| Objective:Establishment a multidrug resistance（MDR）human hepatocellular carcinoma cell line Bel-7402/ADM,detecting the inhibitory effect of antibacterial peptide from Musca domestica Larvae on the cell line.Investigation whether antimicrobial peptide can reverse the drug resistance of Bel-7402/ADM cells,then explore the potentialmechanism.Methods:1.Establishment of a MDR hepatocellular carcinoma cell line Bel-7402/ADM by gradually increasing the concentration of adriamycin in culture medium.2.Plotting the growth curve of cells and calculate the cell doubling time.3.The drug sensitivity of cells was detected by MTT assay.4.The invasiveness of cells was detected by Transwell invasion assay.5.The fluorescence intensity of adriamycin in cells was observed by fluorescence microscope.6.Flow cytometry was used to detect the cell cycle,apoptosis rate,and the content of P-glycoprotein（P-gp）and adriamycin in cells.7.The expression of multidrug resistance gene 1（MDR1）was detected by RT-qPCR.Result:1.A cell line that can be produced in the cell culture medium with 0.5μg/mL adriamycin stably was established and we named it Bel-7402/ADM,the morphology and size of Bel-7402/ADM cells are similar to Bel-7402 cells.The doubling time of Bel-7402/ADM cells was longer than the Bel-7402 cells and the cell proliferation slows down.2.The results of MTT analysis:（1）The resistance index（RI）of Bel-7402/ADM cells against adriamycin（ADM）is 10.97,it was also resistant to other drugs like paclitaxel,vincristine,5-Fu,cisplatin（DDP）and gemcitabine,and Bel-7402/ADM cell line was a MDR cell line.（2）The results of cytotoxicity test showed that,antibacterial peptide fromMusca Domestica Larvae can inhibit the proliferation of Bel-7402 and Bel-7402/ADM cells,and the inhibitory effect was increased with the increase of antimicrobial peptides（AMPs）.We also found that the 50%inhibitory concentration(IC50)of AMPs to Bel-7402/ADM cells was lower than Bel-7402 cells.3.The results of cell cycle:（1）Compared with the Bel-7402 cells,the percentage of Bel-7402/ADMcells in G0/G1 phase was significantly increased（P<0.05）,while the percentage of cells in S phase was markedly decreased（P<0.05）.（2）After treatment with AMPs,the percentage of Bel-7402 andBel-7402/ADM cells in G0/G1 phase was significantly increased,while the percentage in S phase was significantly decreased（P<0.05）.4.The results of apoptosis detection showed that,the early apoptosis rate of Bel-7402 and Bel-7402/ADM cells was increased significantly after treatment with AMPs（P<0.05）.5.The results of cell invasion detection showed that AMPs can inhibit the invasiveness of Bel-7402 and Bel-7402/ADM cells significantly（P<0.05）.6.After treatment with AMPs,the IC500 of ADM on Bel-7402/ADM cells was significantly decreased（P<0.05）,and the RI of Bel-7402/ADM was decreased.While the low-dose AMPs could hardly affect the sensitivity of Bel-7402 cells for ADM.7.Fluorescence intensity detection of ADM in cells:（1）The results of fluorescence microscopy showed that,the fluorescence intensity in Bel-7402/ADM cells was lower than that in Bel-7402 cells.After treatment with AMPs,the fluorescence intensity in Bel-7402 almost did not chang（P=0.61）,while in Bel-7402/ADM cells the intense fluorescence was increased compared with the control group.（2）The results of flow cytometry showed that,the average fluorescence intensity of Bel-7402/ADM cells was significantly lower than that of Bel-7402 cells（P<0.05）.After treatment with AMPs,the average fluorescence intensity of Bel-7402/ADM cells was considerably increased（P<0.05）,while the average fluorescence intensity of Bel-7402 cells was almost not increased（P=0.09）.8.The detection of P-gp content on cells showed that,the fluorescence intensity of P-gp antibody of Bel-7402/ADM cells was significantly higher than Bel-7402 cells（P<0.05）.And the fluorescence intensity of Bel-7402/ADM cells was significantly decreased after treatment with AMPs（P<0.05）.9.The result of RT-qPCR detection showed that,the relative expression of MDR1 in Bel-7402/ADM cells was considerably higher than Bel-7402 cell（P<0.05）.After treatment with AMPs,the relative expression of MDR1 in Bel-7402/ADM cells was decreased significantly compared with the control group（P<0.05）.Conclusion:1.We have established a multidrug resistance human hepatocellular carcinoma cell line——Bel-7402/ADM,Bel-7402/ADM cells also resistant to other chemotherapy drugs like paclitaxel,vincristine,5-Fu,cisplatin,and gemcitabine besides ADM,it was a multidrug resistance（MDR）cell line.2.The morphology of Bel-7402/ADM cells were similar to the parental cells,while the proliferation of Bel-7402/ADM cells was slower than parental cells.3.The crude extract of Musca Domestica Larva antibacterial peptide could inhibit the Bel-7402 and Bel-7402/ADM cells dramatically,and the inhibitory effect on Bel-7402/ADM cells was stronger.4.The crude extract of Musca Domestica Larva antibacterial peptide may play an antitumor role by affecting the cell cycle distribution,inducing apoptosis and inhibiting invasiveness of Bel-7402/ADM cells.5.The crude extract of Musca Domestica Larva antibacterial peptide can reduce the RI of Bel-7402/ADM cells and promote chemotherapeutic drugs enter Bel-7402/ADM cells.AMPs may be able to reverse the resistance of Bel-7402/ADM cell line.6.The crude extract of Musca Domestica Larva antibacterial peptide may decrease the content of P-gp in Bel-7402/ADM cells by inhibiting the expression of MDR1,then reversing the drug resistance of Bel-7402/ADM cell line. |
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