Background It is important to identify the multiple sites of leptin activity in obese women with breast cancer.In this study,we examined the effect of exogenous human leptin on heat shock protein 70 （HSP70） expressi...Background It is important to identify the multiple sites of leptin activity in obese women with breast cancer.In this study,we examined the effect of exogenous human leptin on heat shock protein 70 （HSP70） expression in MCF-7 human breast cancer cells and in a breast carcinoma xenograft model of nude mice.Methods We cultured MCF-7 human breast cancer cells and established nude mice bearing xenograffs of these cells,and randomly divided them into experimental and control groups.The experimental group was treated with human leptin,while the control group was treated with the same volume of normal saline.A real-time reverse transcriptase-polymerase chain reaction （RT-PCR） assay was developed to quantify the mRNA expression of HSP70 in the MCF-7 human breast cancer cells and in tumor tissues.Western blotting analysis was applied to quantify the protein expression of HSP70 in the MCF-7 cells.Immunohistochemical staining was done to assess the positive rate of HSP70 expression in the tumor tissues.Results Leptin activated HSP70 in a dose-dependent manner in vitro：leptin upregulated significantly the expression of HSP70 at mRNA and protein levels in MCF-7 human breast cancer cells （P 〈0.001）.There was no significant difference in expression of HSP70 mRNA in the implanted tumors between the leptin-treated group and the control group （P〉0.05）.Immunohistochemical staining revealed no significant difference in tumor HSP70 expression between the leptin-treated group and the control group （P〉0.05）.Conclusions A nude mouse xenograft model can be safely and efficiently treated with human leptin by subcutaneous injections around the tumor.HSP70 may be target of leptin in breast cancer.Leptin can significantly upregulate the expression of HSP70 in a dose-dependent manner in vitro.展开更多
Background A satisfactory animal model of breast cancer metastasizing to bone is unavailable. In this study, we used human breast cancer stem-like cells and human bone to build a novel “human-source” model of human ...Background A satisfactory animal model of breast cancer metastasizing to bone is unavailable. In this study, we used human breast cancer stem-like cells and human bone to build a novel “human-source” model of human breast cancer skeletal metastasis. Methods Human breast cancer stem-like cells, the CD44^＋/CD24^-/lower subpopulation, was separated and cultured. Before injection with the stem-like cells, mice were implanted with human bone in the right or left dorsal flanks. Animals in Groups A, B, and C were injected with 1×10^5, 1×10^6 human breast cancer stem-like cells, and 1×10^6 parental MDA-MB-231 cells, respectively. A positive control group （D） without implantation of human bone was also injected with 1×10^6 MDA-MB-231 cells. Immunohistochemistry was performed for determination of CD34, CD105, smooth muscle antibody, CD44, CD24, cytokine, CXC chemokine receptor-4 （CXCR4）, and osteopontin （OPN）. mRNA levels of CD44, CD24, CXCR4, and OPN in bone metastasis tissues were analyzed by real-time quantitative polymerase chain reaction （PCR）.Results Our results demonstrated that cells in implanted human bones of group B, which received 1×10^6 cancer stem-like cells, stained strongly positive for CD44, CXCR4, and OPN, whereas those of other groups showed no or minimum staining. Moreover, group B had the highest incidence of human bone metastasis （77.8%, P=0.0230） and no accompaniment of other tissue metastasis. The real-time PCR showed an increase of CD44, CXCR4, and OPN mRNA in metastatic bone tissues in group B compared with those of groups C and D, however the expression of CD24 mRNA in group B were the lowest. Conclusions In the novel “human source” model of breast cancer, breast cancer stem-like cells demonstrated a higher human bone-seeking ability. Its mechanism might be related to the higher expressions of CD44, CXCR4, and OPN, and the lower expression of CD24 in breast cancer stem-like cells.展开更多
目的探讨聚合人脐带血红蛋白氧载体(PolyCHb)对人乳腺癌MCF-7细胞裸鼠皮下移植瘤化疗敏感性的影响及其机制。方法收集处于指数生长期的MCF-7细胞,制成密^度为5×10^(7)个/mL的悬浮细胞,按0.2 mL/只接种于18只BALB/c-nu裸鼠右肢皮下...目的探讨聚合人脐带血红蛋白氧载体(PolyCHb)对人乳腺癌MCF-7细胞裸鼠皮下移植瘤化疗敏感性的影响及其机制。方法收集处于指数生长期的MCF-7细胞,制成密^度为5×10^(7)个/mL的悬浮细胞,按0.2 mL/只接种于18只BALB/c-nu裸鼠右肢皮下,建立裸鼠移植瘤模型,待肿瘤体积达到100 mm^(3)左右时,随机均分为化疗组:多柔比星(DOX)5 mg·kg^(-1),1次/周;化疗+PolyCHb治疗组:除给药(DOX)(同化疗组)外,PolyCHb 600 mg·kg^(-1),3次/周;对照组:生理盐水90 mg·kg^(-1),1次/周;均为经尾静脉连续注射4周。自注射当天(d0)起,每3 d 1次测量各组裸鼠肿瘤体积,据此绘制其各自(组)肿瘤生长曲线。38 d结束肿瘤生长观察,剥瘤并称取瘤重,计算抑瘤率;HE染色观察肿瘤组织病理变化,免疫组化检测肿瘤组织中HIF-1α表达,采用TUNEL法检测肿瘤细胞凋亡情况,荧光染色测定各组肿瘤组织活性氧(ROS)的含量。结果化疗+PolyCHb组、化疗组和对照组至d38时的肿瘤体积(mm^(3))分别为:196.35±103.45 vs 316.29±62.88 vs 519.42±177.33(P<0.05);化疗+PolyCHb组与化疗组抑瘤率(%)分别为62.20 vs 39.11;HE染色和TUNEL检测:化疗+PolyCHb组肿瘤组织生长区域细胞坏死和凋亡增多;免疫组化:化疗+PolyCHb组HIF-1α表达水平降低;荧光染色:化疗+PolyCHb组[活性氧(ROS)]含量增多。结论PolyCHb增加了乳腺癌裸鼠皮下移植瘤化疗的敏感性,其作用机制可能与其提高肿瘤组织内ROS含量,促进肿瘤细胞的凋亡有关。展开更多
基金This study was supported by a grant from the National Natural Science Foundation of China （No.30772121）.
文摘Background It is important to identify the multiple sites of leptin activity in obese women with breast cancer.In this study,we examined the effect of exogenous human leptin on heat shock protein 70 （HSP70） expression in MCF-7 human breast cancer cells and in a breast carcinoma xenograft model of nude mice.Methods We cultured MCF-7 human breast cancer cells and established nude mice bearing xenograffs of these cells,and randomly divided them into experimental and control groups.The experimental group was treated with human leptin,while the control group was treated with the same volume of normal saline.A real-time reverse transcriptase-polymerase chain reaction （RT-PCR） assay was developed to quantify the mRNA expression of HSP70 in the MCF-7 human breast cancer cells and in tumor tissues.Western blotting analysis was applied to quantify the protein expression of HSP70 in the MCF-7 cells.Immunohistochemical staining was done to assess the positive rate of HSP70 expression in the tumor tissues.Results Leptin activated HSP70 in a dose-dependent manner in vitro：leptin upregulated significantly the expression of HSP70 at mRNA and protein levels in MCF-7 human breast cancer cells （P 〈0.001）.There was no significant difference in expression of HSP70 mRNA in the implanted tumors between the leptin-treated group and the control group （P〉0.05）.Immunohistochemical staining revealed no significant difference in tumor HSP70 expression between the leptin-treated group and the control group （P〉0.05）.Conclusions A nude mouse xenograft model can be safely and efficiently treated with human leptin by subcutaneous injections around the tumor.HSP70 may be target of leptin in breast cancer.Leptin can significantly upregulate the expression of HSP70 in a dose-dependent manner in vitro.
基金This work was supported by grants from National Natural Science Foundation of China （No. 300740076）, Jiangsu Six Kinds of Outstanding Talents Foundation （No. 2006B070）, Jiangsu Science and Education for Health Foundation （No. RC2007054） and Jiangsu Province Post-doctor Foundation （No. 0601048B）.
文摘Background A satisfactory animal model of breast cancer metastasizing to bone is unavailable. In this study, we used human breast cancer stem-like cells and human bone to build a novel “human-source” model of human breast cancer skeletal metastasis. Methods Human breast cancer stem-like cells, the CD44^＋/CD24^-/lower subpopulation, was separated and cultured. Before injection with the stem-like cells, mice were implanted with human bone in the right or left dorsal flanks. Animals in Groups A, B, and C were injected with 1×10^5, 1×10^6 human breast cancer stem-like cells, and 1×10^6 parental MDA-MB-231 cells, respectively. A positive control group （D） without implantation of human bone was also injected with 1×10^6 MDA-MB-231 cells. Immunohistochemistry was performed for determination of CD34, CD105, smooth muscle antibody, CD44, CD24, cytokine, CXC chemokine receptor-4 （CXCR4）, and osteopontin （OPN）. mRNA levels of CD44, CD24, CXCR4, and OPN in bone metastasis tissues were analyzed by real-time quantitative polymerase chain reaction （PCR）.Results Our results demonstrated that cells in implanted human bones of group B, which received 1×10^6 cancer stem-like cells, stained strongly positive for CD44, CXCR4, and OPN, whereas those of other groups showed no or minimum staining. Moreover, group B had the highest incidence of human bone metastasis （77.8%, P=0.0230） and no accompaniment of other tissue metastasis. The real-time PCR showed an increase of CD44, CXCR4, and OPN mRNA in metastatic bone tissues in group B compared with those of groups C and D, however the expression of CD24 mRNA in group B were the lowest. Conclusions In the novel “human source” model of breast cancer, breast cancer stem-like cells demonstrated a higher human bone-seeking ability. Its mechanism might be related to the higher expressions of CD44, CXCR4, and OPN, and the lower expression of CD24 in breast cancer stem-like cells.
文摘目的探讨聚合人脐带血红蛋白氧载体(PolyCHb)对人乳腺癌MCF-7细胞裸鼠皮下移植瘤化疗敏感性的影响及其机制。方法收集处于指数生长期的MCF-7细胞,制成密^度为5×10^(7)个/mL的悬浮细胞,按0.2 mL/只接种于18只BALB/c-nu裸鼠右肢皮下,建立裸鼠移植瘤模型,待肿瘤体积达到100 mm^(3)左右时,随机均分为化疗组:多柔比星(DOX)5 mg·kg^(-1),1次/周;化疗+PolyCHb治疗组:除给药(DOX)(同化疗组)外,PolyCHb 600 mg·kg^(-1),3次/周;对照组:生理盐水90 mg·kg^(-1),1次/周;均为经尾静脉连续注射4周。自注射当天(d0)起,每3 d 1次测量各组裸鼠肿瘤体积,据此绘制其各自(组)肿瘤生长曲线。38 d结束肿瘤生长观察,剥瘤并称取瘤重,计算抑瘤率;HE染色观察肿瘤组织病理变化,免疫组化检测肿瘤组织中HIF-1α表达,采用TUNEL法检测肿瘤细胞凋亡情况,荧光染色测定各组肿瘤组织活性氧(ROS)的含量。结果化疗+PolyCHb组、化疗组和对照组至d38时的肿瘤体积(mm^(3))分别为:196.35±103.45 vs 316.29±62.88 vs 519.42±177.33(P<0.05);化疗+PolyCHb组与化疗组抑瘤率(%)分别为62.20 vs 39.11;HE染色和TUNEL检测:化疗+PolyCHb组肿瘤组织生长区域细胞坏死和凋亡增多;免疫组化:化疗+PolyCHb组HIF-1α表达水平降低;荧光染色:化疗+PolyCHb组[活性氧(ROS)]含量增多。结论PolyCHb增加了乳腺癌裸鼠皮下移植瘤化疗的敏感性,其作用机制可能与其提高肿瘤组织内ROS含量,促进肿瘤细胞的凋亡有关。
