A Danshensu-Tetramethylpyrazine Conjugate DT-010 Overcomes Multidrug Resistance in Human Breast Cancer

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.

Keywords: P-glycoprotein; breast cancer; danshensu; glycolysis; resistance; tetramethylpyrazine.

Figure 1

(A) Structures of Danshensu (DSS), tetramethylpyrazine (TMP), and DT-010. A bulky hindrance was introduced on the linker between DSS and TMP. (B and C) Cell viability of MCF-7 and MCF-7/ADR cells after treatment of Dox, DT-010, or their combinations for 24 h. Cell viability was determined using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results are displayed as mean ± SD. (D) Colony formation of MCF-7/ADR cells after treatment of Dox (2 μM), DT-010 (10 μM) or their combinations for 7 days. Cells were stained with 0.1% crystal violet, with colony number counted. Results are displayed as mean ± SD. *p < 0.05, compared to control group.

Figure 2

DT-010 restored Dox-mediated apoptosis in MCF-7/ADR cells. (A) MCF-7/ADR cells were treated with Dox (2 μM), DT-010 (20 μM) or the combination for 24 h, followed by flow cytometric analysis after PI/Annexin-V staining. Early apoptosis rate was calculated based on population of PI−/Annexin-V+ cells. Experiments were performed in triplicate. Results were displayed as mean ± SD. *p < 0.05, compared with control or other groups. (B) Western blot was performed using cell lysate of MCF-7/ADR cells after treatment of Dox (2 μM), DT-010 (20 μM) or the combination for 24 h. Anti-GAPDH was used as loading control.

Figure 3

DT-010 increased Dox concentration in MCF-7/ADR cells. MCF-7/ADR cells were pretreated with DT-010 (0, 5, 10, and 20 μM) for 12 h, followed by incubated with Dox (2 μM) for another 4 h. The intracellular concentration of Dox was analyzed by flow cytometry (A) and observed using Incell Analyzer 2000 (GE Healthcare) (B). MCF-7 cells treated with Dox (2 μM) for 4 h were used for a comparison. Mean fluorescence intensity were recorded in flow cytometry assay. Relative fluorescence intensity (nuclear area) of Dox over Hoechst 33342 was calculated by ImageJ. Data are expressed as mean ± SD. *p < 0.05, compared with MCF-7 cells. ^#p < 0.05, ^##p < 0.001, compared with Dox alone in MCF-7/ADR cells.

Figure 4

Effects of DT-010 on expression and activity of P-gp. (A) Relative messenger RNA expression of ABCB1, ABCC1, ABCG2, and SLC22A16 in MCF-7 and MCF-7/ADR cells. Data are expressed as mean ± SD. *p < 0.05, compared with MCF-7 cells. (B) Multidrug resistance (MDR) transporter activity was detected using a fluorometric MDR assay kit. MCF-7/ADR cells were treated with DT-010 (5, 10, and 20 μM) for 1 h, followed by the addition of 100 μl MDR dye-loading solution and incubated at 37°C for another 1 h. Data are expressed as mean ± SD. *p < 0.05, or **p < 0.01, compared with control. (C) DT-010 (20 μM), Dox (2 μM), or their combination did not affect P-gp expression in MCF-7/ADR cells. Western blot was performed, with anti-GAPDH as loading control. (D and E) The effect of DT-010 on P-gp substrate efflux activity was assessed. MCF-7/ADR cells (5.0 × 10⁴/well) were incubated with the P-gp-specific fluorescent substrate Rh123 at 0.5 μg/ml with or without DT-010 (20 μM) for 1 h, then washed twice with ice-cold phosphate-buffered saline (PBS), and incubated in Rh123-free medium at 37°C for additional 1 h with or without DT-010. Cells were analyzed on flow cytometry to detect Rh123 fluorescence. The PSC-833 (0.1 μM) was used as a positive control. MCF-7 cells were used for confirmation of overexpression of P-gp (increased Rh123 efflux) in MCF-7/ADR cells.

Figure 5

DT-010 inhibited glycolytic pathway of MCF-7/ADR cells. The extracellular acidification rate (ECAR) was measured with XF24 extracellular flux analyzer after 12 h of DT-010 treatment (A). The basal glycolysis (B), glycolytic capacity (C), and glycolytic reserve (D) of MCF-7/ADR cells were calculated. *p < 0.05, compared with DT-010-treated group.

Figure 6

DT-010 inhibited mitochondrial respiration of MCF-7/ADR cells. MCF-7/ADR cells were treated with DT-010 for 12 h, the effects of DT-010 on OCR were monitored with XF24 extracellular flux analyzer (A). The basal respiration (B), ATP production (C) and maximal respiration (D) of MCF-7/ADR cells were calculated. *p < 0.05, compared with DT-010-treated group.

Figure 7

DT-010 inhibited mitochondrial complex II activity in MCF-7/ADR cells. Cells were treated with DT-010 (20 μM), Dox (2 μM), or their combination for 12 h, and complex II activity was measured by Succinate Dehydrogenase Activity Assay kit. *p < 0.05, compared with control. ^# p < 0.05, compared to DT-010-treated group.