NeosedumosideIII induced Apoptosis of Human Hepatocellular Carcinoma HepG2 cells and SMMC-7721 Cells and Related Mechanism

Purpose: NeosedumosideIII (Neo) is a megastigmanes and belongs to monocyclic sesquiterpenoids compound with antioxidant, anti-inammatory and other pharmacological activities. In order to explore the anti-cancer effect and possible mechanism of Neo, the study examined the anti-proliferation and apoptosis effect of Neo against human hepatocellular carcinoma HepG2 cells and SMMC-772 cells and related mechanism in vitro. Methods (cid:0) The anti-proliferation effect of Neo was detected on HepG2 cells and SMMC-772 cells by MTT assay and IC50 with increasing dose and time. Cell cycle and apoptosis were detected by ow cytometer. The changes of Bcl-2, Bax, Caspase-3, Caspase-8 and Caspase-9 proteins were detected by western blotting. Results (cid:0) The results indicated that Neo could inhibited proliferation of HepG2 cells and SMMC-772 cells in vitro and promoted apoptosis, it signicantly induced apoptosis of HepG2 cells and SMMC-772 cells arrested cell cycle at G0/G1 phase in a dose-dependent manner, reduce the expression of Bcl-2 protein, and increase the expression of Bax and Caspase-3, Caspase-8 and Caspase-9 proteins. Conclusion (cid:0) Neo could inhibit proliferation and induce apoptosis of HepG2 cells and SMMC-7721 cells in vivo which suggested that it might be served as a promising candidate for the treatment of liver cancer. To investigate the Neo induced apoptosis of hepatocellular carcinoma HepG2 cells and SMMC-7721 cells, the control group were cultured with DMSO whereas hepatocellular carcinoma HepG2 cells and SMMC-7721 cells were treated by 2, 4, 8, 16, and 32 µmol/L NEO for 48 h, and the results were shown in gure 3 and gure 4. The apoptosis rate of HepG2 cells were caused by the different concentrations of Neo for (2.28 ± 0.68) %, (6.86 ± 1.30) %, (13.01 ± 3.23) %, (20.08 ± 3.10) %, (31.97 ± 2.88) % and (50.67 ± 5.89) %. And the apoptosis rate of SMMC-7721 cells were caused by the different concentrations of Neo for (1.82 ± 0.30) %, (4.82 ± 1.89) %, (11.44 ± 2.54) %, (15.62 ± 2.38) %, (30.55± 2.92) % and (43.82 ± 3.11) %. It was shown that the apoptosis rate of hepatocellular carcinoma HepG2 cells and SMMC-7721 cells increased with the increase of Neo concentration, which also indicated the potential anticancer mechanism of Neo.


Introduction
Liver cancer is one of the most common tumors in the world and mainly caused by chronic hepatitis, fatty liver and other liver diseases [1], it's the second leading cause of cancer-associated high morbidity and mortality all over the worldwide [2,3]. At present, the proportion of mortality and morbidity of liver cancer showed tendency to ascend and the relative survival rate was reduced that indicated a poor prognosis [4]. Liver cancer is not very sensitive to chemotherapy and radiotherapy, and the mainly used treatment methods is surgical resection. however, liver cancer due to its high invasive nature and most of the patients have developed to advanced stage at the time of diagnosis, lost the best time for treatment [5]. Therefore, the most important task is screening potential and effective compounds for the treatment of liver cancer. Natural products isolated from Traditional Chinese Medicines are one of the important sources for anticancer drug which used as effective cancer treatment in China. NeosedumosideIII (Neo) is a megastigmanes and belongs to a class of monocyclic sesquiterpenoids with antioxidant, antiin ammatory and other pharmacological activities [6,7]. A large number of literature reports monocyclic sesquiterpenoids are widely distributed in nature, and have inhibitory activity for human breast cancer cells MDA-MB-231, human colon cancer cells HCT116, Human liver cancer cells HepG2 and SMMC-7721, etc [8, 9,10]. Meanwhile, a megastigmanes has inhibitory activity for human lung adenocarcinoma cells A549, human prostate cancer cells PC3 and human breast cancer cells MCF7 [11]. However, Neo as a megastigmanes compound and the inhibition of Human liver cancer cells HepG2 and SMMC-7721 has not been reported, and the protective mechanism of cos against liver cancer is still unclear. Therefore, in order to explore the anti-cancer effect of Neo and its possible mechanism. The study evaluated the effect of Neo against liver cancer and the mechanism of action of Neo on human liver cancer HepG2 cells and

SMMC-772 cells in vitro.
In this study, the growth inhibit effect of Neo was detected on HepG2 cells and SMMC-7721 cells by MTT assay. Cell cycle and apoptosis analysis were detected by ow cytometry. The expression of Bax, Bcl-2 and Caspase-3, Caspase-8, and Caspase-9 proteins were analyzed by western blotting, the study were used to analyze whether Neo could be used as a promising candidate drug for the treatment of liver cancer. We found that Neo effectively inhibited cell proliferation and induced apoptosis in HepG2 cells and SMMC-7721 cells and its potential mechanisms underlying the effects, it indicate that Neo might be used as a potential drug for the liver cancer therapy.

Reagents
Neo was bought from Herbpurify Company (Chendu, China, purity > 98%) and dissolved in DMSO and Cell viability assay MTT assay were used to assess viability and applied to measure L-O2 cells, HepG2 cells and SMMC-7721 cells viability. Three kinds of cells were plated 5×10 4 cells/well in 96-well culture plates and treated with serial concentrations of Neo (2,4,8,16, 32μmol/L) for 48 h (various time periods) and MTT solution (5 mg/mL) was added to each well, DMSO was used as a control. Subsequently, the cell viability is measured by MTT assay, the IC 50 (The half maximal inhibitory concentration) values were calculated by linear-regression analysis and averages of three independent repeats being calculated.

Cell cycle analysis
After HepG2 cells and SMMC-7721 cells were treated with serial concentrations of Neo (2, 4, 8, 16, 32μmol/L) and DMSO for 48 h, both types of cells were collected and centrifuged at 1000g for 5min to remove the culture medium, the cells were washed twice with PBS for cell cycle analysis. The cells were xed with 70% ethanol at 4 ℃ for 2 h, and the xed solution was discarded, then cells resuspended 5min by PBS and centrifuged at 1000g for 5min, PBS was discarded and stained with propidium iodide (PI), the cells were darkened at 4 ℃ for 30 min, and cell cycle was detected by ow cytometry.

Cell apoptosis assay
After HepG2 cells and SMMC-7721 cells were treated with serial concentrations of Neo (2, 4, 8, 16, 32μmol/L) and DMSO for 48 h and then cells were washed twice by PBS. Cells were resuspended with 500μL binding buffer, Annexin V-FITC (5 µl) and PI solution (5 µl) were added and incubated at room temperature for 15 min in the dark, and the cell apoptosis were analyzed by flow cytometry (ACEA Novo-Cyte, Agilent Technologies Inc., USA). According to the instructions of apoptosis kit, the number of cells were calculated by cell analysis software (BD Inc., USA) for data analysis and the experiments were performed in triplicate.

Western blot assay
Human Hepatocellular Carcinoma HepG2 cells and SMMC-7721 cells were analyzed by western blotting. The total protein fractions were isolation and DMEM medium were removed. Subsequently, cells were washed twice by PBS and then lysed using cell by RIPA lysis buffer. The lysates were collected and centrifuged at 12000 g for 5 min at 4℃, protein concentrations were determined using the BCA protein assay. Samples were separated by 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and then transferred to PVDF membranes, and blots were blocked with 10% skim milk incubated with the primary antibodies Bax, Bcl-2, Caspase-3, Caspase-8, Caspase-9, anti β-actin overnight at 4˚C. Then the membranes were incubated with secondary antibodies conjugated to HRP (Horse Radish Peroxidase), and gray scale analysis used by Image J software platform, the relative protein expression were expressed by the ratio of gray value of Bax, Bcl-2, Caspase-3, Caspase-8, and Caspase-9 protein to βactin.

Statistical analysis
Data were expressed as the mean ± SD. Statistical comparisons of results were made using analysis of variance (ANOVA). Signi cant differences (*P<0.05, **P<0.01) whereas T-tests were used by SPSS software (version 19.0). P values with P < 0.05 was the threshold for a statistical difference, whereas P < 0.01 was the threshold for a signi cant difference. The graphs were generated with Origin version 9.0.

Results
Effect of Neo on the proliferation of HepG2 cells and SMMC-7721 cells in Vitro.
The chemical structure of Neo was shown in gure 1A, which is a megastigmanes and belongs to monocyclic sesquiterpenoids compound. As shown in Figure 1B

Discussion
The sesquiterpenes are widely distributed in nature. The monocyclic sesquiterpenoids are abundant in sesquiterpenes with many physiological activities including anti-in ammatory, antioxidant, anti-ulcer, cytotoxic activities and other physiological activities [12][13][14], among which anti-in ammatory and cytotoxic activities are the most prominent. There are many studies have shown that megastigmanes in different cancer cells have different cytotoxic activities [10] and Neo is a type of megastigmanes compound, in order to further verify the antitumor activity of Neo, the cytotoxic activities of Neo against HepG2 cells and SMMC-7721 cells in vitro was investigated. In this study, it was showed that Neo could inhibit the proliferation of HepG2 and SMMC-7721 cells and in a dose-dependent.
Carcinogenesis is a long and multistep process affected by many factors. The cell cycle is a primary factor which is a very sophisticated process and different compounds have different effects on the cell cycle, the G0/G1 and G2/M phases are critical points in this process. It was found that the volatile oil signi cantly inhibited the proliferation of hepatocellular carcinoma HepG2 cells and SMMC-7721 cells cultured in vitro may be due to the increase of G2/M phase and S phase, accompanied by the decrease of G0/G1 phase [15]. The effect of Rosiglitazone may be prevent the retention of hepatocellular carcinoma HepG2 cells in G0/G1 phase and inhibit the cell migration to G2/M phase to a certain extent [16]. The Drug-containing serum of Clerodendrum Bunge blocks hepatocellular carcinoma MHCC97-H cells from G2/M phase to G0/G1 phase and further reducing S phase, thereby interfering with DNA replication to inhibit cell proliferation [17]. Therefore, it is important to analyze the effect of substances on cell proliferation by detection for the cell cycle, our study found that the Neo could block the cell cycle arrest in G0/G1 phase, it indicated that Neo can block the cell cycle of both types of cells to some extent and it was a great signi cance for the study of anticancer mechanism.
Apoptosis is a physiological process of cells. In recent years, many studies have shown that the occurrence and development of tumors were related and may be caused by abnormalities in the process of cell apoptosis [18,19]. Bcl-2 proteins and Bax proteins belongs to Bcl-2 family and then were key factors in the process of cell apoptosis which were expressed abnormally in various tumors, and then have become one of the targets of anticancer drugs [20]. Cell apoptosis may be induced by inhibition of Bcl-2 or activation of Bax [21], the lower the ratio of Bcl-2/Bax and the higher mortality of the cell, therefore, the up-regulation of Bax protein expression or the down-regulation of Bcl-2 protein expression might be related to the apoptosis of various tumor cells including liver cancer [22]. In addition, the study indicated that there are two apoptotic pathways including the "intrinsic" cytochrome C/Caspase-9 pathway and the "extrinsic" Caspase-8 pathway [23], caspase family proteins were also play a key role of cell apoptosis. Caspase-9 as a promoter of caspase which were necessary for apoptosis signal through mitochondrial pathways and activated on apoptotic complex, and thus activate caspase-3 [24]. Caspase-8 was also as a promoter of caspase and thus activate caspase-3 eventually leads to cell apoptosis [25]. When Caspase-3 was activated by upstream pathway and as a key "killer" of cell apoptosis [26] and induces cascade reactions leading to cells apoptosis [27]. Research suggests that costunolide can activate caspase-3 to mediate apoptosis of esophageal cancer cells by up-regulation of Bax protein expression and down-regulation of Bcl-2 protein expression [28], breast cancer McF-7 cells apoptosis induced by curcumol through regulating the expression of Bcl-2 protein and Bax protein and downregulating the ratio of Bcl-2/Bax [29], and many other studies have shown that inducing apoptosis was important for inhibiting the development of liver cancer [30].Therefore, the study investigated the effect of Neo on the expression levels of the key regulators of apoptotic pathways in HepG2 cells and SMMC-7721 cells including Bax, Bcl-2, Caspase-3, Caspase-8 and Caspase-9. In this study, western blotting analysis showed that Neo induced apoptosis of HepG2 cells and SMMC-7721 cells by up-regulating the expression of Bax, Caspase-3, Caspase-8 and Caspase-9 proteins, and down-regulating the expression of Bcl-2 protein. The results of the study were consistent with those in the literature, and it was indicating that Neo promoted apoptosis in liver cancer cells and have a signi cance for the study of anti-cancer mechanisms.
In conclusion, Neo showed inhibition effect on proliferation of HepG2 cells and SMMC-7721 cells in vitro. The survival rate of the two cells decreased and cell apoptosis rate increased in a dose-dependent and time-dependent way. The mechanism of antitumor effect may be related to cell cycle arresting in the G0/G1 phase .The up-regulating expression of Bax, Caspase-3, Caspase-8 and Caspase-9 proteins and down-regulating the expression of Bcl-2 proteins in both cells may also show the mechanism of cell apoptosis when the dose of Neo is increasing . This study suggested that Neo may be a promising candidate or major compound for the development of liver cancer, providing data support for further animal and clinical trials.