| Objective Cisplatin (CDDP) is frequently used anticancer agent in the treatment of ovarian cancer, but the problem on the drug-resistance to CDDP is becoming more and more obviously in clinic. So it is urgent to find some drugs that can combine with CDDP to therapy the cancer more effectively. Aclarubicin(ACR), one of the topoisomeraseⅡ(topoⅡ) inhibitors, is less used in the ovarian cancer chemotherapy, also it is not cleared about the interaction between ACR and CDDP. This in vitro study on the mechanisms of ACR combined with CDDP to inhibit ovarian cancer cell was carried out on attempt to form a promising class of agents, and hope that might be helpful for improving treatment efficiency over the cancer.Materials and Methods1 Material The SKOV3 ovarian cell line was supplied by the Peking people's hospital. ACR is product of the WanLe Drug Co. Ltd. Shenzhen, China and CDDP is produced by Sanrong Co. Ltd. Kunming, China. The immunocytochemical reagents, a mouse anti-human topoⅡprimary antibody was purchased from Sandcruz Co. American, and a goat anti-mouse biotin-horseradish peroxidase (HRP) secondary antibody was bought from Wuhan Boster Biological Technology Co., Ltd. RPMI-1640 culture medium (Gibco-BRL), microculture tetrazolium (MTT), RNase, Proteinase K, protein marker were purchased from Sino-American Biotechnology Co.2 Methods2.1 Cell culture and the concentration selection of the drugs The SKOV3 cells were cultured in a humidified atmosphere of 5% CO2 at 37 °C in RPMI 1640 medium containing 100U/ml penicillin, 100μg/ml streptomycin and 10% newborn calf serum(NCS)The peak serum concentrations(PSC) of ACR(6μg/ml) and CDDP(3μg/ml) were chosen to form the experimental groups.2.2 MTT Assay Exponentially growing SKOV3 cells were added at 1x 105/ml in 96-well microtiter plates (100 μl/well). The drugs diluted by RPMI 1640 culture medium without NCS were added to the cells in a final volume of 10 μl/well. The PSC of ACR(6μg/ml) was combined with 1/10 PSC(0.3μg/ml), the PSC(3μg/ml) and 10×PSC(30μg/ml) of CDDP, respectively. Three replicates were made for each concentration of the drugs. After drugs exposure for 24h, 20 μl of MTT compound was added to each well and the cells were incubated at 37°C for 4 h. Following the plates were centrifuged and discarded supernatant, the cells were then incubated with 200 μl of dimethyl sulfoxide to stop the reaction. Absorbance (A value) was read at 570 nm using a microplate reader. The inhibition ratios were measured and the synergistic effectiveness of the two drugs was calculated by using the jin's formula. Two separated experiments were made for MTT test. 2.3 Determination of cellular apoptosis by double fluorescent dyes After incubation for 24h, the cells were centrifuged by 1500rpm/min for 5min and suspended in phosphate-buffered saline (PBS). The cells were then mixed with a fluorescent dye mixture (acridine orange 0.5mg/ml and ethidium bromide 0.5mg/ml) and added to slides, which were detected under a fluorescence microscope. To assess the percentage of cells showing features of apoptosis, at least 200 cells were scored in each experiment. The apoptotic ratios were measured by using the formula, numbers of apoptotic cells/ whole cells×100. Statistical significances were determined by the jin's formula.2.4 Colony inhibition assay Briefly, 1×105 /ml cells were seeded on cell culture flasks. Three flasks were prepared for each group. Following the drugs were added to the exponentially growing cells and exposed for 24h, the cells were centrifuged and washed with the culture medium without NCS, then replanted at a density of 1×103 to 60mm2 dishes. The colonies were counted 7d later after staining with 0.5% crystal violet/ethanol. The colony inhibition efficiency (%) were calculated by using a formula of (1-apoptotic cells / planted cells)×100. The differences between the results were tested for statistical significance by the jin's formula. 2.5 DNA Isolation and Electrophoresis... |
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