| Objective1.To establish a method for screening natural compounds that targeting small cell lung cancer with index of the cell proliferation inhibition ratio in vitro;2.To investigate the effect of sanguinarine chloride on cell proliferation of BEAS-2B-As cells transformed by arsenic,and to explore the mechanism of malignant transformation of human lung bronchial epithelial cells induced by arsenic;3.To explore the effect of sanguinarine chloride against small cell lung cancer,and the effect of combination with panobinostat on the growth of small cell lung cancer,and to explore its anti-tumor mechanism.Methods1.A experimental study on natural compounds screening that targeting small cell lung cancer.We established a method for screening natural compounds that targeting small cell lung cancer.The effect of 640 natural compounds on cell proliferation of NCI-H1688 cells was measured by using CCK-8 assay.In addition,the effect of 39 natural compounds on cell proliferation of 16HBE cells was measured by using CCK-8 assay.2.A experimental study on the malignant transformation of human lung bronchial epithelial cells induced by arsenic.We established the arsenic-induced transformed BEAS-2B cells(BEAS-2B-As),BEAS-2B cells were exposed to arsenic(0.25?M)for 72h per passage and cultured for 40 passages.The effect of sanguinarine chloride on cell proliferation of BEAS-2B-As cells was measured by using CCK-8 assay.Meanwhile,the expression level of miR-301a in BEAS-2B-As cells when treated with sanguinarine chloride was measured by using qPCR.To explore the role of miR-301a during arsenic-induced cellular transformation,we firstly measured the expression level of miR-301a between non-transformed BEAS-2B cells and arsenic-induced transformed BEAS-2B cells by using qPCR.Meanwhile,the expression level of miR-301a in BEAS-2B cells when exposed to different concentrations of arsenic(0,2.5,5,or 10?M)for 12h was compared.We then compared miR-301a expression in BEAS-2B cells exposed to 1?M arsenic for 0,10,20,30 passages.Moreover,we measured the level of IL-6 in the culture medium in BEAS-2B cells exposed to 0,5,10?M arsenic and H2O2 as positive control by using ELISA.Then we determined whether IL-6 mediates miR-301a levels via inducing STAT3 activation in BEAS-2B cells,we stimulated BEAS-2B cells with different concentrations of IL-6(0,10,50,and 100ng/mL),and measured miR-301a expression.Next,we determined whether arsenic induced the induction of miR-301a was dependent on IL-6 and STAT3,we also measured miR-301a expression in BEAS-2B cells treated with arsenic in the presence of STAT3 inhibitor and IL-6 neutralizing antibody(anti-IL-6).Then,the expression of miR-301a in BEAS-2B cells was inhibited by using transient transfection assay,we measured the protein expression of phosphor-STAT3,total-STAT3 and I?B? in BEAS-2B cells treated with 5 and 10?M arsenic for 24h.Then,to investigate the function of miR-301a in BEAS-2B-As cells,we measured the cell proliferation,colony formation,cell migration and apoptosis in BEAS-2B-As cells with inhibition of miR-301a.To identify the potential direct target genes of miR-301a in BEAS-2B-As cells,we firstly evaluated the protein levels of several reported miR-301a target genes(SMAD4,RUNX3,PTEN,NKRF,BIM,P63,and PIAS3)and one potential target,GADD45 ?.Furthermore,to test the hypothesis that miR-301a directly suppress SMAD4 expression in BEAS-2B-As cells,we analyzed the binding site for miR-301a and SMAD4 RNA 3' UTR by using the miRNA target prediction software Targetscan(version 5.2).Then,to determine whether miR-301a directly binds the mRNA encoding SMAD4 and down-regulates expression of SMAD4,the luciferase activity and the expression of SMAD4 in BEAS-2B-As cells after co-transfected with pGL3-SMAD4 3' UTR and miR-301a mimics were detected by using a luciferase reporter system and western blot.Next,we determined whether SMAD4 affect the phenotype of BEAS-2B-As cells with miR-301a inhibition.We first designed three shRNA against SMAD4 and measured the cell proliferation,cell migration and apoptosis in BEAS-2B-As cells transformed with miR-301a inhibitor and SMAD4 shRNA.Finally,we subcutaneously injected BEAS-2B-As cells either with Anti-miR-control or Anti-miR-301a into nude mice respectively,after that,the volume of tumor tissues was measured,and the number of SMAD4-positive cells was detected by using immunohistochemistry.Moreover,to define the impact of SMAD4 on tumor growth with miR-301a inhibition,we subcutaneously injected BEAS-2B-As cells with anti-miR-301a plus either shRNA control or shRNA-SMAD4 into nude mice respectively,similarly,the volume of tumor tissues was measured,and the expression of SMAD4,phosphor-STAT3 and total STAT3 was detected by using western blot.Meanwhile,to identify the correlations between miR-301a and SMAD4 in lung cancer of clinical patients from the TCGA databases,we chose two types of lung cancer,adenocarcinoma and squamous cell carcinoma with normal lung tissue samples to analyze the expression of miR-301a and SMAD4.3.A experimental study on the anti-tumor effect of sanguinarine chloride and combination with panobinostat for small cell lung cancer.We observed the effect of sanguinarine chloride on cell morphology and cell proliferation of NCI-H1688 cells.Meanwhile,to investigate the toxic effect of sanguinarine chloride on SCLC cells,NCI-H1688,NCI-H82,and NCI-H526 cells were treated with sanguinarine chloride at various concentrations for 24,48,or 72h.Additionally,colony formation assay was performed to assess the long-term toxicity of sanguinarine chloride on the cell proliferation of small cell lung cancer cell lines NCI-H1688.To further explore the anti-proliferative effect of sanguinarine chloride on SCLC cells,we examined whether sanguinarine chloride treatment induced cell cycle arrest in SCLC cells.NCI-H1688 cells were treated with sanguinarine chloride at each designated concentration for 24h,then cells were stained with PI and analyzed the distribution of cell cycle by using flow cytometry.To determine how sanguinarine chloride induces cellular death in SCLC cells,NCI-H1688 cells were treated with sanguinarine chloride at each designated concentration for 24h.Annexin V-FITC/PI staining and DAPI staining assays were carried out to assess the number of apoptotic cells by using flow cytometry and laser scanning confocal microscopy.Moreover,to further explore the inhibition effect of sanguinarine chloride on cell mobility in SCLC cells,wound-healing assay and transwell migration assay were performed.We performed RNA-sequencing(RNA-seq)using total RNA isolated from the SCLC cell lines NCI-H1688 treated with or without sanguinarine chloride.After quality filtering and normalizing the raw sequencing data,we identified the differentially expressed genes(DEGs).Next,we used Ingenuity Pathway Analysis(IPA)software in conjunction with the DEGs to explore the enriched molecular pathways.The mRNA expression of CDKN1A,CDKN1B,CDKN1C and P53 in NCI-H1688 cells and NCI-H82 cells treated with sanguinarine chloride was measured by using qPCR.Meanwhile,the protein expression of CDKN1A,CDKN1B,CDKN1C and P53 was verified by using western blot.In addition,we subcutaneously injected NCI-H1688 cells into nude mice,and established normal control group and sanguinarine chloride treatment group,after that,the volume and weight of tumor tissues was measured,and the number of CDKNIA-positive cells was detected by using immunohistochemistry.To explore CDKN1A expression in small cell lung cancer,we extract the total mRNA and protein from normal bronchial epithelial cells 16HBE and small cell lung cancer cell lines NCI-H1688,NCI-H82 and NCI-H526,CDKN1A expression was measured by using qPCR and western blot.To further investigate the role of CDKNIA in SCLC,we designed four shRNA against CDKN1A,and measured the ability of colony formation and cell proliferation in NCI-H1688 cells transformed with CDKN1A shRNA.Meanwhile,to identify the expression of CDKN1A in lung cancer of clinical patients from the Oncomine and TCGA databases,we chose three types of lung cancer,small cell lung cancer,adenocarcinoma and squamous cell carcinoma with normal lung tissue samples to analyze the expression of CDKN1A.We determined whether the combination of sanguinarine chloride and chemotherapeutic drugs can promotes cell proliferation in SCLC cells.CCK-8 assay was performed in NCI-H1688 cells treated with sanguinarine chloride and/or panobinostat,gemcitabine,THZ1 and JQ1 at each designated concentration according to their own IC50 values for 24,48,and 72h.Then,we analyzed the cell cycle distribution of NCI-H1688 cells treated with sanguinarine chloride and/or panobinostat.Finally,the protein expression of CDKN1A,CDKN1B,CDKN1C and P53 in NCI-H1688 cells treated with sanguinarine chloride combination with panobinostat was measured by using western blot.Results1.Preliminary screening shows 39 natural compounds have the ability of inhibiting the cell proliferation of small cell lung cancer cell line NCI-H1688.And,sanguinarine chloride can not only inhibit SCLC cells but also have low lethality to normal cells.Therefore,sanguinarine chloride was a potential inhibitor of SCLC.2.BEAS-2B-As,16HBE and BEAS-2B cells were treated with 1?M sanguinarine chloride,we found that sanguinarine chloride not only inhibits the cell proliferation of BEAS-2B-As but also has low lethality to normal cells by comparing with the inhibition ratio.BEAS-2B-As cells were treated with different concentrations of sanguinarine chloride(0,0.8,0.9,1,2,3?M)at different time points,and the inhibition ratio was gradually increased with increased concentrations and prolonged treatment of sanguinarine chloride.BEAS-2B-As cells were treated with different concentrations of sanguinarine chloride(0,0.5,1,1.5/?M),and the miR-301a expression was gradually decreased with increased concentrations of sanguinarine chloride.3.BEAS-2B cells were exposed to arsenic(0.25?M)up to 6 months,and then the cells were undergoing malignant transformation.miR-301a was highly expressed in BEAS-2B-As cells compared with non-transformed cells.In addition,BEAS-2B cells were treated with different concentrations of arsenic(0,2.5,5,10?M),the expression level of miR-301a was significantly increased with increased concentrations of exposure to arsenic.Moreover,BEAS-2B cells were treated with 0.25?M arsenic,the expression level of miR-301a was gradually increased with increased time of exposure to arsenic(10-30 passages).4.BEAS-2B cells were exposed to 0,5,101?M arsenic and H2O2 as positive control for 24h,we found that the level of IL-6 was upregulated at highest level with 10?M arsenic treatment.We stimulated BEAS-2B cells with different concentrations of IL-6(0,10,50,and 100 ng/mL),and miR-301a was significantly increased with IL-6 stimulation,however,the effect was abrogated with STAT3 inhibitor(S31-201,30?M)treatment.In addition,BEAS-2B cells were treated with arsenic in the presence of STAT3 inhibitor and IL-6 neutralizing antibody(anti-IL-6).As we expected,arsenic alone treatment results the higher level of miR-301a,while with inhibition of either STAT3 inhibitor or IL-6 inhibition reduced the expression of miR-301a.Then,BEAS-2B cells were transfected with Anti-control or Anti-miR-301a for 24 hours,we measured the expression of phosphor-STAT3,total STAT3 and I?B? in BEAS-2B cells treated with 5 and 10?M arsenic,we found that arsenic activated STAT3 in BEAS-2B cells,and such activation was markedly inhibited by reduction of miR-301a.Furthermore,there was no significant differences of I?B? expression between BEAS-2B cells transfected with anti-control and with anti-miR-301a with arsenic treatment.5.Down-regulation of miR-301a,by transfection with the miR-301a inhibitor,significantly restrained the cell viability and reduced the ability of colony formation.Meanwhile,miR-301a inhibitor compromised the migratory ability of BEAS-2B-As cells.There was no significant difference of the frequency of apoptotic cells between BEAS-2B-As cells introduced with Anti-control and BEAS-2B-As cells with anti-miR-301a.However,inhibition of miR-301a significantly enhanced the chemosensitivity of doxorubicin in BEAS-2B-As cells.6.SMAD4 and NKRF expression was dramatically downregulated in BEAS-2B-As cells than in non-transformed BEAS-2B cells.Inhibition of miR-301a expression significantly enhanced the expression of SMAD4 in BEAS-2B-As cells and did not affect the expression level of NKRF.BEAS-2B-As cells were co-transfected with full length SMAD4 3' UTR sequence and the miR-301a mimic,the luciferase activity was inhibited by 80%and the protein level of SMAD4 was correspondingly reduced.Neither transfection of miR-301a mimic control with SMAD4 3' UTR nor transfection of miR-301a mimic with WT luciferase into BEAS-2B-As cells affected the luciferase activity and SMAD4 protein levels.7.We found that knockdown of SMAD4 via short hairpin RNA(shRNA)significantly enhanced cell proliferation.Similarly,SMAD4 shRNA promotes the ability of migratory of BEAS-2B-As cells with miR-301a inhibition.In addition,in BEAS-2B-As cells transfected with miR-301a inhibitor,SMAD4 shRNA reduced the cellular apoptotic death.8.In the xenograft model,we found that compared to BEAS-2B-As cells with Anti-miR-control,tumor growth was significantly reduced in BEAS-2B-As cells with miR-301a inhibition.Correspondingly,the number of SMAD4-positive cells and the expression of SMAD4 was distinctly increased in the tumor with miR-301a inhibition.The effect of miR-301a inhibition on restraining tumor growth was reversed by the presence of SMAD4 shRNA in BEAS-2B-As cells.Inhibition of miR-301a leads to highly expressed SMAD4 and dramatically attenuated the activation of STAT3,whereas knockdown of SMAD4 was found to restore the activation of STAT3.In consistent with this,knockdown of SMAD4 leads to increase the activation of STAT3 in BEAS-2B cells with miR-301a inhibition treated with arsenic.9.miR-301a displayed significantly higher expression in both lung adenocarcinoma and lung squamous cell carcinoma compared to normal tissue,whereas its target genes,SMAD4 was significantly downregulated in lung cancer sample compared to normal tissue.Interestingly,miR-301a also show inverse correlation with SMAD4 in lung adenocarcinoma,while it did not display significant anti-correlation to SMAD4 in lung squamous cell carcinoma.10.NCI-H1688,NCI-H82 and NCI-H526 cells were treated with different concentrations of sanguinarine chloride(0,0.9,1,2,3,4?M)at different time points,we found that sanguinarine chloride treatment resulted in dose-and time-dependent growth inhibition in SCLC cells.Compared with the cell morphology of NCI-H1688 cells,sanguinarine chloride treatment markedly reduced eell-to-cell contact.Meanwhile,sanguinarine chloride can inhibits cell colony formation of NCI-H1688 cells dependent on time and concentration.11.NCI-H1688 cells were treated with different concentrations of sanguinarine chloride(0,0.5,1,1.5?M),we found that sanguinarine chloride treatment led to an increased number of cells in G1 phase,and a decreased in the number of cells in S phase and G2/M phase.Notably,sanguinarine chloride can blocked cell cycle in G2/M phase with 1?M treatment.Meanwhile,the apoptotic cells were obviously increased in a dose-dependent manner.NCI-H1688 cells which have been treated with scratches were treated with different concentrations of sanguinarine chloride(0,0.5,1,1.5?M),the wound-healing assay showed that the sanguinarine chloride treated cells failed to occupy the scraped space through migration due to their impaired migration capability.Moreover,NCI-H1688 cells were treated with different concentrations of sanguinarine chloride(0,300,400,500nM),the transwell migration assay shown that the number of cells passing through the transwell chamber was decreased significantly in a dose-dependent manner.Meanwhile,the transwell invasion assay showed that the inhibition of invasion was in a dose-dependent manner.12.We performed RNA-seq using total RNA isolated from the SCLC cell lines NCI-H1688 treated with or without sanguinarine chloride.By comparing the reads per kilobase per million(RPKM)values between the two groups,a total of 1184 DEGs(952 upregulated and 232 downregulated)were identified.Next,we used IPA software in conjunction with the DEGs to explore the enriched molecular pathways.A bar-line graph of canonical pathways showing the effect of DEGs on pathway activity in the data set according to-log(p-value)and Z-score.The activation Z-score of neuroinflammation signaling pathway was found to be activated.According to the Diseases&Functions analysis,cell cycle ranked in the top three of molecular and cellular functions,and 47 DEGs were shown to be associated with a cell cycle signaling network,including CDKN1A.Next,NCI-H1688 cells and NCI-H82 cells were treated with different concentrations of sanguinarine chloride(O,0.5,0.75,1?M),CDKN1A and CDKN1B expression was up-regulated in SCLC cells treated with sanguinarine chloride.Meanwhile,the protein expression of CDKN1A and CDKN1B was highly expressed in SCLC cells after 24h treatment with sanguinarine chloride.13.In the xenograft model,we found that compared to normal control group,tumor growth and weight were significantly reduced in sanguinarine chloride treatment group.Correspondingly,the number of CDKNIA-positive cells was distinctly increased in the tumor with sanguinarine chloride treatment.14.The CDKNIA expression(including mRNA and protein levels)in small cell lung cancer cell lines NCI-H1688,NCI-H82 and NCI-H526 was significantly lower than normal cells.Meanwhile,knockdown of CDKN1A via short hairpin RNA(shRNA)significantly enhanced the ability of colony formation and promoted cell proliferation in NCI-H1688 cells.In addition,CDKN1A was significantly downregulated in lung cancer sample compared to normal tissue.15.NCI-H1688 cells were treated with 0.5?M sanguinarine chloride or 60nM panobinostat or combined usage of these two agents,the combination therapy of sanguinarine chloride and panobinostat led to enhanced inhibition of cell proliferation compared with either agent alone.Moreover,the combination of sanguinarine chloride and panobinostat led to a significantly increased number of cells in G1 phase compared with either agent alone.Meanwhile,the protein expression of CDKN1A was significantly increased in SCLC cells treated with both sanguinarine chloride and panobinostat compared with either agent alone.Conelusions1.39 natural compounds are screened as potential inhibitors of small cell lung cancer;sanguinarine chloride not only inhibits small cell lung cancer,but also has little lethality to normal cells,which is a potential inhibitor of small cell lung cancer.2.miR-301a is a carcinogenic miRNA,sanguinarine chloride can inhibits the cell proliferation of BEAS-2B-As cells and can inhibits the miR-301a expression.SMAD4 is the target gene of miR-301a,miR-301a and SMAD4 play an important role in carcinogenesis induced by arsenic.The activation of STAT3/miR-301a/SMAD4 loop is a key positive regulator in human lung bronchial epithelial cells induced by arsenic.3.Sanguinarine chloride inhibits the growth,proliferation,cell cycle,movement,migration and invasion,and promotes the cell apoptosis of small cell lung cancer.Meanwhile,sanguinarine chloride combined with panobinostat can enhance the anti-tumor effect of small cell lung cancer.Sanguinarine chloride plays an important role of anti-small cell lung cancer by up-regulating the CDKN1A expression. |
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