Antagonistic Pharmacological Interaction between Sirtuin Inhibitor Cambinol and Paclitaxel in Triple-Negative Breast Cancer Cell Lines: An Isobolographic Analysis

Figures

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  Figure 1. The mechanism of CAM action. CAM (SIRT 1/2 inhibitor) inhibits the activity of SIRT 1 and 2 (HDACs), which leads to the relaxation of chromatin and activation of transcription. HDACs–histone deacetylases, HATs–histone acetyltransferases, SIRT–sirtuin. The Figure was created with www.biorender.com (accessed on 22 May 2022).

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  Figure 2. Viability of (A) BT-549 (B) MDA-MB-468 and (C) HCC1937 TNBC cells after 96 h incubation with 0.01–0.1 mM of CAM in the MTT assay. Statistical test: one-way ANOVA, Tukey post-hoc testing. * p < 0.05, *** p < 0.001. Data are presented as mean ± standard deviation (±SD).

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  Figure 3. Log-probit concentration-effect relationship curves (CECs) for PAX and CAM used singly and in combination at the fixed-ratio of 1:1 for (A) BT-549 and (B) MDA-MB-468 TNBC cells. Concentrations of CAM and PAX were transformed into logarithms and the anti-proliferative effects into probits. The dotted line (parallel to the abscissa at 5th probit) indicates in approx. the IC50 values for PAX and CAM. Test of parallelism between PAX and CAM indicated that both lines are not collateral to each other.

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  Figure 4. The proliferation of (A) BT-549 and (B) MDA-MB-468 TNBC cells in the BrdU assay after CAM and PAX treatment. BC cells were incubated for 48 h individually (control) or with the drugs (0.5 = 1/4 IC50; 1.0 = 1/2 IC50 determined in the MTT assay). Statistical test: one-way ANOVA, Tukey post-hoc testing. * p < 0.05, ** p < 0.01, *** p < 0.001. Data are presented as mean ± standard deviation of the mean (±SD). Grey *-data vs. ctr; black *-single treatment vs. combined treatment.

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  Figure 5. Isobolographic analysis of interactions between PAX and CAM in (A) BT-549 and (B) MDA-MB-468 TNBC lines measured in vitro by the MTT assay. The IC50 values for PAX and CAM are plotted on the ordinate and abscissa, respectively. The lower and upper isoboles of additivity represent the curves connecting the IC50 values for PAX and CAM. The points A′ and A″ depict the theoretically calculated IC50 add values, whereas the point M represents the experimentally-derived IC50 mix value. * p < 0.05 and ** p < 0.01 vs. the respective IC50 add values (Student’s t-test with Welch correction).

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  Figure 6. Effect of CAM and PAX alone or in combination on whole cell death induction in (A) BT-549 and (B) MDA-MB-468 TNBC cells in the trypan blue exclusion assay. BC cells were exposed to drugs treatment for 48h using selected ratios of the IC50 determined in the MTT assay (2.0 = IC50, 4.0 = 2IC50). Data are presented as mean ± standard deviation (±SD); one-way ANOVA, Tukey’s post-hoc testing; * p < 0.05, ** p < 0.01, *** p < 0.001. Grey *-data vs. ctr; black *-single treatment vs. combined treatment.

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  Figure 7. Effect of CAM and PAX alone or in combination on caspase-3 activation in (A) BT-549 and (B) MDA-MB-468 TNBC cells. BC cells were exposed to drugs treatment for 48h using selected ratios of the IC50 determined in the MTT assay (2.0 = IC50, 4.0 = 2IC50) and analyzed by FACS. Data are presented as mean ± standard deviation (±SD); one-way ANOVA, Tukey’s post-hoc testing; * p < 0.05, ** p < 0.01, *** p < 0.001. Grey *-data vs. ctr; black *-single treatment vs. combined treatment.

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  Figure 8. Interpolated protein concentration of Annexin V (pg/mL) in 100 µg/mL of protein, extracted from (A) BT-549 and (B) MDA-MB-468 TNBC cells after CAM and PAX treatment, used individually or in combination. BC cells were exposed to drugs treatment for 48h using selected ratios of the IC50 determined in the MTT assay (2.0 = IC50, 4.0 = 2IC50). Concentrations of Annexin V were measured in triplicate and interpolated from the Annexin V standard curve and corrected for sample dilution. The interpolated dilution factor corrected values are plotted (mean ± SD, n = 3).; one-way ANOVA, Tukey’s post-hoc testing; * p < 0.05, ** p < 0.01, *** p < 0.001. Grey *-data vs. ctr; black *-single treatment vs. combined treatment.

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  Figure 9. (A) Influence of CAM and PAX on cell cycle progression in the BT-549 and MDA-MB-468 TNBC cells. BC cells were exposed to an individual or concomitant CAM and PAX treatment for 48 h using selected ratios (2.0 = IC50, 4.0 = 2IC50) of the IC50 determined in the MTT assay, stained with propidium iodide (PI) and analyzed by FACS. The data are presented as the means ± standard deviation (±SD). (B) Representative histograms from the FACS analysis of the BT-549 TNBC cells after a 48 h incubation with a medium (ctr), PAX, CAM and PAX + CAM (2.0 = IC50, 4.0 = 2IC50). M1-pre-G1 phase; M2-G1 phase; M3-S phase; M4-G2 phase.