Ethanol Extract of Tengligen Induces Apoptosis of Colorectal Cancer Cells through AKT/PUMA Pathway

  OBJECTIVE  To investigate the effect and antitumor mechanism of ethanol extract of Tengligen (TLG) on colorectal cancer cells.

  METHODS  The toxicity of TLG on human colorectal adenocarcinoma cells (HCT-15, LoVo) was detected by methyl thiazolyl tetrazolium (MTT). Cell plate cloning assay was used to detect the effect of TLG on colorectal cancer cell proliferation. Hoechst-33258 staining and flow cytometry were used to detect the effect of TLG on colorectal cancer cell apoptosis. Western blot assay was used to detect the effect of TLG on the related proteins Caspase-3, PARP, p65, PUMA, AKT, GSK-3β in HCT-15. Dual luciferase reporter assay was performed to detect the effect of TLG on the activity of related promoter in HCT-15.

  RESULTS  Treatment of colorectal cancer cells (HCT-15, LoVo) with TLG resulted in a time- and dose-dependent decrease (P < 0.01, P < 0.001) in cell viability compared with control cells, and transfection with AKT cDNA, PUMA siRNA or GSK-3β inhibitor treatment was able to restore proliferation (P < 0.01). After treatment with different concentrations of TLG, the rate of apoptosis was increased (P < 0.01, P < 0.001), and cells transfected with AKT cDNA, PUMA siRNA, or GSK-3β inhibitor was able to suppress TLG induced apoptosis (P < 0.01, P < 0.001). Western blot showed that p-AKT/AKT and p-GSK-3β/GSK-3β protein expression in HCT-15 cells decreased (P < 0.001) while Cleaved-Caspase-3/Caspase-3, Cleaved PARP/PARP, p65 and PUMA protein levels increased with the increase of TLG concentrations (P < 0.05, P < 0.01, P < 0.001); simultaneous transfection of AKT cDNA, PUMA siRNA, or GSK-3β inhibitor was able to block the effects of TLG on downstream proteins Cleaved-Caspase-3/Caspase-3, Cleaved PARP/PARP, p65, PUMA (P < 0.05, P < 0.01, P < 0.001). Reporter gene results showed that NF-κB and PUMA promoter activities were enhanced (P < 0.05, P < 0.01) with the increase in TLG concentration, but recovered by simultaneous transfection of AKT cDNA or GSK-3β inhibitor treatment (P < 0.01, P < 0.001).

  CONCLUSION  TLG can significantly induce apoptosis and exert strong tumor suppressive effects in colorectal cancer cells, and the mechanism may be related to the AKT/PUMA signaling pathway.