Effects And Associated Mechanisms Of Demthylation Of MSX1 And DKK3 Genes By Quercetin In Gastric Cancer

Background and purpose:Gastric cancer is second leading cause of cancer-related death in the world, and is common malignant tumor in China, especially in southwest region where people like to eat preserved foods. Good prognoses of gastric patient depend on whether they have early diagnosis and early surgery.As far as the advanced, recurrent and drug resistant patients are concered, Chemotherapies and radiations are usually ineffective and have too much side effects, lacking of effective treatments. So it is an urgent thing to find a specific molecular tumor biomarker for early diagnosis of cancer and to find less toxic also effective drugs for cancer treatment. The epigenetic research brings people novel molecular biomarker and effective anticancer therapies.The tumorigenesis and progression of gastric cancer involves in the abnormal regulation of multiple signaling pathways (such as WNT/ β-catenin, P53 and other signaling pathways), which were associated with activation of oncogene and inactivation of tumor suppressor genes. Tumor suppressor genes epigenetic inactivations through DNA hypermethylation of promoter CpG islands are the important mechanisms of tumor suppressor gene inactivation besides gene deletion and point mutations. Study confirmed that tumor suppressor gene promoter hypermethylation and silencing is a frequent and early event of gastric cancer. It has character of tumor specificity, tissue specificity, and reversity. Activity of tumor suppressor genes can be restored by Demethylation treatment with 5-aza deoxycytidine (5-aza-CdR) and other DMNT inhibitors. So tumor suppressor gene promoter hypermethylation become a novel target for cancer therapy. Therefore, finding and identificating new promoter methylation and silencing of tumor suppressor genes as early gastric cancer-specific diagnostic marker and therapeutic target is of great value and prospects.Recently, some studies have found that multiple tumor suppressor genes in gastric cancer promoter hypermethylation and silencing, including the WNT antagonist DKK3 and the newly discovered candidate gene MSX1 gene. We are concerned about whether MSX1 and DKK3 gene have promoter hypermethylation and silencing in southwest region, which could become a target for cancer therapy.The tumor suppressor gene promoter hypermethylation can be reversible, Decitabine (5-aza-deoxycytidine) can reverse the hypermethylation of tumor suppressor genes and restore the function of tumor suppressor genes, which has been used clinically in myelodysplastic syndrome, leukemia treatment. However,5-aza-CdR not only has toxic side effects, but also has widespread methylation on normal cells and cancer cells. Lacking of specificity of the blocking effect resulted in the instability of the entire genome, which limits it’s widely using in clinical works. Therefore, our goal is to find effective, less toxic and tumor-specific demethylation drug. At this time, we re-examine the anti-cancer effect and mechanism of quercetin.Quercetin is rich in vegetables, fruits, black tea, red wines and herbs, having a wide range of physiological and pharmacological activities. Because of its vessels-dilating and lipid-lowering effects, it has been widely used as a cardiovascular drug; no one reported carcinogenic effects of quercetin until now. Many papers have been reported that quercetin have effects of cancer prevention and treatment, we also previously reported that quercetin inhibited the growth of gastric cancer and induced apoptosis of gastric cancer, decreased WNT downstream target genes (PCNA, VEGF, HSP70) and MTP53 gene. Some literature reported that quercetin could demthlyate tumor suppressor gene in the bladder and colon cancer, suggesting that novel anti-cancer mechanism of quercetin is associated with epigenetic regulation.It is known that MSXland WNT antagonist DKK3 gene was silenced in gastric cancer by their promoter methylation. In this regard, we assumed that the mechanism of quercetin inhibited gastric cancer was associated with the reversal the hypermethylation status of MSXland DKK3 genclf it was relevant, we will further explore the effects and associated mechnism of quercetin on WNT signaling pathway. We want to reveal the novel anti-cancer mechanism of quercetin and provide a theoretical basis for its clinical application as a less toxic and effective drug.Purpose:To explore the relationship between promoter methylation status and its expression of MSXlgene (as a new cancer candidate tumor suppressor gene) and DKK3 gene in gastric cancer; to make clear the demethylation effects and associated mechanism of quercetin in gastric cancer.Method: 1. RT-PCR detected the expression of MSX1 and DKK3 gene mRNA before and after 5-aza-CdR treatment in four cancer cell lines; 2. MSP analysed MSX1, DKK3 promoter methylation status in four gastric cancer cell lines,10 cases of gastric carcinoma and adjacent normal tissues.3. CKK8 detected the growth inhibition of Que on gastric cancer cells;4. Establishing gastric cancer xenograft in nude mice model,twice a week of Que for 28 days, to study growth inhibitory effects of Que on gastric tumor;5. Extraction RNA of gastric cancer cells and tissues, RT-PCR was used to analyse transcription activity of MSX1, DKK3 gene with or without Que treatment;6. Extraction DNA of gastric tissues and cells, MSP analysed gene methylation status of MSX1, DKK3 with or without Que treatment;7. Western blot and immunohistochemical examined the effects of Que on protein expression of the components of WNT (β-catenin, cyclinDl) in gastric cancer cells.Results:1. RT-PCR analysis showed that MSX1, DKK3 gene mRNA expression were silenced in four gastric cancer cell Hnes;5-aza-CdR upregulated MSX1, DKK3 gene mRNA expression; MSP analysis showed that MSX1, DKK3 gene have promoter methylation in gastric biopsy samples after surgery; and compared with adjacent tissues, gastric cancer tissue MSX1, DKK3 gene promoter methylation have significant differences.2. Quercetin could inhibit cancer cell growth in vitro, IC50 (for SGC-7901 48 hours) is 19μmol/L; Quercetin inhibited growth of gastric cancer xenografts in nude mice, the inhibitory effects was 55% for 28 days.3. RT-PCR showed that Que could up-regulate DKK3 and MSX1 gene mRNA in vivo and in vitro.4. MSP confirmed that Que could reverse DKK3 and MSX1 gene promoter methylation status in vivo and in vitro.5. Western blotting and immunohistochemistry confirmed that Que could reduce expression of β-catenin protein, cyclinD1 protein, inhibited the translocation of P-catenin protein from membrane to nucleus.Conclusion:1. The silence of expression of MSX1 and DKK3 gene because of its gene promoter methylation status,5-aza-CdR could reverse the MSX1 and DKK3 gene promoter methylation status in gastric cancer, indicating it might be a good targets for cancer therapy.2. Que inhibited the growth of gastric cancer in vivo and in vitro, the reasons were:firstly, Que reversed DKK3 and MSX1 gene promoter methylation status and restored the tumor suppressor gene function. So quercetin could inhibit WNT singaling pathway from its upstream gene. Secondly, Que inhibited the expression and translocation of P-catenin to stabilize and inhibited the expression of its downstream target genes cyclinD1 protein.Outlook:If conditions permitting, as a new emerging tumor suppressor gene, We should learn more about the promoter methylation status of MSX1 gene in large amounts of tissue samples and cancer cell lines; and should learn its gene function, it will be our next work.