Objective: To explore the correlation between tumor-associated macrophages and the proliferation and invasion of type endometrial carcinoma. Methods: Immunohistochemistry was used to investigate the infiltration of ma...Objective: To explore the correlation between tumor-associated macrophages and the proliferation and invasion of type endometrial carcinoma. Methods: Immunohistochemistry was used to investigate the infiltration of macrophages in normal and different types of hyperplastic endometrial lesions. The proliferation and invasion ability of type endometrial carcinoma cell line RL95-2 influenced by mononuclear macrophage cell line THP-1(constructed M2 type macrophages) was detected by CCK8 and transwell technologies respectively. Transwell was used to evaluate the recruiting ability of RL95-2 on THP-1 cells. Otherwise, the western blot was also used to detect the expression of Cyclin D1 and MMP-2 in RL95-2 with the influence of THP-1. Results: Immunohistochemistry result showed a positive correlation between the number of infiltrating macrophages and the progression of endometrial hyperplasia.THP-1 recruited by RL95-2 could promote its proliferation and invasion and enhance the expression of the Cyclin D1 and MMP-2 protein in a time dependent manner(P<0.05). Conclusions: Increase of the number of infiltrating macrophages and its contribution to the tumor inflammatory microenvironment may result in the development of the type endometrial carcinoma.展开更多
Background Endometrial carcinoma is one of the most common female tract genital malignant tumors. Nifedipine, an L-type calcium channel antagonist can inhibit cell proliferation of carcinomas. Recent studies indicated...Background Endometrial carcinoma is one of the most common female tract genital malignant tumors. Nifedipine, an L-type calcium channel antagonist can inhibit cell proliferation of carcinomas. Recent studies indicated that a rise in the free cytosolic calcium （[Ca2±]c） was a potent inducer of autophagy. Here, we investigated the relationship between nifedipine and autophagy in Hec-IA cells. Methods Cells were cultured with nifedipine （10 μmol/L） and harvested at different times for counting cell number. MTT assay was applied to evaluate the cell viability and transwell assay to reveal cell migration. Apoptotic cells were detected with annexin V/PI assay. Then cells were treated with 3-methyladenine （3-MA） （2.5 mmol/L） for 0, 5, 15, 30, 60, and 120 minutes and the expression of the L-type calcium channel alphalD （Cavl.3） protein was detected. At last, cells were cultured and assigned to four groups with different treatment： untreated （control group）, 10 μmol/L nifedipine （N group）, 2.5 mmol/L 3-MA （3-MA group）, and 10 μmol/L nifedipine plus 2.5 mmol/L 3-MA （N±3MA group）. Autophagy was detected with GFP-LC3 modulation by fluorescent microscopy, and expression of the autophagy-associated proteins （LC3, Beclinl and P70s6K） by Western blotting and monodansylcadaverine （MDC） labeled visualization. Results Proliferation of Hec-lA cells was obviously suppressed by nifedipine compared with that of the untreated cells for 24, 48, and 96 hours （P=0.000 for each day）. The suppression of migration ability of the nifedipine-treated cells （94.0±8.2） was significantly different from that of the untreated cells （160.00±9.50, P=0.021 ）. The level of early period cell apoptosis induced by nifedipine was （2.21_±0.19）%, which was （2.90±0.13）% in control group （P=-0.052）, whereas the late period apoptosis level reached （10.38_±0.96）% and （4.40_±0.60）% （P=0.020）, respectively. The 3-MA group induced a slight increase in the Cavl.3 levels within 15 minutes, but significantly attenuated the Cavl.3 levels after 30 minutes. There were more autophagic vacuoles labeled by MDC in the N group （20.63_±3.36） than the control group （6.29_±0.16, P=-0.015）. GFP-LC3 localization revealed that the LC3 levels of cells in 3-MA group, N±3MA group, 3-MA group were 2.80_±0.29, 2.30_±0.17, and 1.80±0.21, respectively. Cells in the N group showed significant augmentation of autophagy （P 〈0.05）. Western blotting analysis confirmed the down-regulation of LC3 levels in 3-MA group （0.85±0.21） and N±3MA group （1.21±0.12） compared with nifedipine treatment （2.64±0.15, P 〈0.05）. The annexin-V-FITC/PI assay showed that the level of early period cell apoptosis induced in the N＋3-MA group （（11.22±0.91）%） differed significantly from that of the control group （（2.51±0.70）%） and N group （（3.47±0.39）%）. Similarly, the late period level of the N＋3-MA group （（55.19±2.51）%） differed significantly from that of the control group（（15.81±1.36）%） and the N group （（22.09±2.48）%, P 〈0.05）. The down-regulated expression of P70s6k and up-regulated expression of the Beclinl revealed significant differences between the N＋3-MA group and control group （P=0.025; Beclinl： P=-0.015）. Conclusions Proliferation and migration in vitro of endometrial carcinoma Hec-lA cells are significantly suppressed by nifedipine. The nifedipine leads autophagy to oppose Hec-lA cells apoptosis. Autophagy inhibition by 3-MA leads down-regulation of Cavl.3 and enhances nifedipine-induced cell death. The nifedipine-induced autophagy is linked to Beclinl and mTOR pathways.展开更多
文摘Objective: To explore the correlation between tumor-associated macrophages and the proliferation and invasion of type endometrial carcinoma. Methods: Immunohistochemistry was used to investigate the infiltration of macrophages in normal and different types of hyperplastic endometrial lesions. The proliferation and invasion ability of type endometrial carcinoma cell line RL95-2 influenced by mononuclear macrophage cell line THP-1(constructed M2 type macrophages) was detected by CCK8 and transwell technologies respectively. Transwell was used to evaluate the recruiting ability of RL95-2 on THP-1 cells. Otherwise, the western blot was also used to detect the expression of Cyclin D1 and MMP-2 in RL95-2 with the influence of THP-1. Results: Immunohistochemistry result showed a positive correlation between the number of infiltrating macrophages and the progression of endometrial hyperplasia.THP-1 recruited by RL95-2 could promote its proliferation and invasion and enhance the expression of the Cyclin D1 and MMP-2 protein in a time dependent manner(P<0.05). Conclusions: Increase of the number of infiltrating macrophages and its contribution to the tumor inflammatory microenvironment may result in the development of the type endometrial carcinoma.
基金This study was supported by a grant from the National Natural Science Foundation of China （No. 30973182）.
文摘Background Endometrial carcinoma is one of the most common female tract genital malignant tumors. Nifedipine, an L-type calcium channel antagonist can inhibit cell proliferation of carcinomas. Recent studies indicated that a rise in the free cytosolic calcium （[Ca2±]c） was a potent inducer of autophagy. Here, we investigated the relationship between nifedipine and autophagy in Hec-IA cells. Methods Cells were cultured with nifedipine （10 μmol/L） and harvested at different times for counting cell number. MTT assay was applied to evaluate the cell viability and transwell assay to reveal cell migration. Apoptotic cells were detected with annexin V/PI assay. Then cells were treated with 3-methyladenine （3-MA） （2.5 mmol/L） for 0, 5, 15, 30, 60, and 120 minutes and the expression of the L-type calcium channel alphalD （Cavl.3） protein was detected. At last, cells were cultured and assigned to four groups with different treatment： untreated （control group）, 10 μmol/L nifedipine （N group）, 2.5 mmol/L 3-MA （3-MA group）, and 10 μmol/L nifedipine plus 2.5 mmol/L 3-MA （N±3MA group）. Autophagy was detected with GFP-LC3 modulation by fluorescent microscopy, and expression of the autophagy-associated proteins （LC3, Beclinl and P70s6K） by Western blotting and monodansylcadaverine （MDC） labeled visualization. Results Proliferation of Hec-lA cells was obviously suppressed by nifedipine compared with that of the untreated cells for 24, 48, and 96 hours （P=0.000 for each day）. The suppression of migration ability of the nifedipine-treated cells （94.0±8.2） was significantly different from that of the untreated cells （160.00±9.50, P=0.021 ）. The level of early period cell apoptosis induced by nifedipine was （2.21_±0.19）%, which was （2.90±0.13）% in control group （P=-0.052）, whereas the late period apoptosis level reached （10.38_±0.96）% and （4.40_±0.60）% （P=0.020）, respectively. The 3-MA group induced a slight increase in the Cavl.3 levels within 15 minutes, but significantly attenuated the Cavl.3 levels after 30 minutes. There were more autophagic vacuoles labeled by MDC in the N group （20.63_±3.36） than the control group （6.29_±0.16, P=-0.015）. GFP-LC3 localization revealed that the LC3 levels of cells in 3-MA group, N±3MA group, 3-MA group were 2.80_±0.29, 2.30_±0.17, and 1.80±0.21, respectively. Cells in the N group showed significant augmentation of autophagy （P 〈0.05）. Western blotting analysis confirmed the down-regulation of LC3 levels in 3-MA group （0.85±0.21） and N±3MA group （1.21±0.12） compared with nifedipine treatment （2.64±0.15, P 〈0.05）. The annexin-V-FITC/PI assay showed that the level of early period cell apoptosis induced in the N＋3-MA group （（11.22±0.91）%） differed significantly from that of the control group （（2.51±0.70）%） and N group （（3.47±0.39）%）. Similarly, the late period level of the N＋3-MA group （（55.19±2.51）%） differed significantly from that of the control group（（15.81±1.36）%） and the N group （（22.09±2.48）%, P 〈0.05）. The down-regulated expression of P70s6k and up-regulated expression of the Beclinl revealed significant differences between the N＋3-MA group and control group （P=0.025; Beclinl： P=-0.015）. Conclusions Proliferation and migration in vitro of endometrial carcinoma Hec-lA cells are significantly suppressed by nifedipine. The nifedipine leads autophagy to oppose Hec-lA cells apoptosis. Autophagy inhibition by 3-MA leads down-regulation of Cavl.3 and enhances nifedipine-induced cell death. The nifedipine-induced autophagy is linked to Beclinl and mTOR pathways.
