| Background:Gastric cancer (GAC) is under the category of digestive tract malignant tumors, which originated from gastric mucosa epithelium. The incidence stands at the top position in malignant tumors of digestive tract. NQO1 protein is also known as DT-diaphorase, not only a yellow enzyme but also a kind of phase Ⅱ detoxification enzymes. The occurrence of defects, and is closely related to susceptibility to carcinogenesis. Recent studies showed that NQO1 was highly expressed in most human solid tumors. Notably, its role in GAC progression has not yet been reported,β-lapachone is an effective inhibitor of the NQO1 protein, and it is a natural compound that obtained from bark of the South American lapacho tree, and it has been reported to be a natural product that activates apoptotic cell death in several cancer cell lines, including prostate cancer, breast cancer, and leukemia, p-lapachone is also considered to be a good sensitizer of radiotherapy in colon and prostate cancer cells, but what effect and mechanism in gastric cancer is still unclear. Thus, this study is focus on the effect of NQO1 and its inhibitor in the prognostic evaluation and targeted therapy for gastric cancer.Objectives:This study is aimed to investigate the significance of NQ01 protein overexpression on prognostic evaluation of gastric cancer, and the impact on the biological behavior of SGC-7901 and AGS gastric cancer cells after treatment with β-lapachone by the experiments in vitro, to explore the molecular mechanism of P-lapachone on inhibition for gastric cancer, and to provide a possible new biomarker and therapeutic target for gastric cancer.Materials and methods:The NQO1 protein expression in 53 cases of adjacent non-tumor tissues,31 cases of gastric dysplasia and 203 cases of primary gastric cancer were investigated by immunohistochemical (IHC), and the NQO1 mRNA levels in 12 cases of gastric cancer,8 cases of adjacent non-tumor tissue and 8 cases of normal gastric mucosa were detected by qRT-PCR. The relationship between NQO1 expression and clinico-pathological characteristics, the survival rates after tumor removal, and multivariate survival analysis were analyzed to verify the clinical value of NQO1 protein expression in patient prognosis. Gastric cancer cell lines, SGC-7901 and AGS cells, were included in this study. The cell viability was detected using MTT and colony formation assay, the migration ability was determined using scratch assay method, and the apoptosis was examined using flow cytometry (FCM) and Hoechst 33342 staining. Meanwhile, the expression of biomarkers of proliferation, EMT markers and apoptosis were detected using Western blot and immunofluorescence (IF) staining.Results:The mRNA expression level in cancer tissues were significantly higher than in no-tumor tissues by qRT-PCR assays in 12 fresh gastric cancer,8 cases of adjacent non-tumor tissue and 8 cases of normal gastric mucosa. The positive rate and strongly positive rate of NQO1 protein were 75.68%(154/203) and 61.58%(125/203) in gastric cancer, respectively, and both were significantly higher than either in gastric dysplasia and adjacent non-tumor tissues (P<0.05). It can be found that the NQO1 protein overexpression was closely related with patients’tumor size, clinical stage, serosal invasion (P<0.05). Moreover, disease-free survival and 5-years survival rates of the patients with NQO1 protein overexpression were significant lower than the others, and the patients with NQO1 overexpression combined tumor size, differentiation, clinical stage, lymph node metastasis and serosal invasionhad lower 5-years survival rate than the patients with NQO1 overexpression but without the other factors. Clinical stage was an independent risk factor (HR:1.807, P=0.000) for the prognosis of patients with gastric cancer. Determined by MTT and colony formation assays after β-lapachone, the proliferation of gastric cancer cells was significantly decreased (P<0.05), and the mechanism is mainly via down-regulation of Skp2 and DEK proteins expression, which are related to the proliferation and cell cycle; the scratch assay indicated that β-lapachone could inhibit the invasion and motility of SGC-7901 and AGS gastric cancer cells (P<0.05), down-regulate the expression level of MMP-2/9 and Ezrin proteins, and up-regulate the epithelial markers. FCM and Hoechst staining results showed that (3-lapachone enhanced the apoptosis of gastric cancer cells (P<0.05), the mechanism is mainly via down-regulation of Bcl-2/Bax ratio and up-regulation of activated caspase 3/8/9. Additionally, western blot analysis showed that reduced the protein levels related with PI3K/Akt/mTOR signaling pathway.Conclusions:1. High-level expression of NQO1 appears to be associated with gastric cancer progression, and maybe a potential biomarker for poor prognostic evaluation of gastric cancers.2. P-lapachone can effectively inhibit the proliferation and migration, and induce the apoptosis of gastric cancer cells in vitro. β-lapachone inhibits the growth of gastric cancer through down-regulating the expression level of Skp2 and DEK protein;β-lapachone inhibits the migration of SGC-7901 and AGS cells via MMPs and EMT pathways; β-lapachone induces apoptosis through mitochondrial and caspase-dependent pathways in SGC-7901, AGS cells.3. PI3K/Akt/mTOR signaling pathway may participate the molecular mechanism of β-lapachone on the killing effects in gastric cancer in vitro. |
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