Molecular Mechanism Of Lappaconitine Hydrochloride Inducing Apoptosis Of HCT-116 And HepG2 Cells

Colorectal cancer and liver cancer are serious threats to human life and health.Therefore,there is an urgent need for anti-tumor drugs with good curative effects and low side effects.Lappaconitine(LA)is an alkaloid extracted from the root of Aconitum.It has been used in clinic for many years due to its excellent analgesic effect and non-addictive properties.However,its clinical application was constrained by poor solubility.So lappaconitine hydrochloride(LH)of LA derivative was synthesized by introducing chloride ions into LA to reduce side effects,upsurge the solubility and improve the efficacy.Studies have found that LA and its derivatives[Lappaconitine sulfate(LS),Lappaconitine hydrobromide(LAH)and LH]showed good anti-tumor activity in a variety of tumor cells,but the anti-tumor mechanism of LH is still unclear.In this study,HCT-116 cells and HepG2 cells were used as research objects.Cell proliferation was assessed by CCK-8 and Ed U assay.The morphological feature of apoptosis was evaluated with the DAPI staining.Cell cycle distribution and apoptosis rate were detected by flow cytometry.Western blot was used to screen for apoptosis-related proteins(Bax,Bcl-2,CytC,P53,Cleaved caspase-9/7/3,Cleaved PARP)and MAPK pathway related proteins(ERK1/2,p-ERK1/2,JNK,P38,p-JNK,p-P38).The effect of m RNA levels of Bax,Bcl-2,and Cleaved caspase-3 were checked by Rt-PCR assay.JC-1 fluorescent probe was used to determine the changes of cell mitochondrial membrane potential(MMP).Intracellular reactive oxygen species(ROS)was evaluated by DCFH-DA staining assay.HCT-116 tumor-bearing nude mouse model was used to evaluate the anti-tumor effect of LH on HCT-116 tumor-bearing mice in vivo.The pathological changes of transplanted tumor,heart,liver,spleen,lung,and kidney of mice were detected by H&E staining.The possible molecular mechanism of cell apoptosis induced by LH in HCT-116 and HepG2 cells was explored basing on the in vitro and in vivo experimental data.Collectively,our research results provided theoretical basis and valuable experimental data for the development of new anti-tumor drugs.The research results are as follows:(1)LH repressed cell proliferation of HCT-116 and HepG2 cells in a time and dose-dependent way.The inhibitory effect of LH was better than LA.The IC₅₀ of HepG2 and HCT-116 cells treated by LH for 24 h were 596.2?g/mL and 413.1?g/mL.The IC₅₀ of HepG2 and HCT-116 cells treated by LH for 48 h of LH treatment were371.5?g/mL and 174.2?g/mL,respectively.(2)LH could induce S phase cell cycle arrest and apoptosis in HCT-116 cells and HepG2 cells.As the LH treatment concentration increased,the rate of apoptosis also gradually increased.(3)After HCT-116 and HepG2 cells treated with LH,MMP gradually decreased,and the intracellular ROS level gradually increased.LH stimulated mitochondrial apoptosis pathway of HepG2 cells and HCT-116 cells by regulating the expression of CytC,Bcl-2 family(Bax,Bcl-2),P53 and Caspase family(Cleaved caspase-9,Cleaved caspase-7,Cleaved caspase-3).(4)After the treatment of HCT-116 and HepG2 cells with LH,the expression levels of p-P38 and p-JNK was up-regulated,while p-ERK was evidently suppressed,and the protein levels of ERK1/2,JNK,and P38 were basically unchanged.It is suggested that LH might induce apoptosis through the MAPK signaling pathway in HCT-116 and HepG2 cells.(5)LH had a significant inhibitory effect on the tumor growth of the HCT-116Xenograft nude mice,but it did not reduce body weight.Compared with the negative control group,the tumors in the LH treatment group and 5-FU treatment group grew slowly,and the anti-tumor effect of LH was equivalent to that of 5-FU;LH had almost no effect on nude mice heart,liver,spleen,lung,and kidney.The protein expression levels of Bax,Cleaved caspase-9/3,and p-p38 in tumor tissues of nude mice treated with LH were significantly increased,while the protein expression levels of Bcl-2 were decreased,which were consistent with the results of in vitro cell experiments.In summary,LH could significantly inhibit the proliferation of human colorectal cancer HCT-116 cells and human liver cancer HepG2 cells,and induced HCT-116 and HepG2 cell apoptosis through the mitochondrial pathway and MAPK pathway.LH had obvious anti-tumor effect on HCT-116 tumor-bearing mice,and had almost no effect on the body weight and organs of nude mouse,and the tumor suppressing effect was equivalent to that of clinical medication 5-FU.