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Effect of Salinomycin on Expression Pattern of Genes Associated with Apoptosis in Endometrial Cancer Cell Line
Kamil Kiełbasiński1, Wojciech Peszek2, Beniamin O Grabarek3,4
1Department of Obsterics and Gynaecology in Ruda Slaska, Medical University of Silesia, Ruda Slaska, Poland
Background:
Salinomycin is part of a group of ionophore antibiotics characterized by an activity towards tumor cells. To this day, the mechanism through which salinomycin induces their apoptosis is not fully known yet. The goal of this study was to assess the expression pattern of genes and the proteins coded by them connected with the process of programmed cell death in an endometrial cancer cell Ishikawa culture exposed to salinomycin and compared to the control.
Materials And Methods:
Analysis of the effect of salinomycin on Ishikawa endometrial cancer cells (ECACC 99040201) included a cytotoxicity MTT test (with a concentration range of 0.1-100 μM), assessment of the induction of apoptosis and necrosis by salinomycin at a concentration of 1 μM as well the assessment of the expression of the genes chosen in the microarray experiment (microarray HG-U 133A_2) and the proteins coded by them connected with apoptosis (RTqPCR, ELISA assay). The statistical significance level for all analyses carried out as part of this study was p<0.05.
Results:
It was observed that salinomycin causes the death of about 50% of cells treated by it (50.74±0.80% of all cells) at a concentration of 1μM. The decrease in the number of living cells was determined directly after treatment of the cells with the drug (time 0). The average percent of late apoptotic cells was 1.65±0.24% and 0.57±0.01% for necrotic cells throughout the entire observation period.
Discussion:
Microarray analysis indicated the following number of mRNA differentiating the culture depending on the time of incubation with the drug: H_12 vs C = 114 mRNA, H_8 vs C = 84 mRNA, H_48 vs. C = 27 mRNA, whereas 5 mRNAs were expressed differently at all times. During the whole incubation period of the cells with the drug, the following dependence of the expression profile of the analyzed transcripts was observed: Bax>p53>FASL>BIRC5>BCL2L.
Conclusion:
The analysis carried out indicated that salinomycin, at a concentration of 1 μM, stopped the proliferation of 50% of endometrial cancer cells, mainly by inducing the apoptotic process of the cells. The molecular exponent of the induction of programmed cell death was an observed increase in the transcriptional activity of pro-apoptotic genes: Bax;p53;FASL and a decrease in the expression of anti-apoptotic genes: BCL2L2; BIRC5.
Insights
Salinomycin, an ionophore antibiotic, halts endometrial cancer cell proliferation by inducing apoptosis. This study reveals salinomycin increases pro-apoptotic gene expression (Bax, p53, FASL) and decreases anti-apoptotic gene expression (BCL2L2, BIRC5).
Area of Science:
- Oncology
- Molecular Biology
- Pharmacology
Background:
- Salinomycin, an ionophore antibiotic, exhibits activity against tumor cells.
- The precise mechanism by which salinomycin induces apoptosis in cancer cells remains incompletely understood.
- Endometrial cancer is a significant health concern, necessitating research into novel therapeutic agents.
Purpose of the Study:
- To investigate the effect of salinomycin on apoptosis and necrosis in Ishikawa endometrial cancer cells.
- To analyze the gene and protein expression patterns associated with programmed cell death pathways upon salinomycin treatment.
- To elucidate the molecular mechanisms underlying salinomycin-induced cell death in endometrial cancer.
Main Methods:
- Cytotoxicity assessment using MTT assay across a concentration range of 0.1-100 μM salinomycin.
- Apoptosis and necrosis induction evaluation at a salinomycin concentration of 1 μM.
- Gene expression analysis via microarray (HG-U 133A_2) and quantitative real-time PCR (RTqPCR).
- Protein expression analysis using ELISA assays.
Main Results:
- Salinomycin at 1 μM induced cell death in approximately 50% of Ishikawa endometrial cancer cells (50.74±0.80%).
- Microarray analysis revealed differential mRNA expression, with specific transcripts showing altered levels at various incubation times.
- Key apoptosis-related genes exhibited a distinct expression profile: Bax > p53 > FASL > BIRC5 > BCL2L during incubation.
Conclusions:
- Salinomycin effectively inhibits proliferation in endometrial cancer cells, primarily through the induction of apoptosis.
- The pro-apoptotic genes Bax, p53, and FASL showed increased transcriptional activity.
- Conversely, anti-apoptotic genes BCL2L2 and BIRC5 demonstrated decreased expression, confirming their role in salinomycin's mechanism of action.
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