3 and MLKL . Moreover, the ESCRT-III complex has been reported to repair membrane ruptures during necroptosis, thereby preventing cell lysis and promoting survival (Gong et al. 2017 ), indicating a partial reversal of the death process, which is a phenomenon referred to as “abortive necroptosis”. Similarly, in ferroptosis, during the early stages of lipid peroxidation, reactivation of antioxidant systems such as GPX4 and glutathione (GSH) can halt the death process (Doll and Conrad 2017; Jiang, Stockwell, and Conrad 2021), indicating a “reversible ferroptosis”. In contrast, pyroptosis and autophagic cell death are generally considered irreversible, as they involve extensive membrane or lysosomal damage (Y. Liu and Levine 2015). Thus, while the term anastasis has been explicitly defined only for reversal from apoptosis, accumulating evidence suggests that partial or early-stage recovery mechanisms may also exist in necroptotic and ferroptotic pathways. Therefore, in this work, for the first time, we have described a novel method, “anastasis scoring”, to conclude the tendency of cells towards anastatic response since anastasis can result in different cell death phenotypes. This method includes different aspects of the cell death phenomenon, such as apoptotic/necrotic cell discrimination, general cell death by trypan blue and MTT assay, as well as senescence, mitochondrial membrane potential (MMP) (shown as ΔΨm), and pro-apoptotic or anti-apoptotic gene expression profiles. Changes in MMP is one of the processes during cell death (Ly, Grubb, and Lawen 2003 ), therefore, it may also be a significant condition for anastasis. Mitochondria normally operate at a high membrane potential, and this is necessary for ATP production. When apoptosis begins, intracellular stress or damage signals cause increased permeability in the mitochondria. This leads to increased mitochondrial outer membrane permeability and decreased ΔΨm. Pro-apoptotic factors such as cytochrome c are released into the cytosol. These factors initiate the apoptosis process by activating caspases (Green and Kroemer 2004; Karbowski and Youle 2003 ). Therefore, ΔΨm measurement is widely used as a biomarker for monitoring apoptosis (Ly, Grubb, and Lawen 2003 ). In vivo evidence suggests that cells can survive programmed cell death (apoptosis) even after initiating the process, a phenomenon termed anastasis, which has been demonstrated in Drosophila epithelial tissues surviving caspase activation during developmental stages (H. M. Tang, Fung, and Tang 2018)), as well as in mammalian heart cells (cardiomyocytes) that recovered from temporary ischemia (Kenis et al. 2010 ). Further research introduced the related concept of "failed apoptosis" to describe instances where mammalian cells remain viable despite undergoing partial apoptotic signals, such as a limited drop in ΔΨm and restricted caspase activation, following exposure to sub-lethal radiation or chemical insults (X. Liu et al. 2015 ; Ichim et al. 2015 ). There are several studies reporting genes with possible functions during cell survival from death. Analysis of transcriptomic data using microarrays and qRT-PCR during the recovery phase revealed upregulation of genes, such as Bag-3, Bcl-2, Mcl-1, XIAP, MDM2 , and HS proteins (HSPB1, DNAJB1, HSPA1A, HSP27, HSP40, HSP70, and HSP90), showing many genes that are involved in the regulation of apoptosis. Inhibition of factors such as HSP90, MDM2, XIAP , and Bcl-2 disrupted anastasis, indicating the importance of these apoptosis regulators in this process (H. M. Tang et al. 2017 ; H. M. Tang et al. 2022 ). Moreover, investigating the molecular signatures of anastasis in HeLa cells recovering from ethanol exposure demonstrated a notable increase in the expression of c-Fos , c-Jun , Klf4 , and Snail-1 by examining the whole transcriptome through RNA sequencing (Sun et al. 2017 ). SMYD3 protein (a HMT enzyme containing a SET domain), a key epigenetic writer, was found to be involved in the survival of renal cells after acute kidney injury (Du et al. 2025 ). However, the epigenetic regulation of anastasis has not been elucidated before. Our study attempted to understand the regulation of genes functioning in epigenetic mechanisms during positive anastasis. We described the phenomenon of normal cell anastasis, while persistent cell death in cancer cells as a “ positive anastasis ”. Accordingly, we also defined “negative anastasis” in cancer cells with no anastatic response. Epigenetic regulation is expected to play a critical role in the anastasis-switch of the cells against different treatments, such as bee venom in this study. We have confirmed that bee venom resulted in the activation of genes involved in epigenetic regulation in cancer cells ( Fig. 11 ) , while downregulation of epigenetic regulation in normal cells during anastatic response ( Fig. 12 ) . The up- and downregulated epigenetic pathways were described for significantly upregulated and downregulated genes using the String network tool, as mentioned in the methods. These results suggest that normal breast cells may reverse or lose the effects induced by bee venom once the treatment is withdrawn. However, cancer cells showed a prolonged effect of bee venom under the control of a probable epigenetic memory. Declarations Conflict of interest The authors declare that there is no conflict of interest. Funding The experimental section of this study was supported by the Scientific and Technological Research Council of Türkiye (TUBITAK) with grant number 123Z002, and Karadeniz Technical University with the grant number FBA-2022-9176. Author Contribution ST; investigation, SCU; investigation, formal analysis, conceptualization, writing/editing, funding acquisition, methodology, resources, supervision. Acknowledgement The experimental section of this study was supported by the Scientific and Technological Research Council of Türkiye (TUBITAK) with grant number 123Z002, and Karadeniz Technical University with the grant number FBA-2022-9176. Data Availability The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. References Cao, Y. et al. Exploring the Relationship between Anastasis and Mitochondrial ROS-Mediated Ferroptosis in Metastatic Chemoresistant Cancers: A Call for Investigation. Frontiers in Immunology 15 (July). Frontiers Media SA: 1428920. (2024). 10.3389/FIMMU.2024.1428920/XML/NLM Celik-Uzuner, S. Mitochondrial DNA Methylation Misleads Global DNA Methylation Detected by Antibody-Based Methods. Analytical Biochemistry 601. Academic Press: 113789. (2020). 10.1016/J.AB.2020.113789 Doll, S. and Marcus Conrad. Iron and Ferroptosis: A Still Ill-Defined Liaison. IUBMB Life 69 (6). IUBMB Life: 423–434. (2017). 10.1002/IUB.1616 Du, X. et al. and Shougang Zhuang. SMYD3 as an Epigenetic Regulator of Renal Tubular Cell Survival and Regeneration Following Acute Kidney Injury in Mice. The FASEB Journal 39 (9). John Wiley & Sons, Ltd: e70533. 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The Detailed Comparison of Cell Death Detected by Annexin V-PI Counterstain Using Fluorescence Microscope, Flow Cytometry and Automated Cell Counter in Mammalian and Microalgae Cells. Journal of Fluorescence 28 (6). Springer New York LLC: 1393–1404. (2018). 10.1007/S10895-018-2306-4/FIGURES/10 Koressaar, T. and Maido Remm. Enhancements and Modifications of Primer Design Program Primer3. Bioinformatics (Oxford, England) 23 (10). Bioinformatics: 1289–1291. (2007). 10.1093/BIOINFORMATICS/BTM091 Liu, X. et al. and Chuan Yuan Li. Caspase-3 Promotes Genetic Instability and Carcinogenesis. Molecular Cell 58 (2). Cell Press: 284–296. (2015). 10.1016/j.molcel.2015.03.003 Liu, Y. and Beth Levine. Autosis and Autophagic Cell Death: The Dark Side of Autophagy. Cell Death and Differentiation 22 (3). Cell Death Differ: 367–376. (2015). 10.1038/CDD.2014.143 Livak, K. J. & Schmittgen, T. D. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2 – ∆∆CT Method. 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Cell Survival, DNA Damage, and Oncogenic Transformation after a Transient and Reversible Apoptotic Response. Molecular Biology of the Cell 23 (12). The American Society for Cell Biology: 2240–2252. (2012). 10.1091/mbc.E11-11-0926 Tang, H., Lam, H. M., Tang, J. M. & Hardwick and Ming Chiu Fung. Strategies for Tracking Anastasis, A Cell Survival Phenomenon That Reverses Apoptosis. Journal of Visualized Experiments (JoVE) , no. 96 (February). Journal of Visualized Experiments: e51964. (2015). 10.3791/51964 Tang, H., Man, M. C., Fung & Ho Lam, T. Detecting Anastasis In Vivo by CaspaseTracker Biosensor. Journal of Visualized Experiments: JoVE 2018 (132). Journal of Visualized Experiments: 54107. (2018). 10.3791/54107 Tang, H., Man, C., Conover Talbot, M. C. & Fung and Ho Lam Tang. Molecular Signature of Anastasis for Reversal of Apoptosis. F1000Research 6 (February). F1000 Research Limited: 43. (2017). 10.12688/f1000research.10568.2 Tang, H., Man, C., Conover Talbot, M. C., Fung & Ho, L. T. Transcriptomic Study of Anastasis for Reversal of Ethanol-Induced Apoptosis in Mouse Primary Liver Cells. Scientific Data 9 (1). Nature Publishing Group: 1–8. (2022). 10.1038/s41597-022-01470-8 Tetikoglu, S., Akcan, M., Uzuner, U. & Selcen Celik, U. Selective Anastasis Induction by Bee Venom in Normal Cells: A Promising Strategy for Breast Cancer Therapy with Minimal Impact on Cell Viability. Journal of Zhejiang University-SCIENCE B 2025 , September. Springer, 1–11. (2025). 10.1631/JZUS.B2400466 Untergasser, A. et al. Primer3–New Capabilities and Interfaces. Nucleic Acids Research 40 (15). Nucleic Acids Res. (2012). 10.1093/NAR/GKS596 Additional Declarations No competing interests reported. Supplementary Files EpigeneticRegulationSupplementaryData.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8465237","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":585128811,"identity":"9cbd41e3-c67e-4b15-a4e0-2abe4a233d2f","order_by":0,"name":"Sinan TETİKOĞLU","email":"","orcid":"","institution":"Karadeniz Technical University","correspondingAuthor":false,"prefix":"","firstName":"Sinan","middleName":"","lastName":"TETİKOĞLU","suffix":""},{"id":585128812,"identity":"4f927c68-d3ba-4736-b697-8a976f7da34e","order_by":1,"name":"Selcen ÇELİK UZUNER","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYFAC5gYIzc7YzPDhAJhpQEALI1QLM2Mz44wDBlAtCURpYWBm5iFGi3x7Y+OHjzsY5PmZmZuNbc78SWxgb94mwfjjHk4tBmcONkvOPMNgOLOZsTk554ZBYgPPsTIJhoRi3FokEtuYedsYEgwOMzYfzvkA1CKRYwbUgttl8vMftjH/BWqxB2mxAGmRf4NfC8MNxjZmRpAtwBBLZgA5TIIHvxaDM4nNkr1tEoYzgLYY9pwxNm7jSSu2SEjD47D2wwc//Gyzkedvb38s8eOYnGw/++GNNz7Y4HEYBEggmGwggqCGUTAKRsEoGAV4AQA5M08+3qzPJQAAAABJRU5ErkJggg==","orcid":"","institution":"Karadeniz Technical University","correspondingAuthor":true,"prefix":"","firstName":"Selcen","middleName":"ÇELİK","lastName":"UZUNER","suffix":""}],"badges":[],"createdAt":"2025-12-28 10:38:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8465237/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8465237/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101909367,"identity":"f24bfc7d-7cd9-48ec-8622-59554495b915","added_by":"auto","created_at":"2026-02-04 23:13:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":318580,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePPI network of the genes analyzed in this study, indicating epigenetic regulation processes (by the String, KEGG, and Reactome databases) \u003c/strong\u003e(Fabregat et al. 2018; Szklarczyk et al. 2023; Kanehisa et al. 2025)\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8465237/v1/04bb3be4e8ef94e8cbf5d16f.png"},{"id":101909373,"identity":"42bbcae8-ab5d-4d2b-bd05-a087d24e955d","added_by":"auto","created_at":"2026-02-04 23:13:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":250335,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell viability (%) profile in cells after bee venom or cisplatin detected by trypan blue assay. \u003c/strong\u003eBee venom (12µg/mL) induced persistent cell death in MDA-MB-231 breast cancer cells (down panel), but recovery in MCF10A normal breast cells (up panel). Cisplatin treatment resulted in ongoing cell death in both MDA-MB-231 (down panel) and MCF10A (up panel\u003cstrong\u003e)\u003c/strong\u003e cells. \u003cem\u003e+24, incubation with bee venom or cisplatin for 24h; -24, post-incubation in clean media after 24h incubation with bee venom or cisplatin (anastasis incubation); -72, post-incubation in clean media after 72h incubation with bee venom or cisplatin (anastasis incubation).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8465237/v1/d4fe1eae3a8a4a28d71ee677.png"},{"id":101943311,"identity":"bbc6dd87-2e3b-4b78-919b-92e69ed81a53","added_by":"auto","created_at":"2026-02-05 09:41:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":639683,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytotoxicity after Bee Venom and Cisplatin in MCF10A and MDA-MB-231 by MTT assay.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003eCell viability, expressed as arcsine-transformed percentage, of MCF10A and MDA-MB-231 cells following incubations with bee venom (top) or cisplatin (bottom) for three different incubation periods (+24h, -24h, and -48h). Data are presented as mean ±SE. \u003cstrong\u003e(B)\u003c/strong\u003e Corresponding table showing the quantitative cell viability data as average percentages with ±SE.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8465237/v1/78ab72eaa8ec412ec1e1f30d.png"},{"id":101909377,"identity":"c8c2661f-4950-4dae-9f27-f2fed0dfb817","added_by":"auto","created_at":"2026-02-04 23:13:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":600180,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eApoptosis and necrosis profile of MDA-MB-231 and MCF10A cells after bee venom or cisplatin detected by flow cytometry. \u003c/strong\u003eCell percentages for apoptotic and necrotic cells are given in the left panels, with example flow cytometry plots in the right panels\u003cstrong\u003e. (A)\u003c/strong\u003eMDA-MB-231 cells after bee venom, \u003cstrong\u003e(B)\u003c/strong\u003e MCF10A cells after bee venom, \u003cstrong\u003e(C)\u003c/strong\u003eMDA-MB-231 cells after cisplatin, \u003cstrong\u003e(D)\u003c/strong\u003e MCF10A cells after cisplatin. \u003cem\u003e+24, incubation with bee venom or cisplatin for 24h; -24, post-incubation in clean media after 24h incubation with bee venom or cisplatin; -72, post-incubation in clean media after 72h incubation with bee venom or cisplatin.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8465237/v1/8c24996f22739e9e46909ee8.png"},{"id":101943427,"identity":"6b87402c-1610-42cc-9961-aae9db50f426","added_by":"auto","created_at":"2026-02-05 09:41:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":446625,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCellular morphology by forward-scatter (FSC)/side-scatter (SSC) dot plots of flow cytometry detection. \u003c/strong\u003eFSC and SSC represent cell size and cell granularity, respectively\u003cstrong\u003e. (A)\u003c/strong\u003e MDA-MB-231 cells after bee venom, \u003cstrong\u003e(B)\u003c/strong\u003e MCF10A cells after bee venom, \u003cstrong\u003e(C)\u003c/strong\u003e MDA-MB-231 cells after cisplatin, \u003cstrong\u003e(D)\u003c/strong\u003e MCF10A cells after cisplatin. Error bars represent +/- standard error of the mean (s.e.m). \u003cem\u003e+24, incubation with bee venom or cisplatin for 24h; -24, post-incubation in clean media after 24h incubation with bee venom or cisplatin; -72, post-incubation in clean media after 72h incubation with bee venom or cisplatin.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8465237/v1/63deef635f1f15cc31db9468.png"},{"id":102298617,"identity":"90b0f56c-a88b-4220-8240-9b822607f83f","added_by":"auto","created_at":"2026-02-10 10:53:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":281633,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCellular senescence by flow cytometry detection. (A)\u003c/strong\u003e MDA-MB-231 cells after bee venom, \u003cstrong\u003e(B)\u003c/strong\u003e MCF10A cells after bee venom, \u003cstrong\u003e(C)\u003c/strong\u003e MDA-MB-231 cells after cisplatin, \u003cstrong\u003e(D)\u003c/strong\u003e MCF10A cells after cisplatin. Error bars represent +/- standard error of the mean (s.e.m.). \u003cem\u003e+24, incubation with bee venom or cisplatin for 24h; -24, post-incubation in clean media after 24h incubation with bee venom or cisplatin; -72, post-incubation in clean media after 72h incubation with bee venom or cisplatin. p\u003c/em\u003e\u0026lt;0.05 (*)\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8465237/v1/2166965abcf21c9774ba575e.png"},{"id":101943310,"identity":"737b1e54-d7ea-4022-bd5e-205f05c96204","added_by":"auto","created_at":"2026-02-05 09:41:36","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":349221,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMitochondrial membrane potential by flow cytometry detection. (A)\u003c/strong\u003e MDA-MB-231 cells after bee venom, \u003cstrong\u003e(B)\u003c/strong\u003e MCF10A cells after bee venom, \u003cstrong\u003e(C)\u003c/strong\u003e MDA-MB-231 cells after cisplatin, \u003cstrong\u003e(D)\u003c/strong\u003e MCF10A cells after cisplatin. Error bars represent +/- standard error of the mean (s.e.m.). \u003cem\u003e+24, incubation with bee venom or cisplatin for 24h; -24, post-incubation in clean media after 24h incubation with bee venom or cisplatin; -72, post-incubation in clean media after 72h incubation with bee venom or cisplatin. p\u003c/em\u003e\u0026lt;0.05 (*), \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 (**), \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 (***), and \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001 (****).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8465237/v1/8df49eef5eda522904e47b6a.png"},{"id":101943295,"identity":"af052f98-7702-4972-906d-0d4864de3a77","added_by":"auto","created_at":"2026-02-05 09:41:33","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":246576,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of ΔΔCt values for each gene in MDA-MB-231 and MCF10 cells following bee venom or cisplatin treatment (+24h, −24h, and −72h).\u003c/strong\u003e A shows expression profiles in MDA-MB-231 after bee venom, and B shows expression profiles in MCF10A after bee venom. C shows expression profiles in MDA-MB-231 after cisplatin, and D shows expression profiles in MCF10A after cisplatin.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8465237/v1/dace0f84e1685260c5686e1e.png"},{"id":101943509,"identity":"ee92f46a-70b7-411e-86a4-3089ce8341f7","added_by":"auto","created_at":"2026-02-05 09:42:08","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":205970,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeat map of “Fold-change” values ​​in all cell groups and after treatments. \u003c/strong\u003eThe map includes both significant and non-significant values.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8465237/v1/508a381615e22ea71ef54a7c.png"},{"id":101909379,"identity":"f78bd165-e6e7-49ba-a83d-fbf440889fe5","added_by":"auto","created_at":"2026-02-04 23:13:49","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":392895,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeat map including genes significantly changed in each experimental group. \u003c/strong\u003eGenes\u003cstrong\u003e \u003c/strong\u003edown or upregulated significantly are given in order\u003cstrong\u003e \u003c/strong\u003efrom high expression to low expression.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8465237/v1/aeb1a8a6d4a1728eb5bd7dca.png"},{"id":101943516,"identity":"37a66b2a-fb7e-4182-9729-8d7e681a5314","added_by":"auto","created_at":"2026-02-05 09:42:11","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":393332,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUpregulated pathways related to epigenetic regulations after bee venom in MDA-MB-231 cells at +24h, 24h, and -72h (by STRING database).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-8465237/v1/cb489a2ca45a2f9a7aa0611f.png"},{"id":101909375,"identity":"9e81281d-2e54-4d47-a325-a22e6317fc22","added_by":"auto","created_at":"2026-02-04 23:13:49","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":613223,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDownregulated pathways related to epigenetic regulations after bee venom in MDA-MB-231 cells at +24h, 24h, and -72h (by STRING database).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-8465237/v1/7b6f5332feae6da002f79b5b.png"},{"id":103559646,"identity":"3585fc1a-38fc-4298-ae94-bee6e9f26ac2","added_by":"auto","created_at":"2026-02-27 05:25:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5952039,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8465237/v1/14552fe4-423e-4940-859b-44da1ad3b775.pdf"},{"id":101909369,"identity":"e0b391bb-48a6-401f-aa9c-e43d6b20f396","added_by":"auto","created_at":"2026-02-04 23:13:48","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":27582,"visible":true,"origin":"","legend":"","description":"","filename":"EpigeneticRegulationSupplementaryData.docx","url":"https://assets-eu.researchsquare.com/files/rs-8465237/v1/50c25973219e8023172a8956.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Epigenetic Regulation Underlying Bee Venom–Induced Negative Anastasis in Cancer Cells but Positive Anastasis in Normal Cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAnastasis is described as \u0026ldquo;a return\u0026rdquo; from induced cell death. For the first time, this has been defined by a return of ethanol-induced apoptosis, and some genes are related to its regulation (H. M. Tang et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; H. M. Tang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Anastasis is considered a responsive strategy for drug resistance in cancer cells (Mohammed et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, the anastatic response of normal cells to cytotoxic agents has not been elucidated. In our previous study, we revealed that anastasis can occur selectively in normal breast cells after exposure to bee venom. In contrast, it induced persistent cell death in metastatic breast cancer cells (Tetikoglu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Another group of cells exhibited tenacious cell death after treatment with cisplatin, a commonly used chemotherapeutic agent in cancer therapies. Selective anastasis for normal cells only can be defined as \u0026ldquo;positive anastasis,\u0026rdquo; indicating an advantageous cellular regulation in cancer therapies that does not affect normal cell survival (Tetikoglu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The accumulated data on anastasis research suggest the need for a detailed investigation of positive anastasis, focusing on its differential regulation in normal cells versus cancer cells. Epigenetic regulation of positive anastasis is one of the important candidates that needs to be studied, and there is a lack of research on the role of epigenetic (re)programming during positive anastasis, even in regular anastasis (as described initially). This study aimed to elucidate the expression profiles of key genes involved in epigenetic mechanisms, including the reading, erasing, and writing of epigenetic modifications on DNA and histones. This study employed a method called \u0026ldquo;anastasis scoring\u0026rdquo; to define anastasis in each cell after exposure to bee venom or cisplatin. Cisplatin treatment showed persistent cell death in both breast cancer (MDA-MB-231) and normal breast (MCF10A) cells. Therefore, the results suggest that bee venom-mediated positive anastasis is regulated by epigenetic reprogramming, particularly through the deregulation of genes involved in epigenetic regulation, and by distinct patterns of DNA methylation. The findings of this study indicate that cellular memory for bee venom cytotoxicity has been lost, leading to anastasis-mediated cell survival in normal cells but not in cancer cells.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\n\u003ch3\u003e1. Cell culture, bee venom/cisplatin treatment, and anastasis experiment design\u003c/h3\u003e\n\u003cp\u003eMDA-MB-231 (metastatic breast cancer) and MCF10A (normal breast) cell cultures (ATCC) were cultured in RPMI (Wisent, Cat. No. 350-000 RL) and DMEM (Capricorn, Cat. No. HPA) media, respectively. Media were prepared containing 1% penicillin-streptomycin antibiotic and 10% or 20% fetal bovine serum (FBS) for MCF10A and MDA-MB-231 cells, respectively. Media were sterilized by passing through a 0.22 \u0026micro;m pore size filter. Cells in sterile media were incubated in a 37\u0026deg;C incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. Confluent cells were treated with 8 \u0026micro;g/ml or 12 \u0026micro;g/ml of bee venom or cisplatin for 24h (Tetikoglu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Control cells were left untreated. After 24h, bee venom/cisplatin was removed, and cells were washed with 1xPBS. Cells were then cultured in fresh media without bee venom or cisplatin for an extra 24h and 72h to evaluate the anastatic response of the cells. Sequential incubations were classified into 3 groups: i) first 24h with media including bee venom /cisplatin (+\u0026thinsp;24h), ii) 24h with clean media after 24h-treatments (-24h), and iii) 72h with clean media after 24h-treatments (-72h). A batch of cells was used for extended incubations for anastasis (-24h and \u0026minus;\u0026thinsp;72h), and the rest was examined by trypan blue assay, Annexin V-PI assay, cellular morphology assessment, mitochondrial membrane potential, and senescence profiles for each incubation as detailed below.\u003c/p\u003e\n\u003ch3\u003e2. Cell viability by trypan blue staining\u003c/h3\u003e\n\u003cp\u003eTrypan blue is a negatively charged dye used to determine cell viability. Since the membrane structure is intact in living cells, the dye cannot enter them, whereas dead cells absorb the dye and appear blue under the microscope. Healthy and cancerous cells were treated separately with bee venom or cisplatin for 24 hours and then washed once with 1x PBS (phosphate-buffered saline) (Wisent, 311-010-CL). Cells removed with trypsin were collected by centrifugation at 230 rpm. The supernatant was removed, and the cells were resuspended in the medium. 10 \u0026micro;L of cell suspension was mixed with 10 \u0026micro;L of 0.4% trypan blue (Biological Industries, B 103-102-1B) (1:1 ratio) and incubated for approximately 10 minutes at room temperature. The cell and dye mixture was loaded onto the coverslip of the device at 10 \u0026micro;L per well. Cell viability was determined using the Countess FL II automated cell counter (Thermo Fisher). Standard errors of the mean (S.E. +/- standard error of the mean) were calculated using SPSS software. All experiments were performed in at least three independent replicates, and each sample was measured at least three times within a replicate to test the reliability of the device.\u003c/p\u003e\n\u003ch3\u003e3. Cell viability by MTT assay\u003c/h3\u003e\n\u003cp\u003eThe MTT experiment was designed in 3 of 96-well plates for each MDA-MB-231 and MCF10A cells and bee venom or cisplatin (for +\u0026thinsp;24h, -24h, and \u0026minus;\u0026thinsp;72h incubations). 5000 cells per well were cultured in 96-well plates in triplicate and incubated at 37\u003csup\u003eᵒ\u003c/sup\u003eC with 5% CO\u003csub\u003e2\u003c/sub\u003e humidification, and treated with bee venom or cisplatin as mentioned above. Control wells were treated with 0.9% NaCl, the solvent for bee venom, as a negative control. After 24h incubation, the media was removed, and fresh media (190\u0026micro;L) and 10 \u0026micro;L of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, 5mg/mL) (Invitrogen, Cat.No: M6494) was added to each well and incubated at 37\u0026deg;C and 5% CO2 for 4 hours (Meantime, the other 2 plates were washed, fresh media were added, and then left for anastatic incubations, -24h and \u0026minus;\u0026thinsp;72h). After incubation with MTT, the media with MTT was removed, 100 \u0026micro;L of DMSO (Sigma-Aldrich, Cat.No: D4540) was then added to each well, and incubated on a shaker for 30 minutes. After incubation, color development was observed, and the absorbance at 570 nm was measured using a microplate reader. Cell viabilities were then calculated from absorbance values by proportioning untreated control wells (taken as 100% viable). Cell viabilities were then arcsine-transformed for use in the UNIANOVA test. The arcsine transformation is a statistical technique used to stabilize the variance of proportional data. Bar graphs presented data. The y-axis represents cell viability (arcsine-transformed), and the X-axis represents the dose of the treatment agent in microgram/mL.\u003c/p\u003e\n\u003ch3\u003e4. Apoptosis and necrosis determination by flow cytometry\u003c/h3\u003e\n\u003cp\u003eA million cells were separated for Annexin-V and PI staining. Annexin V (FITC)-PI staining protocol (BD, Cat. No. 556547) was applied to separate apoptotic and necrotic cell populations by flow cytometry. Briefly, the protocol includes (i) washing the cells once with cold 1xPBS, (ii) suspending the cells with 1x binding buffer (100 \u0026micro;l), (iii) adding Annexin V (FITC) (5 \u0026micro;l) and PI (5 \u0026micro;l), and finally, (iv) making the volume up to 500 \u0026micro;l with 1x binding buffer. Cells were stained with Annexin V (FITC), and PI was analyzed by flow cytometry at one hour. This analysis was performed according to the untreated control cells by grouping the cells in the FSC-SSC (forward scatter-side scatter) graph, dividing them into four regions (live \u0026ndash; necrotic \u0026ndash; early apoptotic \u0026ndash; late apoptotic), and determining the cell percentages in these regions \u003cem\u003e(UL, upper left: necrotic cells, UR; upper right: cells in late apoptosis, LL, lower left: live cells, LR, lower right: early apoptotic cells)\u003c/em\u003e (Ko\u0026ccedil; et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The accuracy and reliability of the assay were evaluated by measuring cellular autofluorescence in unstained cells (data are provided in the supplementary material \u003cb\u003eTables S1-S4\u003c/b\u003e). Autofluorescence was detected in all assays using flow cytometry (BD Accuri).\u003c/p\u003e\n\u003ch3\u003e5. Cellular morphology assessment by flow cytometry\u003c/h3\u003e\n\u003cp\u003eIn order to quantitatively measure the morphological changes in the cells, a batch of cells after each incubation and dose was washed with PBS, and read by flow cytometry (BD Accuri). FSC-SSC (forward scatter-side scatter) plots were obtained for each sample to morphologically define cells of interest based on cell size and intracellular granularities, respectively.\u003c/p\u003e\n\u003ch3\u003e6. Mitochondrial membrane potential (MMP) by flow cytometry\u003c/h3\u003e\n\u003cp\u003eFor MMP analysis, after bee venom and cisplatin treatments, cells were centrifuged, and a batch of cells was incubated in cell media containing 400 nM MitoTracker Red dye (Invitrogen, Cat. No M7512) at 37\u0026deg;C for 45 minutes. At the end of incubation, the media in the cells was removed by centrifugation and washed once with 1xPBS. Finally, the cells were analyzed by flow cytometry (BD Accuri) in fresh PBS. The amount of mitochondrial membrane potential was measured in the FL-3 channel, where red staining was detected, as before (Celik-Uzuner \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e7. Senescence profile by flow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnother 1 million cells were separated for senescence using CellEvent\u0026trade; Senescence Flow Cytometry Assay Kit (Invitrogen, C10841). The protocol includes the following steps: i) treated cells were washed with PBS and collected with trypsin. Cells were resuspended in 1X PBS at 500,000-1,000,000 cells per 100 \u0026micro;L. Then, 100 \u0026micro;L of the cell suspension was taken into flow cytometry tubes. Cells were pelleted by centrifugation and resuspended in 100 \u0026micro;L of Fixation Solution. The suspension was incubated for 10 min at room temperature in the dark. At the end of the period, cells were washed with PBS containing 1% BSA to remove the fixation solution. Cells were resuspended in 100 \u0026micro;L of working solution and then incubated for 1\u0026ndash;2 hours at 37\u0026deg;C incubator without CO\u003csub\u003e2\u003c/sub\u003e, protected from light. After incubation, the working solution was removed, and cells were washed with PBS containing 1% BSA. Cells were resuspended in 1% BSA in PBS buffer and then read on a BD Accuri device with a 488 nm laser and a 530 nm/30 filter. The mean fluorescence intensity of fluorescence (by FL1-H channel-detected FITC signal) was obtained.\u003c/p\u003e\n\u003ch3\u003e8. Anastasis scoring\u003c/h3\u003e\n\u003cp\u003eWe have developed, for the first time, an extensive method, called \u0026ldquo;anastasis scoring,\u0026rdquo; to determine the tendency of cells towards anastasis. The reasoning for the necessity to develop a comprehensive method for anastasis is based on 1) there is a range of cell death types, 2) it is hard to conclude cell death with only one cellular parameter, and 3) there is also a wide range of methods for detecting cell death. Anastasis is considered a return from apoptosis so far. Tang \u003cem\u003eet al.\u003c/em\u003e have reported a method for tracking anastasis by live cell imaging (H. L. Tang et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). However, apoptosis is not the only type of cell death, and cells have been shown to recover from different cell death mechanisms, such as ferroptosis (Cao et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and necroptosis (Gong et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), suggesting that a range of cellular and molecular parameters should be considered. \u0026ldquo;Anastasis scoring\u0026rdquo; method, therefore, includes scoring cells according to the different parameters as follows: 1) total cell death by trypan blue assay, 2) cell death by MTT assay, 3) apoptosis rate, 4) necrosis rate, 5) cellular morphology, including cell size and cell granularity, 6) mitochondrial membrane potential, 7) senescence, and 8) expression profiles of \u003cem\u003eBAX\u003c/em\u003e, \u003cem\u003eDFFB\u003c/em\u003e and \u003cem\u003eBCL2\u003c/em\u003e genes. The cells were scored as none (score 0, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), less (score 1, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05), more (score 2, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01), high (score 3, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.001), or the highest (score 4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.0001) for each parameter, with a comparison of treated cells and untreated cells. Expected significance (decrease or increase) is given in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. In our case, we compared \u0026ldquo;12\u0026micro;g/ml treated cells for 72h anastatic incubation (-72h)\u0026rdquo; with \u0026ldquo;0\u0026micro;g/ml untreated cells for \u0026minus;\u0026thinsp;72h\u0026rdquo;.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eParameters and significance for anastasis scoring\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCellular Event\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConsidered significance\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eScoring for each\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCell Death (Trypan Blue)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIncreased cell death\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"10\" rowspan=\"11\"\u003e \u003cp\u003enone (score 0, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05),\u003c/p\u003e \u003cp\u003eless (score 1, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05),\u003c/p\u003e \u003cp\u003emoderate (score 2, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01),\u003c/p\u003e \u003cp\u003ehigh (score 3, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.001),\u003c/p\u003e \u003cp\u003ethe highest (score 4, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.0001)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCell Death (MTT)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIncreased cell death\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eApoptosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIncreased apoptosis\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNecrosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIncreased necrosis\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCell size (FSC)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIncrease or decrease in FSC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCell granularity (SSC)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIncrease or decrease in SSC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMitochondrial membrane potential\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIncrease or decrease in MMP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSenescence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIncreased senescence\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBAX gene expression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIncreased expression\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDFFB gene expression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIncreased expression\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCL2 gene expression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDecreased expression\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003e9. RNA isolation and cDNA conversion\u003c/h3\u003e\n\u003cp\u003eAfter bee venom/cisplatin, total cellular RNA was isolated using an RNA isolation kit (BioBasic, BS88003) specifically developed for cultured cells. The steps followed in this kit were as follows: A maximum of 10\u003csup\u003e7\u003c/sup\u003e cells were collected from the flask. Lysis buffer was added, and centrifuged. After centrifugation, the pellet was transferred to an RNA tube. Ethanol was added to the RNA tube and transferred to the RNA column, and after centrifugation, GT and NT solutions were added, respectively, to precipitate the RNA. The purity and concentration of RNAs were measured in the NanoDrop device. The total RNA obtained from the samples was first converted into complementary DNA (cDNA) using a kit from A.B.T. Laboratory Industry (Cat. No: C03-01-05). For cDNA synthesis, 100 ng of isolated total RNA, 5X Reaction Buffer, Reverse Transcriptase (200 U/\u0026micro;l), RNase Inhibitor (10 U/\u0026micro;l), Random Hexamers (50 \u0026micro;M), and 10 mM dNTP Mix were added to the PCR tube. The reaction volume was completed to 20 \u0026micro;l with RNase-Free Water. The tube was kept on ice until the reverse transcriptase reaction took place. The tube was placed in the thermal cycler, run by a cycle of 25\u0026deg;C 10 min, 37\u0026deg;C 120 min, 85\u0026deg;C 5 min, respectively, and kept at 4\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003e10. Primers and Quantitative PCR\u003c/h3\u003e\n\u003cp\u003eWe performed QPCR for a list of genes involved in epigenetic regulation pathways. The genes were \u003cem\u003eTET1, IGF2, H19, EZH1, EP300, BAX, BCL-2, HOXB13, DNMT1, DNMT3A, TP53, HDAC1, KDM6A, KMT2A, DFFB, RBBP4\u003c/em\u003e, and \u003cem\u003eLHX4\u003c/em\u003e. The Primer3 program was used for QPCR primer design (Untergasser et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Koressaar and Remm 2007). Primers used are given in \u003cb\u003eTable S5\u003c/b\u003e. The expression levels of target genes from cDNA samples were analyzed by QPCR reaction. Real-time PCR reactions were performed using A.B.T.\u0026trade; 2X qPCR EVA-Green MasterMix (with ROX) (Atlas Biotechnology, Cat.No. Q02-02-05). The reaction workflow consisted of the following steps: (1) initial denaturation, (2) denaturation, (3) primer annealing, (4) extension, and (5) melting curve analysis. For each qPCR reaction, forward and reverse primers (10 \u0026micro;M), template DNA (cDNA), and RNase-free water were added to PCR tubes. The reaction volume was adjusted to 20 \u0026micro;l. Reagents were kept on ice during preparation, gently mixed, and briefly centrifuged. Reaction tubes were placed into a thermal cycler and amplified under the following cycling conditions: 95\u0026deg;C for 300 seconds (initial denaturation), followed by cycles of 95\u0026deg;C for 10\u0026ndash;30 seconds (denaturation), 55\u0026ndash;68\u0026deg;C for 10\u0026ndash;60 seconds (annealing), and melting curve analysis performed from 65\u0026ndash;95\u0026deg;C with 2\u0026ndash;5 seconds per step, applied sequentially according to the required number of cycles. The amplification curves and threshold cycle (Ct) values were analyzed using the qPCR instrument software. Changes in gene expression were determined using the ΔΔCt (delta\u0026ndash;delta Ct) method by calculating fold-change values. The ΔΔCt method (also known as the comparative Ct method) is widely used for the analysis of qPCR data, particularly for comparing relative gene expression levels between experimental and control samples (Livak and Schmittgen \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The calculation steps are as follows: 1) Ct (Cycle threshold): The PCR cycle number at which the fluorescence signal exceeds the background level for each gene. 2) ΔCt calculation: The difference between the Ct value of the target gene and that of the reference (housekeeping) gene in each sample. In this study, \u003cem\u003eACTB\u003c/em\u003e was used as the reference gene. 3) ΔΔCt calculation: The difference between the ΔCt value of the treated sample and that of the control group. In this study, the control group consisted of untreated cells (neither bee venom nor cisplatin exposure). 4) Relative gene expression (Fold change): The fold-change value indicates how many times the expression of a gene is increased or decreased compared with the control group.\u003c/p\u003e\n\u003ch3\u003e11. The STRING database for protein interactions involved in epigenetics\u003c/h3\u003e\n\u003cp\u003eTo confirm the relationship between the proteins encoded by the genes analyzed using QPCR, the gene list was uploaded to the STRING database (version 12.0; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org\u003c/span\u003e\u003cspan address=\"https://string-db.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to explore potential protein\u0026ndash;protein interactions (PPIs) and predict associated signaling pathways \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. The analysis was performed using the \u0026ldquo;multiple proteins\u0026rdquo; option, restricting the organism to \u003cem\u003eHomo sapiens\u003c/em\u003e. The minimum required interaction score was set to 0.7 (high confidence) to ensure biological relevance. Both known and predicted interactions (from curated databases, experiments, gene co-expression, and text mining) were included in the network. The generated PPI network was visualized within STRING, and clusters of functionally related proteins were identified using the Markov Cluster Algorithm (MCL) with an inflation parameter of 3. Pathway enrichment analyses were conducted using the KEGG (Kanehisa et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) and Reactome databases (Fabregat et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) integrated in STRING (Szklarczyk et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), enabling the identification of up- and down-regulated biological pathways related to the experimental conditions. Functional annotations with the highest enrichment scores (FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were considered significant. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e confirms the PPI of the genes \u003cem\u003eTET1, IGF2, H19, EZH1, EP300, BAX, BCL-2, HOXB13, DNMT1, DNMT3A, TP53, HDAC1, KDM6A, KMT2A, DFFB, RBBP4\u003c/em\u003e, and \u003cem\u003eLHX4\u003c/em\u003e (by the String database). The PPI enrichment \u003cem\u003ep\u003c/em\u003e-value was detected as 3.15\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;14\u003c/sup\u003e, confirming high significance. The pathway of these genes is related to transcriptional misregulation of cancer (KEGG pathway, hsa05202) (Kanehisa et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), and epigenetic regulation of gene expression (by Reactome database HSA-212165, and Gene ontology). The enrichment approaches confirmed the interaction of these genes in epigenetics, and therefore, the expression profiles of these genes were analyzed using QPCR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003e12. Statistical analyses\u003c/h3\u003e\n\u003cp\u003eThe cell percentages obtained from Annexin-V/PI /PI results, mitochondrial membrane potential (as an arbitrary unit of total fluorescence amount), FSC-SSC profiles of cells, trypan blue-assisted cell viability percentages detected by automated cell counter, and the level of senescence were analyzed using the SPSS program UNIANOVA. The levels of significance used were \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05 (*), \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01 (**), \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.001 (***), and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.0001 (****). \u003cem\u003ePost-Hoc\u003c/em\u003e test was applied for pairwise comparisons. Percentage values ​​were first arcsine transformed before tests. All experiments were performed in triplicate.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe general cell death profiles of each cell line after bee venom and cisplatin were analyzed by conventional trypan blue staining, as mentioned in the methods. Bee venom induced continuing cell death in MDA-MB-231 cells but anastasis in MCF10A cells at 72h of anastatic incubation \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Cell viability significantly increased at 72h in MCF10A cells (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000) after bee venom, while the same significance was detected in increased cell death in MDA-MB231 cells. However, cisplatin treatment resulted in persistent cell death in both cells (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. The results confirmed our previous findings (Tetikoglu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). To detail the cell death, we performed Annexin-V and PI dual-staining by flow cytometry.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then performed MTT cytotoxicity assay for +\u0026thinsp;24h, -24h, and \u0026minus;\u0026thinsp;72h in each cell after bee venom and cisplatin. The results demonstrate the concentration- and time-dependent effects of bee venom and cisplatin on the viability of MCF10A (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and MDA-MB-231 cells (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cb\u003etop)\u003c/b\u003e. Both agents induced cytotoxicity in both cell lines, with viability decreasing as the dose and incubation time increased. Cisplatin reduced the viability of both MCF10A cells (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and MDA-MB-231 cells (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cb\u003ebottom)\u003c/b\u003e. Regarding anastasis, the viability of MCF10A cells increased after the maximum bee venom dose (12\u0026micro;g/mL) at -72h anastatic incubation (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) compared to the viability after the same dose at +\u0026thinsp;24h. The viability percentages are also given in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, and importantly, bee venom showed a degree of selective cytotoxicity, reducing the viability of the MDA-MB-231 cancer cells but maintaining viability in MCF10A cells under the same conditions, and significant anastasis in MCF10A cells at -72h \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. This suggests that while cisplatin is more toxic with no specificity, and bee venom may possess a therapeutic advantage due to its preferential toxicity towards the aggressive cancer cell line over the normal epithelial line, which is in an anastatic trend.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBee venom (12 \u0026micro;g/mL) induced necrosis after 24h treatment (+\u0026thinsp;24), followed by a significantly increased apoptosis (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in extended 24h (-24) for anastasis in MDA-MB-231 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Similarly, at the end of 24 hours with bee venom treatment (+\u0026thinsp;24) at a dose of 8 \u0026micro;g/mL, the apoptosis rate increased significantly compared to 24 hours without bee venom (-24) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and 72 hours without bee venom (-72) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). However, at -72h of anastasis incubation, apoptosis decreased in treated cells with 12 \u0026micro;g/mL. Although MCF10A cells tended to undergo necrotic cell death after bee venom for 24h, bee venom did not significantly induce necrosis or apoptosis in comparisons between different doses within each incubation period and in comparisons with different incubation times at the same dose, according to the UNIANOVA test (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eCisplatin treatment provoked significant apoptosis in MDA-MB-231 cells. In general, the apoptosis rate in untreated cells was significantly lower compared to all cisplatin doses. However, there was no significant difference between the doses (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Similarly, the apoptotic cell population was higher after cisplatin in MCF10A compared to their untreated counterparts. But, there was no difference in the rate of apoptotic cells between the doses at each incubation (+\u0026thinsp;24h, -24h, and \u0026minus;\u0026thinsp;72h) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRegarding cellular morphology, bee venom decreased cell size (FSC) in MDA-MB-231 cells at +\u0026thinsp;24h, -24h, and \u0026minus;\u0026thinsp;72h, but increased granularity at -24h and \u0026minus;\u0026thinsp;48h \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. A similar pattern was observed in MCF10A cells, but cell size was not significantly changed at +\u0026thinsp;24h \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. In contrast, cisplatin generally induced a decrease in cell size in MDA-MB-231 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e and MCF10A cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. Granularity (SSC) significantly increased only at +\u0026thinsp;24 after cisplatin in MCF10A \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD\u003cb\u003e).\u003c/b\u003e Statistical analyses confirmed that neither FSC nor SSC changed significantly in MDA-MB-231 or MCF10A cells after bee venom treatment. However, cisplatin induced a significant decrease in FSC, but not in SSC, in both cell lines (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eCellular senescence was only induced in MDA-MB-231 cells after 8\u0026micro;g/ml bee venom for \u0026minus;\u0026thinsp;24h, compared to untreated cells (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and to the higher dose 12\u0026micro;g/ml (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Inconsistent results were obtained for senescence in other groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-D\u003cb\u003e)\u003c/b\u003e. Bee venom did not change the mitochondrial membrane potential \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-B\u003cb\u003e)\u003c/b\u003e. However, the MMP profile was inclined to reduce after bee venom in both cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC-D\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo evaluate anastasis in the cells, anastasis scoring was used as detailed in the methods. According to the scoring, MCF10A cells treated with bee venom were the most anastatic group, followed by MCF10A cells treated with cisplatin, MDA-MB-231 cells treated with bee venom, and MDA-MB-231 cells treated with cisplatin \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eCellular anastasis score of each cell after bee venom or cisplatin\u003c/b\u003e \u003cb\u003e(incubation, -72h and concentration, 12\u0026micro;g/ml, statistically compared with untreated control cells at -72h).\u003c/b\u003e \u003cem\u003eThe lower the score, the higher the anastasis\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCellular Event\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eMCF10A\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eMDA-MB-231\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eBee Venom\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eCisplatin\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eBee Venom\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eCisplatin\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCell Death (Trypan Blue)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCell Death (MTT)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eApoptosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNecrosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCell size (FSC)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCell granularity (SSC)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMitochondrial membrane potential\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSenescence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBAX\u003c/em\u003e gene expression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDFFB\u003c/em\u003e gene expression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBCL2\u003c/em\u003e gene expression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCellular Anastasis Score\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eScoring; 0; none, 1; less, 2; moderate, 3; high, 4; the highest\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFollowing cellular parameters, gene expression profiles including anti-apoptotic \u003cem\u003e(BCL-2)\u003c/em\u003e and anti-apoptotic genes \u003cem\u003e(BAX, DFFB)\u003c/em\u003e were analyzed. Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-D presents the changes in gene expression for each experimental group based on the calculated ΔΔCt values. All values ​​obtained in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e are presented as heat maps in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e. Genes with fold-change values ​​of 2 and above were considered to have significantly increased expression. In contrast, genes with fold-change values ​​of 0.5 and below were considered to have significantly decreased expression. Therefore, among all the findings in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, the most significant data are selected and presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study investigated the epigenetic regulation in selective anastasis in normal breast cells compared to breast cancer cells after bee venom. Normal cells (MCF10A) responded to bee venom by reversing cell death; however, breast cancer cells (MDA-MB-231) showed persistent cell death. On the other hand, cisplatin, a common chemotherapeutic drug, resulted in irreversible cell death in both cells, suggesting its non-selective effect on cancer cells. In recent years, the term \u003cem\u003eanastasis\u003c/em\u003e has been primarily used to describe the recovery of cells from apoptosis. The pioneering study by Tang et al. (H. L. Tang et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) demonstrated that cells displaying apoptotic morphology can revert to a proliferative state once the death stimulus is removed, and this process has since been confirmed by several subsequent studies (H. M. Tang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; H. M. Tang et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, whether similar reversal phenomena occur in other cell death modalities such as necrosis, necroptosis, or ferroptosis remains a matter of debate. In classical necrosis, the irreversible loss of plasma membrane integrity precludes any anastasis-like recovery. Nevertheless, in necroptosis, it has been shown that some cells can survive despite activation of \u003cem\u003eRIPK3\u003c/em\u003e and \u003cem\u003eMLKL\u003c/em\u003e. Moreover, the ESCRT-III complex has been reported to repair membrane ruptures during necroptosis, thereby preventing cell lysis and promoting survival (Gong et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), indicating a partial reversal of the death process, which is a phenomenon referred to as \u0026ldquo;abortive necroptosis\u0026rdquo;. Similarly, in ferroptosis, during the early stages of lipid peroxidation, reactivation of antioxidant systems such as GPX4 and glutathione (GSH) can halt the death process (Doll and Conrad 2017; Jiang, Stockwell, and Conrad 2021), indicating a \u0026ldquo;reversible ferroptosis\u0026rdquo;. In contrast, pyroptosis and autophagic cell death are generally considered irreversible, as they involve extensive membrane or lysosomal damage (Y. Liu and Levine 2015). Thus, while the term \u003cem\u003eanastasis\u003c/em\u003e has been explicitly defined only for reversal from apoptosis, accumulating evidence suggests that partial or early-stage recovery mechanisms may also exist in necroptotic and ferroptotic pathways. Therefore, in this work, for the first time, we have described a novel method, \u0026ldquo;anastasis scoring\u0026rdquo;, to conclude the tendency of cells towards anastatic response since anastasis can result in different cell death phenotypes. This method includes different aspects of the cell death phenomenon, such as apoptotic/necrotic cell discrimination, general cell death by trypan blue and MTT assay, as well as senescence, mitochondrial membrane potential (MMP) (shown as ΔΨm), and pro-apoptotic or anti-apoptotic gene expression profiles. Changes in MMP is one of the processes during cell death (Ly, Grubb, and Lawen \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), therefore, it may also be a significant condition for anastasis. Mitochondria normally operate at a high membrane potential, and this is necessary for ATP production. When apoptosis begins, intracellular stress or damage signals cause increased permeability in the mitochondria. This leads to increased mitochondrial outer membrane permeability and decreased ΔΨm. Pro-apoptotic factors such as cytochrome c are released into the cytosol. These factors initiate the apoptosis process by activating caspases (Green and Kroemer 2004; Karbowski and Youle \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Therefore, ΔΨm measurement is widely used as a biomarker for monitoring apoptosis (Ly, Grubb, and Lawen \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). \u003cem\u003eIn vivo\u003c/em\u003e evidence suggests that cells can survive programmed cell death (apoptosis) even after initiating the process, a phenomenon termed anastasis, which has been demonstrated in Drosophila epithelial tissues surviving caspase activation during developmental stages (H. M. Tang, Fung, and Tang 2018)), as well as in mammalian heart cells (cardiomyocytes) that recovered from temporary ischemia (Kenis et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Further research introduced the related concept of \"failed apoptosis\" to describe instances where mammalian cells remain viable despite undergoing partial apoptotic signals, such as a limited drop in ΔΨm and restricted caspase activation, following exposure to sub-lethal radiation or chemical insults (X. Liu et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Ichim et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere are several studies reporting genes with possible functions during cell survival from death. Analysis of transcriptomic data using microarrays and qRT-PCR during the recovery phase revealed upregulation of genes, such as \u003cem\u003eBag-3, Bcl-2, Mcl-1, XIAP, MDM2\u003c/em\u003e, and HS proteins (HSPB1, DNAJB1, HSPA1A, HSP27, HSP40, HSP70, and HSP90), showing many genes that are involved in the regulation of apoptosis. Inhibition of factors such as \u003cem\u003eHSP90, MDM2, XIAP\u003c/em\u003e, and \u003cem\u003eBcl-2\u003c/em\u003e disrupted anastasis, indicating the importance of these apoptosis regulators in this process (H. M. Tang et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; H. M. Tang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Moreover, investigating the molecular signatures of anastasis in HeLa cells recovering from ethanol exposure demonstrated a notable increase in the expression of \u003cem\u003ec-Fos\u003c/em\u003e, \u003cem\u003ec-Jun\u003c/em\u003e, \u003cem\u003eKlf4\u003c/em\u003e, and \u003cem\u003eSnail-1\u003c/em\u003e by examining the whole transcriptome through RNA sequencing (Sun et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). SMYD3 protein (a HMT enzyme containing a SET domain), a key epigenetic writer, was found to be involved in the survival of renal cells after acute kidney injury (Du et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, the epigenetic regulation of anastasis has not been elucidated before. Our study attempted to understand the regulation of genes functioning in epigenetic mechanisms during positive anastasis. We described the phenomenon of normal cell anastasis, while persistent cell death in cancer cells as a \u0026ldquo;\u003cb\u003epositive anastasis\u003c/b\u003e\u0026rdquo;. Accordingly, we also defined \u003cb\u003e\u0026ldquo;negative anastasis\u0026rdquo;\u003c/b\u003e in cancer cells with no anastatic response. Epigenetic regulation is expected to play a critical role in the anastasis-switch of the cells against different treatments, such as bee venom in this study. We have confirmed that bee venom resulted in the activation of genes involved in epigenetic regulation in cancer cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e, while downregulation of epigenetic regulation in normal cells during anastatic response \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. The up- and downregulated epigenetic pathways were described for significantly upregulated and downregulated genes using the String network tool, as mentioned in the methods. These results suggest that normal breast cells may reverse or lose the effects induced by bee venom once the treatment is withdrawn. However, cancer cells showed a prolonged effect of bee venom under the control of a probable epigenetic memory.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declare that there is no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe experimental section of this study was supported by the Scientific and Technological Research Council of T\u0026uuml;rkiye (TUBITAK) with grant number 123Z002, and Karadeniz Technical University with the grant number FBA-2022-9176.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eST; investigation, SCU; investigation, formal analysis, conceptualization, writing/editing, funding acquisition, methodology, resources, supervision.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe experimental section of this study was supported by the Scientific and Technological Research Council of T\u0026uuml;rkiye (TUBITAK) with grant number 123Z002, and Karadeniz Technical University with the grant number FBA-2022-9176.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCao, Y. et al. 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(2012). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/NAR/GKS596\u003c/span\u003e\u003cspan address=\"10.1093/NAR/GKS596\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"anastasis, epigenetics, cancer, bee venom, cisplatin","lastPublishedDoi":"10.21203/rs.3.rs-8465237/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8465237/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAnastasis\u0026mdash;defined as cellular recovery from the brink of apoptosis\u0026mdash;has been previously characterized in ethanol-induced apoptosis models, yet its regulatory mechanisms and relevance in normal cells remain insufficiently understood. This study investigates the epigenetic regulation underlying divergent anastatic responses in normal breast cells (MCF10A) and breast cancer cells (MDA-MB-231) following bee venom and cisplatin. In our previous work, bee venom induced selective recovery (positive anastasis) in MCF10A cells while promoting persistent cell death in MDA-MB-231 cells. This work explores whether epigenetic mechanisms contribute to this selective response using an anastasis scoring approach to quantify recovery after exposure to bee venom or cisplatin. We found that cisplatin triggered persistent cell death in both cell types, whereas bee venom elicited robust anastatic recovery exclusively in normal cells. Gene expression analyses targeting key epigenetic regulators\u0026mdash;responsible for reading, writing, and erasing DNA and histone modifications\u0026mdash;revealed distinct transcriptional alterations associated with positive anastasis, suggesting that epigenetic reprogramming may facilitate the loss of cellular memory of bee venom cytotoxicity. These findings provide the first evidence that positive anastasis in normal cells is shaped by specific epigenetic mechanisms, highlighting a potential therapeutic advantage in cancer treatment strategies that preserve normal tissue integrity while limiting cancer cell survival.\u003c/p\u003e","manuscriptTitle":"Epigenetic Regulation Underlying Bee Venom–Induced Negative Anastasis in Cancer Cells but Positive Anastasis in Normal Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-04 23:13:40","doi":"10.21203/rs.3.rs-8465237/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"52b1572b-d171-411a-8493-148d7c5abb28","owner":[],"postedDate":"February 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":62248869,"name":"Biological sciences/Cancer"},{"id":62248870,"name":"Biological sciences/Cell biology"},{"id":62248871,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-02-27T05:25:10+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-04 23:13:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8465237","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8465237","identity":"rs-8465237","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}