| BackgroundMalignant melanoma is the most aggressive skin cancer, with early onset age and high transfer rate, and is extremely insensitive to a variety of clinical treatment. Chemotherapy is an important treatment for melanoma, but it is insensitive to chemotherapeutic drug and easy to produce drug resistence. Resistences of tumor cells to apoptosis reduce their sensitivity to chemotherapeutic drugs, apoptosis and its regulatory factors are one of the determining mechanism for drug-resistant of melanoma.Survivin is a new member of the inhibitor apoptosis protein (inhibitorofapoptosis, IAP) family, which is d in the most powerful apoptosis inhibitor currently foun. It plays an important regulatory role in cell mitosis and apoptosis, and is a dual-function protein, not only affects the mitotic apparatus assembly and maintenance of cell ploidy and cytokinesis time, but also regulates apoptosis through its phosphorylation in cell division.Antisense technology can selectively inhibit the target gene expression, play a role in negative regulation of target genes, it has been used to study the biological function of genes, as well as to develop new antisense oligonucleotides drugs which can close harmful gene expression. Antisense technology as a gene therapy method which can selectively close the expression of target genes, enrich and develope the research and treatment of malignant melanoma.Taxol, a compound extracted from the Pacific yew (Taxus) bark, is currently the first line chemotherapy for ovarian cancer, breast cancer, non-small cell lung cancer and many other kinds of tumors, but is rarely reported for the treatment of malignant melanoma in China. Study of paclitaxel on the role of malignant melanoma, with a combination of application of survivin gene therapy, is sure to provide a new attempt for malignant melanoma studies.ObjectiveIn this study, we first observed the effect of taxol promoting apoptosis on malignant melanoma cells, and then detected the effect of survivin antisense oligonucleotide transfection on proliferation and apoptosis of malignant melanoma cell. Further more, we observed the effect of both of their combination on the malignant melanoma cells, so can provide new exploration for the biochemical treatment for malignant melanoma.Methods1. A375 cells were treated by taxol with different concentration (10.50.100. 1000nmol/L) for different time (24h.48h.72h), MTT method was used to assay inhibition of cell proliferation, the change of cell cycle and apoptosis were analyzed by flow cytometry, protein expression of suvivin was detected by western blot.2. A phosphorothioate antisense oligodeoxynucleotide of specific targeting survivin was designed and synthesized and then transferred to A375 cells by Lipofectamine TM2000 .MTT method was used to assay inhibition of cell proliferation, the change of cell cycle and apoptosis were analyzed by flow cytometry, protein expression of survivin was detected by western blot, colorimetric assay was performed to detect the activity of caspase-3.3. Combination the effect of survivin antisense oligonucleotide and taxol on cells. MTT method was used to assay inhibition of cell proliferation, the change of cell apoptosis was analyzed by flow cytometry, protein expression of survivin was detected by western blot.Results1, Effects of taxol on A375 cells (1), the role of different concentrations of taxol on the A375 cell growth inhibition after 24h,48h, and 72h showed that:①With the increase in drug concentration, cell survival rate decreased significantly in a dose-dependent relationship, but when the concentration increased to 1000 nmol/L, the increase of cell growth inhibition rate is not significant.②With extension of time cell survival rate decreased, which was time-dependent. When time was to 48h, the role was most significant.(2). FCM results showed that the cells in G2/M phase increased 24h afterlO nmol/L taxol acting on A375 cells, at the same time the rate of apoptosis increased from 5.23% to 15.23 percent compared with control group.It indicate that paclitaxel has a significant effect on inducing cell cycle arrest and apoptosis. In low concentration range (10～100 nmol/L), cell apoptosis rate increased with the drug concentration increased significantly ((P＜0.05), in a dose-dependent, but the concentration further increased, the apoptosis rate did not increase significantly ((P>0.05).With extension of time in the same concentration of taxol group, the cell apoptotic rate increased significantly ((P<0.05) in a time-dependent manner.(3).The expression of survivin protein was gradually increasing 24h after taxol at different concentrations acting on cells, it was in a dose-dependent manner. With extension of time, the expression of survivin was weakened 48h compared with the control group. The expression of survivin was increased at first and decreased subsequetntly.2. Effects of survivin ASODN on A375 cells(1). Transfection rate of cells in survivin ASODN 600 nmol/L group was more than 80%.(2). Compared with control group, growth inhibition rate of cells increased in a dose and time dependent manner after transfection (P<0.05), the growth inhibition rate of 600nmol/L ASODN group was 60.7% 72h after transfection.(3). The apoptotic rate of cells in ASODN 200.400.600 nmol/L groups(13.5±1.9% vs. 20.1±1.5% vs.32.1±2.9%, respectively) were markedly higher compared with those of sense ODN group, mismatch ODN group, liposome group and control group(6.5±0.6% vs.5.6±0.7% vs.6.4±1.0% vs.6.5±1.3%, respectively) (P＜0.05);The cell number in G2/M phase increased.(4). Compared with control group, survivin protein expression decreased significantly and caspase-3 activity were significantly higher (F=63.1, P<0.05); there were no statistically significant difference in caspase-3 activity of SODN group, MSODN group, liposome group compared with the control group (F=0.512, P>0.05).3. Effects of survivin ASODN and paclitaxel together on A375 cells(1) Changes of cell proliferation inhibition rate Cell proliferation inhibition rates in ASODN+taxol groups 24h were respectively ASODN 200 nmol/L+taxol 50 nmol/L group 34.29±0.85%, ASODN 400 nmol/ L+taxol 50 nmol/L group 36.22±0.26%, ASODN 600 nmol/L+taxol 50 nmol/L group 40.42±0.58%, ASODN 200 nmol/L+taxol 100 nmol/L group 40.15±1.39%, ASODN 400 nmol/L+taxol 100 nmol/L group 44.65±0.37%, ASODN 600 nmol/L+taxol 100 nmol/L group 48.55±0.88%, which were significantly higher compared with the corresponding concentration of ASODN transfection group and taxol group. The rates of 48h group were further improved than those of 24h group.(2) Changes of apoptosis rate of A375 cellsApoptosis rates of cells in ASODN+taxol groups were significantly higher compared with control group (F= 88.7, P<0.05), and with drug concentration increase, the apoptosis rate improved. In the same concentration of paclitaxel group, with the ASODN concentration increased, apoptosis rate increased in a significant dose-dependent manner. Cell apoptosis rate was also increased in a time-dependent manner.(3). Expression of survivin protein of A375 cellsthe expression of survivin protein of cells in ASODN+taxol group decreased compared with the control group, and the decrease was in a dose-dependent manner.Conclusion1, Taxol has a stronger growth inhibition and apoptosis induction effect on human malignant melanoma A375 cells, there is a significant cell cycle arrest. The role of taxol on A375 is time-dose-dependent.2, There is little difference in the apoptosis rate of melanoma cell between low-dose long-time model and high-dose short-time model, but the cytotoxicity of the latter is higher than the former. The low-dose long-time model can be used as a reference for clinical administration.3, Inhibiting the expression of survivin is one of the ways for taxol-induced apoptosis, survivn expression is also relevant to the drug resistance.4, Survivin ASODN can inhibit A375 cells proliferate, promote them apoptosis and produce a significant cell cycle arrest in a dose-time-dependent manner.5, Survivin ASODN transfection can inhibit translation of target gene expression, decrease levels of target protein and promote apoptosis of tumor cells. ASODN acts on the target gene with high specificity.6, Inhibiting the activity of caspase-3 is one important way which survivin can inhibit A375 cell apoptosis.7, survivin ASODN and paclitaxel can significantly inhibit A375 cell proliferation and promote apoptosis.8, survivin ASODN transfected cells can significantly reduce the resistance of A375 cells to taxol, enhancing the sensitivity.9, Expression of survivin protein of A375 cells is one of the mechanisms of taxol resistance. SignificanceTaxol isis currently the first line chemotherapy for many kinds of tumors, but there is little report for the treatment of malignant melanoma in China. Our experiments not only confirmed paclitaxel has strong growth inhibition and apoptosis induction effect on A375 cells, but also made a thorough study for their anti-tumor mechanisms. Thus we can provide new ideas for how to improve paclitaxel efficacy against malignant melanoma. Survivin gene, the most powerful inhibitor of apoptosis, is a hot target for cancer gene therapy. In this study, antisense oligonucleotides was used to transfect melanoma cells to further explore the function of the survivin gene, and the significance as targets for gene therapy in malignant melanoma. At present, the combined gene therapy and conventional chemotherapy was new research direction in cancer treatment, we combined advanced antisense oligonucleotide technology with the classic chemotherapy drugs, and intitiatively used in malignant melanoma cells, detected the effect and found new mechanism,of malignant melanoma cells to taxol-resistant. The combination can induce apoptosis of malignant melanoma more signifcantly. The study provides useful exploration for malignant melanoma treatment. |
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