Low Dose Naltrexone (LDN) Inhibits Malignant Biological Behavior Of Colorectal Cancer And Its Related Mechanisms

Object: Colorectal cancer(CRC)is the third most common cancer in the world.It is also the fourth most common cancer that causes cancer patients to die.The incidence of colorectal cancer is increasing year by year.The incidence of colorectal cancer is expected to double by 2035.At present,surgery is still the main treatment of colorectal cancer,chemotherapy is an auxiliary means.With the development of drugs,targeting drugs anti VEGF and EGFR antibodies have been approved by the U.S.Food and Drug Administration(FDA)for the treatment of metastatic colorectal cancer,but there are always limitations in targeting therapy,such as some drugs need gene detection before treatment,and generally have the disadvantages of high cost and long treatment course.The combination of PD-1 mAb and CTL-A4 inhibitor also showed therapeutic effect,but it was obvious in patients with gene microsatellite instability.Naltrexone(NTX),also known as naltrexone hydrochloride,was first developed by DuPont company in 1963,and passed the review and listing of FDA in 1984.It is mainly used in the treatment of opiate addiction,alcohol dependence and drug withdrawal.Naltrexone is commonly used when the dosage is 50-100 mg / d,when uesd less than5 mg / d,was called low dose naltrexone(LDN).It is found that LDN can alleviate the symptoms of immune related diseases such as multiple sclerosis,inflammatory myofibrodynia,Crohn's disease and inhibit the growth of many tumor cell lines.LDN combined with chemotherapy drugs can reduce the side effects of chemotherapy and enhance the effect of chemotherapy.Although LDN has been reported to be effective in immunotherapy and anti-tumor therapy,its mechanism is still under study.Whether LDN can also inhibit colorectal cancer and its mechanism remains to be further explored.Macrophages play an important role in immune defense and tumor microenvironment.Tumor related macrophages are derived from monocytes,which aggregate near the tumor by cell chemokine 2(CCL2).Influenced by the tumor microenvironment,they undergo the process of gradually developing into mature macrophages.According to the activation state and cell function,they are mainly divided into M1 macrophage(classical activated macrophage)and M2 macrophage(alternativeactivated macrophage).Under the induction of lipopolysaccharide,IFN-? and TNF-?,macrophages can form M1 type macrophages,secrete IL-1,TNF-?,IL-6 and IL-12,which can inhibit tumor growth;under the action of IL-4 and IL-10 TGF-?,macrophages can form M2 type macrophages,secrete TGF-?,EGF and VEGF,which can promote tumor growth.The regulation of macrophages and cytokines may affect the development of tumor.The purpose of this study is to further explore the mechanism of LDN inhibiting colorectal cancer in vitro and in vivo,from the following three aspects: 1.Whether LDN can inhibit the proliferation of colorectal cancer,affect cell cycle,promote apoptosis,and the best concentration and dosage.2.In vivo study on the inhibition of LDN on the proliferation of subcutaneous transplanted tumor of colorectal cancer in nude mice,and the effect on tumor malignancy,and analyze the effect of LDN on different types of macrophages and related cytokines.3.In-depth study of signal pathway,what kind of signal pathway LDN affects to produce the effect of tumor inhibition.Through in vitro and in vivo experiments,the inhibition of LDN on colorectal cancer was further clarified,which laid a theoretical foundation for the clinical use of drugs.Methods: 1.In vitro,CCK-8 kit was used to detect the effect of LDN at different concentrations on colon cancer cell lines(SW480,HCT116)and normal colon mucosa cell line NCM460,to check the inhibition rate of tumor cells,and to determine whether LDN specifically inhibits tumor cells.qRT-PCR and WB methods were used to detect the expression of OGFr in colon cancer cell lines(SW480,HCT116)and the amount of expression.In vitro,different concentrations of LDN(0,0.25,0.5,1,1.5,2mg / ml)were applied to human colon cancer cell lines SW480 and HCT116 respectively,and the effect was detected at the time points of 24 h,48h,72h;the best concentration and time of action were found for the subsequent functional experiments of colon cancer cells.The morphological changes of colon cancer cells were observed by light inverted microscope.The proliferation ability of SW480 and HCT116 cell lines was further detected by clonogenesis experiment;the apoptosis of SW480 and HCT116 cells was observed by Hoechst 33258 staining;the apoptosis and apoptosis stage of SW480 and HCT116 cells were detected by flow cytometry.2.In vivo,the model of colon cancer transplanted subcutaneously in nude mice wasestablished.After subcutaneous inoculation of SW480 cells into the head and neck of nude mice,the nude mice were randomly divided into two groups: LDN group(5mg / 2d)and negative control group(NS).The body weight and subcutaneous tumor volume of nude mice were monitored.After 19 days,the cervical vertebrae were dislocated and the nude mice were killed.The paraffin sections of tumor were prepared,and the following tests were performed: HE was used to detect the histopathology,the expression of OGFr and Ki67,and the expression of tumor associated macrophages(TAMs)surface markers CD68,CD80 and CD163.The expression levels of TNF-? and IL-10 were detected by immunohistochemistry.3.Signal pathway study: LDN of 1mg / ml was used for 48 hours to collect mRNA and protein from colon cancer cells of control group and administration group,mRNA expression of Bax,Bcl-2,Caspase-8,caspase-9,caspase-3 and Survivin was detected by qRT-PCR,and protein expression of Bax,Bcl-2,Caspase-8,caspase-9,caspase-3 and PARP was detected by Western blot.Results: 1.In vitro,LDN significantly inhibited the proliferation of SW480 and HCT116 colon cancer cells,and there was a time-dependent and concentration dependent effect,but it had no significant inhibitory effect on NCM460.LDN increased the expression of OGFr in SW480 and HCT116 cells.The optimal concentration of LDN was 1 mg / ml and the optimal time was 48 hours.Under the action of LDN,the cells of SW480 and HCT116 decreased in size and number,but the cell membrane was intact.LDN induced apoptosis of colon cancer cells.LDN down regulated the expression of Ki67 and decreased the malignant degree of tumor.2.LDN can inhibit the growth of human colon cancer SW480 transplanted subcutaneously in nude mice.After LDN treatment,the tumor volume and weight of subcutaneous transplantation in nude mice decreased significantly,but there was no significant change in the weight of nude mice.Histopathological examination showed that large area necrosis and apoptosis appeared in the central area of the tumor in the LDN treatment group,and nuclear pyknosis and hyperchromatism were seen,while in the control group,the cells were closely arranged,the morphology was neat,and a small number of tumor cells were necrotic.In vivo experiments show that LDN can delay tumor growth by inducing tumor cell apoptosis.The results of immunohistochemistryshowed that OGFr was expressed in the transplanted tumor tissues of nude mice,and LDN could up regulate the expression of OGFr and down regulate the expression of Ki67.The results were consistent with the in vitro experiment.LDN can inhibit colon cancer by inducing tumor associated macrophages(TAMs)to increase the number of M1 macrophages.In the quantitative analysis of immunohistochemistry,it was found that compared with the control group,the number of TAMs labeled with CD68 and M1 macrophages labeled with CD80 increased significantly in LDN treatment group,while M2 macrophages labeled with CD163 had no significant difference.In terms of related cytokines,the expression of TNF-? secreted by M1 increased,while the expression of IL-10 secreted by M2 decreased.Therefore,after LDN treatment,M1 macrophages increased,which promoted the antitumor activity of TAMs.3.LDN group can up regulate the expression of apoptosis related genes and proteins Bax,caspase-9,caspase-3 in SW480 and HCT116 cells,and down regulate the expression of Bcl-2,which is anti apoptosis.LDN can down regulate the protein expression of PARP and significantly reduce the mRNA level of survivin.However,it had little effect on caspase-8 mRNA and protein expression.Conclusions: 1.LDN can inhibit the proliferation of colorectal cancer at the appropriate dose.2.LDN can induce apoptosis of colorectal cancer cells.3.LDN can up regulate the OGFr expression,down regulate Ki67 and reduce tumor malignancy.4.In vivo experiments suggest that LDN may play a role in tumor inhibition by increasing the activity of M1 macrophages and enhancing the antitumor activity of TAMs.5.LDN regulates tumor cell microenvironment,promotes TNF-? secretion and inhibits IL-10 secretion.6.LDN can inhibit colorectal cancer by regulating Bax / Bcl-2 / caspase-9 /caspase-3 / PARP signaling pathway to induce apoptosis.