Indium/Gallium Maltolate Effects on Human Breast Carcinoma Cells: In Vitro Investigation on Cytotoxicity and Synergism with Mitoxantrone

Abstract

In this study, we aimed to investigate in vitro whether the synthetized indium maltolate (InMal) and gallium maltolate (GaMal) could exert either a toxic effect toward breast cancer cell line MDA-MB-231 or an agonistic activity with mitoxantrone (MTX) in comparison to fibroblast cell line NIH-3T3. Both GaMal and InMal reduced viability of MDA-MB-231, and at a lesser extent of NIH3-T3, in a dose- and time-dependent mode, the outcome was more effective in comparison to MTX sole exposure. Both GaMal and InMal toxicity was reverted by iron citrate addition on NIH3-T3, not on MDA-MB-231, showing indirectly that gallium and indium's mechanisms of action may include iron targeting. The agonistic activity against MDA-MB-231 survival was shown pretreating with 100 μM InMal for 24 h followed by medium exchange with MTX at 10 ng mL^-1 or vice-versa but not with co-incubation of both compounds. In particular, InMal pretreating resulted more protective to MTX subsequent exposure.

Figure 1

Dose- and time-dependence cell viability due to the addition of GaMal and InMal alone or each one co-incubated with MTX (10 or 200 ng mL^–1) (Experiment 1, see Experimental Section) on both MDA-MB-231 (a–f) and NIH-3T3 (g–l), respectively. Results of MTT test are expressed as percentage related to untreated cells (no metal complexes or MTX addition) set as 100%. Bars represent the mean values ± SEM (standard error of the means) of results from three experiments (n = 3, p < 0.05).

Figure 2

CLSM images of the apoptosis assay. MDA-MB-231 and NIH-3T3 cells were cultured for 144 h under the following conditions: negative control without substances (a,f); positive control with H₂O₂ (b,g); with 10 ng mL^–1 MTX (c,h); with 50 μM GaMal (d,i); with 50 μM InMal (e,j). PSVue480 reagent was used to evaluate apoptotic cells (in green) in MDA-MB-231 and NIH-3T3 cells, respectively. Nuclei were stained with PI (red). CLSM images were magnified at 40×, scale bar: 50 μm.

Figure 3

CLSM images of the apoptosis assay. MDA-MB-231 and NIH-3T3 cells were cultured for 24 h under the following conditions: negative control without substances (a,f); positive control with H₂O₂ (b,g); with 10 ng mL^–1 MTX (c,h); with 50 μM GaMal (d,i); with 50 μM InMal (e,j). After 24 h, the cell culture media were replaced with metal complexes free media and incubated up to 144 h. PSVue480 reagent was used to evaluate apoptotic cells (in green) in both MDA-MB-231 and NIH-3T3 cells, respectively. Nuclei were stained with PI (red). CLSM images were magnified at 40×, scale bar: 50 μm.

Figure 4

Dose- and time-dependence cell viability exerted by the addition of MTX (10 or 200 ng mL^–1) on either MDA-MB-231 (a–f) and NIH-3T3 cells (g–l) pretreated for 24 h with GaMal (a–c and g–i) or InMal (d–f and j–l), respectively (Experiment 2, see Experimental Section). The control is represented by GaMal pretreated cells for 24 h and then incubated for the indicated time with culture media without MTX. Results of MTT test are expressed as a percentage related to untreated cells (no metal complexes or MTX addition) set as 100%. Bars represent the mean values ± SEM of results from the three experiments (n = 3, p < 0.5).

Figure 5

Dose- and time-dependence cell viability exerted by the addition of GaMal or InMal on MDA-MB-231MDA-MB-231(a–f) and NIH-3T3 (g–l), both pretreated for 24 h with MTX (10 or 200 ng mL^–1), respectively (experiment 3, see Experimental Section). The control is represented by both MTX-pretreated cell types for 24 h and then incubated for the time indicated with culture media without metal complexes. Results of MTT treatments were expressed as a percentage related to untreated cells (no metal complexes or MTX) set as 100%. Bars represent the mean values ± SEM of results from three experiments (n = 3, p < 0.05).