Synergistic or Additive Pharmacological Interactions between Magnoflorine and Cisplatin in Human Cancer Cells of Different Histological Origin

Abstract

Magnoflorine is an aporphine alkaloid present in plant species belonging to the Berberidaceae, Magnoliaceae, Menispermaceae, or Papaveraceae botanical families. The interest of magnoflorine has increased recently due to its multiplicity of pharmacological properties. The aim of this study was the analysis of combined anti-proliferative effect of magnoflorine and cisplatin and the assessment of drug-drug pharmacological interaction between these agents using isobolographic method in MDA-MB-468 human breast, NCIH1299 lung, TE671 rhabdomyosarcoma, or T98G glioblastoma cancer cell lines. Magnoflorine in combination with cisplatin at a fixed ratio of 1:1 augmented their anticancer action and yielded synergistic or additive pharmacological interactions by means of isobolographic method, therefore combined therapy using these two active agents can be a promising chemotherapy regimen in the treatment of some types of breast, lung, rhabdomyosarcoma, and glioblastoma cancers.

Keywords: breast cancer; cisplatin; glioblastoma; isobolographic analysis; lung cancer; magnoflorine; rhabdomyosarcoma.

Figure 1

The purity of the isolated magnoflorine (A) presented in the mass chromatogram, its UV spectrum (B), the isotopic distribution of the parent ion (C), and the fragmentation spectrum (D) obtained at the collision energy of 20 V in the HPLC-MS analysis.

Figure 2

The anti-proliferative effects of magnoflorine (MGN) in (A) TE671 (B) T98G (C) NCIH1299 (D) MDA-MB-468 and (E) all analyzed cancer cell lines after 96 h treatment with various concentrations (10–1000 μg/mL) of an active agent. The cell viability was measured by the MTT assay. Results were analyzed using GraphPad Prism 5.0 software (one-way ANOVA; Tukey post-hoc testing). Statistical differences were considered relevant at p < 0.05 (** p < 0.01, *** p < 0.001). Data are expressed as mean ± standard deviation of the mean (± SD); n = 24 per concentration from three independent experiments.

Figure 3

The anti-proliferative effects of cisplatin (CDDP) in (A) TE671 (B) T98G (C) NCIH1299 (D) MDA-MB-468 and (E) all analyzed cancer cell lines after 96 h treatment with various concentrations (0.01–10 μg/mL) of an active agent. The cell viability was measured by the MTT assay. Results were analyzed using GraphPad Prism 5.0 software (one-way ANOVA; Tukey post-hoc testing). Statistical differences were considered relevant at p < 0.05 (* p < 0.05, *** p < 0.001). Data are expressed as mean ± standard deviation of the mean (± SD); n = 24 per concentration from three independent experiments.

Figure 4

The anti-proliferative effects of combined treatment of magnoflorine (MGN) and cisplatin (CDDP) in (A) TE671 (B) T98G (C) NCIH1299 (D) MDA-MB-468 and (E) all analyzed cancer cell lines after 96 h treatment with 1:1 drug mixture (MGN/CDDP) in increasing concentrations. Cancer cells were exposed to concomitant MGN and CDDP treatment using different ratios of the IC₅₀ (2.0 = IC₅₀ + IC₅₀). The cell viability was measured by the MTT assay. Results were analyzed using GraphPad Prism 5.0 software (one-way ANOVA; Tukey post-hoc testing). Statistical differences were considered relevant at p < 0.05 (*** p < 0.001). Data are expressed as mean ± standard deviation of the mean (± SD); n = 24 per concentration from three independent experiments.

Figure 5

Log-probit analysis of concentration–response relationship lines for MGN and CDDP in TE671, T98G, NCIH1299, and MDA-MB-468 cancer cell lines. (A) Log-probit analysis of concentration–response relationship lines for MGN and CDDP in the TE671 cancer cell line. (B) Log-probit analysis of concentration–response relationship lines for MGN and CDDP in the T98G cancer cell line. (C) Log-probit analysis of concentration–response relationship lines for MGN and CDDP in the NCIH1299 cancer cell line. (D) Log-probit analysis of concentration–response relationship lines for MGN and CDDP in the MDA-MB-468 cancer cell line. Concentrations of MGN and CDDP, when administered singly and in combination at the fixed drug concentration ratio of 1:1, were transformed into logarithms to the base 10, while the anti-proliferative activity of the studied drugs in four cancer cell lines (TE671, T98G, NCIH1299, and MDA-MB-468) were transformed into probits according to Litchfield and Wilcoxon (1949). Each point on each graph illustrates the experimentally derived concentration-effect for the respective drugs (MGN and CDDP) and their mixture at the fixed ratio of 1:1. For each drug and mixture tested, at least 4-5 points create a line reflecting the concentration-effect, whose equation is presented close to the analyzed line on each graph. The dotted line on each graph, which is parallel to X axis and starts from 5 probit, illustrates a 50% anti-proliferative effect for the investigated MGN, CDDP, and their mixture at the fixed ratio of 1:1. This dotted line, by crossing the concentration-effect lines for the drugs and their mixtures, determines in approximation their logarithms of IC₅₀ values. This multipart figure contains equations of concentration–response relationship lines for MGN and CDDP along with their IC₅₀ values, when administered alone. Results from the test of parallelism are placed above the X axis on each graph. This test allows for direct comparison of concentration–response relationship lines for MGN and CDDP (when administered alone) with respect to their mutual parallelism. If MGN and CDDP had their concentration-effect lines nonparallel, the type I isobolographic analysis for nonparallel concentration-effect lines was used. Otherwise, the interaction was analyzed with type I isobolographic analysis for collateral concentration–response relationship lines. At the top of each graph, the IC₅₀ values (± S.E.M.) for MGN and CDDP are presented.

Figure 6

Isobolograms for additive and synergistic interactions between MGN and CDDP in four cancer cell lines. (A) Isobologram for the additive interaction of MGN with CDDP in the TE671 cancer cell line. (B) Isobologram for the additive interaction of MGN with CDDP in the T98G cancer cell line. (C) Isobologram for the synergistic interaction of MGN with CDDP in the NCIH1299 cancer cell line; (D) Isobologram for the additive interaction of MGN with CDDP in the MDA-MB-468 cancer cell line. The IC₅₀ ± S.E.M. for MGN and CDDP are plotted on the abscissa and ordinate of the Cartesian system of coordinates. The dotted line starting from the beginning of the Cartesian plot system illustrates the fixed drug concentration ratio of 1:1 for the mixture of two studied drugs (MGN and CDDP). The hyperbolic curves that connect the IC₅₀ values placed on X- and Y-axes illustrate the lower and upper lines of additivity for nonparallel concentration-effects (a,b,d). For nonparallel concentration-effect relationship lines of MGN and CDDP (a,b,d), the lower and upper isoboles of additivity form the additivity area between the curves connecting the IC₅₀ values for MGN and CDDP administered singly. Intersections of the dotted line for the fixed ratio of 1:1 with lower and upper lines of additivity illustrate, in approximation, the additivity points A’ and A”(a,b,d). The theoretically calculated IC_{50 add} values (± S.E.M.) are placed on the lower and upper isoboles of additivity as points A’ and A”, respectively. The experimentally derived IC_{50 exp} value (± S.E.M.) for the two-drug mixture of MGN and CDDP is placed as the point M, on the dotted line for the fixed ratio of 1:1. Since the point M is placed between the points A’ and A” within the area of additivity (a,d), the interaction between MGN and CDDP is additive in the TE671 and MDA-MB-468 cancer cell lines. Since the point M is placed close to the point A’ for the lower isobole of additivity (b), the interaction is additive with a tendency towards synergy in the T98G cancer cell line. For parallel concentration-effect relationship lines of MGN and CDDP (c), the straight diagonal line of additivity connects the IC₅₀ values placed on X- and Y-axes for MGN and CDDP administered singly. Intersection of the dotted line for the fixed ratio of 1:1 with the line of additivity illustrates, in approximation, the point of additivity A (c). The theoretically calculated IC_{50 add} value (± S.E.M.) is placed on the line of additivity as the point A. The experimentally derived IC_{50 exp} value (± S.E.M.) for the two-drug mixture of MGN and CDDP is placed on the graph as the point M. Since the point M is placed significantly below the point A (c), the interaction between MGN and CDDP is supra-additive (synergistic) in the NCIH1299 cancer cell line. ** p < 0.01 vs. IC₅₀ add.