| Part I: Angiotensin II promotes epithelial-mesenchymal transition in nonsmall cell lung cancer cells and its mechanisms Objective:Metastasis and invasion have become the basic features and important markers of malignant tumors.Epithelial-mesenchymal transition(EMT)has been shown to promote local infiltration of tumor cells and systemic dissemination of cancer.The renin-angiotensin system(RAS)is an important neuro-humoral regulation system,in which Angiotensin II(Ang II)is the main effect molecule.In recent years,it has been reported that Ang II is expressed in local tissues,especially tumor tissues,and has been confirmed to play an important role in the occurrence,development and metastasis processes of tumors,but the effect on EMT in tumor cells of Ang II is rarely reported.The current researches on RAS in tumors mainly focused on the effects of Angiotensinconverting enzyme inhibitor(ACEI)and Angiotensin II receptor antagonist(ARB)on tumor cells,while study on the direct effect of Ang II on tumor cells is less.Therefore,this study explored the effects of Ang II on EMT in non-small cell lung cancer(NSCLC)cells and the related mechanisms,in order to improve the research of RAS in the field of cancer from a new perspective,and to provide a new theoretical foundation and possible treatment for cancer treatment strategy.Methods:The following experiments were carried out with A549 cells and H460 cells:(1)Scratch assay and Transwell migration assay were used to detect the migration motility of cells;(2)Transwell invasion assay was used to detect invasion ability of cells;(3)The expression and distribution of related proteins in cells were detected by Western blot and immunofluorescence;(4)The relative mRNA levels in cells were detected by qRT-PCR;(5)The distribution and metastasis of A549 cells in the tail vein lung metastasis model of nude mice were detected by in vivo bioluminescence.(6)The content of TGF-?1 in the cell supernatant was determined by ELISA kit..Result:(A)The effect of Ang II on EMT in A549 cells1.Scratch assay and Transwell assay results show that Ang II promoted the healing of scratches in A549 cells and H460 cells,increased the number of transmembrane cells in Transwell migration assay,and had no significant effect on cell invasion ability,suggesting that Ang II has a certain promoting effect on the migration ability of NSCLC cells.2.Western blot and qRT-PCR results showed that Ang II could down-regulate Ecadherin protein in A549 cells,up-regulate Vimentin protein,and increase the mRNA levels of Slug,Snail and Zeb1.But Ang II had no effect on EMT-related protein markers in H460 cells,suggesting that Ang II can induce EMT in A549 cells.3.The results of lung vein metastasis of nude mice showed that the lungs in the Ang II group had larger fluorescence area and stronger fluorescence intensity.General images and HE staining showed that the nodules in the lung tissues of Ang II group increased significantly.Results suggested that A549 cells treated with Ang II have a stronger metastasis ability.(B)the mechanism of Ang II induced EMT in A549 cells1.Western blot and qRT-PCR results showed that Ang II up-regulated the expression and mRNA levels of transforming growth factor-?1(TGF-?1).ELISA results showed that Ang II increased TGF-?1 in supernatant of A549 cells.The TGF-? receptor antagonist SB431542 can inhibit the induction of EMT by Ang II,suggesting that Ang II can induce EMT by promoting TGF-?1 transcriptional translation,increasing TGF-?1 protein expression.2.Western blot and qRT-PCR results showed that Ang II up-regulated the protein expression and mRNA levels of sirtuin-related enzyme I(SIRT1);SIRT1 inhibitor EX527 inhibited Ang II-induced EMT;SB431542 inhibited the upregulation of SIRT1 protein expression by Ang II,suggesting that Ang II can increase the expression of SIRT1 protein and induce EMT through TGF?1.Conclusion:Ang II induces EMT in A549 cells through up-regulating SIRT1 by increasing TGF-?1,and enhances the migration and metastasis ability of A549 cells.Part II: Intervention and mechanism of 20(S)-protopanaxadiol on Ang II induced EMT in A549 cells Objective:There are many reports on the inhibitory effects of ACEI and ARB on tumor metastasis,which is considered to be a promising new strategy for anti-tumor metastasis.In the previous study of our laboratory,it was found that ginseng total saponins and 20(S)-protopanaxadiol saponins could increase Ang II degradation and reduce the level of Ang II in local tissues under different conditions,and exerted the protective effect on heart,kidney and liver.20(S)-protopanaxadiol(PPD)is a glycoside of 20(S)-protopanaxadiol saponin extracted from the roots,stems and leaves of ginseng(American ginseng).PPD has been shown to have a good anti-tumor activity,but it is mostly focused on the induction of apoptosis,while the research on inhibition of tumor cell metastasis was very few.Therefore,this study of PPD on Ang II induced EMT in A549 cells,can provide experimental foundation for the selection of PPD clinical indications,and provide a new idea and theoretical foundation for the development of anti-tumor drugs for traditional Chinese medicine.Methods:The following experiments were carried out on A549 cells:(1)Scratch assay and Transwell migration assay were used to detect the migration motility of cells;(2)Transwell invasion assay was used to detect invasion ability of cells;(3)The expression and distribution of related proteins in the cells were detected by Western blot and immunofluorescence;(4)The level of related mRNA in the cells was detected by qRTPCR;(5)The distribution and metastasis of A549 cells in the tail vein lung metastasis model of nude mice were detected by in vivo imaging;6)The content of TGF-?1 in the cell supernatant was determined by ELISA kit.Result:(A)Inhibitory effect of PPD on Ang II induced EMT in A549 cells1.The results of scratch test and Transwell migration experiment showed that PPD could inhibit the effect of Ang II on the scratch healing of A549 cells,and reduce the number of transmembrane cells increased by Ang II in Transwell migration assay,but the migration ability of A549 cells was not obvious.The results suggest that PPD inhibit the promoting effect of Ang II on the migration ability of A549 cells.2.Western blot and qRT-PCR results showed that PPD can antagonize Ang II,upregulate E-cadherin protein,down-regulate Vimentin protein,and decrease the mRNA levels of Slug,Snail and Zeb1,suggesting that PPD can inhibit Ang II-induced A549 cell EMT.3.The results of nude mice showed that PPD can reduce the fluorescence area and intensity of lung in nude mice of Ang II group,and reduce the number and size of nodules in lung tissue,suggesting that it can inhibit the transfer of A549 cells after Ang II treatment.(B)the mechanism of PPD inhibiting Ang II induced EMT in A549 cells1.Western blot results showed that PPD could inhibit the increase of TGF-?1 protein expression induced by Ang II.The results of ELISA showed that PPD could decrease the content of TGF-?1 in A549 cells induced by Ang II,suggesting that PPD can inhibit Ang II.Induced TGF-?1 autocrine.2.Western blot and immunofluorescence showed that PPD could inhibit the upregulation of SIRT1 by AngII;SIRT1 overexpression or EX527 inhibited SIRT1 expression,and up-regulation of SIRT1 protein level could effectively antagonize PPD inhibition of EMT,while PPD synergistic effect with EX527 reversed the effect of Ang II on EMT protein markers and transcription factors,confirming that SIRT1 is one of the important proteins of PPD inhibiting EMT,suggesting that PPD inhibits AngII and induces EMT in A549 cells by down-regulating SIRT1.Conclusion:PPD inhibited the secretion of TGF-?1 by Ang II,down-regulate SIRT1 and inhibited EMT on A549,which is one of the mechanisms of its anti-migration and antimetastatic effects. |
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