Mechanistic Study Of Emodin's Anti-Cancer Effects On Human Liver Cancer HCCLM3 Cell Line

Some traditional Chinese medicinal herbs have anti-tumor effects and less side effects, which can interrupt the progression of tumor through multiple signaling pathways. Therefore, Traditional Chinese medicine gradually becomes the hot spot of research in tumor treatment.Emodin is a natural anthraquinone derivative. It has been reported to inhibit the growth and proliferation of many tumors such as breast, lung, cervical,colorectal, and prostate cancers et al. The high mortality of cancer is associated with the metastatic spread of tumor cells from the original site. Emodin interferes with the progression of tumor metastasis at several potential points. For example,it interferes with cell adhesion, cell invasion and so on.Our experiments further studied the mechanism of emodin's anti-cancer effects on the basis of the predecessors' studies.Objective To study the emodin's effects on proliferation and differentiation of human highly potentially metastatic liver cancer cell line (HCCLM3) and the correlating anti-cancer mechanism of emodin.Methods: 1. MTT assay was used to determine the growth inhibition of HCCLM3 cell by emodin. Flow cytometry was applied to detect the change of cell cycle and expression level of ROS. The laser confocal technique was used to analysis morphological changes of cell nucleus and expression level of ROS of HCCLM3 treated with emodin. The expression of p53 and Bcl-2 were detected by immunocytochemical (ICH) staining.2. The invasive ability, the adhesive ability and the migration ability of HCCLM3 cells treated with emodin were determined by the invasion or trans-well test, adhesion test and immunology experimental technique.3. Reverse transcriptase polymerase chain reaction (RT-PCR), ICH and immunohistochemistry (IHH) staining and western-blot were employed to examine the change of expression of L-FABP and FAS in HCCLM3 treated with emodin and in HCC and its adjacent liver tissues obtained from the surgical specimens of HCC patients.4. Statistical data were analyzed by SPSS13.0. The results obtained from each experiment are expressed as mean±SD. The significance level was set at P＜0.05 for each analysis using Studentt's test and analysis of variance (ANOVA).Result: 1.The results of MTT assay indicated that the proliferation of HCCLM3 cells was significantly inhibited by emodin in a time- and dose-dependent effect.G₂/M phase of cell cycle increased, and the apoptotic peak was observed by flow cytomeric assay. The percentage of apoptotic HCC cells change with the concentration of medicine. The expression of p53 protein in human HCCLM3 cells treated with emodin obviously increased. However, the expression of Bcl-2 protein decreased. Experimental groups were significantly different from the control group (P＜0.05). Furthermore, the ROS content significantly increased (P＜0.05).2. The number of calls adhesion was dramatically lower compared with control group(P＜0.05). The OD value of in lower chamber treated with emodin obviously decreased (P＜0.05). The immunology experiment result showed emodin could enhance the sensitivity of cytotoxin T and nature killing cells to HCCLM3 cells.3. The results of semi-quantitative RT-PCR, IHH, ICH and semi-quantitative Western blot are consistent. FAS and L-FABP compare nearby the cancer in the primary liver cancer expression the organization remarkable enhancement (P＜0.05). After the emodin inhibited the liver cancer cells, FAS and the L-FABP expression drops (P＜0.05).Conclusions: 1. The emodin could induce apoptosis of HCCLM3 cells. The percentage of apoptotic HCCLM3 cells is related to the time and dose. The time-response and dose-response relationship is obvious. The growth of HCCLM3 ceils were suppressed by inducing apoptosis, which may be related to the change of the p53, Bcl-2 protein, the cell cycle and and the level of ROS. It also indicated that emodin probably become a potential anti-tumor drug.2. Emodins can weaken the cellular adhesive effect, weaken the adherence between the cell and the matrix, and decrease the cellular penetration performance and movement performance. Moreover, it also can enhance the specific and nonspecific immune responses leading by circumference uninuclear cell, to prevent the cancer cells from escaping. All results indicated the emodin has the anti-tumor effects on inhibiting invasion and metastasis.3. Our results suggested that emodin could inhibit the expression of FAS and L-FABP in a dose-related manner, that is, emodin could inhibit the synthesis and transportation of fatty acid. This is the first report that emodin has inhibitive effects on both FAS and L-FABP, and this inhibition of FAS in a dose-dependent manner indicated that emodin may suppress the metabolism of fatty acid, and then suppresses the multiplication in hepatocellular carcinoma cell line HCCLM3 cells.Our study strongly suggests that emodin could be a promising candidature for research and development of new anti-tumor drugs. So far, this old remedy has offered encouraging evidence of anti-tumor effects in vitro and in vivo. With the unraveling of more and more molecular mechanisms involved, this anthraquinone can be developed either as chemotherapeutic drug by itself or in combination. To reach the magnitude and specificity of effective cancer treatment, cooperative investigations are necessary to be carried out in the areas of oncology, chemistry, and pharmacology.