Antitumor Activity And Apoptosis Mechanism Of Supercritical Extract Of Typhonium Giganteum Engl.in Human Hepatoma SMMC-7721Cells

Typhonium giganteum Engl. is one of the herbs commonly used in traditional Chinese medicine against cancer. In recent years, it is reported that the anti-tumor activity of T. giganteum Engl. extract mainly concentrated in the alcohol extract and aqueous extract, but the anti-tumor activity of the supercritical extract has not been reported as yet. In our previous studies, we explored the chemical components of supercritical extract of T. giganteum Engl.(SETE) by GC-MS. In this study, SETE exhibited good anti-tumor activity in vitro. The anti-tumor activity and apoptosis mechanism of SETE were further reported on SMMC-7721cell line. The results were as follows:1. Growth inhibition was analyzed by MTT assay. It was found that SETE markedly inhibited proliferation of seven tumor cell lines, including human liver cancer cells (SMMC-7721, IC50:202.37±15.42μg/mL), human lung adenocarcinoma cancer cells (A549,IC50:288.21±16.14μg/mL), human gastric cancer cells(SGC7901,IC50:315.83±19.87μg/mL), human ovarian cancer cells(HO-8910,IC5o:345.86±10.59μg/mL), human colon cancer cells(HCT-116, IC50>500μg/mL), human cervical cancer cells(HeLa, IC50>500μg/mL), human breast cancer cells(MCF-7, IC50>500μg/mL), suggesting that SMMC-7721cell line is the most sensitive.2. Apoptotic population of SMMC-7721cells was analyzed by flow cytometry using annexinV-FITC/PI. The apoptosis results showed that the apoptotic population increased in a concentration-dependent manner with increasing concentrations after the cells treated with SETE.3. The apoptotic cells morphological changes were observed using inverted fluorescence microscope. It was showed that there were significant morphological changes in the nuclear chromatin after treated with200μg/mL SETE for48h.4. The cell cycle distribution was analyzed by flow cytometry to investigate the effects of SETE on the cell cycle. The data showed that SMMC-7721cells were treated with different concentrations of SETE for48h, an accumulation of SMMC-7721cells in the S phase and G2/M phases was observed. It was showed that SETE could induce apoptosis in SMMC-7721cells, which is related to the accumulation of the S phase and G2/M phase cells.5. The level of ROS in SMMC-7721cells was monitored by flow cytometry. These data showed that the level of ROS in SMMC-7721cells treated with SETE was increased in a concentration-dependent manner. ROS production may promote mitochondrial dysfunction and trigger mitochondria-mediated apoptosis.6. We investigated SETE induce alterations in mitochondrial membrane potential of SMMC-7721cells by flow cytometry. Flow cytometry analysis showed that the percentage of cells in high fluorescence produced a dose-dependent decrease. These data showed that SETE induces apoptosis accompanied by the alterationsin the mitochondrial membrane potential.7. Changes in intracellular calcium in SMMC-7721cells were analyzed by flow cytometry using Fluo-3AM. These data showed that the intracellular calcium in SMMC-7721cells treated with SETE was increased in a concentration-dependent manner.8. The changes of Caspase-9and Caspase-3activities in SMMC-7721cells treated with SETE were detected by colorimetric systemic assays. The dose-dependent elevation of Caspase-9and Caspase-3activities was observed in drug treatment group. As a result, SETE can activate Caspase-9and Caspase-3, which in turn may cleave cytoskeletal and nuclear proteins, disrupt mitochondrial function and finally induce apoptosis.9. Western blot analysis showed that the expression of Bcl-2protein was down-regulated, whereas the expression of Bax protein was up-regulated in a concentration-dependent manner in SMMC-7721cells.In summary, SETE had a significant cytotoxic effect and was able to inhibit proliferation and induce apoptosis in SMMC-7721cells. The mechanism of apoptosis induced by SETE may be related to the activation of mitochondrial apoptotic pathway and alteration of apoptotic protein expression. The in vitro findings in the present study offer a significant groundwork for future essential study and clinical application.