Ligustrazine Antitumor Screening And Induced Apoptosis Mechanism Of HepG2 Effect Research

Cancer is one of the major health problems facing mankind. The current treatment of radiotherapy and chemotherapy, there are still side effects, drug resistance and other issues. Therefore, finding effective drugs for cancer prevention and treatment a top priority.TMP has a variety of pharmacological activities, such as antioxidant, improve microcirculation. Recently, TMP has been reported to have anti-tumor effects, but the mechanism around the TMP induced apoptosis needs further exploration.Mitochondria are cellular apoptosis, signal transduction plays a key role in organelles. Mitochondrial pathway of apoptosis is mainly upstream signaling molecules after starting the role of mitochondria in the mitochondrial membrane, mitochondrial membrane functional status change causes the activation of Bcl-2 protein family, mitochondrial PT pore opening, c pigment cells start releasing, causing the cysteine aspartic acid protease activated Caspase family of proteins, Caspase activation prompted apoptosis.Objective Cell Counting Kit-8 assay and screening of target cell proliferation inhibition of tumor cell lines. By High Content System analysis of the amount of apoptosis-effect relationship, when flow cytometry apoptosis-effect relationship, when flow cytometry cell cycle-effect relationship, automatic system to detect protein-related protein expression and function of mitochondrial changes of Ligustrazine on HepG2 cells apoptosis, mitochondrial function and related protein expression, and to explore the molecular mechanisms of HCC TMP inhibited cell proliferation.Methods CCK-8 was observed by different concentrations of TMP on the proliferation of HepG2 cells and the dose-effect relationship; in HepG2 cells as target cells by real-time cell analysis technology detection Chuanxiong affect the application of flow cytometry in HepG2 cell apoptosis and cell cycle; triazine impact on the proliferation of HepG2 cells by automated Western Blot test on HepG2 cells and P53 protein Bcl-2/Bax protein expression. The use of high-content cell imaging analysis system and the impact of the release of cytochrome c HepG2 cells mitochondrial transmembrane potential.Western Blotting experiments by automatic detection TMP on HepG2 cells cleaved-Caspase-3 and cleaved Protein expression -Caspase-9, and to explore the effects of TMP apoptosis mechanisms.Results 1. The proliferation of HepG2 TMP study, a dose-dependent effect of 10μg/mL,25μg/mL,50μg/mL,100μg/mL,200μg/mL and 400μg/mL.There was a significant difference (P<0.01) OD value mL-1 group with the control group, and 200μg/mL and 400μg/mL group was higher than positive drug suppression group. TMP can significantly inhibit the proliferation of HepG2 cells, and in a dose-dependent and time-dependent manner. TMP proliferation (P<0.01) effect on HepG2 12,24 and 48 h after could significantly inhibit HepG2 cells. High content detect apoptosis of HepG2 cells after TMP effect can significantly reduce the size of the nucleus (P<0.05), and a significant increase in Annexin V-FITC (P<0.01) and PI (P<0.01) fluorescence, high doses of fluorescence a lower dose of high value, TMP could significantly induce apoptosis in HepG2 cells, and dose-related.2. TMP hepatoma cell cycle studies show that TMP role HepG2 cells after 12, can significantly increase the G0/G1 phase cells (P<0.01); after treatment for 24 and 48h, TMP significantly induced apoptosis (P<0.01).3. TMP can significantly increase the release of cytochrome c from the mitochondria into the cytoplasm (P<0.01); significant reduction of mitochondrial membrane potential (P<0.05). A significant increase in the expression of cleaved-Caspase-3 and cleaved-Caspase-9 protein (P<0.01). A significant increase in the expression of p53 protein (P<0.01) and significantly decreased Bcl-2/Bax expression ratio (P<0.01).Conclusion TMP of HepG2, A549, SMMC-7721, MCF-7 cells, such as tumor cell proliferation inhibition. While inhibition of MCF-10A, L-O2, and BEAS-2B cells, such as normal some degree of proliferation.2. TMP on HepG2 proliferation in a dose-dependent and time-dependent manner, IC50 dose is low, significant inhibition.3. TMP at 12h, the main role of HepG2 cells induced G0/G1 phase cell cycle arrest; 24 and 48h after effects can significantly induce apoptosis in HepG2 cells.4. TMP can significantly increase the expression of p53 protein, reduce Bcl-2/ Bax expression ratio so that the mitochondrial membrane potential disintegration, can significantly reduce the mitochondrial membrane potential, a significant increase in cytochrome c release from the mitochondria to the cytoplasm, thereby increasing cleaved-Caspase-3 cleaved-Caspase-9 protein expression, suggesting that TMP through Caspase-dependent mitochondrial apoptotic pathway mediated inhibition of proliferation of HepG2 cells.