Study On Anti-Cervical Cancer Mechanism Of Polysaccharides Of Patrinia Heterophylla Bunge

Cervical cancer is one of the most common gynecologic, which is the second most common cancer in women worldwide and threatens the health of women. At present, chemical anticancer drugs are used to heal the patients efficiently, also result in serious side effects. So, many researchers pay attention to exploitation of natural drugs, which is effective and safe. Many botanical polysaccharides have been investigated with a view to their application for inhibiting tumor growth because they possess strong bioactivity and do not result in drug resistance. Patrinia heterophylla Bunge is being used as a traditional medicine against earlier period cervical cancer, typhoid, leucorrhea, metrorrhagia and metrostaxis. The aim of our study was to assess the effects of polysaccharides fraction 1 of Patrinia heterophylla Bunge (PHB-P1) on cervical cancer and to explain its mechanism, which might provide theoretical supports for developing new anti-cervical cancer.Extraction and activity identification of polysaccharides of Patrinia heterophylla Bunge were studied in the first experiment. Crude polysaccharides of Patrinia heterophylla Bunge was got by water-extraction and alcohol precipitation, and a polysaccharides fraction 1 (PHB-P1) was isolated through further purification, which was uniform in molecular weight and contained ketose, uronic acids and so on. Extraction percentage of PHB-P1 was 1.57%. Cytotoxicity to HeLa was found significantly by treatment of PHB-P1 and the IC50 was 52.26μg/mL, and it failed to inhibit significantly the growth of human peripheral blood mononuclear cells by MTT assay.The inhibitory effects of PHB-P1 on HeLa cells proliferation were performed in the second experiment in vitro. HeLa cells were treated with PHB-P1 (50μg/mL) for 24 h and 48 h. The methods were used in the experiment such as fluorescent stainings, analysis of DNA fragmentation, Tdt-mediated dUTP nick end labeling (TUNEL) assay, cell cycle analysis and reverse transcription-polymerase chain reaction (RT-PCR), which assessed the change of cell morphous and biochemistry feature, and detected the change of activities of gelatinase and telomerase, and examined the expression of some genes in HeLa cells. Our results showed that PHB-P1 could induce HeLa cells apoptosis and inhibit significantly the activities of gelatinase and telomerase. Furthermore, cell cycle analysis showed the accumulation of tumor cells in the G0/G1 phase and a relative decrease of the G2/M phase. And the caspase-3 acticity was increased significantly. Meanwhile, PHB-P1 showed the up-regulation of p53, p14ARF and Bax; also PHB-P1 significantly inhibited the expression of Bcl-2 in tumor tissues.The effects of PHB-P1 on antioxidant capability, immunomodulation cell cycle distribution and induced-apoptosis of U14 tumor-bearing mice were investigated in the third experiment. In the study, cyclophosphamide was considered as positive control drug and physiologic saline was used for negative control group. The U14 tumor-bearing mice models were established, and all the animals were treated the drugs for 15 d. The colorimetric method was used in the experiment. Our results showed that the growth of solid tumor was inhibited significantly after administrated U14-bearing mice model with PHB-P1 (80, 40 mg/kg b.w.) and CTX for 15 days, and the life span increasing ratio was 73.28%, 40.46% and 54.20%, respectively. The poison effect of PHB-P1 on liver and kidney of the mice was not found. PHB-P1 (80, 40 mg/kg b.w.) treatment could elevate the level of serum T-AOC (P<0.01, P<0.01) and SOD (P<0.01, P<0.05), but decrease the MDA level in the mice (P<0.05, P<0.05). It also improved the T-AOC level and deducted the MDA level in liver of the mice. Only higher dose PHB-P1 could enhance significantly the level of SOD in liver of the mice. Meanwhile, the level of kidney tissues T-AOC was increased by 79.65% and 59.75% in the mice treated with PHB-P1 (80, 40 mg/kg b.w.), but there was no obvious effect on MDA level. The higher dose PHB-P1 could enhance the level of SOD in the kidney tissue (P<0.05). PHB-P1 (80, 40 mg/kg b.w.) treatment could increase the thymus index to 1.37 and 1.22 from 0.79 compared with the control group, and also the spleen index was increased from 11.71 to 13.58 and 11.88 respectively. However, CTX treatment inhibited significantly the thymus index. Moreover, the level of IFN-γwas increased by 41.22% and 30.22% in the mice treated with PHB-P1 (80, 40 mg/kg b.w.), and higher dose PHB-P1 could inhibit significantly the activity of TNF-α(P<0.05) but the lower dose. Furthermore, the activity of IL-2 was elevated in some degree but the significant difference was not found.The effect mechanism of PHB-P1 on the tumor growth of U14 tumor-bearing mice was investigated in the fourth experiment. In the study, cyclophosphamide was considered as positive control drug and physiologic saline was used for negative control group. The U14 tumor-bearing mice models were established, and all the animals were treated the drugs for 15 d. The colorimetric method, flow cytometry, and immunohistochemistry and so on were used in the experiment. Our results showed that the solid tumor growth was significantly inhibited by treatment of PHB-P1 (80, 40 mg/kg b.w.) and CTX, and the tumor ratio was 42.68%, 31.10% and 54.06%, respectively. Moreover, PHB-P1 (80, 40 mg/kg b.w.) and CTX treatment could induce tumor cells apoptosis and the ratio was 27.85%, 20.96% and 31.03%, respectively. Furthermore, the level of serum LDH was decreased by 23.94% and 18.92% in the mice treatment of PHB-P1 (80, 40 mg/kg b.w.) but did not affect significantly the AKP activity. It showed the accumulation of tumor cells in the G0/G1 phase (P<0.01) and a relative decrease of the S phase (P<0.01) after PHB-P1 treatment. The solid tumor growth was inhibited by PHB-P1 treatment, which might result from the down-regulation of mutant p53 and Bcl-2 and the up-regulation of Bax and p19^ARF protein.The study was performed with HeLa cells in vitro and with U14-bearing mice models in vivo, we analyzed systematically the mechanism of anticancer and regulation pathway, which would provide theoretical supports for further development of PHB-P1 as a natural anti-cervical cancer drug.