Scutellarin Sensitizes Cisplatin Against Non-small Cell Lung Cancer And Its Protective Effect On Cisplatin-induced Renal Toxicity

Objective:Lung cancer is the malignant tumor with the highest morbidity and mortality in China.Non-small cell lung cancer(NSCLC)accounts for 87%of all lung cancers,and 65%-80%of NSCLC patients are diagnosed at an advanced stage,often losing the opportunity for surgery.Cisplatin remains the first-line chemotherapy for the treatment of advanced NSCLC.However,acquired resistance and renal toxicity are produced after cisplatin treatment.At present,the mechanisms of cisplatin resistance and nephrotoxicity have not been fully-elucidated,and it lacks of effective mitigation or reversal strategies,which has become a major problem that plagues NSCLC treatment.Scutellarin is a flavonoid compound found in natural plants,which is the main active ingredient of the traditional Chinese medicine Erigeron breviscapus.It has many pharmacological effects such as improving immunity,anti-inflammatory,anti-oxidation,anti-microbial and anti-tumor.Our previous studies have confirmed that scutellarin effectively inhibited the proliferation of NSCLC cells;scutellarin enhanced the anti-tumor activity of the chemotherapeutic drug bleomycin against hepatocellular carcinoma,and significantly reduced the toxicity of bleomycin-induced pulmonary fibrosis.However,it is unclear whether scutellarin enhances the cytotoxicity of cisplatin on NSCLC and reduces the nephrotoxicity of cisplatin.In this study,we investigated whether scutellarin could synergize with cisplatin to kill NSCLC cells in vitro and in vivo experiments;the protective effect of scutellarin on cisplatin-induced acute kidney injury was investigated by animal experiments and its involved mechanisms were explored.Methods:1.Effect of scutellarin combined with cisplatin on proliferation of A549/DDP cellsEffects of scutellarin combined with cisplatin on proliferation of A549/DDP cells were measured by MTT assay;Using CompuSyn software to evaluate whether scutellarin and cisplatin have synergistic anti-tumor effects;The effect of scutellarin combined with cisplatin on the morphology of A549/DDP cells was observed by microscope;Effect of scutellarin combined with cisplatin on the cell cycle distribution of A549/DDP was determined by flow cytometry.2.Effect of scutellarin combined with cisplatin on apoptosis of A549/DDP cellsEffect of scutellarin combined with cisplatin on the apoptosis of A549/DDP was determined by flow cytometry;Western blotting was used to detect the effects of scutellarin combined with cisplatin on the expression of apoptosis proteins Caspase3,Cleaved caspase3,PARP and p53 in A549/DDP cells;Using p53 inhibitor PFT to investigate the effect of p53 on apoptosis induced by scutellarin combined with cisplatin;Western blotting was used to detect the effects of scutellarin combined with cisplatin on the expression of ERK in A549/DDP cells;Using p53 inhibitor U0126 to investigate the effect of ERK on apoptosis induced by scutellarin combined with cisplatin.3.Effect of scutellarin combined with cisplatin on autophagy of A549/DDP cellsWestern blotting was used to detect the effects of scutellarin combined with cisplatin on the expression of autophagy proteins LC3 and p62 in A549/DDP cells;The number of autophagosomes in A549/DDP cells was observed by transmission electron microscopy;Using autophagy inhibitor HCQ to investigate the role of autophagy in the anti-tumor effect of scutellarin combined with cisplatin;Western blotting was used to detect the effects of scutellarin and cisplatin on the upstream proteins Akt,c-Met and Stat3 in A549/DDP cells;Using Akt or c-Met inhibitor MK-2206 and Crizotinib to investigate the effect of Akt or c-Met on the induction of autophagy and inhibition of cell growth induced scutellarin combined with cisplatin;Using transient transfection over-expressing Stat3 method to investigate the effect of Stat3 on the induction of autophagy and inhibition of cell growth induced by scutellarin combined with cisplatin.4.Inhibitory effect of scutellarin combined with cisplatin on the growth of tumor in nude miceA model of subcutaneous xenograft tumor in nude mice was established.The nude mice were divided into control group,cisplatin group,scutellarin group,cisplatin+scutellarin group.After 21 days of drug treatment,the body weight and tumor volume of nude mice were measured.Fluorescence intensity of nude mice before and after administration was measured by small animal imaging system;After administration,the tumor was removed and weighed;Western blotting was used to detect the expression of p-Stat3,c-Met,p-Akt,p62,LC3,ERK and p53 in tumor tissues of each group.5.Protective effect of scutellarin on cisplatin-induced nephrotoxicityC57BL/6 mice were randomly divided into 6 groups:control group,scutellarin group,cisplatin group,cisplatin+30 mg/kg scutellarin group,cisplatin+60 mg/kg scutellarin group,cisplatin+4 mg/kg dexamethasone group.Mice in the administration group were pretreated with scutellarin or dexamethasone for consecutive 5 days.After administration,except for the control group and the scutellarin group,the other groups received a single dose of intraperitoneal injection of cisplatin.After 72 hours of cisplatin injection,the body weight of the mice was weighed and the animals were sacrificed,and the blood and kidney tissues of mice were collected.The kits were used to detect the concentrations of BUN and CRE in serum of.mice;The histopathological changes of kidney were observed by HE staining;The levels of inflammatory cytokines IL-6 and TNF-? in kidney tissue were determined by ELISA;Western blotting was used to detect IL-6,TNF-a,Bax,Bcl-2,Cleaved Caspase-3,Cleaved PARP,p53,LC3,p62,Atg5,Atg7,Atgl2,p-JNK P-p38,p-ERK,p-Stat3 in the kidney tissues of each group;Immnunohistochemistry was used to detect the expression of p-JNK,p-p38,p-ERK and p-Stat3 in kidney tissues of mice.Results:1.Effect of scutellarin combined with cisplatin on proliferation of A549/DDP cells80,120 ?M scutellarin has no obvious inhibitory effect on A549/DDP cells;80,120 ?M scutellarin synergistically enhanced the inhibitory effect of cisplatin on proliferation of A549/DDP;When the dose of cisplatin was 10 ?g/ml and the dose of scutellarin was 120 ?M,the synergistic index of the two drugs was 0.58,and the combined effect of the two drugs was the most obvious;Compared with the normal group,the cell morphology became significantly longer after cisplatin treatment,and the cell density was significantly reduced,compared with the cisplatin group,the cell morphology of cisplatin+ scutellarin group became significantly shorter and the cell density of the cisplatin+scutellarin group was significantly reduced.Compared with the normal group,cisplatin blocked A549/DDP cells in G2 phase,compared with the cisplatin group,scutellarin combined with cisplatin blocked A549/DDP cells in S phase.2.Effect of scutellarin combined with cisplatin on apoptosis of A549/DDP cellsCompared with the normal group,the apoptosis rate in cisplatin group was significantly increased,compared with the cisplatin group,the apoptosis rate in cisplatin+scutellarin group was significantly increased;Compared with the normal group,cisplatin increased the expression of Cleaved caspase3,PARP,p53 and ERK in A549/DDP cells,compared with the cisplatin group,scutellarin in combination with cisplatin increased the expression of Cleaved caspase3,PARP,p53 and ERK in A549/DDP cells;P53 inhibitor PFT-? significantly inhibited the apoptosis induced by scutellarin combined with cisplatin;ERK inhibitor U0126 could significantly inhibit the expression of p53 protein activated by scutellarin combined with cisplatin;ERK inhibitor U0126 significantly inhibited apoptosis induced by scutellarin combined with cisplatin.3.Effect of scutellarin combined with cisplatin on autophagy of A549/DDP cellsCompared with the normal group,the expression of LC3II in cisplatin group was significantly increased,the level of p62 and the number of autophagosomes increased,compared with the cisplatin group,scutellarin in combination with cisplatin increased the expression of LC3? and the number of autophagosomes,and reduced the level of p62 in A549/DDP cells;The use of autophagy inhibitor HCQ significantly inhibited the sensitization of scutellarin to cisplatin;Compared with the normal group,cisplatin inhibited the level of c-Met,compared with the cisplatin group,scutellarin in combination with cisplatin inhibited the expression of p-Akt and c-Met in A549/DDP cells;Using the Akt inhibitor MK-2206 significantly enhanced the autophagy induced by scutellarin combined with cisplatin;Using c-Met inhibitor Crizotinib significantly enhanced autophagy induced by scutellarin combined with cisplatin,and inhibited the expression of p-Akt;MK-2206 or Crizotinib significantly enhanced the inhibitory effect of scutellarin combined with cisplatin on proliferation of A549/DDP cells;Compared with the normal group,cisplatin reduced the expression of p-Stat3,compared with the cisplatin group,scutellarin in combination with cisplatin inhibited the expression of p-Stat3;Overexpression of p-Stat3 significantly inhibited the autophagy induced by scutellarin combined with cisplatin,and significantly reduced the proliferation inhibition effect of scutellarin combined with cisplatin on A549/DDP.4.Inhibitory effect of scutellarin combined with cisplatin on the growth of tumor in nude miceAnimal studies have shown that,after 21 days of administration,cisplatin injection significantly increased the fluorescence intensity,volume and weight of nude mice,compared with the cisplatin group,scutellarin in combination with cisplatin reduced the fluorescence intensity,volume and weight of nude mice;Compared with the normal group,the body weight of the cisplatin group was significantly decreased,compared with the cisplatin group,the weight of the cisplatin+scutellarin group was significantly increased;Compared with the normal group,cisplatin significantly down-regulated the expressions of p-Stat3,c-Met and p-Akt in the tumor tissues,and up-regulated the expression of LC3II,compared with the cisplatin group,cisplatin+scutellarin down-regulated the expressions of p-Stat3,c-Met,p-Akt and p62 in the tumor tissues,up-regulated the expression of p-ERK,p53 and LC3II.5.Protective effect of scutellarin on cisplatin-induced nephrotoxicityCompared with the normal group,cisplatin injection significantly increased the level of BUN and CRE in serum of mice,compared with the cisplatin group,high dose of scutellarin or Dex significantly reduced the level of BUN and CRE in serum of mice;Compared with the normal group,the weight of mice in the cisplatin group decreased significantly,compared with the cisplatin group,high dose of scutellarin or Dex significantly increased the weight of mice;Compared with the normal group,mice in the cisplatin group showed increased erythrocytosis in the cortex of mouse kidney,obvious tubular degeneration and necrosis(cytoplasmic vacuolization),and a large number of damaged casts in the lumen,and deciduous epithelial cells was seen in the lumen,the cells are arranged in disorder,the cortical vessels were dilated and congested,compared with the cisplatin group,the pathological damage of mice was significantly improved after high dose of scutellarin or Dex administration.Compared with the normal group,the level of IL-6 and TNF-? in the kidney tissue of cisplatin group were significantly increased,pretreatment of high dose of scutellarin or Dex significantly inhibited the inflammatory factors IL-6 and TNF-? level.Compared with the normal group,the ratio of Bax/bcl-2 and the expressions of Cleaved caspase-3,Cleaved PARP,and p53 in the kidney tissue of cisplatin group were significantly increased,pretreatment of high dose of scutellarin or Dex significantly inhibited the ratio of Bax/bcl-2 and the expressions of Cleaved caspase-3,Cleaved PARP,and p53 in the kidney tissue.Compared with the normal group,cisplatin injection reduced the ratio of LC3?/? and the level of Atg7,and increased the expression of p62,however,pretreatment of high dose of scutellarin or Dex significantly increased the ratio of LC3?/? and the level of Atg7,and reduced the expression of p62.Compared with the normal group,the expressions of p-JNK,p-p38,p-ERK and p-Stat3 in the kidney tissue of cisplatin group were significantly increased,pretreatment of high dose of scutellarin or Dex significantly inhibited the expressions of p-JNK,p-p38,p-ERK and p-Stat3.Conclusions:1.Scutellarin could synergistically increase the inhibitory effect of cisplatin on proliferation of A549/DDP cells;Scutellarin combined with cisplatin arrested A549/DDP cells in S phase;2.Scutellarin synergistically enhanced the pro-apoptotic and pro-autophagic effects of cisplatin on A549/DDP cells;3.Scutellarin combined with cisplatin induced apoptosis by activating ERK/p53 signaling pathway in A549/DDP cells;4.Scutellarin combined with cisplatin induced autophagic cell death in cells by inhibiting c-Met/Akt or Stat3 signaling pathway in A549/DDP cells;5.Animal experiments confirmed that scutellarin significantly enhanced the inhibitory effect of cisplatin on solid tumor growth in nude mice;6.Scutellarin significantly alleviated cisplatin-induced renal dysfunction and kidney tissue damage;Scutellarin inhibited cisplatin-induced inflammatory response;Scutellarin inhibited cisplatin-induced renal cell apoptosis;Scutellarin increased autophagy in renal cells inhibited by cisplatin;Scutellarin inhibited MAPK and Stat3 signaling pathways in mouse kidney tissues.