Inhibition Of Proliferation Induced By Anticancer Drugs And Its Mechanism In Human Pancreatic Cancer Cells

Objective: Pancreatic cancer is a aggressive malignancy, the morbidity of pancreaticcancer is ascending year by year recently. But it’s very difficult to have early diagnosis ofpancreatic cancer, and the the operation result after diagnosis is not so good, neither doesthe chemotherapy and other adjunctive therapy. In recent years, the study of naturalanti-tumor drugs was performed actively, people paid more attention to the anti-tumoreffect of traditional Chinese drug and it’s monomer. The anticarcinogen of traditionalChinese drug including arsenic trioxide had a very good clinical effect. Silibinin andHarmine hydrochloride have already been studied a lot, especially Silibinin, many researchproved its growth inhibitory effect to many tumors. But there’s little reports of the effect ofSilibinin and Harmine hydrochloride to the pancreatic cancer. With the purpose to findnatural anti-tumor drugs of high effect and low side effect, we used human pancreaticcancer cell line AsPC-1and SW1990as objects, investigated the molecular mechanism ofthe two drugs, which can inhibit tumor cells’ growth, induce their apoptosis, autophagyand arrest cell cycle.Methods:1. MTT assay was used to investigate the inhibition effect of Silibinin on normalliver cell L02and human pancreatic cancer cell line AsPC-1and SW1990. Changes inapoptotic cell percentage were calculated by flow cytometry. Western blotting assay wasused to determine cell apoptosis and the protein of JNK pathway. Changes in cell cyclepercentage was calculated by flow cytometry. The expression of cycle related proteins wasanalyzed by Western blot.2. MTT and cell colony formation inhibitory assay were used to investigate the inhibitioneffect of Harmine hydrochloride on normal liver cell L02and human pancreatic cancer cellline AsPC-1. Changes in apoptotic cell percentage were calculated by flowcytometry.Western blotting assay was used to determine cell apoptosis and autophagy.Results:1. MTT showed that Silibinin with different concentrations inhibited pancreatic cancer AsPC-1and SW1990cells in a dose-dependent manner（P＜0.05）, but have littleeffect on L02cells. The IC₅₀for Silibinin against pancreatic cancer AsPC-1and SW1990cells for48hours and72hours was224.20,87.25μmol/L and218.41,86.91μmol/Lrespectively. Annexin V-FITC/PI staining analysis also showed a dose-dependent effect onapoptotic cells. Western blot assay showed that Bcl-2, Bcl-xl, Mcl-1, Caspase-3andCaspase-9were decreased and Bax increased. Bcl-xs, Bax, Bid and Bim was increased.The cut bands of PARP were detected. Meanwhile, JNK is activated. PI staining analysisshowed that cell cycle was arrested in G1phase. Western blot assay showed that CyclinD1,CyclinE2, CyclinA, CyclinB1, CDK6were decreased, P15and p21was increased.2．MTT showed that Harmine hydrochloride could inhibited human pancreatic cancerAsPC-1cells in a dose-dependent manner, but on L02cells,there is not too much effect. IC₅₀of Harmine hydrochloride on AsPC-1cells at24h,48h and72h was116.5μmol/L,66.0μmol/L and22.3μmol/L respectively；Cell clone formation inhibition assay demonstratedthat cell clones were decreased with increase of the concentration of Harmine hydrochloride.Annexin V-FITC/PI staining also showed a dose-dependent effect on late apoptotic andnecrotic cells. Western blotting assay showed that the apoptosis and autophagy landmarkprotein Caspase-3, PARP, LC3-II and P62protein expression levels changed gradually withincreasing of Harmine hydrochloride.Conclusion:1. Apoptosis was induced by Silibinin on the AsPC-1and SW1990cells throuth themitochondrial pathway, and the JNK pathway was activation. Silibinin treatment led theAsPC-1and SW1990cell cycle was arrested in G1phase.2. Harmine hydrochloride inhibits the cell survival of human pancreatic cancer cell AsPC-1in a dose-dependent manner. Harmine hydrochloride inhibits cell growth by inducingapoptotic and autophagy.