Investigation Of The Anti-tumor Activity And The Underlying Mechanisms Of Polysaccharide From Taxus Chinensis Var. Mairei

Objective:Cancer is the most common disease worldwide with which death is often occurred as a result of metastasis. Metastasis, one of the most demonstrated hallmarks of cancer, is responsible for more than 90 percent of cancer-associated mortality in clinics. Thus, interfering with metastasis has been regarded as one of the most promising point of penetration to improve the current cancer treatment. Therefore, whether in the field of biology or medicine, the research on the mechanisms of tumor metastasis and drug discovery are hotspots of enduring. In our previous work, a complex water-soluble polysaccharide named PSY-1, which was demonstrated to be the most abundant constituent, was isolated, purified and characterized from the leaves of T. chinensis var. mairei. In this study, researches focusing on the anti-cancer and anti-metastasis activity and mechanisms of PSY-1 were carried out. The effect on proliferation of different cancer cell lines, the anti-metastasis effect and the related mechanisms were investigated. It was expected to provide data foundation for the subsequent development of new drug, provide basis for the medicine design and clinical medication and provide insight for the exploitation of Taxus vegetation.Methods:(1) For cell proliferation assay, the anti-proliferation activities were determined using the sulforhodamine B(SRB) assay. Taxol was used as the reference drug. The effect of PSY-1 on 6 cell lines, including the proliferation of the human ovary cancer cell lines A2780 and SKOV-3, human liver cancer cell lines HepG2 and SMMC-7721, human prostatic cancer cell line PC-3, human colon cancer cell line HT-29 and mouse melanoma cell line B16-F10 by exposing them to a serial concentrations of PSY-1 for 72 h were investigated. The optical density (OD) was obtained using a Multiskan Spectrum at a wavelength of 535 nm and IC50 values were then calculated.(2) The effects of PSY-1 on B16-F10 cells migration and invasion were examined using wound-healing assay and Transwell assay respectively.(3) Real-Time PCR was conducted to exam the mRNA expression of MMPs family proteins at the level of transcription. Western blot was used to exam the expression of MMPs proteins and the activation levels of the related proteins which effected by PSY-1 on AKT, MAPK and NF-κB cell signaling pathways. MMPs Gelatin Zymography was performed to detect the enzymic activity of MMPs proteins.(4) The S180 sarcoma, Hep A Hepatic carcinoma and Lewis lung cancer cell xenografted mice model were utilized to evaluate the anti-tumor activity of PSY-1 in vivo.Results:(1) The results showed that PSY-1 had the most potent anti-proliferative activity, with IC50 values of 11.97 mg/mL for the B10-F16 murine melanoma cells. Their IC50 values against six human carcinoma cell lines ranged from 1.55 mg/mL to more than 15.03 mg/mL. The anti-proliferative activity was the strongest in the HepG2 and the weakest in the SMMC-7721, respectively.(2) The Wound-healing assay showed that PSY-1 (0.2 mg/mL,1 mg/mL and 5 mg/mL) could effectively suppresse the B16-F10 cells healing of the scratched wound in a concentration-dependent manner. The Transwell assay further demonstrated that B16-F10 cells treated with 1 or 5 mg/mL of PSY-1 resulted in inhibitory effects on cellular invasion in a concentration-dependent manner. PSY-1 at 1 and 5 mg/mL significantly inhibited melanoma cell migration by approximately 35% and 50%, respectively.(3) The results of Real-Time PCR assay showed that PSY-1(1 mg/mL and 5 mg/mL) could down-regulate the mRNA expression level of MMP-2 in a concentration-dependent manner. The Zymography assay implyed that PSY-1 (1 mg/mL and 5 mg/mL) could inhibit the enzymic activity of MMP-2 and MMP-9. Western Blot assay uncovered that PSY-1 could decreased the expression of MMP-2 and MMP-9 remarkably, and the further results showed that PSY-1 (1 mg/mL,5 mg/mL) could decrease the phosphorylation of IκB, without influncing the AKT, p38 and ERK signaling pathway.(4) PSY-1 could inhibit tumor growth and metastasis significantly. Three doses of the polysaccharide PSY-1 could inhibit the tumor growth in S180 sarcoma, HepA Hepatic carcinoma and Lewis lung cancer cell xenografted mice models.Conclusion:This study showed that PSY-1 could significant inhibit the proliferation of human and carcinoma tumor cell lines in vitro. PSY-1 treatment of non-toxic dose (0-5 mg/mL) exhibited a concentration-dependent inhibition effect on the invasion and migration of the B16-F10 cell. In vivo studies also showed that PSY-1 could inhibit the tumor growth in S180 sarcoma, HepA Hepatic carcinoma and Lewis lung cancer cell xenografted mice models. The present study suggested that PSY-1 exhibited anti-metastatic effect on B16-F10 cell via down-regulating of two important MMPs family members, MMP-2 and MMP-9, as well as decreasing of phosphorylated IκB. PSY-1 could be considered as a potential therapeutic reagent against the metastasis of invasive B16-F10 melanoma cells. To conclude, our current study demonstrated that the extract of Taxus chinensis var. mairei possess the potential anti-tumor and anti-metastasis activities. Those discoveries could provide foundation data for new drug discovery in clinical and the further development and utilization of natural plant resources such as Taxus.