Effects And Mechanism Of Pinus Massoniana Pollen Polysaccharides On Tumor Cell Growth And Metastasis

Cancer remains the second cause of deaths and major public health problem in human and animals.In recent years,with the rise of pet industry,the average life expectancy of canines has increased,and therefore the incidence of canine tumor has also increased.At present,the main treatment of canine malignant tumor in veterinary clinic is surgical resection,with adjuvant therapy such as chemotherapy and radiotherapy.However,these methods usually have short-term or long-term side effects on sick canines,posing their lives serious threats.Therefore,searching for more safe,effective,non-toxic,and tolerable anti-cancer single or combined therapies is one of the major challenges for researchers.Natural products are considered as noval anti-cancer drugs because of their considerable antitumor effects,rich source and low cytotoxicity.Among all kinds of natural products,plant polysaccharides are satisfactory products which could be developed for the prevention and treatment of cancer.Previous studies have shown that Pinus massoniana pollen polysaccharides(PPPS)can inhibit the growth of various tumor cells.However,the targets of PPPS and its antitumor mechanism remain unclear.Therefore,in this study,we firstly evaluated antitumor effects of PPPS in vivo and in vitro,screened and validated key signaling pathway of PPPS acting on antitumor effect through transcriptome analysis,and further explored the potential antitumor targets of PPPS,and revealed the role of target protein in occurrence and development of tumor.The purpose of this study is to provide theoretical basis for the prevention and treatment of PPPS in malignant tumors and targeted therapy of antitumor drugs.This study is mainly divided into the following four parts:1.Evaluation of antitumor effect of PPPS in vitro.To explore whether PPPS has satisfactory antitumor effect,several common tumor cell lines were selected to evaluate the effect of PPPS on tumor cell proliferation through cytotoxicity tests,and it was found that PPPS significantly inhibited HCT-116 cells at safe concentrations.Second,we selected four common polysaccharides and compared their anti-colon cancer effects,the results showed that the anti-colon cancer effect of PPPS is second only to Lentinan.Therefore,we selected two common colon cancer cell lines,HCT-116 and HT-29 cells.It was found that PPPS significantly inhibited the growth of HCT-116 and HT-29 cells in a time-dependent manner.Cell colony formation assay showed that PPPS significantly inhibited HCT-116 cell proliferation.We further evaluated the anti-colon cancer effect of PPPS by morphological analysis.Hoechest 33342 staining showed that colon cancer cells treated with PPPS showed intense stained nucleus and fragmental chromatin.Transmission electron microscopy was used to observe the subcellular structure of HCT-116 cells.It was found that 400 ?g/mL PPPS treatment contributed to chromatin condensation and margination,and the cristae of mitochondria structure destruction and vacuolation in HCT-116 cells.Subsequently,we analyzed the effects of PPPS on apoptosis and cell cycle of colon cancer cells by flow cytometry,and the results showed that PPPS can significantly promote apoptosis of colon cancer cells and arrest the cell cycle at G0/G1 phase.In addition,transwell cell migration assay showed that PPPS significantly inhibited the migration of HCT-116 and HT-29 cells,and down-regulated the expression level of MMP-9 protein in cell supernatant and cell lysates.Quantitative real time PCR(qRT-PCR)results showed that PPPS inhibited EMT in HCT-116 cells.Therefore,PPPS showed satisfactory anti-colon cancer effect in vitro.2.Anti-tumor Effects of PPPS in HCT-116 xenograft tumor model.In this study,twenty nude mice aged four weeks were selected and reared in an independent ventilation cage,and randomly divided into PPPS therapy group(PPPS-Ther group),PPPS prevention group(PPPS-Prev group),the positive drug group(5-FU group),and the negative Control group.The xenograft tumor model of colon cancer was established by subcutaneous inoculation of HCT-116 cells.The mice in PPPS-Prev group were intraperitoneally injected with 400 mg/kg PPPS twice a week for three weeks.At the fourth week,mice in PPPS-Ther group were intraperitoneally injected with 400 mg/kg PPPS,and mice in 5-FU group were intraperitoneally injected with 50 mg/kg 5-FU twice a week.The body weight and tumor sizes of nude mice were recorded.After six weeks,all mice were euthanized,the serum was collected,and the tumor tissues were stripped from the nude mice.The growth curve of mice showed that the body weight of mice in PPPS-Ther group was significantly higher than that of the other three groups,and PPPS had no liver and kidney toxicity in mice by detecting LDH,AST,and ALT levels in serum.Tumor growth curve showed that compared with the Control group,PPPS-Ther group,PPPS-Prev group,and 5-FU group could significantly inhibit tumor growth,and therapy effect of PPPS was better than that of 5-FU group.H?E staining results showed that compared with the Control group,the other three groups had less mitotic counts and fibrous tissues.Subsequently,the expression level of Ki67 in tumor tissues was detected by immunohistochemistry,and the results showed that the positive signals of PPPS-Ther group and PPPS-Prev group were decreased but not significantly.Immunofluorescence results of TUNEL tissue sections showed that tumor tissues in the PPPS-Ther group and 5-FU group presented apoptosis.Western blot results showed that Cleaved Caspase-3,-6,-7,-9,and-PARP expression levels were significantly increased in PPPS-Ther group compared with the Control group.Our results showed that 400 mg/kg PPPS can significantly inhibit tumor growth in mice in vivo without hepatorenal toxicity in mice.3.Transcriptome analysis and verification of PPPS inhibiting the growth of colon cancer cells.To explore the anti-colon cancer mechanism of PPPS,we used transcriptome analysis to explore differentially expressed genes(DEGs)in HCT-116 cells treated with or without 400?g/mL PPPS at 24 h.We selelcted the differentially expressed genes according with log2FC>1 and P value<0.01,and therefore 20 up-regulated genes and 49 down-regulated genes were enriched.KEGG enrichment results showed that DEGs was significantly enriched in TGF-?signaling pathway.To further explore the role of TGF-? signaling pathway in tumor development,we first detected the expression of TGF-?1 in canine tumor tissues,and the results showed that the expression of TGF-?1 in tumor tissues was significantly higher than that in para-cancer tissues.Subsequently,we selected TMAO,a TGF-?/Smad2 signaling pathway activator,and western blotting showed that 100 ?M TMAO increased the expression levels of TGF-?1,pSmad2,and pSmad3 in HCT-116 cells,that activate the TGF?/Smad2/3 signaling pathway.TMAO could increase the expression level of MMP-9 and promote the wound healing of colon cancer cells,and thus promoting the migration of colon cancer cells,but not inducing the EMT of colon cancer cells.We further demonstrated by western blotting and indirect immunofluorescence that PPPS antagonizes the activation of TGF-? signaling pathway by TMAO in colon cancer cells.In addition,detection of MMP-9 expression level and wound healing assay showed that PPPS can counteract the pro-metastasis effect of 100?M TMAO in colon cancer cells.Therefore,PPPS inhibits the metastasis of colon cancer cells through TGF?/Smad2/3 signaling pathway.4.NR5A2 promotes colon cancer cells metastasis by activating TGF-?/SMAD2/3 signaling pathway.To identify the potential target of PPPS inhibiting the metastasis of colon cancer,we first verified the 5 genes with the most significant differences in down-regulated DEGs(NR5A2,LCA5L,EXO5,DCDC1,and ZNF493)by qRT-PCR in three common colon cancer cells(HCT-116,HT-29,and SW480 cells).The results showed that NR5A2 was significantly down-regulated after PPPS treatment.Subsequently,indirect immunofluorescence and western blot confirmed that NR5A2 protein level was also significantly down-regulated after PPPS treatment in colon cancer cells.To verify the role of NR5A2 in tumor development,the expression difference of NR5A2 in colon cancer cells(HCT-116,HT-29,SW480,and Caco2 cells)and normal colon epithelial cells(NCM460)was detected by qRT-PCR,and the results showed that NR5A2 expression level in HCT-116 and SW480 cells was significantly higher than that in NCM460 cells.We detected the expression of NR5A2 in canine tumor tissues by immunofluorescence,and the results showed that the expression of NR5A2 in tumor tissues was significantly higher than that in para-cancer tissues.Therefore,to further verify the role of NR5A2 in anti-CRC of PPPS,the expression of mRNA and protein levels of NR5A2 were significantly decreased by interfering NR5A2 expression.The results showed that the protein expression levels of TGF-?1,p-Smad2,and p-Smad3 in HCT-116 cells were decreased significantly after interfering NR5A2 expression,the protein expression level of MMP-9 was also decreased.Wound healing assay and qRT-PCR showed that interfering NR5A2 expression reduced the migration ability of colon cancer cells,suggesting that NR5A2 can mediate TGF-?/Smad2/3 signaling pathway to promote the metastasis of colon cancer cells.In conclusion,PPPS,as a natural medicinal plant polysaccharides,has favourable anti-colorectal tumor effect in vivo and in vitro.Transcriptomic analysis and mechanism exploration suggested that PPPS inhibited colon cancer cells metastasis through TGF-?/Smad2/3 signaling pathway,and NR5A2 was the key target of PPPS in its anti-colon cancer effect.Interference of NR5A2 expression can decreased the key protein expression levels in TGF-?/Smad2/3 signaling pathway and thus inhibited colon cancer cells metastasis.In addition,TGF-?1 and NR5A2 expressions in canine malignant tumor tissues were significantly higher than those in paracancer tissues.Therefore,PPPS can be used as a candidate drug for tumor therapy or chemopropition,and TGF-?1 and NR5A2 have the potential to become new therapeutic targets for the treatment of canine malignant tumor,providing theoretical basis for the development of novel drugs.