Killing Effects And Preliminary Mechanisms Of PAMs To Human Liver Cancer HepG2 Cells

According to the latest statistics, malignant tumors have become the diseases with the highest mortality rate around the world and the number of new cancer cases are increasing by years. Therefore, it is of great importance to find safe and effective drugs to achieve the prevention or treatment of cancers with low side effects. No need of surgery and little side effect have brought natural plant drugs to new hot spots in the field of anti-tumor drug development. The natural plant antimicrobial solution (PAMs), provided by the Yunnan folk medicine Co.Ltd, is the ethanol extract of complex Chinese natural folk medicinal plants including Carthamus tinctorius L., Lithospermum erythrorhizon., Solanum indicum L. and Cymbopogon distans (nees) wats.Folk experience and previous studies have confirmed that PAMs has antibacterial, anti-inflammatory, wound healing and ulcer repairing effects. What’s more PAMs has shown strong killing effects in vitro studies to a wild range of malignant tumor cell linessuch as leukemia, liver cancer, breast cancer and cervical cancer.lt is proved that the PAMs as a traditional medicine of ethnic minorities has greatpotential to be a efficient anticancer drug. However, as a kind of compound medicaments with a variety of plant components, PAMs still needs further investigation to have a better understanding of its antitumor mechanism In this study, HepG2 cells were used to explore the inhibitory effect and molecular mechanism of PAMs in vitro and in vivo. The in vitro effects of PAMs on MCF-7 and MDA-MB-231 of breast cancer cells were also demonstratedin this paper.First of all, morphological observation and CCK-8 assay were employed to study the killing effect of PAMs to the HepG2, MCF-7 and MDA-MB-231 cells,which indicated that PAMs could inhibit the proliferation of these three kinds of cells, in a time-and dose-dependent manner. Results of cell scratch test and Transwell experiments confirmed that PAMs could effectively inhibit the migration of all the cells in vitro. Futhermore, using HepG2 cells as the research object, Western Blot was performed to detectthe expression level of apoptosis-related FoxMl protein, and tumor suppressor P53 protein; the results shown that FoxMl protein, expression was down regulated, while expression level of p53 protein was up-regulated. Combined with earlier results, it is easy to draw a conclusion that that the tumor-killing effect of PAMs had a close correlation with apoptotic pathway of HepG2.The BALB/c (nu/nu) nude mice were used to establish a HepG2 transplanted tumor models, which were construction by injecting HepG2 cells into the nude mice left axillary subcutaneous part. All the tumor model mice were divided into four groups separately treated with alcohol (negative control group), PAMs (experimental group) pre-PAMs(experimental group in advance) and 5-Fu (positive control group). After three weeks, the nude mice were sacrificed and the tumor tissues were dissected to observe and calculate tumor growth, by tumor weight, liver weight, spleen weight, tumor inhibition rate, liver index and spleen index. Tumor pathological changes and tumor cell apoptosis were observed separately by HE staining and TUNEL. In addition, Immunohisto chemical method was used to determine expression changes of caspase3 and caspase9 protein in different experimental groups of tumor cells, then We performed ELISA asssy to measure changes of serum IL-12, TNF-a and VEGF in nude mice after drug treatment. Western Blot, FQ-PCR were conducted to explore inhibitory effect of PAMs from mRNA level and protein level on liver cancer. Results revealed that, tumor suppression rates of PAMs group, Pre-PAMs group and 5-Fu group were 55.13%,57.43% and 56.58% compared with negative control group, showing a extremely significant statistical difference (**P<0.01). Liver index and spleen index in 5-FU group were abnormal, showing significant differences (*P<0.05) to control group, and there was no significant difference between PAMS group, Pre-PAMs group and control group. The toxicity and side effect were quite low compared to positive drug, hence it indicated that PAMS had no harm on nude mice immune system. Expressions of caspase3 and caspase9 protein in PAMs and Pre-PAMs groups increased significantly, while mRNA and protein expressions of MMP-2, FoxMl and P53 were down-regulated. However, VEGF in these two groups declined to a certain extent, with highly significant difference (**P<0.01) to negative control group. IL-12 and TNF-a appeared arise to a certain extent, with significant differences (*P<0.05) to negative control group.Conclusion:All results from experimental studies indicated that PAMs could inhibit HepG2 cells growth in vitro and in vivo through pathways of different levels to HepG2 cells. On the one hand, PAMs could down-regulate FoxM1 transcription factor as well as factors related to apoptosis, to induce cancer cell apoptosis and activate TNF-a factor to achieve directly killing of tumor cells. On the other hand, PAMs inhibited melanoma angiogenesis by down-regulation of VEGF and up-regulation of IL-12, meanwhile, inhibited migration of cancer cells by down regulation of MMP2 expression. Other molecular mechanisms should be discussed in further study.