The Effects And Mechanisms Of Huaier On Human Cervical Cancer Cells

Cervical cancer is the most common cancer of the gynecologic malignancies. The recurrence and metastasis of tumor are still threatening women’s lives, in spite of early diagnosis and treatment due to the screening of cervical cancer. The main treatments of cervical cancer are surgery, radiotherapy and chemotherapy.The quality of life should be considered when treating younger patients, such as fertility sparing, sexual activity and function of ovary. Although the enlarged extent of surgery and adjuvant radiotherapy can reduce recurrent rate under certain circumstance, the treatments may not be accepted by young patients attribute to the complications including stenosis of vagina, fistula of bladder and rectum. It may also do harm to the relationship between the patients and their husbands. Although platinum-based chemotherapy has activity for the treatment of metastatic disease, cisplatin resistance is still a big problem. Therefore efforts should be devoted to search new anti-tumor agents from natural products.Huaier is a kind of fungi and has been used for the treatment of various diseases in China for many years. However its anti-tumor effects have not been found until recently. The effective ingredient of Huaier extract is proteoglycan. The anticancer activity of Huaier extract has been demonstrated in vitro and in vivo in hepatocarcinoma, breast cancer, colorectal cancer, melanoma, and ovarian cancer. Huaier inhibits different tumors through various mechanisms, including inhibition of cell growth, induction of tumor cell apoptosis, inhibition of tumor-induced angiogenesis and inactivation of the epithelial-mesenchymal transition (EMT).It is unknown what effects Huaier will have on cervical cancer cells. We investigated the anti-tumor mechanisms of Huaier in cervical cancer cells including three parts.PART Ⅰ The role of Huaier on proliferation of human cervical cancer cellsBACKGROUND AND OBJECTPrevious studies have advocated that Huaier could obviously inhibit the proliferation of breast cancer, hepatocarcinoma, ovarian cancer cells, and so on. Huaier played the role in a variety of ways including induction of tumor cell apoptosis, autophagy and cell-cycle arrest. It is unknown about the effects of Huaier on cervical cancer cells. In this part of study we put different doses of Huaier extract into different types of cervical cancer cell lines, and observed the inhibitory effects using various methods. Furthermore the underlying mechanisms were explored.METHODSCell viability was measured by MTT assay and colony assay. The distribution of cell cycle and cell apoptosis were analyzed by flow cytometry. The expression of proteins related to special molecular signaling pathway was assessed using western blot.RESULTS1.The effects of Huaier on cervical cancer cell morphologyCompared with the untreated cells, the majority of the Huaier-treated SiHa and CaSki cells changed from spindle to star-shaped with sharp outlines, and some of which become the special ’wiredrawing’ morphology. But most of the C33A cells did not show obvious changes and were killed directly to death.2. The inhibitory effects of Huaier on cervical cancer cells by MTT assayThe proliferation of both SiHa and C33A cells which treated with different concentrations of Huaier for 48 and 72 h was inhibited in a time- and dose-dependent manner. The C33A cells were more sensitive to Huaier treatment than SiHa cells. The IC50 values in SiHa and C33A cells treated with Huaier for 48 h were approximately 5.20 mg/ml and 3.95 mg/ml, respectively. Huaier could also inhibit the proliferation of CaSki cells in a dose-dependent manner, but not does-dependent manner. In addition, the cell numbers did not decrease when Huaier concentration was less than 2mg/ml.3. The inhibitory effects of Huaier on cervical cancer cells by colony formation assayAfter 8 days continuous culture with tumor cells, Huaier at concentrations of 1 mg/ml and 2 mg/ml significantly inhibit the colony forming abilities of SiHa, C33A and CaSki cells.4. The effects of Huaier on cervical cancer cell cycleIn order to determine whether the anti-proliferative effect of Huaier attributes to its induction of cell-cycle arrest, we treated cells with different concentrations of Huaier for 24 h and analyzed the cell cycle distribution using flow cytometry. The result showed that Huaier increased the percentage of cells at the G2/M phase in a dose-dependent manner in C33A cells, while it had no effects on SiHa and CaSki cells at any tested concentration.5. The effects of Huaier on cervical cancer cell apoptosis rateHuaier could not induce the apoptosis of SiHa and CaSki cells.6. Huaier exerts its anti-cervical cancer activity via MAPK signaling pathways.The levels of phosphorylated ERK (p-ERK) in both C33A and SiHa cells treated with Huaier extract were decreased in a time-and does-dependent manner. On the other hand, the expressions of phosphorylated p-JNK and p-p38 were increased with treatment of increasing concentrations of Huaier extract in both SiHa and C33A cells.CONCLUSIONS1. Huaier extract could inhibit the proliferation of the cervical cancer cells.2. Huaier extract increased the percentage of cells at the G2/M phase in a dose-dependent manner in C33A cells.3. Huaier extract exerts its anti-cervical cancer activity in C33A and SiHa cells via p-JNK/p-p38 signaling pathway.PART II The role of Huaier on migration and invasion of human cervical cancer cellsBACKGROUND AND OBJECTRecurrence and metastasis of cervical cancer are the main causes of poor prognosis. Previous studies have reported that Huaier can inhibit the abilities of cell mobility and invasion in vitro and vivo in various cancers, such as breast cancer, hapetocarcinoma and ovarian cancer. The mechanisms include anti-angiogenic function, inactivation of EMT, and so on. It is unknown whether or not Huaier may also play an important role on cervical cancer cells’invasion capacity. In this part we studied the effects of Huaier extract on cell mobility and invasion in cervical cancer cells and explored the underlying mechanism.METHODSMigration and invasion assays were performed using the 24-well transwell chambers. After incubated with Huaier (2mg/ml) for 24h～48h, the cells which moved through the membrane to the lower surface were counted. The expression of proteins related to special molecular signaling pathway was assessed using western blot.RESULTS1. Huaier extract could inhibit the abilities of migration of cervical cancer cells.The mobility of SiHa、C33A and CaSki cells were all inhibited after incubated with low concentration Huaier extract (2mg/ml). CaSki cells were more sensitive than the other two cells.2. Huaier extract could inhibit the abilities of invasion of cervical cancer cells.SiHa and CaSki cells were studied in this part and the results showed both cells moved through the membrane obviously decreased in Huaier group compared with control group.3. Huaier exerts its anti-invasion activity via ERK/β-catenin signaling pathwaysThe levels of phosphorylated ERK (p-ERK) in CaSki cells treated by Huaier extract decreased in a time-dependent manner. In addition, the expression of β-catenin also reduced with treatment of increasing concentrations of Huaier (0,1,2,4 mg/ml).CONCLUSIONS1.Huaier extract could inhibit the abilities of migration and invasion of cervical cancer cells.2. Huaier extract exerts its anti-invasion activity via ERK/β-catenin signaling pathwaysPART III Huaier extract decreased the tumorigenic ability of cervical cancer cellsBACKGROUND AND OBJECTAlthough experiments in vitro have a lot of advantages including environment control, repeated experiments, minor error and lower cost, it cannot consider other systems related to tumor, such as immune system and neuroendocrine system. However, animal experiments are most important methods which can overcome these disadvantages, and can explain the occurrence and progress of tumor and evaluate the efficacy of new agent. As we have confirmed above that Huaier played an important role on proliferation and invasion of cervical cancer cells, then we will investigate the anti-cancer effects of Huaier in vivo.METHODSSiHa cells were subcutaneously injected into the right flank of each BALB/c nu/nu female mouse. After 2 days, each mouse was given 100μl solution containing 50 mg Huaier extract (test group) or medium only (control group) by gavage daily. The mouse body weight and tumor size were measured regularly. Tumor volume (mm3) was determined by the length (a) and the width (b) as V=ab2/2.RESULTS1.There was no difference in mouse body weight between the test group and control group.As the tumors growing, all the mice drank much and looked a little fatigued. The body weight of mice increased slightly at the end of the test. But there was no obvious difference between the two groups.2. Huaier inhibited tumor growth in vivo.a slight reduction of tumor volume in the mice treated with Huaier occurred one week after the last gavage and the significant reduction was found four weeks later, compared with the untreated group (141.57±98.44 mm3 versus 88.71±66.97 mm3,P= 0.0093).CONCLUSIONSTumorigenic assays in mice showed that Huaier prevented tumor growth.