Screening Of Combined Anti-tumor Strategies For Cannabinoid Receptor Agonists Based On Synergistic Lethal Theory

Malignant tumors are about to become the number one killer of human health in the 21 st century.With the advancement of modern biological technology and the deep understanding of the pathophysiological mechanism of tumors,the research on anti-tumor drugs has developed rapidly,and the level of tumor treatment has been significantly improved.At present,tumors have become a curable disease,and tumor survival has become a new normal for cancer patients.Therefore,while maximizing the efficacy of patients with malignant tumors,improving the quality of life of cancer patients has become an increasingly concerned issue in the clinical treatment of cancer.Tumor heterogeneity,tumor treatment,drug resistance,metastasis and recurrence,etc,make the combination of the two drugs and even multi-drugs become the necessary measures to maximize the efficacy of malignant tumors.In view of the fact that cancer patients are suffering from the pain and nausea,loss of appetite,anorexia,depression and anxiety caused by the disease itself and drug treatment,it provides a kind of toxic and side effects and adverse reactions.New drug combination strategies that improve patient quality of life have important clinical value.However,the associated joint scheme is still blank.Synergistic lethal chemistry screening is an unbiased preclinical screening method for synergistic lethal screening to expand into anti-tumor drug combination screening.Unlike clinical drug combinations,which are limited to marketed drugs,synergistic lethal screening can discover new joint targets across the genome,combined with an increasingly rich small-molecule-targeted drug library,to maximize possible drug combinations.Among the current tumor adjuvant therapies,the cannabinoid receptor(CBR)agonist is the most comprehensive treatment for adverse reactions in patients,such as: Cesamet treatment of chemotherapy-induced nausea and vomiting,Sativex for the treatment of neuralgia and In addition,a large number of studies have shown that CB1 R agonists have the effect of stimulating appetite and improving bad mood;many clinically found that CBR agonists have anti-tumor effects in various tumor cell lines and mouse xenografts.Based on the unique advantages of CB1 R agonists in the treatment of tumors,this study aimed to find a new anti-tumor combination combination of CB1 R agonists.The research work is divided into two parts: In the first part,based on the kinase group gene RNAi screening,there is a synergistic anti-tumor kinase inhibitor with WIN55212-2.First,two methods of HCA and CCK-8 cell viability assays using self-installed cell microarrays were used to screen for 675 genes in the human kinase group and combined with the CB1 R agonist WIN55212-2 to cause HepG2 cell death or decreased viability by RNA interference(RNAi).The kinase gene is a possible positive gene for the siRNA group relative cell proliferation ratio <1.09 and the relative cell proliferation ratio difference of the siRNA group and the combined group(siRNA+WIN)is >0.09,a total of 49 possible positive genes,through the literature and genes.In the function manual search,six genes with strong tumor correlation,including TNK1,AXL,MKNK1,PRKCi,PDGFRL and MINK1,were selected.Then,on the 7 CB1 high expression tumor cell lines(HepG2,HCT-8,DU145,SKOV3,MCF-7,PLC,MD-MB-231),the 6 genes identified by the primary screening were combined and tested by MTT method.After rescreening and sieving,it was found that AXL gene siRNA combined with WIN55212-2 had synergistic lethal activity on HCT-8 and HepG2 cells,and HCT-8 was the most active,thus confirming that AXL kinase is effective in combination with WIN55212-2;second,select AXL kinase specific inhibitor TP-0903 in 12 CB1 R and / or AXLR high expression cells(NCI-H446,NCI-H460,NCI-H462,HT-29,HepG2,BxPC3,PC3,Synergistic anti-tumor screening on SKOV3,PLC,MD-MB-231,Caco-2,HCT-8),and found that TP-0903 and WIN55212-2 have a combined lethal effect only on a certain concentration of TP-0903 on HepG2 cells,in HT-29,Caco-2 and HCT-8 cells have a combined lethal effect,of which HCT-8 cells have the best combined activity,the synergy index is: 0.90,0.85,0.82,0.68,0.66;third,using HCT-8 tumor cell colony formation and 3D tumor sphere proliferation inhibition assay validate the combined efficacy of TP-0903 and WIN55212-2,and transplanted in HCT-8 mice The model demonstrated that TP-0903(0.13mg/kg)combined with CBR agonist WIN55212-2 significantly inhibited tumor growth in tumor-bearing nude mice(P<0.05 vs TP-0903 or WIN55212-2 alone),and the inhibition rate was greater than The sum of the inhibition rates of the two drugs alone was low,and the toxicity was low.Fourth,flow cytometry showed that TP-0903 and WIN55212-2 significantly increased the apoptosis rate of HCT-8 cells compared with TP-0903 alone(P<0.01),leading to G0/G1 arrest in HCT-8 cells;immunohistochemical analysis of transplanted tumor tissue Tunnel and CD31+ confirmed that WIN55212-2 combined with TP-0903 can also enhance TP-0903 the ability to induced HCT-8 to apoptosis,to inhibit angiogenesis of transplanted tumors in vivo.The second part,based on drug-drug combination screening,has a synergistic anti-tumor activity combination with a CB1 R agonist.First,through high-throughput screening(HTS)based on in vitro tumor cell proliferation and viability:on 7 CB1 R highly expressed tumor cell lines(HepG2,DU145,HCT-8,MD-MB-231,MCF-7,SKOV3,SKOV3/T),a combination of synergistic antitumor activity with the CBR1 agonist WIN55212-2 was screened from 25 anticancer drugs for the corresponding tumor indications and found on HepG2,DU145 and HCT-8 cells,8 drugs(exemestane,crizotinib,reguginib,raloxifene,pemetrexed,flevis,celecoxib,and everolimus)with WIN55212-2 have good synergistic activity,and the synergistic activity of exemestane combined with WIN55212-2 on HepG2 cells is the best,the synergistic index is: 0.45,0.54,0.58,0.82,0.96;after that,adopting MTT method exemestane and WIN55212-2 combined was used to screen on 9 CB1 R high expression tumor cells(H4,BEL,HepG2,PLC,MX-1,DU145,U251,PC3 and HCT-8)to confirm synergistic antitumor activity of exemestane and WIN55212-2,the synergistic effect was best on the HepG2 cell line,and the synergy index was: 0.42,0.48,0.59,0.72,0.75;In the HepG2 cell colony formation assay and the 3D tumor sphere proliferation inhibition test,the combination of exemestane and WIN55212-2 was confirmed,and the preliminary effect was observed in the pre-experiment of the HepG2 subcutaneous xenograft model,due to the small number of animals,did not show statistical differences;preliminary mechanism of action studies,in vitro and in vivo,WIN55212-2 and exemestane combined can promote exemestane-induced apoptosis(P <0.01);WIN55212-2 and Low and high concentrations of exemestane combined induced cell G2/M and S phase arrest,respectively.Based on the above research results,the subject obtained the following research conclusions: 1.AXL kinase inhibitor TP-0903 and CB1 R agonist WIN55212-2 have synergistic anti-HCT-8 cell proliferation in vitro;AXL kinase inhibitor TP-0903 and CB1 R agonist WIN55212-2 also synergistically inhibit HCT-8 mouse xenograft model;2.Irreversible steroidal aromatase inactivator Exemestane and WIN55212-2 synergistically enhance the anti-HepG2 cells in vitro.It also has a certain effect on the HepG2 mouse xenograft model,but the effect is not obvious,and further optimization of the dose to be administered is needed;3.In vitro and in vivo CB1 R agonist WIN55212-2 combined with AXL kinase inhibitor TP-0903 or aromatase inactivator exemestane enhanced the latter’s induction of apoptosis and cell cycle arrest.It is suggested that the anti-tumor effects of both combinations are closely related to apoptosis;4.In vivo CB1 R agonist WIN55212-2 can enhance the inhibition of TP-0903 to tumor tissue angiogenesis.In conclusion,based on the synergistic lethal chemical screening and drug-drug combination in vitro screening strategy,this study first discovered two targeted drug combinations that synergistically anti-colorectal cancer and liver cancer with CB1 R agonists.Tumor treatment strategies for the efficacy of oncology drugs and improving the quality of life of cancer patients provide new ideas.