Protective effects of mangafodipir against chemotherapy-induced ovarian damage in mice

Abstract

Background: Given the seriousness of chemotherapy-induced ovarian injury in female cancer patients, the preservation of fertility, including through the use of cryopreservation technology and pharmaceuticals, requires investigation. Previous studies have shown that damage to the ovaries is related to oxidative stress caused by anticancer drugs. Therefore, superoxide dismutase (SOD) may represent a key factor in the pharmacological protection of the ovaries. The aim of our study was to identify the effects of mangafodipir, a manganese chelate and SOD-mimetic, on suppression of apoptosis in granulosa cells and primordial follicle activation induced by anticancer drugs.

Methods: Cell viability assays using methyltrichlorosilane solutions and immunoblotting for cleaved caspase-3 were performed in in vitro experiments with the simultaneous addition of mangafodipir to human non-luteinized granulosa cell line (HGrC) cultures treated with hydrogen peroxide (H₂O₂), cisplatin, or paclitaxel. Count and morphological analyses of follicles at each developing stage in the ovaries and immunohistochemistry for cleaved caspase-3, Ki67 and 4-hydroxynonenal, a marker for oxidative stress, were also performed using mangafodipir-injected 6-week-old female ICR mice treated with cisplatin or paclitaxel. Further, mangafodipir was injected into 6-week-old female BALB/c mice inoculated with ES-2 to analyze whether mangafodipir inhibits the anti-tumor effects of cisplatin or paclitaxel treatment.

Results: Mangafodipir attenuated apoptosis induced by H₂O₂ and anticancer drugs in vitro. Mangafodipir also decreased the expression of 4-hydroxynonenal and reduced cisplatin- and paclitaxel-induced apoptosis in granulosa cells in vivo. In addition, mangafodipir inhibited the loss of primordial follicles. Tumor xenograft studies in mice showed that mangafodipir did not affect anticancer drug antitumor effects.

Conclusions: Oxidative stress might be one of the mechanisms of cisplatin- and paclitaxel-induced the loss of primordial follicles. Mangafodipir can reduce cisplatin- and paclitaxel-induced apoptosis in granulosa cells and primordial follicle activation partially via its SOD activity. At the same time, mangafodipir might have other potential mechanisms to inhibit the activation of primordial follicles. Further, mangafodipir attenuated the ovarian damage caused by cisplatin and paclitaxel without affecting their antitumor activities. Mangafodipir, therefore, though its efficacy might be limited, may be a new option for the preservation of fertility during anticancer treatment.

Keywords: Anticancer drug; Follicle; Mangafodipir; Ovary; Oxidative stress.

Fig. 1

Mangafodipir inhibits apoptosis in granulosa cells induced by H₂O₂ and anticancer drugs. a A MTS assay was performed after treatment with H₂O₂ (0, 0.1, or 1 mM) without (m0) or with the administration of mangafodipir (40 μM: m40; 200 μM: m200; 1000 μM: m1000). Mean ± SEM, *, p < 0.01 vs. each control (m0). b Apoptosis was assessed via immunoblotting using anti-cleaved caspase-3 antibody. Mangafodipir did not increase cleaved caspase-3 levels (upper panels). Cleaved caspase-3 induced by 1 mM H₂O₂ was suppressed by the simultaneous addition of 1000 μM/L mangafodipir (lower panels). c The relative intensity of cleaved caspase-3 (CC3) to β-actin was measured to assess the results of the immunoblot in the H₂O₂ in vitro experiments. Mean ± SEM, *, p < 0.01 vs. each control (m0). d A MTS assay was performed after 48-h treatment with 10 or 60 μM cisplatin (CIS) or 10 or 50 μM paclitaxel (PTX) with mangafodipir (200 μM: m200; 1000 μM: m1000) or without mangafodipir (m0). Mean ± SEM, *, p < 0.05 vs. each control (m0). e Apoptosis was assessed via immunoblotting using anti-cleaved caspase-3 antibody. Cleaved caspase-3 was induced with 10 or 60 μM cisplatin (CIS) and 10 μM paclitaxel (PTX) and was suppressed by the simultaneous addition of 1000 μM mangafodipir. f The relative intensity of cleaved caspase-3 (CC3) to β-actin was measured to assess the results of the immunoblot in the anticancer drug-based in vitro experiments. Mean ± SEM, *, p < 0.05 vs. each control (m0). g ROS production was assessed by immunoblotting using anti-4HNE antibody. 4-HNE induced by 10 or 60 μM cisplatin (CIS) or 10 or 50 μM paclitaxel (PTX) was suppressed by the simultaneous addition of 200 μM and 1000 μM mangafodipir. h The relative intensity of 4-HNE to β-actin was measured to assess the results of immunoblotting in anticancer drugs in vitro experiments. Mean ± SEM, *, p < 0.05 vs. each control (m0)

Fig. 2

Effects of mangafodipir on the number of follicles at each development stage and on the rate of healthy follicles after anticancer treatment. a Representative images of ovaries on day 7 after the administration of 7.5 or 20 mg/kg cisplatin (CIS), or 7.5 or 20 mg/kg paclitaxel (PTX), with or without mangafodipir (+/− Mangafodipir, respectively). Bar, 100 μm. b The total number of follicles at each developing stage (primordial, primary, secondary, antral) after the administration of 7.5 (CIS7.5) or 20 (CIS20) mg/kg cisplatin, 7.5 (PTX7.5) or 20 (PTX20) mg/kg paclitaxel, with or without mangafodipir (+/− Mangafodipir, respectively). Mean ± SD, *, p < 0.01. c The rates of healthy follicles after the administration of cisplatin and paclitaxel, with or without mangafodipir. Mean ± SD, *, p < 0.05

Fig. 3

Mangafodipir rescues follicles from anticancer drug-induced apoptosis. a Representative images of immunohistochemistry for cleaved caspase-3 in ovaries treated with 7.5 (CIS7.5) or 20 (CIS20) mg/kg cisplatin, or 7.5 (PTX7.5) or 20 (PTX20) mg/kg paclitaxel, with or without mangafodipir (+/− Mangafodipir, respectively). Bar, 100 μm. b Cleaved caspase-3 (CC3) immunoreactive intensity scores. Mean ± SD, *, p < 0.05. c Mangafodipir (m.10 mg/kg) significantly reduced cleaved caspase-3 levels induced by 7.5 (CIS7.5) or 20 (CIS20) mg/kg cisplatin, 7.5 (PTX7.5) mg/kg paclitaxel, but not by 20 (PTX20) mg/kg paclitaxel. The relative intensities of cleaved caspase-3 (CC3) signals were normalized to those of β-actin to assess the results of immunoblot in mice ovaries. Mean ± SEM, *, p < 0.05 vs. each control (m0)

Fig. 4

Mangafodipir rescues follicles from anticancer drug-induced oxidative stress. a Representative images of immunohistochemistry for 4-HNE in ovaries treated with 7.5 (CIS7.5) or 20 (CIS20) mg/kg cisplatin, or 7.5 (PTX7.5) or 20 (PTX20) mg/kg paclitaxel, with or without mangafodipir (+/− Mangafodipir, respectively). Bar, 100 μm. b 4-HNE immunoreactive intensity scores. Mean ± SD, *, p < 0.05. c Mangafodipir (m.10 mg/kg) reduced 4-HNE induced by 7.5 (CIS7.5), 20 (CIS20) mg/kg cisplatin, 7.5 (PTX7.5) or 20 (PTX20) mg/kg paclitaxel. The relative intensities of 4-HNE signals were normalized to those of β-actin to assess the results of the immunoblot in mice ovaries. Mean ± SEM, *, p < 0.05 vs. each control (m0)

Fig. 5

Expressions of 4-HNE and Ki67 in primordial follicles. a Representative images of immunohistochemistry for 4-HNE in primordial follicles of murine ovaries treated with 7.5 (CIS7.5) or 20 (CIS20) mg/kg cisplatin, 7.5 (PTV7.5) or 20 (PTX20) mg/kg paclitaxel, with or without mangafodipir (+/− Mangafodipir, respectively). Bar, 100 μm. b Representative images of immunohistochemistry for Ki67 in primordial follicles of murine ovaries treated with 20 (CIS20) mg/kg cisplatin, or 7.5 (PTX7.5) or 20 (PTX20) mg/kg paclitaxel, with or without mangafodipir (+/− Mangafodipir, respectively). Bar, 100 μm. c The percentage of Ki67-positive primordial follicles. Mean ± SEM, *, p < 0.05

Fig. 6

Mangafodipir does not impair the antitumor effects of anticancer drugs. Nude mice were injected with 15 mg/kg cisplatin or 7.5 mg/kg paclitaxel with or without mangafodipir (day 0, PTX, CIS, PTX + M, and CIS + M) and sacrificed 5 days after drug injection (day 5). a Increase rate of tumor sizes of ES-2 tumors in nude mice after drug’s treatment. Mean ± SD. b Representative pictures showing tumors in nude mice prior to treatment (day 0) and after treatment (day 5)

Fig. 7

Mechanism of mangafodipir on maintaining the primordial follicle reserve. Excessive ROS produced by cisplatin (CIS) and paclitaxel (PTX) led to apoptosis of developing follicles. Apoptosis in developing follicles reduced the amount of inhibitory factors secreted by granulosa cells that activate primordial follicles (such as AMH and inhibin B), and accelerated the activation of primordial follicles and the loss of ovarian reserve. Mangafodipir (M), that possesses SOD2 activity (S), attenuated ROS-induced damage to follicles. Thus, inhibitory factors secreted by developing follicles can inhibit the activation of primordial follicles and maintain normal ovarian reserve. Cisplatin and paclitaxel might have other mechanisms to activate primordial follicles (pink dotted line). And mangafodipir might have other mechanisms that can directly inhibit the activation of primordial follicles (green dotted line)