Pharmacological Effect And Molecular Mechanism Of Apoptosis Induced By Pseudo-G-Rh2in Gastric Carcinoma SGC Cells

BackgroundGinseng is a Chinese traditional medicine that has multiple pharmacological effects.Ginsenoside is the major effective component that has many anti-tumor effects. In this study,we detected the anti-tumor effect of pseudo-ginsenoside Rh2(pseudo-G-Rh2) on severalhuman tumor cell lines and we found it could inhibit tumor cell growth, especially on humangastric carcinoma SGC cells. The IC50value was32.9μg/mL24hours after pseudo-G-Rh2treatment on SGC cells, and the inhibition was dose-and time-dependent. However, themechanism of pseudo-G-Rh2inhibiting the growth of SGC cells is still unknown. In thisstudy, we used molecular biological methods to investigate the traditional Chinese medicine;we developed a new anti-tumor drug and such in-depth exploration on pseudo-G-Rh2inhibiting SGC cells is prospective and valuable.ObjectsTo investigate the mechanism of peudo-G-Rh2on inhibiting SGC cells.Methods1. The inhibition effect of pseudo-G-Rh2on SGC cells and the IC50value was detectedby MTT assay.2. The morphological changes of pseudo-G-Rh2treated SGC cells were observed byfluorescence microscope after Hoechst33342or AO/EB staining.3. The apoptotic ratio, mitochondrial transmembrane potential and ROS inpseudo-G-Rh2treated SGC cells were measured by flow cytometry.4. The activity of caspase-3,-9in pseudo-G-Rh2treated SGC cells were detected bySpectrophotometry.5. The expression and location of CytC, AIF, Smac, Bcl-2, Bax, caspase-3,-9and PARPwere detected by western bloting.6. The effects of pseudo-G-Rh2on PI3K/AKT and MAPK pathway were detected bywestern bolting.Results1Effects of pseudo-G-Rh2on inhibiting tumor cells growth1.1MTT assay was applied to detect the effects of pseudo-G-Rh2on the growths of humangastric cell SGC, human non-small cell lung cancer cell A549, human fibrosarcoma cellHT1080, human uterine cervix cancer cell Hela, human chronic myelogenous leukemia cellK562, human melanoma cell A375, human breast cancer cell MCF-7, human acutemyelogenous leukemia cell HL-60, human hepatoma cell SMMC and huaman oophoromacell SK-OV-3. Different doses (5,10,20,40and80μg/mL, respectively) of pseudo-G-Rh2were treated for24hours, and then we found it inhibited the growth of all these cells,especially on human gastric cell SGC, with time-and-dose dependency.1.2Pseudo-G-Rh2has stronger effect on inhibiting tumor cell growth than G-Rh2.2Effects of pseudo-G-Rh2on inducing SGC cell apoptosis2.1Morphological studies2.1.1Hoechst33342stainingSGC cells were treated with different doses of pseudo-G-Rh2for24h, then the cellswere observed by fluorescence microscope. After stained by Hoechst33342, nuclei innormal cells were round and light blue. However, the cells treated with pseudo-G-Rh2showed chromatin agglutination, and apoptotic bodies could be seen in these cells. And sucheffect is dose dependent.2.1.2AO/EB stainingSGC cells were treated with different doses pseudo-G-Rh2for A24h, then the cells wereobserved by fluorescence microscope. We found that with the dose of pseudo-G-Rh2increased, more and more cells became shrinking, the membrane became rough, and the cellsdisplayed flavo-green fluorescence.2.2Apoptosis detected by flow cytometryFITC-AnnexinV/PI double staining After being treated by different doses ofpseudo-G-Rh2for24h, SGC cells were found to become apoptosis dose-dependently,comparing with untreated ones. These results indicated that pseudo-G-Rh2could induceapoptosis in SGC cell.3Molecular mechanisms of pseudo-G-Rh2on inducing SGC apoptosis3.1Effects of pseudo-G-Rh2on mitochondrial fouction3.1.1Effects of pseudo-G-Rh2on mitochondrial transmembrane potentialAfter being treated with different doses of pseudo-G-Rh2for24h, the mitochondrialtransmembrane potential in SGC cells decreased significantly in a dose-dependent manner,comparing with untreated ones, indicating pseudo-G-Rh2may induce apoptosis by altering the mitochondrial transmembrane potential.3.1.2Effects of pseudo-G-Rh2on Bcl-2and BaxPseudo-G-Rh2could depress the expression of Bcl-2and increase the ratio ofBax/Bcl-2, in dose dependent manner, indicating pseudo-G-Rh2induced apoptosis byaltering Bax/Bcl-2ratio to depress the mitochondrial transmembrane potential.3.1.3Effects of pseudo-G-Rh2on Cyt C, AIF and Smac expressionPseudo-G-Rh2could enhance the release of Cyt C, AIF and Smac to cytoplasm, in dosedependent manner.3.1.4Effects of pseudo-G-Rh2on the expression and activities of caspase-3,-9and PARPPseudo-G-Rh2could increase the activities of caspase-3and-9, activate caspase familycascade and PARP cleavage.These results indicated that pseudo-G-Rh2induced apoptosis in SGC cells throughmitochondrial pathway. Pseudo-G-Rh2increased Bax/Bcl-2ratio, depressed mitochondrialtransmembrane potential, enhance the release of Cyt C, AIF and Smac, activate caspaseprotein family, and eventually induced cell apoptosis.3.1.5Effects of pseudo-G-Rh2on ROSAfter being treated with different doses of pseudo-G-Rh2, the production of ROS inSGC cells increased significantly in dose-dependent manner, comparing with untreated ones,and indicating pseudo-G-Rh2induced apoptosis with ROS production.3.2Effects of pseudo-G-Rh2on PI3K/AKT pathwayPseudo-G-Rh2could significantly inhibit the phosphorylation of AKT in dosedependent manner, indicating the participation of PI3K/AKT pathway in the course ofapoptosis induced by pseudo-G-Rh2.3.3Effects of pseudo-G-Rh2on MAPK pathwayPseudo-G-Rh2could significantly increase the phosphorylation of ERK in dosedependent manner, indicating the participation of MAPK pathway in the course of apoptosisinduced by pseudo-G-Rh2.Conclusion1. Pseudo-G-Rh2could significantly inhibit the growth of SGC cells, in dose and timedependent manner;2. Pseudo-G-Rh2could induce apoptosis in SGC cells;3. Pseudo-G-Rh2induced apoptosis through mitochondrial pathway;4. Pseudo-G-Rh2induced apoptosis with production of ROX; 5. PI3K/AKT pathway and MAPK pathway participated in the course of pseudo-G-Rh2induced apoptosis.