| Background:The incidence and mortality of lung cancer are high.According to the histopathological characteristics of lung cancer,it can be divided into two categories:small cell lung cancers(SCLC)and non-small cell lung cancers(NSCLC),of which NSCLC is the most common type.About 85%of lung cancer patients are NSCLC.The 5-year survival rate of NSCLC is only 10%-15%.Continuously exploring new tumor-related genes and possible molecular mechanisms,understanding their impact on the occurrence and development of lung cancer and finding new effective therapeutic drugs are urgently needed in the treatment of lung cancer.Signal converter and activator of transcription 3(STAT3)is an important signal transduction protein involved in intracellular cytokine and growth factor-related reactions,and is important for cell differentiation,growth,proliferation and apoptosis.The process plays a regulatory function.Studies have shown that STAT3 is closely related to the occurrence,development and prognosis of tumors.In recent years,research on STAT3 inhibitors has become a hot topic in the development of anti-tumor drugs.Through virtual drug screening technology,a variety of STAT3inhibitors have been discovered,including peptides and peptidomimetics,natural products and small molecule drugs,etc.However,because peptides and peptidomimetic compounds are easily metabolized in the body and their bioavailability is also low,the drug performance is not ideal.Therefore,researchers are focusing on natural STAT3 inhibitors.Natural products in nature contain a large number of active monomers that can inhibit the activity of STAT3,of which terpenoids and flavonoids are the main ones.Sesquiterpene lactones are a new type of natural terpenoids with a 15-carbon skeleton structure,mainly from the genus Compositae and is one of the cores in medicinal plants.It has been confirmed that a variety of sesquiterpene lactones have various biological functions such as anti-inflammatory,anti-tumor,deworming,antibacterial,hypoglycemic and analgesic,etc.,and eupatolide is an important part of it.It can be extracted from Inula.Existing in vitro studies have proved that eupatolide has anti-inflammatory,anti-aortic vascular smooth muscle cell proliferation and migration effects.There are also studies suggesting that eupatolide can inhibit epithelial-mesenchymal transition(EMT)process through the TGF-β1 signaling pathway.In addition,eupatolide can also inhibit the EMT process mediated by TGF-β1 by down-regulating Smad3 phosphorylation and reducing the expression of TGF-βtype I receptor.At present,there is no relevant research on the role of eupatolide isolated from natural plants in the treatment of NSCLC and whether it participates in regulating the activation of STAT3.The research focus of this article is to explore the therapeutic effect and possible mechanism of eupatolide on NSCLC,and whether it participates in the regulation of STAT3.The research results of this article are divided into the following two parts.Part I:Effect of eupatolide on cell proliferation and apoptosis of non-small cell lung cancerObjective:To study the effect of eupatolide on cell proliferation and apoptosis of non-small cell lung cancer.Methods:1.The ffect of different concentrations of eupatolide on the viability of NSCLC cell lines:human bronchial epithelial cell line(HBE)and NSCLC cell lines(A549,H460,H1299,H1975)were treated with increasing concentration(0,5,10,20μM) for 24 h.The cell viability was measured by CCK-8.2.The effect of different time treated with eupatolide on NSCLC cell viability:A549,H1975 cells were treated with increasing concentration(0,5,10,20μM)for 24 h,48 h,72 h.CCK-8 was used to detect the cell viability at different time points.3.Synergistic experiment of eupatolide combined with cisplatin:A549,H1975 cells were treated with 20μM cisplatin,5μM eupatolide,20μM cisplatin+5μM eupatanide respectively for 24 h.CCK-8 was used to detect the cell viability,and the effect of eupatolide combined with cisplatin on NSCLC cell viability was observed.4.Synergistic experiment of eupatolide combined with 5-FU:A549,H1975 cells were treated with 50μg/ml 5-FU,5μM eupatolide,50μg/ml 5-FU+5μM eupatolide for 24 h.CCK-8 was used to detect the cell viability,and the effect of eupatolide combined with 5-FU on NSCLC cell viability was observed.5.Effect of eupatolide on PARP cleavage level of NSCLC cells:A549,H1975 cells were treated with increasing concentration(0,5,10,20μM)for 24 hours.The cleavage level of ribose polymerase(PARP)in A549 and H1975 cell lines was detected by Western blot.6.The effect of eupatolide on the expression of Bcl2 and Mcl1 in NSCLC cell lines:A549,H1975 cells were treated with increasing concentration(0,5,10,20μM)for 24 h.The m RNA expression levels of Bcl2 and Mcl1 in A549 and H1975 cells were detected by q RT-PCR;the protein expression levels of Bcl2 and Mcl1 in A549 and H1975 cells were detected by Western blot.7.Effect of Eupatolide on apoptosis of NSCLC cell lines:A549 cells were treated with increasing concentration(0,5,10μM)of Eupatolide for 12 h,and then were directly stained with annexin V-FITC and propidium iodide(PI)apoptosis detection kit to verify whether eupatoride induced apoptosis of NSCLC cells.8.Animal transplantation tumor test:after establishing xenograft tumor model by subcutaneous injection of A549 and H1975 lung cancer cell lines in nude mice,the experimental group was treated with Eupatolide(30 mg/kg,once a day,for three weeks)and the control group was given oral solvent.The tumor volume was monitored every 3 days,and the tumor growth curve was drawn.After 3 weeks,the weight of tumor tissue was measured and the antitumor activity in vivo was observed.Results:1.The higher the concentration of eupatolide,the lower the cell viability of A549,H460,H1299 and H1975 cells(P<0.05);however,the cell viability of human bronchial epithelial cell lines(HBE)was not significantly affected by different concentrations of eupatolide.2.The longer the treatment time was,the lower the A549 and H1975 cell viability was.That is to say,the longer the treatment time was,the stronger the inhibition of proliferation of tumor cells was(P<0.05).3.The combination of 20μM cisplatin and 5μM eupatolide had stronger inhibitory effect on A549 and H1975 cells than the control group(P<0.01).4.50μg/ml 5-FU and 5μM eupatolide combined treatment for 24 h had stronger inhibitory effect on A549 and H1975 cell proliferation than the control group(P<0.01).5.The cleavage of PARP in NSCLC A549,H1975 cells was significantly induced by eupatolide,and the cleavage level of PARP was gradually increased with the increase of the dose of eupatolide,showing a positive correlation.6.The expression levels of Bcl2 and Mcl1 m RNA and protein in A549,H1975 cells were significantly decreased by eupatolide,and the expression levels showed a negative correlation with the increase of the dose of eupatolide.7.A549 treated with eupatolide for 24 h,the proportion of early apoptotic cells (annexin V~+/PI~-)and late apoptotic cells and necrotic cells(annexin V~+/PI~+)were significantly increased.8.The tumor volume and weight of nude mice fed with eupatolide were significantly lower than those of the control group(P<0.01).Part II:Eupatolide induces apoptosis of NSCLC through STAT3 signaling pathwayObjective:To analyze the mechanism of eupatolide inducing apoptosis of NSCLC cells.Methods:1.The effect of eupatolide on the expression of p-STAT3 and STAT3 in NSCLC cells:NSCLC cell lines(A549,H1299,H1975)were treated with 20μM eupatolide for 24 h,and the expression levels of p-STAT3 and STAT3 were detected by Western blot.2.The effect of different concentrations of eupatolide on the expression of p-STAT3 and STAT3 in NSCLC cells:the expression levels of p-STAT3 and STAT3 were detected by Western blot after A549,H1975 cells were treated with increasing concentration(0,5,10,20μM).3.The expression of p-STAT3 and STAT3 was detected by Western blot after treatment of A549 cells with 20μM eupatolide for 4 h,8 h,12 h,24 h.4.The effect of EGF on STAT3 phosphorylation induced by eupatolide:A549,H1975 cells were treated with increasing concentration(0,5,10,20μM)for 12 h,and then treated with 50 ng/ml EGF for 20 min.The expression levels of p-STAT3 and STAT3 were detected by Western blot.5.The effect of STAT3 overexpression on the inhibition of proliferation of NSCLC cells by eupatolide:NSCLC cells were transfected with STAT3 overexpression vector,and the cells were treated with increasing concentration(0,5,10,20μM) for 24 h,and the cell viability was detected by CCK-8.6.The effect of STAT3 knockdown on the inhibition of NSCLC cell activity by eupatolide:A549 cells were silenced with STAT3 by sh RNA technique,and the cells were treated with increasing concentration(0,5,10,20μM)for 24 h,andthe cell viability was detected by CCK-8.Results:1.The protein expression of p-STAT3 in NSCLC cells(A549,H1299,H1975)was decreased after treated with 20μM eupatolide for 24 h,but the protein expression level of STAT3 did not change significantly.2.A549 and H1975 cells were treated with increasing concentration(0,5,10,20μM) of eupatolide for 24 h.The protein expression level of p-STAT3 in A549 and H1975 cells decreased with the increase of eupatolide concentration,but the total protein expression level of STAT3 did not change significantly.3.The protein expression of p-STAT3 in A549 cells treated with 20μM zeylactone for different time(4 h,8 h,12 h,24 h)decreased with the increase of treatment time,while the total expression of STAT3 did not change significantly.4.The protein expression of p-STAT3 in A549 and H1975 cells was decreased by increasing concentration(0,5,10,20μM)of eupatolide for 12 h,but the stimulation of 50 ng/ml EGF did not induce the expression of p-STAT3.With the increase of the concentration of eupatolide,the inhibitory effect on EGF activation was further enhanced.5.Compared with the control group,the inhibition of proliferation of A549 cells was significantly reduced after STAT3 overexpression(P<0.01).6.Compared with the control group,after STAT3 was silenced,the inhibitory effect of eupatolide on A549 cell proliferation was significantly enhanced(P<0.01).Conclusion:1.Eupatolide can inhibit the cell proliferation and induce the apoptosis of non-small cell lung cancer,and has synergistic effect with cisplatin/5-FU.2.Eupatolide can inhibit the proliferation of NSCLC cells by inhibiting the activation of STAT3 in NSCLC cells.3.Eupatolide may become a new STAT3 inhibitor for the treatment of NSCLC in the future. |
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