Study On The Effects Of NQO1 And Its Inhibitor On The Proliferation And Migration Of Breast Cancer

Background:Breast cancer is one of the most common malignancies in women worldwide and the second leading cause of cancer death among women. NAD(P)H-quinone oxidoreductase 1 (NQO1) is a xenobiotic metabolizing enzyme that detoxifies chemical stressors and antioxidants, providing cytoprotection in normal tissues. Recent studies showed that NQO1 was highly expressed in most human solid tumors, including melanoma, pancreatic cancer, cholangiocarcinoma, lung cancer and breast cancer. Notably, its role in breast cancer 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 the effect and mechanism of β-lapachone in breast cancer is still unclear.Objectives:This study is aimed to investigate the significance of NQO1 protein overexpression on prognostic evaluation of breast cancer, and the impact on the biological behavior of MCF-7 breast cancer cells after treatment with β-lapachone by the experiments in vitro.Materials and methods:A total of 176 breast cancer patients with strict follow-up,45 DCIS,22 hyperplasia and 52 adjacent non-tumor tissues were selected for immunohistochemical staining of NQO1 protein. Immunofluorescence staining was also performed to detect the subcellular localization of NQO1 protein in MCF-7 breast cancer cells. Eight fresh breast cancers paired with adjacent non-tumor tissues were quantified using real time RT-PCR (qRT-PCR) and Western blot. 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. Breast cancer cell lines, MCF-7 cell, were included in this study. The cell viability was detected using MTT, and the migration ability was determined using scratch assay method. Meanwhile, the expression of biomarkers of proliferation and EMT markers were detected using Western blot.Results:Both of NQO1 protein and mRNA level in cancer tissues were significantly higher than in no-cancerous tissues by RT-PCR and Western blot assays in 8 fresh breast cancers and paired adjacent normal breast tissues. NQO1 protein showed a mainly cytoplasmic staining pattern in breast cancer. The immunohistochemical testing in 176 breast cancer patients with strict follow-up,45 DCIS,22 hyperplasia and 52 adjacent non-tumor tissues showed that the strongly positive rate of NQO1 protein was 61.9%(109/176) in breast cancer, and was significantly higher than in DCIS (31.1%,14/45), hyperplasia tissues (13.6%,3/22) and adjacent non-tumor tissues (13.5%,7/52)(P<0.05). It can be found that the NQO1 protein overexpression was closely related with late clinical stage, poor differentiation, lymph node metastasis, Her2 expression and disease-free and 10-year overall survival rates in breast cancer(P< 0.01). Moreover, multivariate analysis suggested that NQO1 emerged as a significant independent prognostic factor along with clinical stage and Her2 expression status in patients with breast cancer. Determined by MTT assay after β-lapachone, the proliferation of breast 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 P-lapachone could inhibit the invasion and motility of MCF-7 breast cancer cells, and up-regulate the epithelial markers (P<0.05). 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 breast cancer progression, and may be a potential biomarker for poor prognostic evaluation of breast cancers.2. β-lapachone can effectively inhibit the proliferation and migration of breast cancer cells in vitro.β-lapachone inhibits the growth of breast cancer through down-regulating the expression level of Skp2 and DEK protein; P-lapachone inhibits the migration of MCF-7 cell via EMT pathways.3. PI3K/Akt/mTOR signaling pathway may participate the molecular mechanism of β-lapachone on the killing effects in breast cancer in vitro.