Moran Tzabari

Doxorubicin-induced apoptosis in germinal vesicle (GV) oocytes.

Ovarian failure is a-known side-effect observed in women treated for cancer. Doxorubicin (DXR) was found to be detrimental to MII mouse oocytes. We aimed at characterizing the effect of DXR on germinal vesicle (GV) oocytes that comprise the majority of oocytes within ovaries encountering chemotherapy. Mouse follicles and oocytes were exposed to DXR in vitro. DXR localization and its possible cellular targets were examined by fluorescence confocal microscopy. We demonstrated that DXR crosses the blood-follicle barrier and accumulates in oocytes and granulosa cells. The mechanism of DXR-induced apoptosis involves chromosomal disintegration, activation of the mitochondria followed by activation of PERK and caspase-12 and inactivation of PARP. The follicular GV oocytes were more vulnerable to the toxic effect of DXR than ovulated MII oocytes. We suggest that DXR elicits apoptotic signal within GV oocytes that involves activation of the mitochondria, induction of ER-stress and a possible increase in intracellular calcium.

Doxorubicin-Induced Vascular Toxicity – Targeting Potential Pathways May Reduce Procoagulant Activity

Introduction Previous study in mice using real-time intravital imaging revealed an acute deleterious effect of doxorubicin (DXR) on the gonadal vasculature, as a prototype of an end-organ, manifested by a reduction in blood flow and disintegration of the vessel wall. We hypothesized that this pattern may represent the formation of microthrombi. We aimed to further characterize the effect of DXR on platelets’ activity and interaction with endothelial cells (EC) and to examine potential protectants to reduce DXR acute effect on the blood flow. Methods The effect of DXR on platelet adhesion and aggregation were studied in vitro. For in vivo studies, mice were injected with either low molecular weight heparin (LMWH; Enoxaparin) or with eptifibatide (Integrilin©) prior to DXR treatment. Testicular arterial blood flow was examined in real-time by pulse wave Doppler ultrasound. Results Platelet treatment with DXR did not affect platelet adhesion to a thrombogenic surface but significantly decreased ADP-induced platelet aggregation by up to 40% (p<0.001). However, there was a significant increase in GPIIbIIIa-mediated platelet adhesion to DXR-exposed endothelial cells (EC; 5.7-fold; p<0.001) reflecting the toxic effect of DXR on EC. The testicular arterial blood flow was preserved in mice pre-treated with LMWH or eptifibatide prior to DXR (P<0.01). Conclusions DXR-induced acute vascular toxicity may involve increased platelet–EC adhesion leading to EC-bound microthrombi formation resulting in compromised blood flow. Anti-platelet/anti-coagulant agents are effective in reducing the detrimental effect of DXR on the vasculature and thus may serve as potential protectants to lessen this critical toxicity.

Dexrazoxane exacerbates doxorubicin-induced testicular toxicity

Infertility induced by anti-cancer treatments pose a major concern for cancer survivors. Doxorubicin (DXR) has been previously shown to exert toxic effects on the testicular germinal epithelium. Based upon the cardioprotective traits of dexrazoxane (DEX), we studied its potential effect in reducing DXR-induced testicular toxicity. Male mice were injected with 5  mg/kg DXR, 100  mg/kg DEX, combination of both or saline (control) and sacrificed either 1, 3 or 6 months later. Testes were excised and further processed. Glutathione and apoptosis assays were performed to determine oxidative stress. Immunohistochemistry and confocal microscopy were used to study the effects of the drugs on testicular histology and on spermatogonial reserve. DXR and the combined treatment induced a striking decline in testicular weight. DEX prevented DXR-induced oxidative stress, but enhanced DXR-induced apoptosis within the testes. Furthermore, the combined treatment depleted the spermatogonial reserve after 1 month, with impaired recovery at 3 and 6 months post-treatment. This resulted in compromised sperm parameters, testicular and epididymal weights as well as significantly reduced sperm motility, all of which were more severe than those observed in DXR-treated mice. The activity of DEX in the testis may differ from its activity in cardiomyocytes. Adding DEX to DXR exacerbates DXR-induced testicular toxicity.

Cetuximab intensifies cisplatin-induced testicular toxicity

Epidermal growth factor receptor (EGFR) has proliferative properties in the testis. Cetuximab, an anti-EGFR, is administered together with chemotherapy to patients with various types of cancer. This studies aim was to investigate the effect of cetuximab on testicular function. Adult male mice were injected with cetuximab (10 mg/kg), cisplatin (8 mg/kg) or a combination of both, and killed one week or one month later. The doses were chosen by human equivalent dose calculation. Testicular function was evaluated by epididymal-spermatozoa total motile count and sperm motility, weights of testes and epididymides, and the level of anti-Müllerian hormone (AMH) in the serum. Immunohistochemistry was performed to examine germ cell proliferation (Ki-67), apoptosis (Terminal transferase-mediated deoxyuridine 5-triphosphate nick-end labelling), reserve (DAZL-Deleted in azoospermia-like, Promyelocytic leukaemia zinc-finger), blood vessels (CD34) and Sertoli cells (GATA-4). Administration of cetuximab alone increased testicular apoptosis and decreased epididymal-spermatozoa total motile count over time. When added to cisplatin, cetuximab exacerbated most of the recorded testicular parameters, compared with the effect of cisplatin alone, including testis and epididymis weights, epididymal-spermatozoa total motile count, AMH concentration, meiosis and apoptosis. In conclusion, cetuximab has only a mild effect on testicular reserve, but when added to cisplatin, it exacerbates cisplatin-induced testicular toxicity.

Abstract 3840: Cetuximab-induced testicular toxicity

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Cetuximab, an anti-EGFR monoclonal antibody, is the only targeted therapy approved for the treatment of head and neck squamous cell carcinoma (HNSCC) in patients with locally advanced tumors, in association with radiotherapy, and in patients with recurrent or metastatic disease, in association with cisplatin-based chemotherapy. We aimed to study the effect of cetuximab on testicular function and reserve. Methods: Male mice were injected intraperitoneally with cetuximab (1mg), or cisplatin (8mg/kg) or the combination of both and sacrificed either 1 week or 6 weeks later. Saline-injected mice served as controls. Testes were excised, weighed and further processed. Glutathione and apoptosis assays were performed on 6 weeks samples for determination of oxidative stress. Immunohistochemistry and confocal microscopy were used to study the effect of cetuximab, cisplatin and their combination on the testis histology as well as on the spermatogonial reserve. Results: Cetuximab induced mild apoptosis in the testis. Cisplatin induced enhanced apoptosis and depleted spermatogonial reserve, reflected by reduced DAZL staining (early spermatocyte marker). The combined treatment resulted in further decline in testicular weight compared with cisplatin-only treated mice at 1-month post treatment (p<0.05). Cetuximab enhanced cisplatin-induced oxidative stress, and further depletion of spermatogonial reserve (p<0.05). Conclusions: Cetuximab has a mild effect on testicular reserve. Nevertheless, the addition of cetuximab to cisplatin exacerbates cisplatin-induced testicular toxicity. Citation Format: Mattan Levi, Aaron Popovtzer, Moran Tzabari, Salomon M. Stemmer, Ruth Shalgi, Irit Ben-Aharon. Cetuximab-induced testicular toxicity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3840. doi:10.1158/1538-7445.AM2014-3840

Abstract A229: Novel in vivo imaging of chemotherapy‐induced gonadotoxicity

Introduction: Advances in cancer therapy have improved the long‐term survival of cancer patients who may then face infertility and premature gonadal failure due to chemotherapy. It has been demonstrated that the chemotherapeutic agent doxorubicin (DXR) induces apoptosis in germ cells, though its role in inflicting potential gonadal failure remains obscure. We had previously shown ovarian damage in mice following DXR treatment, manifested by reduced ovarian size and weight and by apoptosis. We aimed at establishing a real‐time in vivo molecular imaging platform in mice designated to evaluate chemotherapy‐induced toxicity and to assess potential vascular insult in both ovaries and testes. Methods: The volume of both ovaries and testes was measured in vivo by ultrasound biomicroscopy (Vevo2100) and by high resolution MRI, over a period of one month after adminstration of 10mg/kg intraperitoneal DXR. Ovaries were imaged by ultrasound using microbubbles as a contrast agent for evaluating the effect of DXR on ovarian blood flow. Results: We have established a platform of visualizing the gonads by innovative high resolution ultrasound biomicroscopy and MRI that enabled in vivo detection of chemotherapy‐induced effects in the same individuals over time. We observed a constant, significant reduction in gonadal volume, evident by both MRI and ultrasound. The ovary volume was reduced to 52% (N=10, p Conclusions: Using high resolution, state of the art imaging modules enabled us to trace chemotherapy‐induce gonadotoxicity by in vivo imaging of the same individuals throughout an extended period of time. The acute reduction of ovarian blood flow following chemotherapy treatment may suggest a role for vascular injury in inflicting ovarian damage. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A229.