The Anti-Tumor Activities And Underlying Mechanisms Of Polysaccharides From Lentinus Edodes

Cancer has become the leading cause of death in the global, and searching anti-cancer drugs with high efficiency and low toxicity becomes a new strategy for cancer therapy. Journals of "Science", "Nature" and "Cell" have reported one after another that polysaccharides are the third molecular chain carrying important life information after protein and nucleic acid. Polysaccharides play an important role in cells and participate in various life activities, including cell differentiation, cell adhesion and recognition, cellular communication, and cell infection etc. In particular, polysaccharides possess immunomodulatory and anti-tumor activity due to its complex structure, which thus has attracted much attention. As a result, research on structure and function of polysaccharides has become the third major milestone of human beings in the exploration of the mysteries of life. It is well known that polysaccharide macromolecules can be synthesized and metabolized by almost all organisms, and they thus have good biocompatibility and safety. As a biological macromolecule, the polysaccharide from mushroom of Lentinus edodes coded as Lentinan has significant anti-tumor activity, which is closely correlated to its molecular weight, chain conformation and so on. Studies have shown that Lentinan adopts triple helical conformation in water, and its anti-tumor activity significantly depends on the triple helix structure. Moreover, Lentinan has been used for clinical treatment of advanced gastric cancer and lung cancer as an anti-cancer drug in Japan, but the not fully clarified anti-tumor mechanism has become the obstacle greatly preventing the global clinical application of Lentinan. Therefore, this thesis aims at setting up several cancer models to study the anti-tumor activity of Lentinan in vivo and in vitro, and exploring the underlying mechanism, which will provide an important scientific basis for clinical anti-tumor application of Lentinan.The main innovations of this thesis are summarized as follows:(1) In vitro and in vivo experimental results revealed that Lentinan (LNT) significantly inhibited the growth of S-180 sarcoma. It was found that LNT activated the immune system to induce tumor cells apoptosis, targeted tumor suppressor gene p53 to suppress tumor cell proliferation, and inhibited tumor angiogenesis, leading to tumor growth inhibition. (2) It was demonstrated that LNT interacted with cervical cancer HeLa cells followed by inhibiting proliferation of HeLa cells through targeting tumor suppressor gene p53, inducing Hela cell apoptosis through activating caspase- dependent signaling pathway, and suppressing angiogenesis in tumor tissues, all of which led to HeLa tumor growth inhibition. (3) It was clarified that LNT significantly inhibited growth of the transplanted MCF-7 tumors through both intraperitoneal and tail vein injection of LNT. Additionally, it was found that the LNT sample prepared in this thesis showed higher anti-tumor activity against MCF-7 cancer cells than the commercially available LNT injection. (4) It was revealed that LNT inhibited the proliferation of MCF-7 breast cancer cells by targeting tumor suppressor gene p53 and estrogen receptor ERa, inhibiting activation of PI3K-Akt-mTOR signaling pathway and angiogenesis, as well as induced MCF-7 cells apoptosis through activating caspase-dependent pathway. (5) It was demonstrated that LNT could inhibit the growth of human hepatoma HepG2 and squamous cell carcinoma SCC-7 in vivo possibly via activation of p53-or caspase-dependent signaling pathways as well as inhibiting activation of PI3K/Akt/mTOR signaling pathway.The main research contents and conclusions in this thesis are summarized as follows. A water soluble beta-glucan (LNT) was obtained from fruiting bodies of Lentinus edodes, and it was a beta-(1,3)-D-glucan with beta-(1,6) branched chain, and it was a three helical conformation in water and a compliant single stranded conformation in DMSO. In vitro and in vivo experiments demonstrated that LNT exhibited significant inhibition against the growth of S-180 sarcoma with the inhibition rate of 75% at the dosage of 1 mg/kg, which was much higher than anti-tumor effect of the positive drug cyclophosphamide. In addition, the potential anti-tumor mechanism was revealed by using western blot, histochemical and immunohistochemical staining and immunofluorescence staining. In brief, LNT induced immune cell infiltration into the tumor microenvironment through activating the immune system, activated caspase-dependent cell apoptosis pathway and p53-dependent cell proliferation pathway, leading to S-180 tumor growth inhibition. Additionally, LNT blocked S-180 tumor angiogenesis by inhibiting the expression of angiogenic factors such as Stat 3 and VEGF to inhibit tumor growth.It was demonstrated that LNT interacted with cervical cancer HeLa cells to induce ROS production, as well as inhibit proliferation of HeLa cells with almost no toxicity to normal cells by using confocal microscopy, MTT assay and trypan blue staining method. In vivo animal experiments indicated that LNT significantly inhibited growth of the transplanted HeLa tumor with the tumor inhibition rate of 61.2%. At the same time, by using western blotting, siRNA transfection and histochemical methods, it was further revealed that LNT targeted p53 to enhance the expression of cyclin p21, leading to blocking cell division cycle and thus inhibiting HeLa cell proliferation. Moreover, LNT induced HeLa cell apoptosis through targeting p53 to up-regulate Bax expression and down-regulate Bcl-2 expression, and activating caspase-dependent apoptosis pathway, leading to tumor growth inhibition. In addition, LNT blocked tumor angiogenesis by inhibiting the expression of angiogenic factors such as Stat 3 and VEGF to inhibit tumor growth.The results from MTT method and trypan blue staining showed that LNT exhibited remarkably inhibitory effect on the proliferation of estrogen receptor positive breast cancer MCF-7 cells, and almost no inhibitory effect on estrogen receptor negative breast cancer cells (MDA-MB-231 and MDA-MB-468 cells) and normal cells, indicating that LNT showed selectivity on the proliferation of breast cancer cells. The transplanted MCF-7 tumor test revealed that LNT showed anti-tumor activity against MCF-7 tumor through either intravenous injection or intraperitoneal injection, and anti-tumor effect was equal to the positive drug, indicating that LNT inhibited MCF-7 tumor with high efficiency. Different from the positive anti-cancer drug, LNT had almost no toxic side effects on the body, revealing its biological safety. Besides, it was proved that the anti-tumor effect of LNT was better than that of the commercially available LNT injection in the transplanted MCF-7 tumor test in nude mice. Therefore, LNT can be used as anti-breast cancer drug candidates for clinical breast therapy.Based on laser confocal microscopy, western blot, histological and immunohistochemical staining, immunofluorescence staining and flow cytometry technique, the underlying mechanism by which LNT inhibited MCF-7 breast tumor growth in vivo was revealed herein. Briefly, (1) LNT interacted with breast cancer MCF-7 cells to induce ROS production and promote the phosphorylation of ERK1/2, leading to cell cycle arrest at G2/M phase; (2) LNT inhibited the proliferation of MCF-7 cells by targeting p53 to up-regulate the expression of p53 and MDM2, and by down-regulating ERa expression; (3) LNT inhibited the proliferation of MCF-7 cells also through suppressing the activation of PI3K-Akt-mTOR signaling pathway; (4) LNT inhibited tumor angiogenesis, leading to suppression of MCF-7 cell proliferation and tumor progression; (5) LNT induced MCF-7 breast cancer cells apoptosis through activating caspase-dependent signaling pathway.It was demonstrated that LNT significantly inhibited the proliferation of human hepatocarcinoma cell HepG2 in vitro, and significantly inhibited HepG2-and SCC-7-transplanted tumor growth in vivo. The inhibition rate was 63.6% and 48.8%, respectively. Western blotting results showed that LNT markedly up-regulated the expression of p53, Bax and caspase 3, and significantly reduced the expression of PARPl in HepG2 and SCC-7 tumor tissues, revealing that LNT inhibited HepG2 and SCC-7 tumor growth possibly through the p53-and caspase-dependent signaling pathways. Moreover, LNT might inhibit SCC-7 tumor growth closely associated with the repression of PI3K/Akt/mTOR signaling pathway activation.In summary, LNT showed significant inhibitory effects on different tumor cell lines including sarcoma S-180, human cervical carcinoma HeLa, human breast cancer MCF-7, human hepatocellular carcinoma HepG2, and human squamous cell carcinoma SCC-7 cells with almost no cytotoxicity and equivalent anti-tumor effect of commercially available and clinically used anti-cancer drugs. Moreover, LNT showed the common mechanism in different tumor cells. Namely, LNT targeted tumor suppressor gene p53 to inhibit tumor cell proliferation, activated caspase-dependent pathway to induce tumor cells apoptosis, and inhibited tumor angiogenesis, all of which led to tumor growth inhibition. This thesis involved the interdisciplinary field of polymer physics, molecular biology, biochemistry and basic medicine, and the research results provide an excellent basis for the clinically anti-tumor application of Lentinan, which have important academic value and application potentials.