N-Methylcantharidinimidum Induces Apoptosis in Human Hepatocellular Carcinoma Cell HepG2

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors and the prognosis of patients was poor. Cantharidin is an effective anti-tumor component of Chinese traditional medicine. N-methylcantharidinimidum is a novel cantharidin derivative with better curative effect and less side effect. In this study, the effects of N-methylcantharidinimidum on proliferation, apoptosis and cell cycle of human hepatocellular carcinoma cell line HepG2 were examined. The results demonstrated that N-methylcantharidinimidum significantly induced apoptotic cell death in a dose and time-dependent man-ner and arrested cell cycle in HepG2 cells. N-methylcantharidinimidum also suppressed cyclin D1. This study suggests that N-methylcantharidinimidum may serves as a potential chemopreventive agent for live cancer.


Cell Proliferation Assay
The effect of N-methylcantharidinimidum on cell proliferation was assessed by MTT assay. HepG2 cells were seeded at a density of 1 × 10 4 cells per well in triplicate in 96-well plates and cultured for 24 h to allow the cells for attachment. N-methylcantharidinimidum was dissolved in DMSO and diluted in MEM/EBSS. The cells were treated with 5, 6, 7 and 8 mg/ml of N-methylcantharidinimidum for 24, 48 and 72 h. After treatment, 20 μl of 5 mg/ml MTT solution was added to each well and incubated for 4 h at 37˚C. The supernatant was removed and 200 μl of DMSO was added to each well. The absorbance was quantified using enzyme-linked immunosorbent assay (ELISA) reader at 570 nm.

Morphological Analysis
According to the results of MTT assay, HepG2 cells (5 × 10 4 /ml) were inoculated in 24-well plates with presetted slides, the cells were treated with 5, 6 and 7 mg/ml of N-methylcantharidinimidum for 1 to 6 days. Then the slide were removed and stained with Wright's stain, and observed under light microscope.

Flow Cytometric Analysis
HepG2 cells at 5 × 10 5 cells/ml were inoculated into 6-well culture plate and incubated at 37˚C. The next day, after the medium was removed, 2 ml of MEM/EBSS complete medium with 6 mg/ml N-methylcantharidinimidum was added to each well. After cultured for 3, 4 and 5 d, cells were harvested by trypsinization, washed three times with PBS, and suspended in 500 μl binding buffer. PI (50

TUNEL Assay
In situ cell death, detection kit-POD (Boehringer Mannheim, German) was used to detect the late-stage apoptosis. Cells were seeded in 96-well plates and treated with 6 mg/ml of N-methylcantharidinimidum. Cells were fixed with 4% paraformaldehyde. Fixed cells were penetrated with 0.5% Triton X-100 for 2 min on the ice and then incubated with TUNEL reaction mixture for 1 h at 37˚C. All the slides were stained by DAB coupling and counterstained with hematoxylin. The cells were then examined using a light microscope.

Western Blot Analysis
HepG2 cells were treated with 6 mg/ml N-methylcantharidinimidum for 3 d.

Statistical Analysis
All statistical analyses were evaluated using SPSS 17.0 software. The significance of difference between the groups was analyzed with two-way ANOVA test or two-tailed unpaired Student's t-test. P-values < 0.05 were considered as statistically significant.

N-Methylcantharidinimidum Inhibits Cell Proliferation
The effect of N-methylcantharidinimidum on HepG2 cells was investigated by MTT assay. The results showed that N-methylcantharidinimidum inhibited the proliferation of HepG2 cells in dose-and time-dependent manners ( Table   1). The cytostatic does of 6 mg/ml was taken for further study of N-methylcantharidinimidum on HepG2 cells. After treated with N-methylcantharidinimidum, vacuoles appeared in the cy- 3) In situ determination of apoptosis by TUNEL. The TUNEL assay was used to detect DNA fragmentation characteristic for apoptosis, seen as nuclei stained dark brown. A number of TUNEL positive cells substantially increased after 6 mg/ml N-methylcantharidinimidum treated for 4 days, dark brown granules were seen in the nucleus, indicated that DNA strand breaks had occurred.

N-Methylcantharidinimidum Decreased the Expression of Cyclin D1
The protein level of cyclin D1 was determined by immunoblotting. As shown in Figure 1, expression of cyclin D1 decreased in HepG2 cell after treated with 6 mg/ml N-methylcantharidinimidum for 3 days. β-actin served as an internal control.

Discussion
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors and the third leading cause of cancer-related mortality worldwide [5] [6]. Chemotherapy is one of the therapeutic methods for HCC treatment [7], but the effects are poor due to the relapse of disease and resistance to chemotherapeutics [8]. Recent years, many natural products with stronger antitumor activity were  compounds inhibit cell proliferation in numerous cancer cell lines such as hepatic, bladder, pancreatic, colorectal, leukemic, oral, and breast cancers [9] [10] [11]. In order to improve pharmacokinetic properties, increase efficacy and reduce toxic side effects, some new cantharidin derivatives were synthesized and be studied. Cantharidin showed inhibitory effects on murine ascites hepatoma and ascites reticulum cell sarcoma [12]. Cantharidin sodium and Shenmai injection combined with chemotherapy significantly reduced the incidence of side effects in postoperative breast cancer patients in clinical trial [13]. Norcantharidin, a demethylated analogue of cantharidin, induced cell apoptosis in human oral cancer cells through a mitochondria-mediated pathway [14]. Here, we investigated the anti-tumor effect and its mechanism of N-methylcantharidinimidum, a novel cantharidin derivative, in hepatocellular carcinoma cell line HepG2.
In this study, N-methylcantharidinimidum was shown to exert strong cyto- In conclusion, our study demonstrated that N-methylcantharidinimidum treatment inhibited cell proliferation in HepG2. N-methylcantharidinimidum inhibited HepG2 cells mainly through apoptosis and cell cycle arrest. This work provides a novel insight that N-methylcantharidinimidum may serve as a potential candidate for chemoprevention of hepatocellular carcinoma in the future.