Galaxamide alleviates cisplatin-induced premature ovarian insufficiency via the PI3K signaling pathway in HeLa tumor-bearing mice

Abstract

Background

It is challenging to improve the effects of chemotherapy and reduce its adverse impact on the ovaries. Our previous study suggested that the combination of galaxamide could enhance the antitumor effect of cisplatin (CIS) in HeLa cell xenograft mice. However, their potential effects on ovarian tissues remain unknown.

Methods

The Hela tumor-bearing female BALB/c mice model was established and randomly divided into three groups: control group (PBS group), CIS group (0.3 mg/kg CIS group) and galaxamide group (0.3 mg/kg CIS + 3 mg/kg galaxamide-treated group). The serum sex hormones levels, ovarian morphology, functional and molecular characterisation were determined and compared with those of the control group.

Results

The hormonal effects indicated premature ovarian insufficiency (POI) associated with CIS-induced tumor-bearing mice. CIS induces the apoptosis in primordial and developing follicles and subsequently increases follicular atresia, eventually leading to follicle loss. After cotreatment, galaxamide significantly increased anti-Mullerian hormone (AMH) and follicle-stimulating hormone receptor (FSHR) expression and prevented the CIS-induced PI3K pathway, which triggers follicle activation, apoptosis or atresia.

Conclusion

These findings demonstrate that galaxamide could attenuate CIS-induced follicle loss by acting on the PI3K signaling pathway by stimulating AMH and/or FSHR and thus provides promising therapeutic options for patients with cervical cancer.

Background

Cervical cancer is a leading cause of cancer-related mortality in women in China and represents a major global health challenge [1]. In particular, the majority of new cases of cervical cancer occur in women at reproductive age. For patients with cervical cancer, cisplatin (CIS)-based chemotherapy is regarded as a first-line standard scheme [2]. Unfortunately, although it is moderate, the cytotoxic effect of CIS could damage the ovary, leading to premature ovarian insufficiency (POI), which interferes with fertility potential and is also related to poor quality of life as a result of irregular menstruation, osteoporosis and cardiovascular disorders. Moreover, evidence supports that the pregnancy does not affect the the oncological prognosis of mothers and the outcomcoms of newborn in cervical cancer [3]. Thus, there is a growing concern about ovarian toxicity caused by oncotherapy since treatment options for cancer that cause infertility are increasing, resulting in a decrease in the quality of life.

The main mechanisms of POI pathogenesis, such as oxidative and inflammation are related to phosphoinositide 3- kinase (PI3K)-Akt signaling pathway [4]. PI3K/Akt is considered to play a central role on the ovarian function including primordial follicle activation and growing follicle development [5]. Many studies have confirmed PI3K/Akt inhibition can have a therapeutic effect on POI animals [6]. Additionally, CIS induced POI is related to overactivation of PI3K pathway, the pharmacological inhibition of PI3K signaling is a promising strategy for POI caused by CIS treatment.

Marine organisms have emerged as a potential anticancer drug, and many studies have shown that natural products are effective and have less toxic effects than chemotherapy alone [4]. In particular, the identification of natural compounds that have protective effects on the ovary holds great promise in developing chemopreventive strategies to abrogate the risk of POI [5]. We recently reported that galaxamide, a cyclopeptide extracted from the seaweed Galaxaura filamentosa, showed strong inhibitory effects on cervical cancer in vivo and in vitro [6, 7]. In particular, a synergistic effect of galaxamide and CIS was observed in a HeLa xenograft mouse model [6]. This has further heightened interest in the synergistic effect of galaxamide on ovaries and whether galaxamide treatment is beneficial for the ovary in CIS-based chemotherapy in cervical cancer. Additionally, this has clear relevance to the prevention and treatment of CIS-induced POI. In this study, starting from the modeling and treatment of CIS-induced tumor-bearing mice, the effects of galaxamide on the follicular growth pattern in CIS-induced tumor-bearing ovaries were determined. This study aimed to explore the pathogenesis and molecular mechanisms of CIS-induced POI and investigate the effects and mechanisms of galaxamide on ovarian reserve capacity.

Methods

Cell culture and in vivo xenograft models

Female BALB/c nude mice (n = 60; 5–6 weeks; 20.0 ± 0.1 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd (China), and ovarian tissues were obtained from HeLa-xenografted female BALB/c nude mice from our previous study [6]. Briefly, HeLa cells were subcutaneously injected into the armpit of nude mice. HeLa-xenografted mice were monitored for the development of primary xenograft tumors and sacrificed when tumors reached 10% of body weight.

After successful modeling, mice were randomly divided into three groups: control group (PBS-treated group), CIS group (0.3 mg/kg CIS-treated group) and galaxamide group (0.3 mg/kg CIS + 3 mg/kg galaxamide-treated group).All group were given treatment via i.p. injection every other day for 20 days (n = 20/ group). The body weights of the mice were measured before each administration. On day 21, the mice were anaesthetized with an intraperitoneal injection of sodium pentobarbital (40 mg/kg) and subsequently cervical dislocation. The ovaries were weighed after careful removal of adipose tissue and other tissues, and the ovarian index was calculated by dividing bilateral ovarian wet weight by body weight.

Hormone assays

Blood samples were collected via the abdominal aorta. Serum levels of estradiol (E2), progesterone (P), Mullerian hormone (AMH), follicle-stimulating hormone (FSH), luteinizing hormone(LH) and prolactin (PRL) were detected using enzyme-linked immunosorbent assay (ELISA) kits as previously described [8].

Estrus cycle

During administration, the vaginal smears of five mice in each group were evaluated as previously described [8]. The estrous cycle stage was determined according to the cell types observed by Wright’s Giemsa staining.

Histology

The mouse ovaries were fixed, embedded in paraffin, sectioned (5 µm thick) and stained with hematoxylin and eosin (H&E) routinely. The follicles in the ovarian histological sections were counted as primordial, primary, secondary or antral follicles. Atretic preantral follicles were recognized according to the morphologic criteria as described [8]. Every fifth and sixth histological section was selected for comparison and evaluation.

Immunohistological staining and qualification

Immunohistochemistry and immunofluorescence staining were performed as previously described [9]. Antibodies are presented in Table 1. The intensity was quantified and compared between each group with the follicles only at the same development.

Table 1 List of Antibodies

Western blotting analysis

Western blotting was performed as previously described [9]. The antibodies are presented in Table 1. The expression of genes was normalized to β-actin. The results are representative of three independent experiments.

Statistical analysis

GraphPad Prism 9 Software was used for all analyses. Comparisons between groups were performed by unpaired t test, and data with unequal variance were compared with the Mann‒Whitney U test. Normally distributed data were compared with ANOVA for more than two groups. The results are presented as the means ± SEM, and P < 0.05 was considered statistically significant.

Result

The effects of galaxamide on follicle development

To determine the impact of galaxamide on the molecular and cellular properties of the ovary, xenograft tumors of the cervical cancer line HeLa were established in female BALB/c nude mice with and without galaxamide followed by treatment with CIS for 20 days (Fig. 1A). Mice treated with CIS alone showed a significant decrease in body weight compared with controls, and the effect was ameliorated in mice treated with CIS combined with galaxamide (Fig. 1B). The tumor weight and volume were significantly reduced in both the CIS with and without galaxamide-treated groups (Supplemental Figure S1). According to the different cell morphologies in the vaginal smears, the estrous cycle of the mice in each group was detected. The tumor-bearing mice displayed prolonged metestrus and diestrus stages, as revealed by microscopic analysis of the predominant cell types in the vaginal smears (Fig. 1C and D).

Fig. 1

Effects of galaxamide on folliculogenesis. A Experimental design of CIS and galaxamide treatment in a HeLa tumor-bearing model. B Average mouse weight (C)Crystal violet staining of vaginal smears from mice in four different stages of the estrous stage. D Representative estrous cycle of mice during 20 consecutive days and quantitative analysis of estrous cycles. E Representative ovary gross images and statistics of the ovary/body weight ratio in tumor-bearing mice. F Representative histology images of ovary sections from tumor-bearing mice and bar chart showing the quantification of total, primordial, primary, secondary and antral follicles in tumor-bearing mice. Scale bars: 50 µm. PDF, primordial follicle; PF, primary follicle; SF, secondary follicle; AF, antral follicles

After 20 days, CIS-induced tumor-bearing mice exhibited a smaller ovary size. The ovarian index was significantly lower in the CIS group than in the control group (P < 0.001) (Fig. 1E). The size and weight of ovaries from galaxamide-treated mice were rescued compared with those of the control (Fig. 1E). Follicles are the morphofunctional unit of the ovary. The CIS group displayed a decrease in the number of total follicles, particularly primordial and secondary follicles and galaxamide treatment significantly reduced CIS-induced total, primordial and growing follicle loss (Fig. 1F).

The effect of galaxamide on the hormone levels

In tumor-bearing mice, the E2 levels of the CIS group were significantly lower than those of the control group (15.55 ± 0.19 vs. 10.96 ± 0.31, P < 0.0001), while the FSH levels of the CIS group were significantly higher (27.63 ± 1.98 vs. 58.07 ± 4.19, P = 0.0006), thus indicating POI in the CIS-induced tumor-bearing mouse model. The serum progesterone and AMH levels were decreased by 38% and 21%, whereas the serum LH and PRL levels were elevated by 29% and 28%, respectively. Galaxamide treatment significantly increased progesterone, E2 and AMH levels (P = 0.0326, P = 0.0024 and P = 0.0374, respectively) and reduced FSH levels (P = 0.0135) (Fig. 2A).

Fig. 2

Effects of galaxamide on serum hormones and ovarian reserve-related protein expression. A Serum hormone levels in mice were measured by enzyme-linked immunosorbent assay. B and C Immunostaining for AMH and FSHR expression in GCs in follicles at different developmental stages and quantification of the percentages of positive GCs per follicle within the ovaries of each group. D Representative immunoblot analysis and quantification of AMH and FSHR expression in the ovaries of each group. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 indicate significant differences between the control and experimental groups. Scale bars: 50 µm (B). AMH, anti-Mullerian hormone; FSHR, follicle stimulating hormone receptor; GC, granulosa cells

The effect of galaxamide on AMH and FSHR expression

The expression of anti-Mullerian hormone (AMH) and follicle stimulating hormone receptor (FSHR) was detected by immunohistochemical analysis. AMH protein was mainly present in granulose cells (GCs) from the preantral to small antral follicles and was faint to absent in GCs in atretic follicles (Supplemental Figure S2). The data also showed that the expression of AMH and FSHR was decreased in the GCs of CIS-induced tumor-bearing ovaries compared to the control. In contrast, the majority of follicles were observed in the ovaries from the galaxamide group, with a significantly increased secretion of AMH and FSHR (Fig. 2B and C).

Furthermore, the expression of AMH in primordial follicles was weak in control ovaries but absent in tumor-bearing ovaries, suggesting that reduced AMH expression results in a higher proportion of primordial follicle activation in CIS and that galaxamide increases AMH, which regulates CIS-induced primordial follicle activation (Fig. 2C). Furthermore, the protein expression of AMH and FSHR in the ovaries was measured by Western blot analysis. The data showed that AMH and FSHR expression in the ovarian tissues of the POI group was decreased extensively compared to that in the control group. After galaxamide treatment, AMH and FSHR expression was significantly increased in the CIS-induced group (Fig. 2D).

The effects of galaxamide on the PI3K pathway

Immunofluorescence staining showed that positive DEAD-box helicase 4 (DDX4) staining was present in oocytes within follicles of developing ovaries, with an obvious decrease in the number of primordial follicles in the CIS group compared to controls (Fig. 3A), suggesting CIS-induced damage of primordial follicles. Furthermore, the stain of nuclear proliferating cell nuclear antigen (PCNA) was intense in the GCs of growing follicles of ovaries. The proportion of overall positive follicles was decreased in the CIS group (64% versus 36%) but increased in the galaxamide group, suggesting that galaxamide treatment caused follicles to grow (Fig. 3B). Immunofluorescence staining for PTEN was expressed in the cytoplasm of GCs at all stages of follicles in ovaries, with a relative lower expression level in the CIS group compared with that in controls (Fig. 3C), as determined by Western blot analysis (Fig. 3D). The results showed that CIS activates PI3K pathway genes, which are responsible for primordial follicle growth initiation. For FOXO3a, immunohistochemistry analysis demonstrated that FOXO3a was expressed in the oocyte nuclei of primordial follicles in controls, and there was increased nuclear exclusion of FOXO3a in the ovaries of the CIS group. Galaxamide treatment inhibited shunting of FOXO3a to the cytoplasm and reduced nuclear phosphorylated FOXO3a expression (Fig. 3E). Consistent with these data, Western blot analysis results showed that galaxamide treatment increased the phosphorylation of the activation proteins Akt and RPS6 compared with that in the CIS group (Fig. 3F). These results suggest that galaxamide suppresses CIS-induced toxicity through the PI3K pathway.

Fig. 3

Galaxamide suppresses follicle loss via the PI3K signaling pathway. A Images of immunofluorescence with dashed lines were taken that showed the nuclei of the oocytes of primordial follicles using an anti-DDX4 antibody in each group. All of the nuclei were stained with DAPI. B Immunofluorescence staining of PCNA-positive GCs in follicles at different developmental stages and quantification of the positive GC rate per follicle in the three groups. C Immunofluorescence staining of PTEN in GCs in primordial follicles and quantification of the percentages of PTEN⁺ GCs (white arrows) per follicle within ovaries in each group. Right panels: Higher magnification of the dotted boxed area in the middle panels. D Western blot analyses of PTEN in the ovaries of the three groups; protein levels were analyzed by normalization to β-actin. E Immunostaining for nuclear shuttling of FOXO3a and pFOXO3a expression and increased cytosolic localization of pFOXO3a in activated primordial follicles (red circles). Bottom left panels: Higher magnification of the dashed boxed areas. F Representative immunoblot analysis and quantification of key proteins of PI3K-Akt and mTOR signaling in the ovaries of the three groups. *P < 0.05, **P < 0.01 and ***P < 0.001 indicate significant differences between the control and experimental groups. Scale bar: 50 µm (A, B and E). PTEN, phosphatase regulator and tensin homolog

The effects of galaxamide on follicular apoptosis and atresia.

Follicle atresia is the main process responsible for the loss of ovarian follicles, and apoptosis is the underlying mechanism. Atresia was further confirmed by immunostaining for cleaved caspase-3, which showed atretic follicles, including primordial follicles and growing follicles (Fig. 4A). The CIS group displayed an incresse in the number of total atretic follicles, particularly the primordial and secondary follicles compared to the control group. Compared with the CIS group, the galaxamide group showed dense cortical tissue, decreased follicular atresia (P = 0.0036), and decreased atretic follicle number at the primordial and secondary stages (Fig. 4B). This atresia was further confirmed by immunofluorescence for cleaved caspase-3, suggesting that atretic follicles were found, including primordial follicles and growing follicles, in both groups, suggesting that the apoptosis signaling pathway was hyperactivated in the CIS group but suppressed in the galaxamide group (Fig. 4C). In addition, the expression of the proapoptotic proteins cleaved caspase-3 was significantly increased in the CIS group compared to the controls but significantly decreased in the galaxamide group, as determined by Western blotting (Fig. 4D). These findings suggest that CIS treatment results in the activation of follicle growth and triggers an apoptotic response.

Fig. 4

Effects of galaxamide on apoptosis. A Representative H&E (top panel) and cleaved caspase-3 staining (bottom panel) of atretic follicles (dashed line) at different developmental stages in control ovaries. Insets: Higher magnifications of the dotted boxed areas. B Bar chart showing the quantification of atretic follicles within total, primordial, primary, secondary and antral follicles from the three groups. C Immunofluorescence staining and (D) Western blotting showing the expression of cleaved caspase-3. granulosa cells, GC. PDF, primordial follicle. SF, secondary follicle. *P < 0.05 and ****P < 0.0001 indicate significant differences between the control and experimental groups. Scale bar: 200 µm (A, G and H); 50 µm (D, E and I)

Discussion

For fertility preservation, it is challenging to improve the effects of chemotherapy and reduce its adverse impact on ovaries. Our previous study suggested that the combination of galaxamide could synergistically enhance the antitumor effect of CIS in vivo and hence may have great potential for therapeutic intervention for cervical cancer [6]. However, their potential effects on ovarian tissues are still unknown. The current study demonstrates that hormonal effects indicated POI associated with CIS-induced HeLa tumor-bearing mice. The CIS-induced tumor-bearing ovaries had low follicle density, with activation of the PI3K pathway in tumor-bearing mice, but toxicity was largely avoided after galaxamide treatment. CIS induces the apoptosis of oocytes in primordial follicles and GCs in developing follicles and subsequently increases follicular atresia. In addition, the increases in AMH and FSHR expression and inactivation of the PI3K pathway by galaxamide could cause an increase in follicle numbers, improve follicle health and hormone function in tumor-bearing mice following CIS treatment and provide promising therapeutic options for patients with cervical cancer.

There is a need to identify the effects of marine natural products, an ideal synergistic candidate, on ovarian function in various cancers [6, 10]. Although the mechanisms are not fully understood, studies show that several seaweed-derived compounds are beneficial to premenopausal women by regulating abnormal hormonal status [11] and may be critical determinants of POI risk. POI patients vary in their hormonal secretion, with an association between hormone levels and follicle development [12]. Ovarian reserve can be detected by hormonal markers, such as FSH and AMH. Studies in human ovaries have shown that CIS causes primordial follicle depletion in cervical cancer patients at a young age [13], and increased FSH levels and reduced estradiol levels correlate with follicular depletion after CIS treatment in cervical cancer [14]. In this study, the elevation of FSH and decrease in estradiol and AMH levels indicated POI-associated CIS-induced tumor-bearing mice, and galaxamide treatment led to a marked reduction in serum FSH levels and an increase in estradiol and AMH levels in tumor-bearing mice that exhibited CIS-induced POI prior to the intervention. Thus, galaxamide has a role in CIS-induced ovarian dysfunction, which confers protection against cervical cancer.

Various studies have successfully established a mouse model of POI through the application of CIS. However, the degree of ovarian damage and risk of infertility depends on the dose and type of chemotherapeutic agent. Previous studies have demonstrated that 2 mg/kg CIS induces oocyte death and depletion of ovarian reserve in mice [15]. The PI3K pathway is critical for the fates of primordial follicles related to POI [16, 17]. PTEN is an upstream negative regulator, and Foxo3 (forkheadboxO3) is a downstream inhibitory transcriptional factor of PI3K signaling pathway. Activation of the PI3K pathway by inhibition of PTEN causes depletion of the primordial follicle pool through excessive follicle activation. PI3K activates the downstream serinethreonine kinase, Akt. Mammalian target of rapamycin (mTOR) is one of downstream signals that can be regulated by the activation of Akt, and the S6 ribosomal protein (S6RP) is phosphorylated by the mammalian target of mTOR [15, 16]. In this study, a lower dosage of CIS (0.3 mg/kg) activated PI3K/Akt signaling cascade in tumor-bearing ovaries, suggesting primordial follicle activation via CIS induction. Indeed, cancer patients had lower pretreatment AMH levels than noncancer patients, indicating that ovarian aging occurs earlier [18], which may support our belief that a tumor-induced alteration occurs in the ovaries [8], reinforcing the sensitivity of the cells to the toxicity of CIS at a lower dosage, as evidenced by the numbers of follicles.

Consistent with our previous findings, follicle loss in cancers is involved in initiation, subsequent growth and follicular atresia [8]. Indeed, abnormal hormone levels are caused not only by a significant loss in the number of follicles but also by reduced hormonal production in the GCs of growing follicles [19]. It is thought that AMH and FSHR are closely related in GC, and both affect ovarian response outcome [20]. The downregulation of AMH expression in patients upon chemotherapy is considered a marker of reduced proliferation of GCs in growing follicles [8, 21]. The PI3K signaling pathway is one of the canonical effectors of FSHR or AMH, and the abnormal expression of key components could lead to primordial follicle activation or increased atresia [22, 23]. Moreover, in AMH-null mice, more atretic follicles were observed, accompanied by a blunted FSH surge [24]. AMH is considered to have a stronger effect on follicle atresia than primordial follicle activation [25]. In this study,the effects of CIS showed a decrease in the expression of AMH and FSHR in GCs of growing follicles, particularly in atretic follicles, while treatment with galaxamide prevented follicle loss, accompanied by elevated expression of AMH and FSHR.

Apoptosis is thought to be a key contributor and mechanism in CIS-induced follicular atresia [6] and in human granulosa cell lines [26]. For proliferative activities, GCs are always the first cells to be affected during chemotherapy [27]. In vitro, natural products from Dicathais orbita selectively induce apoptosis in cancer cells but not in normal GCs, which confer protection to the ovary in cancer treatment [26]. Galaxamide has been shown to induce apoptosis through ROS generation and inhibit tumor growth [28]. PTEN negatively regulates the PI3K pathway, thereby inducing cell apoptosis through FOXO3a [29]. In GCs, galaxamide upregulates PTEN activity, increases PTEN protein levels, and enhances nuclear PTEN localization, inhibiting CTX-induced cellular apoptosis through the inactivation of the PI3K signaling pathway. Thus, the anti-apoptotic effect of galaxamide is attributed to PTEN-mediated FoxO3a in GCs.

In conclusion, the present study demonstrates that in a xenograft mouse model, galaxamide has a similar antitumor efficacy as CIS but without associated CIS-related ovarian toxicity in cervical cancer. The underlying mechanism was possibly through the inhibition of cell apoptosis and follicle atresia through the activation of the PI3K/Akt signaling pathway. Whether galaxamide acts on the PI3K signaling pathway by stimulating AMH and/or FSHR needs further verification by in vitro experiments.