The effect of MKP-1 inhibition on the cytotoxicity of chemotherapeutic drugs in breast cancer

[摘要] ENGLISH ABSTRACT: Introduction: Cancer is an emerging health problem in South Africa, with breast cancer being one of the leading cancers affecting women globally. Therefore, there is a need to find novel targets to improve the therapeutic options for these patients. A recently proposed target is the mitogen-activated protein kinase phosphatase-1 (MKP-1). Studies have suggested that mitogen-activated protein kinase phosphatases are involved in the development of cancer and play an important role in the response of cancer cells to chemotherapy. Additionally, numerous studies have indicated that there is increased expression of MKP-1 in breast cancers where its over-expression is proposed to be a significant mediator in chemo-resistance. We propose that inhibition of MKP-1 will increase the cytotoxic effect of doxorubicin in breast cancer cells, thus making the cells more responsive to treatment leading to increased cell death through autophagy and apoptosis.Methods: In MDA-MB231 cells, MKP-1 was inhibited using sanguinarine or MKP-1 siRNA and this was compared to a known inducer of MKP-1, dexamethasone. MDA-MB231 cells were treated with doxorubicin alone or in combination with MKP-1 inhibitors or an inducer. Following treatment, cell death was determined by trypan blue and a caspase glo assay as well as with western blotting. Autophagy was determined by western blotting and flow cytometry. LC3 and p62 were used as markers of autophagy and caspase 3 and PARP as apoptosis markers. Likewise, the level of MKP-1 expression under conditions of MKP-1 induction, inhibition or silencing was evaluated by means of western blotting. C57BL6 tumour bearing mice was used to analyse apoptosis and autophagy in vivo under conditions of MKP-1 inhibition, using sanguinarine, together with doxorubicin treatment. Western blotting was used to determine levels of caspase 3, LC3, p62 and MKP-1 expression.Results and discussion: A concentration and time curve indicated that 5 μM doxorubicin reduced cell viability in the MDA-MB231 cells significantly after 24 hours of treatment. MKP-1 expression was significantly reduced with sanguinarine and MKP-1 siRNA. Furthermore, our results indicate a significant increase in apoptosis in MDA-MB231 cells when treated with doxorubicin, under conditions of MKP-1 inhibition or MKP-1 silencing. Also, an increase in autophagic activity was observed following treatment with doxorubicin in combination with sanguinarine. Whole excised tumours of C57BL6 mice also showed an increase in apoptosis and autophagy following treatment with sanguinarine in combination with doxorubicin. This indicates that the inhibition of MKP-1 with sanguinarine sensitized the MDA-MB231 cells and E0771 cell tumours to doxorubicin-induced-apoptosis through a mechanism involving autophagy.Conclusion: This is an encouraging finding that could hopefully be used in future studies to overcome doxorubicin-resistance in breast cancer cells overexpressing MKP-1. Targeting MKP-1 can have potential therapeutic benefits for breast cancer patients by making chemotherapy more effective. Sanguinarine thus has potential to be developed as a clinically relevant inhibitor of MKP-1 which could provide a novel avenue for therapeutic intervention in combination with chemotherapy in breast cancer patients.

[发布机构] Stellenbosch University