A Study On The Inhibitory Effects Of Tubeimoside I On Cultured Human Hepatoma (HepG2) Cells And Associated Mechanisms

Hepatoma is one of the most prevalent malignant tumor types and the third leading cause of cancer-related death worldwide. Each year, approximately 550,000 new cases of hepatoma are reported worldwide, representing more than 5% of all human cancers. Despite the development and use of multimodality therapies including chemotherapy, the clinical outcome of hepatoma treatment remains unsatisfactory, with usually less than 7% of the 5-year overall survival rate. Thus, it is imminent to find more effective methods and/or agents for hepatoma treatment. Traditional Chinese medicinal herbs, owing to its intrinsic advantages, may provide a great source for such search.Indeed, one herb that may have great potential is tubeimoside (TBMS), or the tuber of Bolbostemma paniculatum (Maxim.) Franquet (Cucurbitaceae). In traditional Chinese medicine TBMS has long been widely used for treatment of illness such as inflammation and snake venoms, and was listed in the Supplement to the Compendium of Materia Medica published in early 1765. In the 1980's TBMS was first reported to show potent anti-tumor activity. Such anti-tumor activity in part motivated the successful isolation of tubeimoside I (TBMS I), a triterpenoid saponin. And subsequent studies confirmed that TBMS I can indeed inhibit growth of cultured cancer cells of several human cancer cell lines including human promyelocytic leukemia (HL-60), nasopharyngeal carcinoma CNE-2Z cell line (CNE-2Z) and HeLa cells. These studies appear to suggest that TBMS I may be a potential candidate as a novel anti-tumor drug, However, TBMS I so far has not been well studied for its anti-tumor activity against hepatoma, even though that TBMS I is known to preferentially distribute in the liver during in vivo metabolism, and thus might better target liver cancer, or hepatoma.Therefore, we evaluated TBMS I for its cytotoxicity to cultured human hepatoma cells or normal liver cells, from HepG2 or L-02 cell lines, respectively. Because the genesis and progression of cancer are known to involve not only abnormal proliferation and differentiation, but also abnormal apoptosis of the cells, we thus investigated apoptosis-associated molecular events as potential mechanisms responsible for the cytotoxic effects of TBMS I on HepG2 cells. Furthermore, we evaluated the effects of TBMS I on migration, invasion, F-actin structuer, and cytoskeletal stiffness of the cultured HepG2 cells. All together, this study may serve as a foundation for more comprehensive understanding of the pharmacology of TBMS I with HepG2 cells, and provide a scientific basis for further development of TBMS I as a novel drug for treating hepatoma in clinical practice. The main findings of this study are as follows:①TBMS I inhibited growth of cultured cells. The results from MTT assay demonstrated that TBMS I inhibited growth of HepG2 cells, HCCLM3 cells, L-02 cells and QSG-7701 cells in a typical concentration- and time-dependent manner. However, as compared to the normal human liver cells, hepatoma cells appeared to be more sensitive to TBMS I, particularly at the dose range of 10-30μM. Cell cycle analysis by flow cytometry demonstrated that exposure to TBMS I inhibited HepG2 cell proliferation via G2/M phase arrest in a dose-dependent manner.②TBMS I induced apoptosis in HepG2 cells. When HepG2 cells were examined for cell and nucleus morphology by phase contrast microscopy and Hoechst 33258 staining, it was found that after exposure to TBMS I induced cell shrinkage and even detachment, nuclear condensation and fragmentation. The cytoplasmic microtubules labeled with Tubulin-Tracker fluorescent probe also changed from bright fluorescence and appeared radiating from center to periphery of the cells without TBMS I to an apparent decrease in the fluorescence intensity, membrane translocation, and amorphous appearance, all suggesting tubulin depolymerization. Analysis of the cells double stained with Annexin V/PI by flow cytometry demonstrated that the percentage of the early apoptotic cells induced by TBMS I exposure seemed indifferent from that of the late ones when TBMS I concentration was low (e.g.≤15μM), but at high concentrations of TBMS I (e.g. =30μM), the exposure induced much greater percentage of the early apoptotic cells as compared to the late apoptotic cells. This suggests that exposure to TBMS I at high concentrations may predominately promote early apoptosis in HepG2 cells. TBMS I induced a similar trend of apoptotic process in L-02 cells, but with less total apoptotic cells, which was consistent with the results of proliferation inhibition.The mitochondrial apoptotic pathway has been described as an important signaling pathway of apoptosis. In the present study, we examined whether this pathway was involved in the TBMS I-induced apoptosis. Results obtained from flow cytometry and Western blotting demonstrated that exposure of HepG2 cells to TBMS I resulted in loss of mitochondrial membrane integrity, release of cytochrome c from mitochondria to the cytoplasm, marked activation of caspase-9, and -3, decrease in Bcl-2 expression and increase in Bax expression that led to increase of Bax/Bcl-1 ratio. All these results show that mitochondrial apoptotic pathways participate in TBMS I-induced apoptosis. ③TBMS I effectively inhibited migration and invasion of HepG2 cells. Cell migration and invasion are major biological behaviors that characterize tumore malignancy, and important determinanats of therapeutic efficacy and prognosis in clinical treatment of hepatoma patients. In this study, we used Transwell assay to evaluate the effect of TBMS I on the migration and invasion of HepG2 cells. The results showed that as the cells were treated with increasing concentration of TBMS I, both the migration and invasion of HepG2 cells were increasingly inhibited. In parallel, the cells treated with TBMS I show destruction and desolution of the F-actin cytoskeleton labeled with FITC-Phalloidin, and also decreased expression of matrix metalloproteinase (MMP)-2 and -9 as measured by Gelatin zymography. These results suggest that F-actin cytoskeleton and MMPs signal pathways may be alternative targets of TBMS I through which TBMS I may regulate cytoskeleton dynamics and cell-matrix interactions, then inhibit migration and invasion of the cells.④TBMS I affected biomechanical properties of HepG2 cells. Since TBMS I induced destruction and desolution of F-actin cytoskeleton, we proposed that TBMS I may affect the biomechanical properties of the HepG2 cells. To test this idea, we measured the stiffness of HepG2 cells before and after exposure to TBMS I using optical magnetic twisting cytometry (OMTC). The results showed that TBMS I indeed affected the stiffness of HepG2 cells. Exposure of the HepG2 cells to increasing concentration of TBMS I resulted in decreases in both cell stiffness (G') and the value of exponent (α) for the power-law between the cell stiffness and the loading frequency (G~fα). In addition, while the baseline stiffness of HepG2 cells was lower than that of L-02 cells, the response of HepG2 cells to TBMS I was faster than than of L-02 cells. This was the first time that biomechanical properties of HepG2 cells were studied with regards TBMS I.Taken together, we conclude that TBMS I was a potent agent to suppress the growth of hepatoma cells in vitro. And the growth inhibition was in large part mediated via apoptosis-associated mitochondrial dysfunction and microtubule pathways. On the other hand, TBMS I also showed destruction and desolution of the F-actin cytoskeleton, then affected the stiffness and further inhibited migration and invasion of HepG2 cells. So TBMS I not only inhibited the growth of hepatoma cells but also inhibited the migration and invasion, together with its preferential distribution in the liver and its origin of natural medicinal plant, suggest that TBMS I may be a preferred drug candidate for treating liver cancer.