| BackgroundLung cancer has been addressed to be the main element of global cancer deaths. And nonsmall cell lung cancer(NSCLC) accounts for about four fifths of lung cancer. The tumor metastasis and drug resistance make most of the patients with nonsmall cell lung cancer suffered more from disease than the basic tumor death. Despite the recent improvement in this territory of lung cancer metastasis, rigid underlying mechanisms persist elusive. Therefore, it is important to define the molecular mechanism of nonsmall cell lung cancer metastasis to develop innovative effective treatment strategies.Drug resistance is the one of the major reasons why the patients with NSCLC can not be treated efficiently. From the cellular aspects, drug resistance can be considered to be the reslult of the ways which made the cells failure to apoptosis and non-apoptosis death. Anti-apoptosis death is the major hallmark of maligant tumors. Among the numerous symbols of cancer, epithelial-to-mesenchymal transition(EMT) is an innate program that affects the transformation of polarized epithelial cancer cells to more unstationary mesenchymal cells. This phenotypic metamorphosis is straightly associated with metastasis and is controlled by numerous transcriptional factors. In this conversion, there is a suppression of the epithelial marker, E-cadherin, and initiation of the mesenchymal genes, N-cadherin, and vimentin. EMT is customarily provoked by spur from the multifarious matrix proteins outside the cells or dissoluble factors like transforming growth factor-β1(TGF-β1), EGF, LPS, Wnt proteins, IL-6, tumor necrosis factor-α and other cytokines. Among these, TGF-β1 and Wnt pathways are quite active in lung cancer cells and thus play an important role in the induction of EMT in NSCLC. TGF-β1 is a polymorphous cytokine that regulates a heterogeneous cellular faculty including induction of EMT. It exerts its effect through transcription factor Snail.Supplementally, the canonical Wnt/β-catenin signaling pathway plays an substantial role in numerous cancers including NSCLC. The secreted Wnt antagonists Wnt inhibitory factor-1(WIF-1), frizzled-related protein and dickkopf proteins are important regulators in the canonical Wnt signaling pathway. Wnt/β-catenin signaling pathway has been verified to play a crucial role in the development of EMT and cancer metastasis. The activity of β-catenin, which also acts in linking E-cadherin to the cytoskeleton at the cell-cell adhesion junctions to form the epithelial phenotype, is regulated by these secreted Wnt proteins.Curcumin, a mixture of compounds extracted from turmeric, has been reported to induce autophagy, inhibit growth, invasion and metastasis in a variety of tumor cells. In addition, it has also been reported to induce EMT in pancreatic cancer cells. Bisdemethoxycurcumin(BDMC) is one of the three dominant effective chemical components of curcumin. However, the basic mechanism of its action is largely unknown. In order to better understand its mechanisms of actions in NSCLC, we focused on a process of EMT to explain BDMC regulation of biological behaviors including tumor growth, invasion and metastasis.Methods:Therefore, in this study, Western blotting,CCK8 assay, wound healing, Matrigel invasion, RT-PCR, flow cytometry and so on are used to detect the function of BDMC on the biological behaviors and the related mechanisms in NSCLC cells. And we explored the related mechanisms whereby BDMC induced autophagy in NSCLC cells. We also examined the relationship between BDMC and Wnt signaling pathway in the process of EMT induction in NSCLC cells. The status of Wnt signaling pathway and expression of WIF-1 in 95 D cells was analyzed in this study. Furthermore, we investigated the function of the Wnt/β-catenin signaling pathway and WIF-1 in TGF-β1-induced EMT in the presence of BDMC.Results: And we have got the following results and conclusions.We found that the viability of the NSCLC including A549 and 95 D cells was significantly repressed in a dose- and time-dependent manner, it had no destructive properity on normal lung cells. In the process whereby programmed cell death was stimulated by BDMC. Apoptotic cell death stirred up by BDMC was in consort with the stimulation of autophagy in NSCLC cells. In order to understand the role of autophagy in cancer cell death, 3-methyladenine(3-MA) which can inhibit autophagy. CCK8 assay and flow cytometry were used to test some indexes. And the result told us the autophagy induced by BDMC played a positive role in promoting apoptosis. BDMC significantly attenuated the cell viability of tumor. Exceptionally, Smoothened(SMO) is one of the important members o the Hedgehog pathway. Cyclopamine is an chemical agent which can inhibit the effect of SMO. And the transcription factor glioma-associated oncogene 1(Gli1) protein was also one crucial member of the Hedgehog. The mRNA and protein expression of SMO and Gli1 were are down–regulated by BDMC. Furthermore, autophagic cell death can be stimulated by depletion of Gli1 through si RNA. The specific SMO inhibitor, Cyclopamine, was also proved to induce autophagy.We found that TGF-β1 stimulated EMT in 95 D cells. In the EMT process in 95 D cells stimulated by TGF-β1, the Wingless-type(Wnt)/β-catenin pathway was repressed by BDMC. To prove the assumption that BDMC inhibited the Wnt pathway via restorating the protein expression of Wnt inhibitory factor-1(WIF-1). Western blotting was used to test the alteration of WIF-1 expression under the condition with or not with BDMC. And the result showed that WIF-1 expression was increased. TGF-β1-mediated EMT in 95 D cells accompanied with the inhibition of migration and invasion induced by TGF-β1. Furthermore, we explored the contribution of WIF-1 in mediating BDMC response towards TGF-β1-induced EMT. Finally we showed that the inhibitory effect of BDMC on the EMT induced by TGF-β1 was mediated by WIF-1.Furthermore, inhibition of WIF-1 by WIF-1 siRNA significantly decreased epithelial marker E-cadherin expression, increased mesenchymal markers vimentin and fibronectin. In addition, inhibition of WIF-1 increased the nucleus β-catenin levels while decreased cytosol β-catenin. Meanwhile, the important EMT regulator, Snail expression was down-regulated by WIF1 si RNA.Conclusions:The disorders of the Hedgehog(Hh) signaling has been considered to be implicated in many human cancers including lung cancers. The findings from our research work indicated that autophagy can be induced by BDMC. And the autophagy induced by BDMC contributes to cell death in NSCLC. To some txtent, BDMC can kill tumor cells by inducing autophagy partly through modulating the Hedgehog pathway. It can be summarized that EMT induced by TGF-β1 was restrained by BDMC. And the inhibitory effect of BDMC on TGF-β-induced EMT in NSCLC can be taken as the result of mediation of WIF-1... |
PDF Full Text Request