Preliminary Study On The Mechanism Of CAI Anti-tumor Action And Combination Therapy

BackgroundLung cancer is the leading cause of cancer-related deaths in the world.Due to the insufficient of third/fourth line therapies are available at present,novel strategies are urgently required.Carboxyamidotriazole is a novel antitumor drug,and it inhibits several tumor lines both in vivo and in vitro.However,in some clinical trials CAI exerts moderate antitumor activity but exhibits mild side effects.Sorafenib is an oral multi-kinase inhibitor which effectively inhibits cancer proliferation.In addition,sorafenib increased progression-free survival(PFS)in patients with non-small-cell-lung-cancer(NSCLC),but it did not change the overall survival(OS).Thus,we investigated the effectsand possible mechanism of the combination of CAI and sorafenib in NSCLC.Moreover,cancer cachexia is a complex disorder characterized by inflammatory responses and it is associated with poor performance status and high mortality rate of cancer patients.CAI exerts anti-inflammatory features in the treatment of many diseases.Therefore,we investigated the preventive and therapeutic effects of CAI on muscle loss occurred in mice with advanced Lewis lung carcinoma(LLC).In addition,in the present research we found CAI promoted the protein content of phosphor-ACC(phosphor-acetyl-CoA carboxylase),the first key enzyme of fatty acid synthesis.The phosphorylation of ACC(Ser 79)is the marker of AMPK activity and indicates the inhibition of fatty acid synthesis.To explore the potential mechanism of CAI affecting bioenergetic signaling in cancer cells,we investigated on the changes ofupstream of ACC under CAI treatment.MethodsThe proliferation of NSCLC cells were measured by SRB staining and combination indices were calculated.Cell apoptosis and ROS production were detected by flow cytometry using corresponding dyes.RNA and protein levels of NANOG were tested by qPCR and western blot analysis.The bFGF as a stimulator of NANOG was applied to identify the possible mechanism that downregulation of NANOG is involved in the accumulation of ROS by CAI and sorafenib.The anti-tumor effect of CAI and sorafenib was investigated using C57BL/6J mice bearing LLC cell model.LLC cell line was treated with CAI,and cell proliferation was determined using the SRB assay.The in vivo effectiveness of CAI was evaluated in a LLC xenograft model.Protein and mRNA expressions were assessed by Western blotting and qPCR.Pro-inflammatory cytokines in serum were examined by ELISA.The in vitro activation of SIRT1 by CAI was analyzed by a fluorescence-based method.Western blot was performed to analyze the expression of pACC under the treatment of CAI in different concentrations.STO609,the inhibitor of CaMKK?,was added in cotreatment with indicated drug in LLC and A549(LKB1 mutant)and western blot was performed to test the expression of pACC.Ex 527 and nicotinamide,the inhibitors of SIRT1,as well as H-89,the inhibitor of PKA,were added in cotreatment with indicated drug and western blot was performed to test the expression of pACC.Results1.CAI synergized with sorafenib inhibited NSCLC cells(LLC,A549 and NCI-H1975)proliferation.2.The combination of CAI and sorafenib promoted apoptosis as well as elevated the protein content of cleaved-caspase 3 and cleaved-PARP.The apoptotic percentage of cells co-treated with Z-VAD-FMK,a pan-caspase inhibitor,was significantly decreased in comparison with treatment of indicated drugs alone.3.Combined therapy induced accumulation of ROS and mitochondrial associated superoxide.4.CAI and sorafenib could decrease mRNA and protein expressions of NANOG in vitro.Concomitantly using bFGF with indicated drugs could stimulate NANOG expression and synchronously promote cell proliferation.5.Adding bFGF can attenuate ROS production followed combined therapy treatment.6.Co-treated 40 ng ml-1 bFGF and 4 mM GSH with indicated drugs could partially revert the effect of sorafenib and/or CAI on cell apoptosis.7.CAI in combination with sorafenib(10 mgkg-1)significantly suppressed tumor growth in vivo and the efficacy of the combination group was equivalent to sorafenib(30 mgkg-1)monotherapy group.In addition,the average carcass weight in combination group was heavier than sorafenib(30 mgkg-1)monotherapy group.8.Combination group showed down-regulation of NANOG expression and increase MDA concentration in vivo.9.The carcass weights of CAI-treated group were significantly higher than the vehicle group from Day 19 to the end of study.The gastrocnemius and epididymal adipose tissue weights were increased by CAI treatment.10.Furthermore,CAI treatment inhibited the proteolysis in muscles by decreasing expressions of muscle specific Fox03 transcription factor and ubiquitin E3 ligases(MuRF1 and atrogin 1).Moreover,CAI restricted the NF-?B signaling,downregulated the level of TNF-? in muscle and both TNF-? and IL-6 levels in serum,directly stimulated SIRT1 activity in vitro and induced SIRT1 content in muscle.11.CAI increased the protein level of pACC in a dose-dependent manner in vitro.12.CAI inhibited ACC in a CaMKK? and LKB1 independent manner,but partial dependent on SIRT1 and PKA.ConclusionCAI enhances the antitumor activity of sorafenib in NSCLC which may be associated with the induction of apoptosis and inhibition of NANOG.Moreover,CAI can alleviate muscle wasting and is a promising drug against lung cancer cachexia.