Experimental Study Of Chrysin Enhanced The Sensitivity Of Gemcitabine In Pancreatic Cancer By Inhibiting Autophagy

Part ? Chrysin inhibited pancreatic cancer cell proliferation and induced mitochondrial pathway of apoptosisObjective Study the effects of chrysin, a naturally occurring flavonoid, on proliferation and apoptosis of human pancreatic cancer cells and its possible mechanism.Methods Cell viability was detected by using CCK-8 assay kit and colony formation assay; cell apoptosis was detected by Annexin V/PI double staining flow cytometry; cell cycle was detected by PI staining flow cytometry; the expressions of apoptosis related proteins were detected by Western blot.Results Chrysin inhibited cell proliferation and colony formation of human pancreatic cancer cell lines in a time-and dose-dependent manner; Chrysin caused the cell cycle arrested at Go/G1 phase; Western blot analysis showed that Chrysin could significantly enhanced the expression of cleaved PARP, cleaved caspase-9 and cleaved caspase-3, and increased the ratio of Bax/Bcl-2 in pancreatic cancer cell lines.Conclusion Chrysin could inhibit cell proliferation and colony formation of human pancreatic cancer cells, and caused the cell cycle arrest at Go/G1 phase; Chrysin also induced mitochondrial pathway of apoptosis in pancreatic cancer cells.Part ? Chrysin blocked autophagy by inhibiting lysosomal enzyme activity in pancreatic cancer cellsObjective Study the effects of chrysin on autophagy of human pancreatic cancer cells and its possible mechanism.Methods Construction of a lentivirus vector carrying GFP-tagged LC3 and stable transfected pancreatic cancer cells, the number of intracellular GFP-LC3 punctate aggregation was observed under a confocal microscope; the expression of autophagy related proteins were detected by Western blot; the expression of autophagy substrate protein p62 was detected by immunofluorescence assay; The super-microstructural changes were observed by transmission electron microscopy; Construction of a lentivirus vector carrying GFP-mRFP-LC3 and stable transfected cell lines, the co-location of GFP-LC3 and mRFP-LC3 in the cells was observed under a confocal microscope; the spatial location relationship between the lysosomes and GFP-LC3 punctate was detected by using the lysosomal probe LysoTracker Red under a confocal microscope; the AO (acridine orange) staining was used to observe the number of intracellular acidic autophagic vacuoles; The catalytic activities of cathespins were determined by CTSB and CTSD activity fluorometric assay kits.Results Confocal microscope show that chrysin could induce GFP-LC3 stable transfected pancreatic cancer cells appeared a large number of GFP-LC3 dots aggregates in a time-and dose-dependent manner, and the GFP-LC3 dots colocalized with Lysotracker red; Western blot analysis and immunofluorescence results show that chrysin also significantly increased the expression of LC3B-II as well as autophagy substrate protein p62; Transmission electron microscope showed that a large number of autophagic vacuoles were observed in chrysin-treated pancreatic cancer cells; Western blot analysis showed that a combined effect of using autophagic lysosome inhibitors CQ and chrysin dramatically increased the accumulation of LC3-II compared with either agent alone; similar to the CQ, chrysin induced GFP-mRFP-LC3 stable transfected pancreatic cancer cells appeared the GFP-LC3 dots colocalized with RFP-LC3 dots; Conversely, in rapamycin-treated cells, only parts of GFP-LC3 dots colocalized with RFP-LC3 dots; AO staining showed chrysin and CQ could significantly increase the number of acidic autophagic vesicles in pancreatic cancer cells; cathepsin activity assay and Western blot analysis showed, the enzymatic activity of CTSB and CTSD were dramatically inhibited upon chrysin treatment.Conclusion Chrysin blocked autophagy by inhibiting lysosomal enzyme activity in pancreatic cancer cells.Part III chrysin enhanced the sensitivity of gemcitabine in pancreatic cancer by inhibiting autophagyObjective Investigate the effect and mechanism of combination of chrysin and gemcitabine on proliferation and apoptosis in human pancreatic cancer cells.Methods Cell viability was detected by using CCK-8 assay kit and colony formation assay; cell apoptosis was detected by Annexin V/PI double staining flow cytometry; inverted microscope to observe the influence of single or combined use of drugs for cell morphology; the expressions of autophagy and apoptosis related proteins were detected by Western blot; RNA interference technique was used to detect the effect of gemcitabine on proliferation and apoptosis of pancreatic cancer cell line after silencing Beclinl, ATG5 and ATG7.Results Gemcitabine partially inhibited cell proliferation of human pancreatic cancer cell lines in a time-and dose-dependent manner; Western blot analysis showed that gemcitabine significantly enhanced the accumulation of LC3-II, but the expression of autophagy substrate protein p62 was decreased; the expression of LC3-II was significantly increased after the combination of gemcitabine and lysosomal inhibitor CQ compared with the single use of CQ; inhibition of autophagy, by either lysosomal inhibitors CQ or Beclinl, ATG5 and ATG7 silencing, enhanced gemcitabine-induced cell death; similar to the CQ, the expression of LC3-? was significantly increased after the combination of gemcitabine and chrysin compared with either agent alone; both CQ and chrysin were able to sensitize cells to gemcitabine when applied alone, the addition of CQ together with chrysin had no additional sensitizing effect on gemcitabine-induced toxicity compared to chrysin alone; Western blot analysis showed that the combination of gemcitabine and chrysin could further enhanced the expression of cleaved PARP, cleaved caspase-9 and cleaved caspase-3, and increased the ratio of Bax/Bcl-2 when compared with either agent alone.Conclusion Gemcitabine partially inhibited the proliferation of pancreatic cancer cells and induces protective autophagy; chrysin enhanced the sensitivity of gemcitabine in pancreatic cancer by inhibiting autophagy.