CSE1L/CAS, a microtubule-associated protein, inhibits taxol (paclitaxel)-induced apoptosis but enhances cancer cell apoptosis induced by various chemotherapeutic drugs

CSE1L/CAS, a microtubule-associated, cellular apoptosis susceptibility protein, is highly expressed in various cancers. Microtubules are the target of paclitaxel-induced apoptosis. We studied the effects of increased or reduced CAS expression on cancer cell apoptosis induced by chemotherapeutic drugs including paclitaxel. Our results showed that CAS overexpression enhanced apoptosis induced by doxorubicin, 5-fluorouracil, cisplatin, and tamoxifen, but inhibited paclitaxel-induced apoptosis of cancer cells. Reductions in CAS produced opposite results. CAS overexpression enhanced p53 accumulation induced by doxorubicin, 5-fluorouracil, cisplatin, tamoxifen, and etoposide. CAS was associated with alpha-tubulin and beta-tubulin and enhanced the association between alpha-tubulin and beta-tubulin. Paclitaxel can induce G2/M phase cell cycle arrest and microtubule aster formation during apoptosis induction, but CAS overexpression reduced paclitaxel-induced G2/M phase cell cycle arrest and microtubule aster formation. Our results indicate that CAS may play an important role in regulating the cytotoxicities of chemotherapeutic drugs used in cancer chemotherapy against cancer cells.     

Cardiotonic steroids attenuate ERK phosphorylation and generate cell cycle arrest to block human hepatoma cell growth

Recent studies revealed the potential of Na(+)/K(+)-ATPase as a target for anticancer therapy and showed additional modes of action of cardiotonic steroids (CSs), a diverse family of naturally derived compounds, as inhibitors of Na(+)/K(+)-ATPase. The results from epidemiological studies showed significantly lower mortality rates in cancer patients receiving CSs, which sparked interest in the anticancer properties of these drugs. The present study was designed to investigate the anticancer effect of CSs (ouabain or cinobufagin) and to elucidate the molecular mechanisms of CS activity in hepatoma cell lines (HepG2 and SMMC-7721). Ouabain and cinobufagin significantly inhibited cell proliferation by attenuating the phosphorylation of extracellular regulated kinase (ERK) and down-regulating the expression of C-myc. These CSs also induced cell apoptosis by increasing the concentration of intracellular free calcium ([Ca(2+)](i)) and induced S phase cell cycle arrest by down-regulating the expression of Cyclin A, cyclin dependent kinase 2 (CDK2) and proliferating cell nuclear antigen (PCNA) as well as up-regulating the expression of cyclin dependent kinase inhibitor 1A (p21(CIP1)). Overexpression of ERK reversed the antiproliferation effect of ouabain or cinobufagin in HepG2 and SMMC-7721 cells. Currently, the first generation of CS-based anticancer drugs (UNBS1450 and Anvirzel) are in Phase I clinical trials. These data clearly support their potential use as cancer therapies.     

Microtubule dysfunction induced by paclitaxel initiates apoptosis through both c-Jun N-terminal kinase (JNK)-dependent and -independent pathways in ovarian cancer cells

The antineoplastic agent paclitaxel (TaxolTM), a microtubule stabilizing agent, is known to arrest cells at the G2/M phase of the cell cycle and induce apoptosis. We and others have recently demonstrated that paclitaxel also activates the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) signal transduction pathway in various human cell types, however, no clear role has been established for JNK/SAPK in paclitaxel-induced apoptosis. To further examine the role of JNK/SAPK signaling cascades in apoptosis resulting from microtubular dysfunction induced by paclitaxel, we have coexpressed dominant negative (dn) mutants of signaling proteins of the JNK/SAPK pathway (Ras, ASK1, Rac, JNKK, and JNK) in human ovarian cancer cells with a selectable marker to analyze the apoptotic characteristics of cells expressing dn vectors following exposure to paclitaxel. Expression of these dn signaling proteins had no effect on Bcl-2 phosphorylation, yet inhibited apoptotic changes induced by paclitaxel up to 16 h after treatment. Coexpression of these dn signaling proteins had no protective effect after 48 h of paclitaxel treatment. Our data indicate that: (i) activated JNK/SAPK acts upstream of membrane changes and caspase-3 activation in paclitaxel-initiated apoptotic pathways, independently of cell cycle stage, (ii) activated JNK/SAPK is not responsible for paclitaxel-induced phosphorylation of Bcl-2, and (iii) apoptosis resulting from microtubule damage may comprise multiple mechanisms, including a JNK/SAPK-dependent early phase and a JNK/SAPK-independent late phase.     

A Synthetic Podophyllotoxin Derivative Exerts Anti-Cancer Effects by Inducing Mitotic Arrest and Pro-Apoptotic ER Stress in Lung Cancer Preclinical Models

Some potent chemotherapy drugs including tubulin-binding agents had been developed from nature plants, such as podophyllotoxin and paclitaxel. However, poor cytotoxic selectivity, serious side-effects, and limited effectiveness are still the major concerns in their therapeutic application. We developed a fully synthetic podophyllotoxin derivative named Ching001 and investigated its anti-tumor growth effects and mechanisms in lung cancer preclinical models. Ching001 showed a selective cytotoxicity to different lung cancer cell lines but not to normal lung cells. Ching001 inhibited the polymerization of microtubule resulting in mitotic arrest as evident by the accumulation of mitosis-related proteins, survivin and aurora B, thereby leading to DNA damage and apoptosis. Ching001 also activated pro-apoptotic ER stress signaling pathway. Intraperitoneal injection of 2 mg/kg Ching001 significantly inhibited the tumor growth of A549 xenograft, while injection of 0.2 mg/kg Ching001 decreased the lung colonization ability of A549 cells in experimental metastasis assay. These anti-tumor growth and lung colonization inhibition effects were stronger than those of paclitaxel treatment at the same dosage. The xenograft tumor tissue stains further confirmed that Ching001 induced mitosis arrest and tumor apoptosis. In addition, the hematology and biochemistry tests of blood samples as well as tissue examinations indicated that Ching001 treatment did not show apparent organ toxicities in tested animals. We provided preclinical evidence that novel synthetic microtubule inhibitor Ching001, which can trigger DNA damage and apoptosis by inducing mitotic arrest and ER stress, is a potential anti-cancer compound for further drug development.     

Paclitaxel affects cytosolic calcium signals by opening the mitochondrial permeability transition pore.

We have characterized the effects of the antimitotic drug paclitaxel (Taxol(TM)) on the Ca(2+) signaling cascade of terminally differentiated mouse pancreatic acinar cells. Using single cell fluorescence techniques and whole-cell patch clamping to record cytosolic Ca(2+) and plasma membrane Ca(2+)-dependent Cl(-) currents, we find that paclitaxel abolishes cytosolic Ca(2+) oscillations and in more than half of the cells it also induces a rapid, transient cytosolic Ca(2+) response. This response is not affected by removal of extracellular Ca(2+) indicating that paclitaxel releases Ca(2+) from an intracellular Ca(2+) store. Using saponin-permeabilized cells, we show that paclitaxel does not affect Ca(2+) release from an inositol trisphosphate-sensitive store. Furthermore, up to 15 min after paclitaxel application, there is no significant effect on either microtubule organization or on endoplasmic reticulum organization. The data suggest a non-endoplasmic reticulum source for the intracellular Ca(2+) response. Using the mitochondrial fluorescent dyes, JC-1 and Rhod-2, we show that paclitaxel evoked a rapid decline in the mitochondrial membrane potential and a loss of mitochondrial Ca(2+). Cyclosporin A, a blocker of the mitochondrial permeability transition pore, blocked both the paclitaxel-induced loss of mitochondrial Ca(2+) and the effect on Ca(2+) spikes. We conclude that paclitaxel exerts rapid effects on the cytosolic Ca(2+) signal via the opening of the mitochondrial permeability transition pore. This work indicates that some of the more rapidly developing side effects of chemotherapy might be due to an action of antimitotic drugs on mitochondrial function and an interference with the Ca(2+) signal cascade.     

Cardiac glycosides induce autophagy in human non-small cell lung cancer cells through regulation of dual signaling pathways

Na(+)/K(+)-ATPase targeted cancer therapy has attracted increasing interests of oncologists in lung cancer field. Although multiple anti-cancer mechanisms of cardiac glycosides as Na(+)/K(+)-ATPase inhibitors are revealed, the role of autophagy and related molecular signaling pathway for the class of compounds in human non-small cell lung cancer (NSCLC) cells has not been systematically examined. We herein investigated the anti-cancer effects of two representative cardiac glycosides, digoxin and ouabain, in A549 and H460 cell lines. Both agents caused significant growth inhibition at nanomolar level. The cardiac glycosides were found to induce moderate G(2)/M arrest but not apoptosis at IC(50) level in the NSCLC cell lines. Moreover, autophagy was markedly induced by both agents, as evidenced by the time- and dose-dependent increase of LC3-II, up-regulation of Atg5 and Beclin1, as well as by the observations through acridine orange staining, transmission electron microscopy and quantification of GFP-LC3 fluorescence. Importantly, AMP-activated protein kinase (AMPK) pathway was activated, resulting in mammalian target of rapamycin (mTOR) deactivation during autophagy induction. Moreover, extracellular-signal-regulated kinase 1/2 (ERK1/2) activation was simultaneously found to be involved in the autophagy regulation. Co-treatment with respective inhibitors or siRNAs could either block the autophagic phenotypes and signals, or significantly increase the cellular viability, indicating the drugs-induced autophagy plays tumor-suppressing role. This work provides first evidence showing that the cardiac glycosides induce autophagy in human NSCLC cells through regulation of both mTOR and ERK1/2 signaling pathways. The autophagy may at least partially account for the growth inhibitory effects of the compounds in human NSCLC cells.     

Crosstalk between autophagy and apoptosis in the regulation of paclitaxel-induced cell death in v-Ha-ras-transformed fibroblasts

The previous studies by this author group has shown that paclitaxel, a mitotic inhibitor used in breast cancer chemotherapy, inhibits cell growth via induction of Raf-1-dependent apoptosis. In this article, the role of autophagy in paclitaxel anticancer action was investigated using v-Ha-ras-transformed NIH 3T3 cells. Paclitaxel induced a notable increase in the number of fluorescent particles labeled with monodansylcadaverine (MDC), a specific marker for autophagic vacuoles. MDC-labeled vacuoles clearly exhibited the fluorescent-tagged LC3 in cells transiently overexpressing GFP-LC3 (a protein that associates with autophagosome membranes). However, autophagy inhibition with 3-methyladenine (3-MA) failed to rescue v-Ha-ras-transformed NIH 3T3 cells from paclitaxel-induced cell death. More interestingly, the apoptosis inhibition by overexpression of the X-linked inhibitor of apoptosis (XIAP) did not fully block the cell death by paclitaxel, implying that apoptosis inhibition might accelerate the autophagic components of the paclitaxel response. Conversely, Raf-1 shRNA expression protected against paclitaxel-induced cell death through the simultaneous inhibition of both autophagy and apoptosis. These results suggest that both autophagy and apoptosis act as cooperative partners to induce cell death in v-Ha-ras-transformed NIH 3T3 cells treated with paclitaxel.     

Ouabain,a Cardiac Glycoside,Inhibits the Fanconi Anemia/BRCA Pathway Activated by DNA Interstrand Cross-Linking Agents

Modulation of the DNA repair pathway is an emerging target for the development of anticancer drugs. DNA interstrand cross-links (ICLs), one of the most severe forms of DNA damage caused by anticancer drugs such as cisplatin and mitomycin C (MMC), activates the Fanconi anemia (FA)/BRCA DNA repair pathway. Inhibition of the FA/BRCA pathway can enhance the cytotoxic effects of ICL-inducing anticancer drugs and can reduce anticancer drug resistance. To find FA/BRCA pathway inhibitory small molecules, we established a cell-based high-content screening method for quantitating the activation of the FA/BRCA pathway by measuring FANCD2 foci on DNA lesions and then applied our method to chemical screening. Using commercial LOPAC1280 chemical library screening, ouabain was identified as a competent FA/BRCA pathway inhibitory compound. Ouabain, a member of the cardiac glycoside family, binds to and inhibits Na⁺/K⁺-ATPase and has been used to treat heart disease for many years. We observed that ouabain, as well as other cardiac glycoside family members―digitoxin and digoxin―down-regulated FANCD2 and FANCI mRNA levels, reduced monoubiquitination of FANCD2, inhibited FANCD2 foci formation on DNA lesions, and abrogated cell cycle arrest induced by MMC treatment. These inhibitory activities of ouabain required p38 MAPK and were independent of cellular Ca²⁺ ion increase or the drug uptake-inhibition effect of ouabain. Furthermore, we found that ouabain potentiated the cytotoxic effects of MMC in tumor cells. Taken together, we identified an additional effect of ouabain as a FA/BRCA pathway-inhibiting chemosensitization compound. The results of this study suggest that ouabain may serve as a chemosensitizer to ICL-inducing anticancer drugs.     

Bim is reversibly phosphorylated but plays a limited role in paclitaxel cytotoxicity of breast cancer cell lines

The chemotherapeutic drug, paclitaxel, induces mitotic arrest and then activates the cellular apoptotic program. Although paclitaxel has been in clinical use for over 10 years for the treatment of breast, ovarian, and lung cancer, the molecular mechanisms of paclitaxel-induced cytotoxicity are ill defined. We decided to investigate the regulatory mechanism of the pro-apoptotic BH3-only protein Bim, which is known to play a role in paclitaxel cytotoxicity. We discovered that paclitaxel induces reversible phosphorylation of Bim. Bim initially displays enhanced phosphorylation during paclitaxel-induced mitotic arrest, and then undergoes de-phosphorylation as cells become apoptotic. This dynamic phosphorylation is dependent on mitotic checkpoint signaling. However, while these results suggest that reversible phosphorylation of Bim may contribute to the transmission of a mitotic checkpoint-to-apoptosis signal, we did not observe a strong correlation between Bim protein levels and cellular sensitivity to paclitaxel. Indeed, in contrast to the well-defined role of Bim in paclitaxel-induced cell death in mouse model cells, our depletion studies demonstrate that Bim is not absolutely required for paclitaxel cytotoxicity in breast cancer cell lines. Clearly it is imperative to define the contribution of Bim in paclitaxel-induced apoptosis of clinically relevant targets in order to rationally develop enhanced treatment strategies.     

Stimulation of erythrocyte phosphatidylserine exposure by paclitaxel.

Side effects of cytostatic treatment include development of anemia resulting from either decreased generation or accelerated clearance of circulating erythrocytes. Recent experiments revealed a novel kind of stress-induced erythrocyte death, i.e. eryptosis, which is characterized by enhanced cytosolic Ca(2+) levels, increased ceramide formation and exposure of phosphatidylserine at the cell surface. The present study explored whether cytostatic treatment with paclitaxel (Taxol) triggers eryptosis. Blood was drawn from cancer patients before and after infusion of 175 mg/m2 Taxol. The treatment significantly decreased the hematocrit and significantly increased the percentage of annexin-V-binding erythrocytes in vivo (by 37%). In vitro incubation of human erythrocytes with 10 microM paclitaxel again significantly increased annexin-V-binding (by 129%) and augmented the increase of annexin-V-binding following cellular stress. The enhanced phosphatidylserine exposure was not dependent on caspase-activity but paralleled by erythrocyte shrinkage, increase of cytosolic Ca(2+) activity, ceramide formation and activation of calpain. Phosphatidylserine exposure was similarly induced by docetaxel but not by carboplatin or doxorubicin. Moreover, eryptosis was triggered by the Ca(2+) ionophore ionomycin (10 microM). In mice, ionomycin-treated eryptotic erythrocytes were rapidly cleared from circulating blood and sequestrated into the spleen. In conclusion, our data strongly suggest that paclitaxel-induced anemia is at least partially due to induction of eryptosis.     

Cardiac glycosides as novel cancer therapeutic agents

The class of steroid-like compounds designated cardiac glycosides includes well-known drugs such as digoxin, digitoxin, and ouabain. Their continued efficacy in treatment of congestive heart failure and as anti-arrhythmic agents is well appreciated. Less well known, however, is the emerging role of this category of compounds in the prevention and/or treatment of proliferative diseases such as cancer. New findings within the past five years have revealed these compounds to be involved in complex cell-signal transduction mechanisms, resulting in selective control of human tumor but not normal cellular proliferation. As such, they represent a promising form of targeted cancer chemotherapy. New clinical studies of their anticancer potential as single or adjuvant treatments may provide insight into these potentially valuable therapeutic options. This review focuses on recent findings on cellular pharmacology of cardiac glycosides as they relate to treatment of human cancer and attempts to explain why these agents have been overlooked in the past.     

Ouabain decreases sarco(endo)plasmic reticulum calcium ATPase activity in rat hearts by a process involving protein oxidation

The effect of cardiac glycosides to increase cardiac inotropy by altering Ca(2+) cycling is well known but still poorly understood. The studies described in this report focus on defining the effects of ouabain signaling on sarcoplasmic reticulum Ca(2+)-ATPase function. Rat cardiac myocytes treated with 50 microM ouabain demonstrated substantial increases in systolic and diastolic Ca(2+) concentrations. The recovery time constant for the Ca(2+) transient, tau(Ca(2+)), was significantly prolonged by ouabain. Exposure to 10 microM H(2)O(2), which causes an increase in intracellular reactive oxygen species similar to that of 50 microM ouabain, caused a similar increase in tau(Ca(2+)). Concurrent exposure to 10 mM N-acetylcysteine or an aqueous extract from green tea (50 mg/ml) both prevented the increases in tau(Ca(2+)) as well as the changes in systolic or diastolic Ca(2+) concentrations. We also observed that 50 microM ouabain induced increases in developed pressure in addition to diastolic dysfunction in the isolated perfused rat heart. Coadministration of ouabain with N-acetylcysteine prevented these increases. Analysis of sarcoplasmic reticulum Ca(2+)-ATPase protein revealed increases in both the oxidation and nitrotyrosine content in the ouabain-treated hearts. Liquid chromatography-mass spectrometric analysis confirmed that the sarcoplasmic reticulum Ca(2+)-ATPase protein from ouabain-treated hearts had modifications consistent with oxidative and nitrosative stress. These data suggest that ouabain induces oxidative changes of the sarcoplasmic reticulum Ca(2+)-ATPase structure and function that may, in turn, produce some of the associated changes in Ca(2+) cycling and physiological function.     

Discovery of thalicthuberine as a novel antimitotic agent from nature that disrupts microtubule dynamics and induces apoptosis in prostate cancer cells

We report for the first time the mechanism of action of the natural product thalicthuberine (TH) in prostate and cervical cancer cells. TH induced a strong accumulation of LNCaP cells in mitosis, severe mitotic spindle defects, and asymmetric cell divisions, ultimately leading to mitotic catastrophe accompanied by cell death through apoptosis. However, unlike microtubule-binding drugs (vinblastine and paclitaxel), TH did not directly inhibit tubulin polymerization when tested in a cell-free system, whereas it reduced cellular microtubule polymer mass in LNCaP cells. This suggests that TH indirectly targets microtubule dynamics through inhibition of a critical regulator or tubulin-associated protein. Furthermore, TH is not a major substrate for P-glycoprotein (Pgp), which is responsible for multidrug resistance in numerous cancers, providing a rationale to further study TH in cancers with Pgp-mediated treatment resistance. The identification of TH's molecular target in future studies will be of great value to the development of TH as potential treatment of multidrug-resistant tumors.     

Combining Paclitaxel with ABT-263 Has a Synergistic Effect on Paclitaxel Resistant Prostate Cancer Cells

We assessed the capability of paclitaxel, one of the taxanes, to induce death in two prostate cancer lines, LNCaP and PC3. Paclitaxel drove an apoptotic pathway in LNCaP, but not in PC3 cells, in response to G2/M arrest. An examination of the levels of anti-apoptotic proteins revealed that Bcl-xl was much higher in PC3 cells than in LNCaP cells and Bcl2 could be detected only in PC3 cells, not in LNCaP cells. Knocking down Bcl-xl enhanced paclitaxel-induced apoptosis in LNCaP cells, while we were unable to knock down Bcl-xl efficiently in PC3 cells. Significantly, a comparison of ABT-263, a specific inhibitor of Bcl2 and Bcl-xl, with ABT-199, a Bcl2 selective inhibitor, disclosed that only ABT-263, not ABT-199, could induce apoptosis in LNCaP and PC3 cells. The results indicate that Bcl-xl has a protective role against paclitaxel-induced apoptosis in LNCaP and PC3 cells, and its overexpression causes the paclitaxel resistance seen in PC3 cells. Interestingly, combined paclitaxel with ABT-263 to treat LNCaP and PC3 cells demonstrated synergistic apoptosis activation, indicating that ABT-263 could enhance paclitaxel-induced apoptosis in LNCaP cells and overcome Bcl-xl overexpression to trigger paclitaxel-induced apoptosis in PC3 cells. We also observed that the activation of apoptosis in LNCaP cells was more efficient than in PC3 cells in response to paclitaxel plus ABT-263 or to ABT-263 alone, suggesting that the apoptosis pathway in PC3 cells might have further differences from that in LNCaP cells even after Bcl-xl overexpression is accounted for.     

Short exposure to paclitaxel induces multipolar spindle formation and aneuploidy through promotion of acentrosomal pole assembly

Paclitaxel is a widely used microtubule drug and cancer medicine. Here we report that by short exposure to paclitaxel at a low dose, multipolar spindles were induced in mitotic cells without centrosome amplification. Both TPX2 depletion and Aurora-A overexpression antagonized the multipolarity. Live cell imaging showed that some paclitaxel-treated cells accomplished multipolar cell division and a portion of the daughter cells went on to the next round of mitosis. The surviving cells grew into clones with varied genome content. The results indicated that an aneuploidy population could be induced by short exposure to paclitaxel at a low dose, implicating potential side effects of paclitaxel.     

Microtubule-interfering activity of parthenolide

Parthenolide is an active sesquiterpene lactone present in a variety of medicinal herbs, well known as anti-inflammatory drug. It has recently been proposed as a chemotherapeutic drug, but the pharmacological pathways of its action have not yet been fully elucidated. Firstly, we explored whether the anticancer properties of parthenolide may be related to a tubulin/microtubule-interfering activity. We additionally compared bioactivities of parthenolide with those checked after combined treatments with paclitaxel in human breast cancer MCF-7 cells. Parthenolide exerted in vitro stimulatory activity on tubulin assembly, by inducing the formation of well-organized microtubule polymers. Light microscopy detections showed that parthenolide-induced alterations of either microtubule network and nuclear morphology happened only after combined exposures to paclitaxel. In addition, the growth of MCF-7 cells was significantly inhibited by parthenolide, which enhanced paclitaxel effectiveness. In conclusion, the antimicrotubular and antiproliferative effects of parthenolide, well known microtubule-stabilizing anticancer agent, may influence paclitaxel activity. The tubulin/microtubule system may represent a novel molecular target for parthenolide, to be utilized in developing new combinational anticancer strategies.     

Microtubules: a dynamic target in cancer therapy

The tubulin/microtubule system is an important target for anticancer therapy. Two of the most clinically valuable groups of these agents are the vinca alkaloids and taxanes. In recent years, new tubulin-binding agents have been under preclinical or clinical development. One of these classes of agents, epothilones, has shown great promise in phase III clinical trials. What all these agents share in common, is that they bind to beta-tubulin and disrupt microtubule function during mitosis which in turn leads to mitotic arrest and cell death. In addition, these agents can inhibit angiogenesis. Not withstanding their effectiveness, drug resistance can pose a major clinical problem. This review provides an overview of the mechanisms mediating resistance to tubulin-binding agents related to the cellular target and discusses strategies to overcome this important clinical problem.     

Molecular basis for the insensitivity of the Monarch (Danaus plexippus) to cardiac glycosides.

The Monarch (Danaus plexippus) sequesters cardiac glycosides for its chemical defence against predators. Larvae and adults of this butterfly are insensitive towards dietary cardiac glycosides, whereas other Lepidoptera, such as Manduca sexta and Creatonotos transiens are sensitive and intoxicated by ouabain. Ouabain inhibits the Na+,K(+)-ATPase by binding to its alpha-subunit. We have amplified and cloned the DNA sequence encoding the respective ouabain binding site. Instead of the amino acid asparagine at position 122 in ouabain-sensitive insects, the Monarch has a histidine in the putative ouabain binding site, which consists of about 12 amino acids. This change may explain the ouabain insensitivity.     

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

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.