Abstract：
OBJECTIVE: To what extent and via what mechanism does the concomitant administration of rapamycin (a follicle activation pathway inhibitor and antitumour agent) and cyclophosphamide (a highly toxic ovarian anticancer agent) prevent cyclophosphamide-induced ovarian reserve loss and inhibit tumour proliferation in a breast cancer xenograft mouse model?
CONCLUSIONS: Daily concomitant administration of rapamycin and a cyclic regimen of cyclophosphamide, which has sufficient antitumour effects as a single agent, suppressed cyclophosphamide-induced primordial follicle loss by inhibiting primordial follicle activation in a breast cancer xenograft mouse model, suggesting the potential of an additive inhibitory effect against tumour proliferation.
BACKGROUND: Cyclophosphamide stimulates primordial follicles by activating the mammalian target of the rapamycin (mTOR) pathway, resulting in the accumulation of primary follicles, most of which undergo apoptosis. Rapamycin, an mTOR inhibitor, regulates primordial follicle activation and exhibits potential inhibitory effects against breast cancer cell proliferation.
METHODS: To assess ovarian follicular apoptosis, 3 weeks after administering breast cancer cells, 8-week-old mice were randomized into three treatment groups: control, cyclophosphamide, and cyclophosphamide + rapamycin (Cy + Rap) (n = 5 or 6 mice/group). Mice were treated with rapamycin or vehicle control for 1 week, followed by a single dose of cyclophosphamide or vehicle control. Subsequently, the ovaries were resected 24 h after cyclophosphamide administration (short-term treatment groups). To evaluate follicle abundance and the mTOR pathway in ovaries, as well as the antitumour effects and impact on the mTOR pathway in tumours, 8-week-old xenograft breast cancer transplanted mice were randomized into three treatment groups: vehicle control, Cy, and Cy + Rap (n = 6 or 7 mice/group). Rapamycin (5 mg/kg) or the vehicle was administered daily for 29 days. Cyclophosphamide (120 mg/kg) or the vehicle was administered thrice weekly (long-term treatment groups). The tumour diameter was measured weekly. Seven days after the last cyclophosphamide treatment, the ovaries were harvested, fixed, and sectioned (for follicle counting) or frozen (for further analysis). Similarly, the tumours were resected and fixed or frozen.
METHODS: Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) was performed to examine ovarian follicular apoptosis in the short-term treatment groups. All subsequent experiments were conducted in the long-term treatment groups. Tumour growth was evaluated using the tumour volume index. The tumour volume index indicates the relative volume, compared to the volume 3 weeks after tumour cell injection (at treatment initiation) set to 100%. Tumour cell proliferation was evaluated by Ki-67 immunostaining. Activation of the mTOR pathway in tumours was assessed using the protein extracts from tumours and analysed by western blotting. Haematoxylin and eosin staining of ovaries was used to perform differential follicle counts for primordial, primary, secondary, antral, and atretic follicles. Activation of the mTOR pathway in ovaries was assessed using protein extracts from whole ovaries and analysed by western blotting. Localization of mTOR pathway activation within ovaries was assessed by performing anti-phospho-S6 kinase (downstream of mTOR pathway) immunohistochemistry.
RESULTS: Ovaries of the short-term treatment groups were resected 24 h after cyclophosphamide administration and subjected to TUNEL staining of apoptotic cells. No TUNEL-positive primordial follicles were detected in the control, Cy, and Cy + Rap groups. Conversely, many granulosa cells of growing follicles were TUNEL positive in the Cy group but negative in the control and Cy + Rap groups. All subsequent experimental results were obtained from the long-term treatment groups. The tumour volume index stabilized at a mean of 160-200% in the Cy group and 130% in the Cy + Rap group throughout the treatment period. In contrast, tumours in the vehicle control group grew continuously with a mean tumour volume index of 600%, significantly greater than that of the two treatment groups. Based on the western blot analysis of tumours, the mTOR pathway was activated in the vehicle control group and downregulated in the Cy + Rap group when compared with the control and Cy groups. Ki-67 immunostaining of tumours showed significant inhibition of cell proliferation in the Cy + Rap group when compared with that in the control and Cy groups. The ovarian follicle count revealed that the Cy group had significantly fewer primordial follicles (P < 0.001) than the control group, whereas the Cy + Rap group had significantly higher number of primordial follicles (P < 0.001, 2.5 times) than the Cy group. The ratio of primary to primordial follicles was twice as high in the Cy group than in the control group; however, no significant difference was observed between the control group and the Cy + Rap group. Western blot analysis of ovaries revealed that the mTOR pathway was activated by cyclophosphamide and inhibited by rapamycin. The phospho-S6 kinase (pS6K)-positive primordial follicle rate was 2.7 times higher in the Cy group than in the control group. However, this effect was suppressed to a level similar to the control group in the Cy + Rap group.
METHODS: None.
CONCLUSIONS: The combinatorial treatment of breast cancer tumours with rapamycin and cyclophosphamide elicited inhibitory effects on cell proliferative potential compared to cyclophosphamide monotherapy. However, no statistically significant additive effect was observed on tumour volume. Thus, the beneficial antitumour effect afforded by rapamycin administration on breast cancer could not be definitively proven. Although rapamycin has ovarian-protective effects, it does not fully counteract the ovarian toxicity of cyclophosphamide. Nevertheless, rapamycin is advantageous as an ovarian protective agent as it can be used in combination with other ovarian protective agents, such as hormonal therapy. Hence, in combination with other agents, mTOR inhibitors may be sufficiently ovario-protective against high-dose and cyclic cyclophosphamide regimens.
CONCLUSIONS: Compared with a cyclic cyclophosphamide regimen that replicates human clinical practice under breast cancer-bearing conditions, the combination with rapamycin mitigates the ovarian follicle loss of cyclophosphamide without interfering with the anticipated antitumour effects. Hence, rapamycin may represent a new non-invasive treatment option for cyclophosphamide-induced ovarian dysfunction in breast cancer patients.
BACKGROUND: This work was not financially supported. The authors declare that they have no conflict of interest.
CONCLUSIONS: Daily concomitant administration of rapamycin and a cyclic regimen of cyclophosphamide, which has sufficient antitumour effects as a single agent, suppressed cyclophosphamide-induced primordial follicle loss by inhibiting primordial follicle activation in a breast cancer xenograft mouse model, suggesting the potential of an additive inhibitory effect against tumour proliferation.
BACKGROUND: Cyclophosphamide stimulates primordial follicles by activating the mammalian target of the rapamycin (mTOR) pathway, resulting in the accumulation of primary follicles, most of which undergo apoptosis. Rapamycin, an mTOR inhibitor, regulates primordial follicle activation and exhibits potential inhibitory effects against breast cancer cell proliferation.
METHODS: To assess ovarian follicular apoptosis, 3 weeks after administering breast cancer cells, 8-week-old mice were randomized into three treatment groups: control, cyclophosphamide, and cyclophosphamide + rapamycin (Cy + Rap) (n = 5 or 6 mice/group). Mice were treated with rapamycin or vehicle control for 1 week, followed by a single dose of cyclophosphamide or vehicle control. Subsequently, the ovaries were resected 24 h after cyclophosphamide administration (short-term treatment groups). To evaluate follicle abundance and the mTOR pathway in ovaries, as well as the antitumour effects and impact on the mTOR pathway in tumours, 8-week-old xenograft breast cancer transplanted mice were randomized into three treatment groups: vehicle control, Cy, and Cy + Rap (n = 6 or 7 mice/group). Rapamycin (5 mg/kg) or the vehicle was administered daily for 29 days. Cyclophosphamide (120 mg/kg) or the vehicle was administered thrice weekly (long-term treatment groups). The tumour diameter was measured weekly. Seven days after the last cyclophosphamide treatment, the ovaries were harvested, fixed, and sectioned (for follicle counting) or frozen (for further analysis). Similarly, the tumours were resected and fixed or frozen.
METHODS: Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) was performed to examine ovarian follicular apoptosis in the short-term treatment groups. All subsequent experiments were conducted in the long-term treatment groups. Tumour growth was evaluated using the tumour volume index. The tumour volume index indicates the relative volume, compared to the volume 3 weeks after tumour cell injection (at treatment initiation) set to 100%. Tumour cell proliferation was evaluated by Ki-67 immunostaining. Activation of the mTOR pathway in tumours was assessed using the protein extracts from tumours and analysed by western blotting. Haematoxylin and eosin staining of ovaries was used to perform differential follicle counts for primordial, primary, secondary, antral, and atretic follicles. Activation of the mTOR pathway in ovaries was assessed using protein extracts from whole ovaries and analysed by western blotting. Localization of mTOR pathway activation within ovaries was assessed by performing anti-phospho-S6 kinase (downstream of mTOR pathway) immunohistochemistry.
RESULTS: Ovaries of the short-term treatment groups were resected 24 h after cyclophosphamide administration and subjected to TUNEL staining of apoptotic cells. No TUNEL-positive primordial follicles were detected in the control, Cy, and Cy + Rap groups. Conversely, many granulosa cells of growing follicles were TUNEL positive in the Cy group but negative in the control and Cy + Rap groups. All subsequent experimental results were obtained from the long-term treatment groups. The tumour volume index stabilized at a mean of 160-200% in the Cy group and 130% in the Cy + Rap group throughout the treatment period. In contrast, tumours in the vehicle control group grew continuously with a mean tumour volume index of 600%, significantly greater than that of the two treatment groups. Based on the western blot analysis of tumours, the mTOR pathway was activated in the vehicle control group and downregulated in the Cy + Rap group when compared with the control and Cy groups. Ki-67 immunostaining of tumours showed significant inhibition of cell proliferation in the Cy + Rap group when compared with that in the control and Cy groups. The ovarian follicle count revealed that the Cy group had significantly fewer primordial follicles (P < 0.001) than the control group, whereas the Cy + Rap group had significantly higher number of primordial follicles (P < 0.001, 2.5 times) than the Cy group. The ratio of primary to primordial follicles was twice as high in the Cy group than in the control group; however, no significant difference was observed between the control group and the Cy + Rap group. Western blot analysis of ovaries revealed that the mTOR pathway was activated by cyclophosphamide and inhibited by rapamycin. The phospho-S6 kinase (pS6K)-positive primordial follicle rate was 2.7 times higher in the Cy group than in the control group. However, this effect was suppressed to a level similar to the control group in the Cy + Rap group.
METHODS: None.
CONCLUSIONS: The combinatorial treatment of breast cancer tumours with rapamycin and cyclophosphamide elicited inhibitory effects on cell proliferative potential compared to cyclophosphamide monotherapy. However, no statistically significant additive effect was observed on tumour volume. Thus, the beneficial antitumour effect afforded by rapamycin administration on breast cancer could not be definitively proven. Although rapamycin has ovarian-protective effects, it does not fully counteract the ovarian toxicity of cyclophosphamide. Nevertheless, rapamycin is advantageous as an ovarian protective agent as it can be used in combination with other ovarian protective agents, such as hormonal therapy. Hence, in combination with other agents, mTOR inhibitors may be sufficiently ovario-protective against high-dose and cyclic cyclophosphamide regimens.
CONCLUSIONS: Compared with a cyclic cyclophosphamide regimen that replicates human clinical practice under breast cancer-bearing conditions, the combination with rapamycin mitigates the ovarian follicle loss of cyclophosphamide without interfering with the anticipated antitumour effects. Hence, rapamycin may represent a new non-invasive treatment option for cyclophosphamide-induced ovarian dysfunction in breast cancer patients.
BACKGROUND: This work was not financially supported. The authors declare that they have no conflict of interest.
摘要：
目的：在乳腺癌异种移植小鼠模型中，雷帕霉素（一种卵泡激活途径抑制剂和抗肿瘤药物）和环磷酰胺（一种高毒性的卵巢抗癌剂）的同时给药在何种程度上和通过何种机制预防环磷酰胺诱导的卵巢储备丧失和抑制肿瘤增殖？作为单一药物具有足够的抗肿瘤作用，在乳腺癌异种移植小鼠模型中，通过抑制原始卵泡激活来抑制环磷酰胺诱导的原始卵泡损失，提示对肿瘤增殖的累加抑制作用的潜力。
背景：环磷酰胺通过激活哺乳动物雷帕霉素靶标（mTOR）途径刺激原始卵泡，导致初级卵泡的积累，其中大多数经历凋亡。雷帕霉素,一种mTOR抑制剂,调节原始卵泡的激活，并表现出对乳腺癌细胞增殖的潜在抑制作用。
方法：评估卵巢卵泡凋亡，施用乳腺癌细胞后3周，将8周龄小鼠随机分为三个治疗组：对照组，环磷酰胺,和环磷酰胺+雷帕霉素(Cy+Rap)(n=5或6只小鼠/组)。用雷帕霉素或媒介物对照治疗小鼠1周，然后是单剂量的环磷酰胺或媒介物对照。随后，环磷酰胺给药后24h切除卵巢(短期治疗组).为了评估卵巢中卵泡丰度和mTOR通路，以及抗肿瘤作用和对肿瘤中mTOR通路的影响，将8周龄的异种移植乳腺癌移植小鼠随机分为三个治疗组：载体对照，Cy,和Cy+Rap(n=6或7只小鼠/组)。每天施用雷帕霉素(5mg/kg)或载体,持续29天。环磷酰胺(120mg/kg)或载体每周施用三次(长期治疗组)。每周测量肿瘤直径。最后一次环磷酰胺治疗后七天，卵巢被收获，固定,并切片（用于卵泡计数）或冷冻（用于进一步分析）。同样，肿瘤被切除并固定或冷冻。
方法：在短期治疗组中进行末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）以检查卵泡凋亡。所有后续实验均在长期治疗组中进行。使用肿瘤体积指数评估肿瘤生长。肿瘤体积指数表示相对体积，与肿瘤细胞注射后3周的体积相比（在治疗开始时）设定为100％。通过Ki-67免疫染色评估肿瘤细胞增殖。使用来自肿瘤的蛋白质提取物评估肿瘤中mTOR途径的激活并通过蛋白质印迹分析。卵巢的苏木素和伊红染色用于进行原始卵泡的差异计数，小学,次要,窦,和闭锁卵泡。使用来自整个卵巢的蛋白质提取物评估卵巢中mTOR途径的激活并通过蛋白质印迹分析。通过进行抗磷酸-S6激酶(mTOR途径的下游)免疫组织化学来评估mTOR途径激活在卵巢内的定位。
结果：在环磷酰胺给药后24小时切除短期治疗组的卵巢，并对凋亡细胞进行TUNEL染色。在对照组中未检测到TUNEL阳性原始卵泡，Cy,和Cy+Rap组。相反，在Cy组中许多正在生长的卵泡的颗粒细胞为TUNEL阳性,但在对照组和Cy+Rap组中为阴性。所有后续实验结果均从长期治疗组获得。在整个治疗期间，肿瘤体积指数在Cy组中稳定在160-200％的平均值，在CyRap组中稳定在130％。相比之下,媒介物对照组的肿瘤以600%的平均肿瘤体积指数持续增长，显著大于两个治疗组。根据肿瘤的蛋白质印迹分析，与对照组和Cy组相比,载体对照组的mTOR通路被激活,Cy+Rap组的mTOR通路下调.与对照组和Cy组相比，肿瘤的Ki-67免疫染色显示CyRap组细胞增殖显着抑制。卵泡计数显示，Cy组的原始卵泡明显少于对照组（P<0.001）。而Cy+Rap组的原始卵泡数明显高于Cy组(P<0.001,2.5倍)。Cy组的初级卵泡与原始卵泡的比率是对照组的两倍；然而，对照组与Cy+Rap组之间无显著差异。卵巢的Western印迹分析显示mTOR途径被环磷酰胺激活并被雷帕霉素抑制。Cy组的磷酸化S6激酶（pS6K）阳性原始卵泡率为对照组的2.7倍。然而,在Cy+Rap组中,这种效应被抑制到与对照组相似的水平.
方法：无。
结论：与环磷酰胺单一疗法相比，雷帕霉素和环磷酰胺联合治疗乳腺癌肿瘤可引起细胞增殖潜能的抑制作用。然而,对肿瘤体积没有观察到统计学上显著的累加效应。因此，雷帕霉素给药对乳腺癌的有益抗肿瘤作用尚未得到明确证实.尽管雷帕霉素具有保护卵巢的作用,它不能完全抵消环磷酰胺的卵巢毒性。然而，雷帕霉素作为卵巢保护剂是有利的，因为它可以与其他卵巢保护剂联合使用，比如荷尔蒙疗法。因此，与其他代理商结合，mTOR抑制剂可能对高剂量和环状环磷酰胺方案具有足够的卵巢保护作用。
结论：与环磷酰胺方案相比，环磷酰胺方案在乳腺癌条件下复制人类临床实践，与雷帕霉素合用可减轻环磷酰胺的卵泡丢失,而不干扰预期的抗肿瘤作用.因此，雷帕霉素可能是环磷酰胺所致乳腺癌患者卵巢功能障碍的一种新的非侵入性治疗选择.
背景：这项工作没有得到财政支持。作者声明他们没有利益冲突。
背景：环磷酰胺通过激活哺乳动物雷帕霉素靶标（mTOR）途径刺激原始卵泡，导致初级卵泡的积累，其中大多数经历凋亡。雷帕霉素,一种mTOR抑制剂,调节原始卵泡的激活，并表现出对乳腺癌细胞增殖的潜在抑制作用。
方法：评估卵巢卵泡凋亡，施用乳腺癌细胞后3周，将8周龄小鼠随机分为三个治疗组：对照组，环磷酰胺,和环磷酰胺+雷帕霉素(Cy+Rap)(n=5或6只小鼠/组)。用雷帕霉素或媒介物对照治疗小鼠1周，然后是单剂量的环磷酰胺或媒介物对照。随后，环磷酰胺给药后24h切除卵巢(短期治疗组).为了评估卵巢中卵泡丰度和mTOR通路，以及抗肿瘤作用和对肿瘤中mTOR通路的影响，将8周龄的异种移植乳腺癌移植小鼠随机分为三个治疗组：载体对照，Cy,和Cy+Rap(n=6或7只小鼠/组)。每天施用雷帕霉素(5mg/kg)或载体,持续29天。环磷酰胺(120mg/kg)或载体每周施用三次(长期治疗组)。每周测量肿瘤直径。最后一次环磷酰胺治疗后七天，卵巢被收获，固定,并切片（用于卵泡计数）或冷冻（用于进一步分析）。同样，肿瘤被切除并固定或冷冻。
方法：在短期治疗组中进行末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）以检查卵泡凋亡。所有后续实验均在长期治疗组中进行。使用肿瘤体积指数评估肿瘤生长。肿瘤体积指数表示相对体积，与肿瘤细胞注射后3周的体积相比（在治疗开始时）设定为100％。通过Ki-67免疫染色评估肿瘤细胞增殖。使用来自肿瘤的蛋白质提取物评估肿瘤中mTOR途径的激活并通过蛋白质印迹分析。卵巢的苏木素和伊红染色用于进行原始卵泡的差异计数，小学,次要,窦,和闭锁卵泡。使用来自整个卵巢的蛋白质提取物评估卵巢中mTOR途径的激活并通过蛋白质印迹分析。通过进行抗磷酸-S6激酶(mTOR途径的下游)免疫组织化学来评估mTOR途径激活在卵巢内的定位。
结果：在环磷酰胺给药后24小时切除短期治疗组的卵巢，并对凋亡细胞进行TUNEL染色。在对照组中未检测到TUNEL阳性原始卵泡，Cy,和Cy+Rap组。相反，在Cy组中许多正在生长的卵泡的颗粒细胞为TUNEL阳性,但在对照组和Cy+Rap组中为阴性。所有后续实验结果均从长期治疗组获得。在整个治疗期间，肿瘤体积指数在Cy组中稳定在160-200％的平均值，在CyRap组中稳定在130％。相比之下,媒介物对照组的肿瘤以600%的平均肿瘤体积指数持续增长，显著大于两个治疗组。根据肿瘤的蛋白质印迹分析，与对照组和Cy组相比,载体对照组的mTOR通路被激活,Cy+Rap组的mTOR通路下调.与对照组和Cy组相比，肿瘤的Ki-67免疫染色显示CyRap组细胞增殖显着抑制。卵泡计数显示，Cy组的原始卵泡明显少于对照组（P<0.001）。而Cy+Rap组的原始卵泡数明显高于Cy组(P<0.001,2.5倍)。Cy组的初级卵泡与原始卵泡的比率是对照组的两倍；然而，对照组与Cy+Rap组之间无显著差异。卵巢的Western印迹分析显示mTOR途径被环磷酰胺激活并被雷帕霉素抑制。Cy组的磷酸化S6激酶（pS6K）阳性原始卵泡率为对照组的2.7倍。然而,在Cy+Rap组中,这种效应被抑制到与对照组相似的水平.
方法：无。
结论：与环磷酰胺单一疗法相比，雷帕霉素和环磷酰胺联合治疗乳腺癌肿瘤可引起细胞增殖潜能的抑制作用。然而,对肿瘤体积没有观察到统计学上显著的累加效应。因此，雷帕霉素给药对乳腺癌的有益抗肿瘤作用尚未得到明确证实.尽管雷帕霉素具有保护卵巢的作用,它不能完全抵消环磷酰胺的卵巢毒性。然而，雷帕霉素作为卵巢保护剂是有利的，因为它可以与其他卵巢保护剂联合使用，比如荷尔蒙疗法。因此，与其他代理商结合，mTOR抑制剂可能对高剂量和环状环磷酰胺方案具有足够的卵巢保护作用。
结论：与环磷酰胺方案相比，环磷酰胺方案在乳腺癌条件下复制人类临床实践，与雷帕霉素合用可减轻环磷酰胺的卵泡丢失,而不干扰预期的抗肿瘤作用.因此，雷帕霉素可能是环磷酰胺所致乳腺癌患者卵巢功能障碍的一种新的非侵入性治疗选择.
背景：这项工作没有得到财政支持。作者声明他们没有利益冲突。
