Abstract
Background: We previously demonstrated that a Danshensu-Tetramethylpyrazine conjugate DT-010 enhanced anticancer effect of doxorubicin (Dox) in Dox-sensitive human breast cancer cells, and protected against Dox-induced cardiotoxicity. This work was designed to see whether DT-010 overcomes Dox resistance in resistant human breast cancer cells. Methods: The effects of DT-010, Dox or their combination on cell viability of Dox-resistant human breast cancer MCF-7/ADR cells were conducted using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was examined by flow cytometry after Annexin V-FITC/PI co-staining. Dox accumulation in MCF-7/ADR cells was detected by flow cytometry and fluorescence microscopy. A fluorometric multidrug resistance (MDR) assay kit was used to evaluate the effect of DT-010 on MDR transporter activity. P-glycoprotein (P-gp) expression and activity were analyzed by Western blot and rhodamine 123 (Rh123) efflux assay, respectively. The effects of DT-010 on glycolysis and mitochondrial stress were detected using an Extracellular Flux Analyzer. A Succinate Dehydrogenase Activity Assay kit was used to measure mitochondrial complex II activity. Results: At non-cytotoxic concentrations, DT-010 in combination with Dox led to a significant growth inhibition of MCF-7/ADR cells, suggesting a synergy between DT-010 and Dox to reverse Dox resistance. DT-010 restored Dox-mediated apoptosis and p53 induction in MCF-7/ADR cells. DT-010 increased Dox accumulation in MCF-7/ADR cells via inhibiting P-gp activity, but without changing P-gp expression. Further studies showed that DT-010 significantly inhibited glycolysis and mitochondrial function of MCF-7/ADR cells. Mitochondrial complex II activity was inhibited by DT-010 or DT-010/Dox combination, but not by Dox. The DT-010-mediated suppression of metabolic process may render cells more vulnerable to Dox treatment and thus result in enhanced efficacy. Conclusions: The results indicate that DT-010 overcomes Dox resistance in human breast cancer cells through a dual action via simultaneously inhibiting P-gp-mediated drug efflux and influencing metabolic process.
Highlights
Breast cancer is the most frequent cancer and the leading cause of cancer-associated death among women worldwide (Siegel et al, 2018)
The primary antibodies against poly ADPribose polymerase (PARP), P-gp, p53, Bax, and glyceraldehyde-3phospate dehydrogenase (GAPDH) and secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA)
In consistent with previous study (Wang et al, 2016b), we showed that DT-010 at 10 and 20 μM showed slight growth-inhibitory effect in Dox-sensitive MCF-7 cells, and DT-010 significantly enhanced Dox sensitivity in MCF-7 cells (Figure 1B)
Summary
Breast cancer is the most frequent cancer and the leading cause of cancer-associated death among women worldwide (Siegel et al, 2018). Progress in early diagnosis and improved therapeutic strategies has greatly prolonged overall survival of patients with breast cancer. Chemotherapy, hormonal therapy, and targeted therapy are commonly used for the treatment of breast cancer. Patients are responsive to initial drug treatment, progressive disease occurs invariably, and the response rate becomes decreased due to the occurrence of multidrug resistance (MDR). Multiple MDR impairments may simultaneously occur, which gives rise to challenge in successful therapies in cancer. Strategies to inhibit or bypass MDR processes are highly advocated in cancer therapy. We previously demonstrated that a Danshensu-Tetramethylpyrazine conjugate DT-010 enhanced anticancer effect of doxorubicin (Dox) in Dox-sensitive human breast cancer cells, and protected against Dox-induced cardiotoxicity. This work was designed to see whether DT-010 overcomes Dox resistance in resistant human breast cancer cells
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