Study On The Efficacy Of Morinda Officinalis Against Colorectal Cancer In Mice And Its Preliminary Mechanism

Colorectal cancer(CRC)is the third most common cancer in China after lung cancer and gastric cancer,whose prevention and control situation are in grave difficulties.However,there is a long process between precancerous lesion to malignant tumors in CRC.And CRC is one of the few malignant tumors that can be detected and cursed early by screening.The key to controlling the incidence of colorectal cancer is to control the precancerous lesions.The key to controlling the incidence of CRC is to control precancerous lesions.Hence,it is very important to find more effective drugs with low toxicity and side effects for preventing CRC.In previous study,we found that ethanol extract of the roots of Morinda officinali(EEMO)had an excellent effect on curing chronic colitis in mice.As there had been no reports on the effect of Morinda officinalis(MO)for treating colorectal cancer,we aimed to investigate whether Morinda officinalis could suppress colorectal cancer in mice models.ObjectiveThe main purpose of this study was to investigate the effects of EEMO and the crude drug of Morinda officinali(CDMO)on AOM/DSS-induced colitis-associated cancer(CAC)models and HCT-116 xenograft tumor model,and evaluated the underlying mechanisms.Methods1.Therapeutic effect and of MO on AOM/DSS induced CAC model and HCT-116 subcutaneous xenograft tumor modelFirst,AOM/DSS-induced CAC model could be divided into three cycles.First cycle,mice were injected intraperitoneally(i.p.)with 10 mg/kg azoxymethane(AOM),and supplied drinking water including 2%dextran sulfate sodium(DSS)for 7 days.Then mice were supplied distilled water for 14 days.Intragastric administration(i.g.)of 5-ASA(100mg/kg),CDMO(200 mg/kg)and EEMO(12 mg/kg)started after first DSS treatment cycle.Then two additional DSS treatment cycles were followed with modification.The mice of control group were given distilled water all the time.During the experiment,the status of the mice was observed daily,and the weight of the mice was recorded.After the experiment,we were supposed to recorded the survival rate of the mice.Mice were subsequently fasted overnight and euthanized with anesthetic.We recorded the length,thickness,and number of tumors of the colon tumor area and non-tumor area.Histopathological examination of the colon was used to evaluate the effect of MO against colorectal.And the effect on curing CRC was accessed by the comparison between groups.HCT-116 subcutaneous xenograft tumor model was used as the second model.After 7 days of adaptive feeding,each nude mouse was subcutaneously inoculated with 106 HCT-116 cells.After the tumor volume of the animals reached 200m3-300mm3,mice were grouped randomly according to the tumor volume and started the administration of 5-FU(30mg/kg),EEMO(12mg/kg),CDMO(200mg/kg),5-FU combined with EEMO(5-FU+EEMO,dose as above)and 5-FU combined with CDMO(5-FU+CDMO,dose as above).During the experiment,the state of the mice,the body weight and tumor volume of the mice were recorded daily.After the experiment,mice were subsequently fasted overnight and euthanized with anesthetic.The weight and volume of the tumors of the mice were recorded,and the tumors were analyzed pathologically to evaluate the effect of MO on HCT-116 xenograft tumor.2.Mechanism of MO on AOM/DSS induced CAC model and HCT-116 subcutaneous xenograft tumor modelThe indicators needed to be detected of tumor tissue in each group was following.First,western blot,immunohistochemistry and ELISA were used to detect the expression of HIF-α/COX-2/PGE2 pathway.Flow cytometry was used to detect the population of myeloid derived suppressor cells(MDSCs)and mononuclear myeloid derived suppressor cells(M-MDSCs)at the tumor tissues.By means of immunofluorescence,flow cytometry and western blot,the regulatory effect of MO on the polarization of tumor associated macrophages was evaluated.Finally,the level of VEGF at the tumor tissues was detected by immunohistochemistry and western blot.Results1.Suppressive effect and of MO on AOM/DSS induced CAC model and human colon cancer HCT-116 subcutaneous xenograft tumor modelIn AOM/DSS-induced CAC mice,EEMO and CDMO had no effect on the body weight of the mice,but both of them could improve the loose stools and bloody stools of the mice,especially EEMO.Additionally,EEMO and CDMO both could improve the survival rate of mice.In terms of tumor incidence,EEMO and CDMO effectively reduced tumor incidence.In addition,EEMO and CDMO both could improve colon swelling,shortening,and tumor growth caused by AOM/DSS.Pathological results showed that EEMO and CDMO had ability to reduce colon damage caused by AOM/DSS.In the xenograft model of HCT-116,MO had no significant effect on the body weight of the mice.What’s more,EEMO and MO could significantly inhibit the growth of tumors.Pathological examination showed that tumor angiogenesis could be inhibited by EEMO and CDMO.2.MO suppresses colorectal cancer in mice may be related to its inhibition of HIF-1α/COX-2/PGE2 pathway and increase of M1 macrophages in tumor microenvironmentFrom the tumor tissue of two model,we found that the expression of HIF-1α/COX-2/PGE2 pathway was reduced by MO.The decreased population of MDSCs and M-MDSCs were found in the tumor tissues.As the level of iNOS+macrophages increased and the level of CD206+ macrophages decreased,which finally caused the proportion of M1/M2 macrophages increased.That meant the polarization of TAMs was regulated by MO toward M1-like macrophages.The decreased expression of VEGF in the tumor tissues was detected.ConclusionEEMO and CDMO had ability to down-regulate the HIF-1α/COX-2/PGE2 signaling pathway,thereby reducing MDSCs and M-MDSCs and improving immune suppression of tumor microenvironment.And through regulating the polarization of tumor-associated macrophages,the proportion of M1 macrophages was increased accompanied with the decreased proportion of M2 macrophages,which exerted anti-tumor effects.In addition,EEMO and CDMO also inhibited tumor angiogenesis by lowering VEGF to inhibit the growth and development of tumors.