| Objective:To establish a chemotherapy resistant human salivary gland adenoid cystic carcinoma cell line, then detect the multidrug resistant characteristics of drug resistant cells and explore the effect of artesunate on the proliferation and drug resistance of chemotherapy resistant cell line.Method:A cisplatin resistant human salivary gland adenoid cystic carcinoma cell line was established from the parental cell line NACC by periodically exposing it to 3μM cisplatin; the cellular morphology of cisplatin resistant cells were observed by inverted microscope and electron microscop; the sensitivity of resistance for parental and cisplatin resistant cells to DDP, 5-FU, BLM and VCR were evaluated by MTT assay;the proliferation ability of cell lines was measured by clone formation experiment; the growth curve of cell lines was measured by MTT assay; Flow cytometry(FCM) was performed to determine the cell cycle distribution of parental and cisplatin resistant cells;the expression of survivin, MRP2, ERCC1 and CTR1 mRNA was detected by real time quantitative PCR; the expression of survivin, MRP2 and CTR1 protein was detected by Western Blot. MTT assay was conducted to detect the effect of artesunate on the proliferation of cisplatin resistant cells and evaluate the IC50 and IC05 concentrations of artesunate on cisplatin resistant cells; after parental and cisplatin resistant cells were exposed to artesunate for 48 hours, the morphological changes of cells were observed by inverted microscope and electron microscop; the cisplatin resistance reversal effects of 37.5,75,150μM artesunate were detected by MTT assay; the cell cycle distributions of parental and cisplatin resistant cells were detected by FCM after the cells were treated with 37.5,75,150μM artesunate for 48 hours; the apoptosis rates of parental and cisplatin resistant cells were analyzed by Annexin V/PI double staining after the cells were treated with artesunate at the concentration of 37.5,150,600μM for 48 hours; after cisplatin resistant cells were exposed to artesunate at the concentration of 37.5,75,150,600μM for 48 hours, the expression of survivin, MRP2 and ERCC1 mRNA was detected by real time quantitative PCR and the survivin and MRP2 protein was measured by Western Blot. Results:1.A cisplatin resistant adenoid cystic carcinoma cell line which was named NACC/DDP was established with stable growth level after exposingNACC to 3μM cisplatin over a period of 7 months, the morphology of cells were changed, inverted microscope showed that the size of NACC/DDP cells were bigger than that of NACC cells, and the resistant cells were multangularer and coenocyticer than that of parental cells. While observed by transmission electron microscope, the number of nucleolus, rough endoplasmic reticulum, lipid droplet and lysosome in resistant cells were increased.2. NACC/DDP cells developed drug resistance not only to cisplatin but also to other chemotherapeutic drugs, such as bleomycin and vincristine, the resistance index (RI) of the cells to DDP, BLM and VCR was 2.16,1.99 and 1.97, respectively. The colony formation rate of NACC/DDP cells (32.08±2.82) % was significantly higher than that of NACC cells (23.52±2.72)% (P<0.01).The cell growth curve showed that proliferation ability of resistant cells increased significantly than that of parental cells. The ratio of NACC/DDP cells in G0/G1 phase increased while that of S and G2/M phase decreased when compared with NACC cells.3. Real time quantitative PCR revealed that the expression of survivin, MRP2, ERCC1 and CTR1 mRNA in NACC/DDP cells were up-regulated when compared with NACC cells. Western Blot showed that the expression of survivin, MRP2 and CTR1 protein in resistant cells were also increased when compared with parental cells.4. Artesunate inhibited the proliferation of NACC/DDP cells in a dose dependent manner. According to the MTT result, the IC50 and IC05 concentrations of artesunate on NACC/DDP cells were 1258.52±36.87μM and 166.86±49.84μM, respectively. Then 37.5,75,150 μM artesunate were chosen to reverse the cisplatin resistance of NACC/DDP cells and 600 μM artesunate was regarded as positive control. Inverted microscope showed that most NACC/DDP cells in reverse concentration groups did not have significant morphological changes, while cell death could be easily found after NACC/DDP cells were treated with 600μM artesunate for 48 hours. More swell of mitochondria and bubble of cytoplasm was observed by transmission electron microscope in both kinds of cells after treated by 150 μM artesunate for 48 hours, but the microvillus was decreased, and mitochondrial swollen were more serious in parental cells when compared with resistant cells. The MTT assay demonstrated that 75 and 150 μM artesunate could partly reverse cisplatin resistance of NACC/DDP cells in different degree, the IC50 values of cisplatin was decreased after NACC/DDP cells were treated with 75 and 150 μM artesunate for 48 hours, and the reversal index was 1.29 and 1.70, respectively.5. After the cells were cultured with artesunate at the concentration of 37.5, 75 and 150 μM for 48 hours, the FCM result showed that the ratio of cells in G0/G1 phase decreased while that of S and G2/M phase increased in both kinds of cells, meanwhile, the effect of artesunate at the same concentration on the cell cycle distribution of both kinds of cells were similar. After treated with 37.5, 150 and 600μM artesunate for 48 hours, the apoptosis rate in NACC andNACC/DDP cells significantly increased gradually with the concentration of artesunate, the result showed that artesunate could remarkably promote the apoptosis of NACC and NACC/DDP cells in dose dependent manners, also, the apoptosis rate in NACC cells was slightly higher than that of NACC/DDP cells after treated with artesunate at the same concentration, but there was no statistical significant between them.6. After the NACC/DDP cells were treated with different concentrations of artesunate for 48 hours, real time quantitative PCR showed that the expression of survivin, MRP2 and ERCC1 mRNA were dose-depressed by artesunate. Similarly, the result of Western Blot showed that artesunate significantly decreased the expression of survivin and MRP2 protein.Conclusion:1.NACC/DDP cell line has the multidrug resistant characteristics, it exhibited low resistance (R<5x) to DDP, BLM and VCR. Compared with parental cell line NACC, the colony formation rate and proliferation ability of NACC/DDP cells increased, the ratio of cells in G0/G1 phase increased while that of S and G2/M phase decreased. The up-regulated expression of survivin, MRP2 and ERCC1 genes may be related to the multidrug resistance of NACC/DDP cells.2. Artesunate could efficiently inhibit the proliferation of NACC/DDP cells, reverse the cisplatin resistance of NACC/DDP cells in vitro, block the cell cycle at S and G2/M phase, induce the apoptosis of cells, the mechanism of artesunate reversing the cisplatin resistance of NACC/DDP cells may be associated with suppressing the survivin, MRP2 and ERCC1 genes and inhibiting the expression of survivin and MRP2 proteins. |
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