Investigation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced chemoresistance in breast cancer

Abstract

According to the Global Cancer Statistics, breast cancer is the most commonly diagnosed cancer in women. In Hong Kong, breast cancer is the third commonly diagnosed cancer, representing 26.6% of all cancers in women. Cigarette smoking is one of the risk factors in breast cancer that received much attention in the past decades. In 2013, World Health Organization estimates that 250 million women were current smokers globally. Epidemiological studies revealed that current smokers have a higher risk of breast cancer than ex-smokers. The rate of breast cancer recurrence and mortality were increased in current smokers compared with non-smokers. 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), one of the most important components in cigarette smoke, has been shown to cause carcinogenesis in breast epithelial cells. However, the effect of NNK on the development of breast cancer is still unclear. This study aims to investigate the effect of NNK on breast cancer cells and the underlying mechanism on breast carcinogenesis. Serial concentrations of NNK treatment were examined in the assays of migration, invasion, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation. The chemoresistance effect was also examined in different cell lines by MTT, flow cytometry and western blot. The underlying mechanisms were explored to investigate the molecular targets using drug transporter array, real-time polymerase chain reaction (PCR), small interfering RNA transfection, western blot and MTT assay. Finally, mouse model was used to confirm the effects of NNK on tumor growth. MTT assay results showed that NNK increased cell viability in breast cancer cells. However, there were no significant changes on cell migration and invasion. Colony formation ability was increased and cell apoptosis was decreased upon NNK treatment. Expression levels of mechanistic target of rapamycin (mTOR) and nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) were increased by NNK. Results from the drug transporter array revealed that NNK upregulated SLC7A5 expression. Transfection of SLC7A5 siRNA reversed NNK-induced chemoresistance in breast cancer cells. In animal study, mice bearing NNK pretreated cells formed larger tumors when compared with control cells, and was resistant to cisplatin treatment. In summary, this study proved that NNK increased breast cancer cell viability and decreased apoptosis through mTOR pathway, which lead to chemoresistance in breast cancer. The effect of NNK in chemoresistance was reversed by the blockade of SLC7A5. Hence, targeting SLC7A5 may be a potential strategy to enhance chemotherapy effect in clinical treatment of breast cancer.