Effect And Mechanism Of Nitric Oxide Donor NO-2 Against Drug Resistant Tumor

Objective:To investigate the anti-multidrug resistant tumor activity and mechanism of nitric oxide donor NO-2,as well as the role of NO-2 in inhibiting invasion and metastasis and related mechanisms.Methods:(1)The ability of Glutathione to release NO on NO-2 and JS-K was assessed by Nitric oxide assay;Flow cytometry and laser confocal microscopy were used to detect the changes of NO content in K562/A02 and MCF-7/ADM cells after different doses and times of NO-2 incubation.(2)The toxicity of NO-2 to K562,K562/A02,MCF-7,MCF-7/ADM and 7702 was determined by MTT assay;Flow cytometry and confocal laser microscopy were used to detect the change of Mito SOX,JC-1,Annexin V-FITC/PI,MDC fluorescence intensity with concentration,which reflected the effect of NO-2 on cell mitochondrial ROS,mitochondrial membrane potential,apoptosis and autophagy;Western blot was used to detect the effects of different concentrations of NO-2 on the expression of apoptosis-related proteins and autophagy related proteins in drug-resistant tumor cells.(3)MTT assay was used to detect the effect of NO-2 on the survival rate of drug-resistant tumor cells at short-term subtoxic dose and the synergistic effect of different doses of NO-2 and adriamycin in long-term;Western blot was used to detect the effect of different doses of NO-2 on P-gp expression of K562/A02 and MCF-7/ADM cells;Flow cytometry was used to detect the effects of different concentrations NO-2 and JS-K on intracellular accumulation of adriamycin and rhodamine 123 in drug-resistant tumor cells for a short period;Na~+K~+-ATPase kit was used to detect the effects of NO-2 and JS-K on Na~+K~+-ATPase content in drug-resistant tumor cells under short-term and long-term conditions.(4)The effect of NO-2 and JS-K on scratch healing of MCF-7/ADM cells was detected by cell scratch assay;Transwell assay was used to detect the effects of NO-2 and JS-K on invasion and metastasis of K562/A02 and MCF-7/ADM resistant tumor cells;Western blot was used to detect the effects of NO-2,JS-K and inhibitors on expression of i NOS and metastasis-related proteins in drug-resistant tumor cells.(5)MCF-7/ADM cells were used to establish drug-resistant animal models of breast cancer in nude mice,and the changes of tumor volume,body weight and viscera index in different groups were detected;HE staining was used to detect the histological changes of heart,liver,spleen,lung and kidney tissues;Western blot was used to detect the expression of P-gp and apoptosis-related proteins in tumor tissues;Blood routine test blood index changes;BALB/C mouse model of lung metastasis was established with 4T-1 cells,and the number of lung metastasis nodules and organ index of different groups of mice were detected;HE tissue staining was used to detect the changes of metastatic nodules in lung tissue;Western blot was used to detect the effect of i NOS expression levels and the expression of transfer-related proteins in lung metastasis tissues of mice.Results:(1)The total NO release of NO-2 and JS-K in PBS solution increased with the increase of GSH concentration;In K562/A02 and MCF-7/ADM cells,NO-2 could increase the release of NO in a concentration-dependent manner;its release was time dependent and reached the peak at 6 h.(2)NO-2 and JS-K had good antitumor effect on tumor cells of different germ lines and drug-resistant tumor cells;NO-2can dose-dependently promote mitochondrial ROS and apoptosis rates in drug-resistant tumor cells,increase mitochondrial membrane potential and decrease P62 and Beclin-1 mediated autophagy activation.(3)In short term low dose,NO-2 has little toxicity to K562/A02 and MCF-7/ADM,and has no significant effect on P-gp expression,but can significantly increase the accumulation of ADM and Rh123 in drug-resistant tumor cells,and the content of Na~+K~+-ATPase gradually decreased with the increase of NO-2concentration,indicating that NO-2 can inhibit the efflux function of P-gp and increase the accumulation of drugs in cells;NO-2 significantly inhibited the expression of P-gp and the content of Na~+K~+-ATPase in drug-resistant tumor cells at a long-term toxic dose,and the accumulation of adriamycin was significantly increased in NO-2+ADM group compared with ADM group.The accumulation of ADM in cells was significantly increased,and NO-2 and ADM had a good synergistic effect in the range of 1～50μM,and the synergistic indexes were all less than 1(CI<1).(4)NO-2 inhibited the healing of cell scratches and the number of cells invaded and migrated to the lower ventricle.NO-2 significantly decreased the expression of i NOS.(5)MCF-7/ADM xenograft tumor animal experiments showed that both NO-2 and JS-K had good antitumor effects,and the combination of NO-2 and ADM showed synergistic and attenuated effects;NO-2significantly inhibited the expression of P-gp and apoptosis proteins Cleaved caspase-8 and Bcl-2,and increased the expression of Cleaved caspase-9 and Bax in dissected tumor tissues;Animal lung metastasis model experiments showed that NO-2 significantly reduced lung metastasis,and the number of pulmonary nodules was less in the prevention group((Pre)NO-2);NO-2 decreased the expression of i NOS,which inhibited the expression of MMP-9 and MMP-2,and increased the expression of TIMP-1 and TIMP-2.Conclusions:(1)NO-2 has the role of GSH sensitive release of NO,through which NO can produce oxidative stress and play an anti-drug resistant tumor effect.(2)NO-2 can reverse tumor multidrug resistance by inhibiting P-gp activity at low dose for a short time;In the long term,high dose can inhibit the expression of P-gp and reduce the activity of Na~+K~+-ATPase,while exerting its own cytotoxicity,so it has a good synergistic effect with ADM against drug-resistant tumors.(3)NO-2 can inhibit tumor metastasis,and the mechanism may be related to the decreased expression of i NOS.