The Hypomethylation Agent Bisdemethoxycurcumin Acts On The WIF-1 Promoter And Inhibits The Canonical Wnt Pathway And Induction Of Apoptosis In Human Non-small-cell Lung Cancer

Background and ObjectiveLung cancer is the most common cause of cancer-related mortality worldwide. The overall five-year survival rate for the patients with lung cancer is still below 15% although the great progress in the early diagnosis and chemotherapy, radiotherapy, immune therapy. In recent years, small-molecule inhibitors, most of which are epidermal growth factor receptor (EGFR)-targeted drugs such as gefitinib or erlotinib, have improved the treatment of lung cancer with confirmed curative results and fewer adverse effects. Although the effects of EGFR-targeted drugs are widely accepted, primary drug resistance exists within the EGFR(-) patient population. New therapeutic agents, especially those that work by novel mechanisms in other aberrant signal pathways within lung cancer,are urgently needed.Sharma found wingless gene in the study about the development of Drosophila in 1973. In 1982, Nusse found the integrated gene (int) in breast tumors of mice. Later research confirmed that the int gene is the Drosophila wingless gene. So the relevant gene had been named as wnt gene family. Recent studies have found that the protein of Wnt gene family can transmit signals to control cell development and regulate growth of cell. Wnt signaling pathway pass from the extracellular to the intracellular. It mainly includes: Wnt gene family (mainly encoded secreted protein), Wnt receptor protein on the cell membrane,β-catenin, APC complex protein (one is GSK-3βkinase, another is axin or conductiin), Lef/Tcf(lymphoid enhancer factor/T-cell factor)transcription factor family, MYC gene and so on. Wnt signaling pathway normally expressed during embryonic development, while the body is mature there is no Wnt signal. Many studies found that, Wnt signaling will destroy the abnormal expression of tumor suppressor gene, activation of oncogenes, and through the Wnt pathway leads to cancer.Deregulation of the Wingless/int (Wnt) signaling pathway is believed to play an important role in the pathogenesis of various cancers. Although genetic mutations of APC orβ-catenin are rarely observed in human lung cancer, compelling evidence has indicated an important role for the Wnt/β-catenin pathway in the development and the progression of lung cancer. Upregulation of the Wnt ligand has been demonstrated in several lung cancer cell lines and tissues.Furthermore,it was shown that treatment of prostate cancer cells with Wnt5A significantly enhanced cell growth. Overexpression of Wnt ligands results in aberrant cytoplasmic/nuclearβ-catenin localization in lung cancer cells and leads to the activation of downstream target genes that mostly encode proto-oncogenes and cell cycle regulators. The reason for Wnt ligand overexpression in lung cancer has not been fully investigated; however, the absence of Wnt antagonists proved to be an important reason for the deregulation of Wnt. Wnt inhibitory factor-1 (WIF-1) is a Wnt antagonist that inhibits Wnt signaling by directly binding to Wnt molecules. In previous studies, the WIF-1 promoter was cloned, and it was shown that the silencing of WIF-1 in non-small-cell lung cancer (NSCLC) is caused by promoter hypermethylation. In this study, we tried to identify WIF-1 promoter hypomethylation agents from natural products that have been proven effective in the treatment of lung cancer and then investigate their effect on Wnt signaling.Natural products are of increasing interest and importance to cancer patients. Curcumin, a mixture of compounds extracted from turmeric and derived from a member of the ginger family Curcuma longa, has been reported to suppress lung cancer cell growth and induce apoptosis. There are three major active chemical components within curcumin: curcumin (compound 1), demethoxycurcumin (compound 2), and bisdemethoxycurcumin (compound 3). Recently, computer modeling has indicated that these three compounds within curcumin are potent hypomethylation agents. However,the hypomethylation activity of curcumin when applied to a cell and its effect on promoter demethylation of WIF-1 need to be further investigated.To test the above hypothesis, this study is divided into three parts. In first part, we constructed gene silencing DNMT1 siRNAi by gene technique. By RT-PCR, Western blot and gene sequencing techniques we observe the effects of siRNA on mRNA and protein of DNMT1 and whether it could affect the hypermethylation of WIF-1 promoter. Secondly, we compare the capacity of hypomethylation in curcumin. We observed whether curcumin had the capacity of hypomethylation in the non-small cell lung cancer and restored mRNA and protein expression of WIF-1.Finally, we used Western blot, apoptosis measured and other molecular biology techniques to explore the demethylation of bisdemethoxycurcumin on activation of DNMT1 , WIF-1 and Wnt signaling pathways , affect apoptosis-related gene and whether it could promote apoptosis of tumor cells. At last, animal experiments had been done to verify the anti-tumor effect of bisdemethoxycurcuminMethodsThe present study includes in vivo and in vitro experiments. In vitro experiments, we chose several cell lines of non-small cell lung cancer as the research object to to detect the effects of hypomethylation in curcumin. We used gene technology (including the plasmid to a modification, methylation PCR, gene sequencing, etc.) to find the role of curcumin in WIF-1 and Wnt signaling pathway. In vivo parts, non-small cell lung cancer A549 cells were seeded into mice. We used different means to interfere the mice (regular diet and bisdemethoxycurcumin diet ) to verify the anti-tumor effect of bisdemethoxycurcumin.Selected non-small cell lung cancer A549, H460 and SPC-A-1 as the object of study, using methylation PCR to observe the methylation status of tumor cells.Selected non-small cell lung cancer A549, H460 and SPC-A-1 as the object of study, using different types of curcumin to interfere cells to observe the hypomethylation of them.Selected non-small cell lung cancer A549, H460 and SPC-A-1 as the object of study, using different concentration of curcumin to interfere cells to observer the efftcts on mRNA and potein in WIF-1.Using RT-PCR, Western Blot, methylation PCR and gene sequencing technique, we observed different concentrations of bisdemethoxycurcumin affected the mRNA and protein expression of WIF-1. We compared the hypomethylation effects to WIF-1 promter using different concentration of bisdemethoxycurcumin.Selected non-small cell lung cancer A549, H460 and SPC-A-1 as the object of study, using ELISA, electrophoretic mobility shift assay and western blot to observe the effects of bisdemethoxycurcumin on DNMT1 and Wnt signaling pathway.Using the apoptosis detection techniques to observe the effects of bisdemethoxycurcumin on A549 non-small cell lung cancer cells. Using PCR technique to observe the changes in apoptosis-related genes.Using NOD / SCID mice as research subjects, interventions by different means (normal diet and bisdemethoxycurcumin diet) to verify the anti-tumor effect of bisdemethoxycurcumin. Using the ELISA method, specific oligonucleotide probe to detect the hypomethylation effects of curcumin, demethoxycurcumin and bisdemethoxycurcumin.DNMT1 gene silencing siRNAi was constructed by gene technique. Using RT-PCR, Western blot and gene sequencing to observe the mRNA and potein of DNMTS, and observe the hypomethylation effects in WIF-1 promter.Results:1. Curcumin, Dmc, and Bdmc possess different hypomethylating potent.2. WIF-1 methylation state in A549, H460, and SPC-A-1 cell lines.3. WIF-1 promoter demethylation by curcuminoids in A549, H460 and SPC-A-1 cell lines.4. Bisdemethoxycurcumin restored normal WIF-1 expression by promoter demethylation.5. Restoration of WIF-1 mRNA and protein expression in A549 cell line6. Down-regulation of the Wnt canonical pathway in A549 cells after bisdemethoxycurcumin treatment.7. Bisdemethoxycurcumin induced apoptosis in the A549 cell line.8. Bisdemethoxycurcumin down-regulates genes associated with apoptosis.9. In vivo tumor formation was inhibited by dietary bisdemethoxycurcumin, with accompanying spontaneous necrosis. 1. Gene silencing DNMT1 siRNAi significantly reduced the mRNA of DNMT1.10. Bisdemethoxycurcumin can not suppress the mRNA expression of DNMT1, however, can suppress the acitivty of DNMT1 directly.11. Gene silencing DNMT1 siRNAi significantly reduced the protein expression of DNMT1.12. Gene silencing DNMT1 siRNAi reduced the expression of the DNMT1 gene and protein, then reversed hypermethylation level in the WIF-1 gene promoter.13. Bisdemethoxycurcumin can not induce further demethylation in A549(DNMT1/KO) cells.14. Inhibition of DNMT1 activity by bisdemethoxycurcumin in vitro.15. Bisdemethoxycurcumin induce WIF-1 demethylation by targeting DNMT1...