| Kidney cancer is a common malignant tumor of the urinary system,and the incidence rate is increasing.Traditional chemotherapeutic drugs will damage to the autoimmune system when killing cancer cells.Weak patients may have weaker immune systems,which can accelerate the spread of cancer cells.Therefore,it is urgent to develop new drugs,which can not only inhibit tumor proliferation,but also improve immunity and the patients’ life quality.As the only alkaline amino oligosaccharide with positive charge in nature,chitosan oligosaccharide has excellent biological activities such as anti-inflammatory,anti-tumor and immune enhancement.In the present work,water-soluble chitosan oligosaccharide（COS）were used as the research object to evaluate the immune enhancement ability of COS,and its mechanisms on renal cancer inhibition.Three different immune model were used to systematically study the immunoenhancement ability of COS both in vitro and in vivo.Based on the transcriptome sequencing results,we indicated that COS enhanced the phagocytosis in COSstimulated RAW264.7 and increased the levels of inflammatory factors,such as NO、TNF-α、IL-6 and IL-1β.In the cyclophasphamide-induced immunosuppression model,it was found that COS could significantly restore phagocytic index（P<0.01）,lymphocyte proliferation（P<0.05）,and natural killer cell activity（P<0.05）to relieve the immunosuppression.COS can also significantly increase the spleen indices（P<0.05）,up-regulated the CD4+/CD8+ ratio in splenocytes（P<0.05）,and improve the survival rate in 60Co-γ-induced irradiation injury mice.In the S180 subcutaneous xenograft tumor model,COS significantly increased spleen indices（P<0.05）,enhanced the activation of T lymphocytes and natural killer cell activity in splenocytes（P<0.05）,and promoted TNF-α expression in plasma.Moreover,the combination of COS and cyclophosphamide has a more significant inhibitory effect on subcutaneous transplanted tumors（P<0.01）,up to 74.5% and 89.3%.Based on the anti-tumor function and in vivo biodistribution of COS,an orthotopic xenograft tumor model was designed to evaluate the antitumor efficacy of COS.The kidney was found to be a major target organ through synthesis of cyanine-7 labeled COS（COS-Cy7）and IVIS technology.COS repressed tumor growth in a dose-dependent manner,and the inhibiton rate of highest dose（40 mg/kg）reached to 55.8%.The results of immunohistochemisty and immunofluorescence staining showed that COS induced apopotosis and ROS accumulation in tumors.As an immunopotentiator,COS promoted caspase-3 activation and played a sensitizing role in adjuvant radiotherapy.In the case of adjvant chemotherapy,a synergistic effect was observed when COS combined with equal-dose chemotherapeutic drugs.Taking COS can also reduce the amount of chemotherapeutic drugs（5-Fu,from 30 mg/kg to 15 mg/kg）,but achieve the same therapeutic effect.The mechanism of COS inhibiting the growth of renal carcinoma was studied by molecular biology and signal pathway analysis.COS can inhibit the growth of renal carcinoma cells in dose-dependent manner,causing G2/M phase arrest,DNA damage and inducing apoptosis of renal carcinoma cells.Differential genes were screened in renal cancer cell KCC853 after COS treatment by transcriptome sequencing technology.After GO and KEGG analysis,it was found that COS was mainly involved in the metabolic pathway,PI3K-Akt pathway,and NF-κB pathway,etc.COS broke the redox balance of renal cancer cells,and changed the ROS levels,which activated the endogenous antioxidant defense mechanism in cell s.COS induced endoplasmic reticulum stress in renal cancer cells by activating GRP78-PERK-e IF2α-ATF4-CHOP pathway,promoted mitochondrial releases of Cyt c,decreased mitochondrial membrane potential,stimulated ROS accumulation,and induced apoptosis in tumor cells.The inhibition of COS on renal carcinoma involved multiple signaling pathways,such as endoplasmic reticulum stress,endogenous apoptosis,immune enhancement,etc.,which jointly exerted the biological activity of antagonizing renal carcinoma. |
