β-lapachone Inhibits Invasion And Metastasis Of Breast Cancer Through Epithelial-mesenchymal Transition

Background:Breast cancer is one of the most common malignancies in women worldwide and the second leading cause of cancer death among women. NQO1, also known as DT-diaphorase, is not only a flavin enzyme, but also a kind of phase Ⅱ detoxification enzymes,which is closely related to tumorous susceptibility after the occurrence of defects. Recent studies have shown that NQO1 is highly expressed in malignant tumors, and related to the prognosis of disease. β-lapachone, a novel radiosensitizer with potent anti-tumor efficacy selectively kills tumor cells that overexpress NAD(P)H:quinone oxidoreductase 1 (NQO1). However, the inhibitory effect of β-lapachone on breast cancer is not clear.Objectives:The purpose of this study is to investigate the significance of NQO1 protein over-expression in metastasis of breast cancer, and the impact of P-lapachone on the biological behavior of breast cancer cells, and analyze the mechanism of P-lapachone inhibition on metastasis of breast cancer for providing the possible new therapeutic target.Materials and methods:The expression of NQO1 in 45 cases of adjacent non-tumor tissues and 176 paraffin embedded breast cancer samples were investigated by immunohistochemistry(IHC),62 breast cancers paired with adjacent non-tumor tissues were quantified by using real-time PCR (qRT-PCR) and western blotting. In addition, the correlation between the abnormal expression of NQOlprotein and distant metastasis in breast cancer were also analyzed. Experiments in vitro:NQO1-positive breast cancer cell line, MDA-MB-231 and MCF-7, were cultured in this study. The cell viability was detected by using MTT and colony formation assay. The migration ability was determined by using scratch assay method, migration assay, and invasion assay. the expression of biomarkers of proliferation, EMT markers and Akt/mTOR signaling pathway related proteins were detected by using Western blot. Experiments in vivo:mices transplanted tumor model of MDA-MB-231 and MCF-7 cell line were randomly divided into P-lapachone-treated groups and the control group. After 4 weeks of continuous administration, expression of Ki67, E-cadherin, p-Akt, p-S6, and p-4EBP1 protein were analyzed by immunohistochemistry to detect the inhibition abilities of β-lapachone in the tumor.Results:1. The mRNA and protein expression levels of NQO1 in cancer tissues were significantly higher than in no-tumor tissues by qRT-PCR and Western blot assays in 62 fresh breast cancer and adjacent non-tumor tissue. The mRNA and protein expression levels of NQO1 in metastasis tissues were significantly higher than in non-metastasis tissues of breast cancer. Moreover, the consistant results were also found by qRT-PCR and Western blot assays in breast cancer cell lines. The positive and strongly positive rate of NQO1 protein were 23% and 62% in breast cancer, respectively, which were significantly higher than in adjacent non-tumor tissues (20% and 0.0%)(P<0.05).2. Determined by MTT and colony formation assays after treatment with P-lapachone, the proliferation of breast cancer cells was significantly decreased (P<0.05), which mechanism is mainly regulated via down-regulation of Skp2 and DEK proteins expression; The scratch, migration, and invasion assays indicated that β-lapachone could inhibit the migration and invasion of MDA-MB-231 and MCF-7 cells (P<0.05). Western blot and IF staining showed that β-lapachone could down-regulate the expression level of MMP-9, Snail, Vimentin, Twist, Slug proteins, and up-regulate the epithelial markers.3. Additionally, Western blot analysis showed that β-lapachone can reduce the protein levels related with Akt/mTOR signaling pathway, such as p-AKT, p-S6, and p-4EBP1, and up-regulate the expression level of p-PTEN protein.4. β-lapachone inhibited MDA-MB-231 cell tumorigenetic ability in mice. After the treatment with β-lapachone, the necrosis of tumor was increased. And the expression of Ki67, p-AKT, p-S6, and p-4EBP1 were down-regulated, while the expression of E-cadherin was up-regulated.Conclusions:1. The expression of NQO1 protein was significantly correlated with metastasis in breast cancer.2. β-lapachone inhibits the growth of breast cancer cells through down-regulating the expression level of Skp2 and DEK protein.3. β-lapachone inhibits migration and invasion abilities of breast cancer cells via EMT pathway.4. Akt/mTOR signaling pathway may participate the molecular mechanism of β-lapachone on the inhibition effects in breast cancer.