Curcumin inhibits telomerase activity through human telomerase reverse transcritpase in MCF-7 breast cancer cell line

The inhibitory effect of curcumin, the yellow-colored pigment from turmeric, on telomerase activity was analyzed in human mammary epithelial (MCF-10A) and breast cancer (MCF-7) cells. Telomerase activity in MCF-7 cells is 6.9-fold higher than that of human mammary epithelial cells. In MCF-7 cells, telomerase activity decreased with increasing concentrations of curcumin, inhibiting about 93.4% activity at 100 microM concentration. The inhibition of telomerase activity in MCF-7 cells may be due to down-regulation of hTERT expression. Increasing concentrations of curcumin caused a steady decrease in the level of hTERT mRNA in MCF-7 cells whereas the level of hTER and c-myc mRNAs remained the same. Our results suggest that curcumin inhibits telomerase activity by down-regulating hTERT expression in breast cancer cells and this down-regulation is not through the c-myc pathway.     

EGCG down-regulates telomerase in human breast carcinoma MCF-7 cells, leading to suppression of cell viability and induction of apoptosis

Telomerase is elevated in >90% of breast carcinomas and therefore has received much attention as a target for breast cancer therapy and cancer diagnostic research. Dietary components that are capable of inhibiting the growth of cancer cells without affecting the growth of normal cells are receiving considerable attention in developing novel cancer-preventive approaches. Studies have shown that (-)-epigallocatechin-3-gallate (EGCG) from green tea imparts a growth inhibitory effect on cancer cells. Here, we show that treatment of EGCG dose-dependently inhibited (20-100%) the reproductive or colony forming potential, and also decreased cell viability at different time points studied ( approximately 80% inhibition) in human breast carcinoma MCF-7 cells but had no adverse effect on the growth of normal mammary cells. Treatment of EGCG for 48 and 72 h markedly increased the percentage of apoptotic cells (32-51%) in MCF-7 cells compared to that of non-EGCG treated cells (8-14%). In order to identify the possible mechanism of decreased cell viability and induction of apoptosis in breast carcinoma cells by EGCG, we found that treatment of MCF-7 cells with EGCG dose-dependently inhibited telomerase activity (40-55%), and also inhibited the mRNA expression (40-55%) of hTERT, a catalytic subunit of telomerase. Additional studies demonstrated that EGCG also inhibited the protein expression of hTERT, which indicated that inhibition of telomerase was associated with down-regulation of hTERT. Together, our results indicate that EGCG down-regulates telomerase in human breast carcinoma MCF-7 cells, leading to the suppression of cell viability and induction of apoptosis, thus providing the molecular basis for the development of EGCG as a novel chemopreventive and pharmacologically safe agent against breast cancer.     

瘦素上调乳腺癌细胞端粒酶的活性及其分子机制研究

  目的  观察瘦素(leptin)对乳腺癌细胞端粒酶的活性影响, 并探讨其可能的分子机制。  方法  采用ELISA检测瘦素对人乳腺癌细胞MCF-7端粒酶活性的影响。应用实时荧光定量PCR和Western blot法测定瘦素对MCF-7细胞人端粒酶逆转录酶(hTERT)及STAT3 mRNA和蛋白表达水平的影响。  结果  瘦素可增加MCF-7端粒酶的活性, 且呈剂量依赖性, 当浓度是10 nmol/L时, 活性增加2.8倍; 而采用瘦素受体单克隆抗体ZMC-2时则可明显抑制端粒酶的活性; 端粒酶的活性与hTERT紧密相关, 瘦素不仅增强了端粒酶的活性, 同时也增加了hTERTmRNA的表达水平, 且呈剂量依赖性, 经瘦素10 nmol/L处理后, hTERT蛋白表达水平增加2.9倍; 在瘦素处理MCF-7细胞后, hTERT的表达水平与磷酸化STAT3的水平呈现相关性上升, 提示STAT3途径可能参与了瘦素诱导hTERT的表达。  结论  在乳腺癌MCF-7细胞中, 瘦素可能通过STAT3途径促进hTERT的表达, 并最终上调端粒酶的活性。      

Ribozyme cleavage of telomerase mRNA sensitizes breast epithelial cells to inhibitors of topoisomerase

Telomerase activity is necessary and sufficient for immortality in many cells and hence represents a prime target for antitumor strategies. Here, we show that a hammerhead ribozyme cleaves human telomerase (hTERT) mRNA in vitro. Stable transfection in clones of the human breast tumor line MCF-7 and the immortal breast cell line HBL-100 results in expression of the ribozyme, diminishes the abundance of hTERT mRNA, and inhibits telomerase activity. This led to shortened telomeres, inhibition of net growth, and induction of apoptosis. In HBL-100 mass cultures infected with a ribozyme-expressing adenovirus diminution of hTERT mRNA, attenuation of telomerase activity, inhibition of net growth, and induction of apoptosis was found as well. Attenuation of telomerase activity increased the sensitivity of HBL-100 and MCF-7 clones specifically to inhibitors of topoisomerase. Likewise, expression of exogenous telomerase in originally telomerase-negative human fibroblasts decreased their sensitivity to topoisomerase poisons but not to a number of other cytotoxic drugs. The data validate a ribozyme approach for telomerase inhibition therapy in cancer and suggest that it might be combined advantageously with topoisomerase-directed chemotherapy.     

Antiproliferative effects of the histone deacetylase inhibitor FR901228 on small-cell lung cancer lines and drug-resistant sublines

FR901228 is a novel histone deacetylase (HDAC) inhibitor, and its antiproliferative effects on non-small cell lung cancer cells have been shown in vitro. However, there have been no reports concerning the effects on small-cell lung cancer (SCLC). We have recently demonstrated that the HDAC inhibitors trichostatin A and sodium butyrate inhibit expression of the catalytic subunit of telomerase (hTERT) mRNA and telomerase activity in prostate cancer cells. The present study was designed to evaluate the effects of FR901228 on proliferation and telomerase activity in SCLC cells in vitro. FR901228 at 5 to 10 nM increased the fraction of cells in the G(2)/M and sub-G(1) phases of the cell-cycle, and inhibited the growth of H69, H526 and H82 cell lines. The expression of hTERT mRNA was inhibited 6 hr after treatment, prior to obvious inhibition of cell growth or cell-cycle distribution shifts. The inhibition of hTERT mRNA expression and telomerase activity was not a consequence of cell-growth arrest or apoptosis. Cycloheximide blocked the suppression of hTERT mRNA induced by FR901228, and the inhibition of hTERT mRNA by FR901228 required newly synthesized proteins. FR901228 also effectively inhibited growth of etoposide-resistant UMCC-1/VP-16, irinotecan-resistant PC-6/SN2-5H and cisplatin-resistant H526/CDDP cells having decreased expression of hTERT mRNA and telomerase activity, as well as their parental cells. This implies that SCLC resistant to these key drugs are not cross-resistant to FR901228. The present study suggests that FR901228 may be a promising drug for chemotherapy of cancers including SCLC, even for refractory or relapsing tumors after conventional chemotherapy.     

Telomerase as a Possible Candidate Targeting Therapy in Different Breast Cancer Cell Lines

Background: Telomerase activity is up regulated in most breast cancer subtypes but not in the adjacent normal tissues. Thus, it is a promising target for anticancer therapy. The present work investigated the effects of telomerase inhibition by siRNA on breast cancer cell lines and studied the feasibility of whether the combined effect of doxorubicin with siRNA treatment on breast cancer cells potentiates a rapid cellular response to the cytotoxic effect of chemotherapy.  Methods: This study was performed on Luminal A (MCF-7), triple negative (MDA-MB-468), and HER-2/neu (SKBR-3) human breast cancer cell lines, wherein telomerase activity inhibition by hTERT siRNA and doxorubicin was detected by measuring telomerase activity using Telomeric Repeat Amplification Protocol (TRAP assay), assessing cell viability through MTT assay, and evaluating apoptosis through scanning electron microscopy (SEM) and through estimating caspase-3 and -8 activities using enzyme-linked immunosorbent assay (ELISA). Results: In the present study, hTERT siRNA effectively reduced telomerase activity and cell viability to more than 90% and 60%, respectively, in most breast cancer cell lines within 72 hours after transfection. The combination of hTERT siRNA and doxorubicin showed a cumulative effect compared with either treatment alone (P < 0.05). Meanwhile, SEM demonstrated apoptotic morphologic cell changes. Conclusion: Telomerase inhibition is a promising strategy for the effective treatment of breast cancer. When used in combination with doxorubicin, it could potentiate the cytotoxic effect of the drug on breast cancer cells.     

肿瘤选择性增殖腺病毒CNHK300 治疗乳腺癌的体外实验研究

Objective： To evaluate the tumor selectivity and therapeutic efficiency of replication-competent adenovirus CNHK300 on human breast cancer cells. Methods： RT-PCR was used to detect the hTERT mRNA activity in various breast cancer and normal fibroblast cell lines. Virus proliferation assay, cell viability assay and Western blot were applied to evaluate the proliferation and cytolysis selectivity of CNHK300. Results： The telomerase activity of MCF-7, BT-549 and SK-BR-3 was positive, while telomerase in MRC-5 and BJ was negative. The progeny virus titers in MCF-7, BT-549 and SK-BR-3 after 48 h of CNHK300 exposure was 40 625, 1 265 and 20 000 fold higher than those of 0 h, even slightly higher than those of wtAd5 （except in SK-BR-3）. ONYX-015 virus proliferation ability was weaker than that of CNHK300 in cancer cells. However, CNHK300 exhibited attenuated replicative ability as compared with wtAd5 in MRC-5 and BJ. The CNHK300 replicatative multiple was 63 and 192 fold at 48 h respectively, while the wtAd5 still multiplied 3 160-4 846 fold. CNHK300 could cause about half of breast cancer cells to die within 7 days at MOI 10 pfu/cell and below, whereas the IC50 in BJ and MRC-5 was as high as MOI 100 pfu/cell. CNHK300 E1A protein could be detected in breast cancer cells and 293 cells but not in normal fibroblast cells. Conclusion： hTERT promoter can successfully modulate the CNHK300 to be selectively replicated in breast cancer cells positive for telomerase, which may be a potential treatment strategy in breast cancer.     

双基因干扰对MCF-7细胞基因表达抑制及细胞凋亡影响的研究

背景与目的：肿瘤细胞具有端粒酶高表达及端粒稳定性强两大特点。人端粒酶逆转录酶（hTERT@是端粒酶的催化亚单位,端粒重复序列结合因子2（TRF2@对维持端粒长度及端粒稳定性非常重要。本实验研究针对hTERT和TRF2的特异性RNAi腺病毒表达载体单独和联合转染乳腺癌MCF-7细胞后,对两基因的抑制效率以及对细胞凋亡的影响,探索针对端粒的多基因干扰对乳腺癌基因治疗的可行性。方法：构建RNA干扰腺病毒载体rAd-hTERT和rAd-TRF2,单独和联合转染MCF-7细胞后实时荧光定量PCR检测hTERT和TRF2基因表达情况,流式细胞仪检测各组细胞凋亡率。结果：①转染后48h,与PBS组相比,rAd-hTERT对hTERT基因mRNA的抑制率约86%,对TRF2基因表达无明显抑制（P〉0.05@;rAd-TRF2对TRF2基因mRNA的抑制率约80%,对hTERT基因表达无明显抑制（P〉0.05@;rAd-hTERT/rAd-TRF2联合干扰组对hTERT基因表达抑制率约88%,TRF2基因表达抑制率约85%,联合干扰与单基因干扰相比,对hTERT和TRF2基因的表达抑制差异无显著性（P〉0.05@。②干扰组转染后1～6d均能促进细胞凋亡。单基因干扰组转染后3～5d凋亡最明显,第5天达凋亡高峰;凋亡率rAd-hTERT组为46.2%,rAd-TRF2组为43.5%。联合组转染后第1天凋亡率为46.2%,第2天为68.5%,第3～6天均维持在较高水平,第6天达77.6%。转染后1～6d,联合干扰细胞凋亡率与单基因干扰细胞凋亡率相比,差异均有显著性（P〈0.05@。结论：①rAd-hTERT和rAd-TRF2转染MCF-7细胞48h后可分别显著抑制hTERT和TRF2基因mRNA表达,但rAd-hTERT和rAd-TRF2在抑制hTERT和TRF2基因mRNA表达上无相互协同或相互抑制作用。②rAd-hTERT和rAd-TRF2均能有效促进MCF-7细胞凋亡,且两者联合干扰效果具有累加效应。因此利用联合干扰技术靶向抑制端粒酶活性和端粒稳定性相关的多基因的表达能更高效地促进乳腺癌细胞凋亡,抑制肿瘤细胞的增殖与生长。     

人端粒酶逆转录亚单位(hTERT)基因特异性siRNA对MCF-7细胞生长抑制作用的研究

目的 研究人端粒酶逆转录亚单位（hTERT）特异性siRNA对MCF-7细胞体外生长及体内肿瘤形成能力的抑制作用。方法 设计并化学合成针对hTERT的siRNA，用脂质体转染法将其导入MCF-7细胞内，通过软琼脂克隆形成试验及接种裸鼠的肿瘤形成实验，观察hTERT-siRNA对人乳腺癌MCF-7细胞体外及体内的生长抑制作用。结果 软琼脂克隆形成试验显示，hTERT-siRNA转染的MCF-7细胞克隆形成数量明显少于对照组，抑制率达到84．1％。裸鼠体内实验结果显示，hTERT-siRNA转染组肿瘤生长速度也明显慢于对照组。结论 hTERT-siRNA在体内外均显示可以有效、特异地抑制肿瘤细胞的生长。     

肿瘤选择性增殖腺病毒CNHK300治疗乳腺癌的体外实验研究(英文)

目的观察肿瘤选择性增殖腺病毒 CNHK300对乳腺癌的选择性杀伤作用。方法用 RT-PCR 方法检测各种细胞株的端粒酶活性;CNHK300、ONYX-015(E1B 55 KDa 蛋白缺失的2型和5型嵌合型腺病毒)、wtAd5(野生型腺病毒)分别行病毒增殖实验和细胞生长抑制实验,验证 CNHK300选择性复制和杀伤能力;Western Blot 检测腺病毒E1A 在细胞中的表达。结果乳腺癌细胞株 MCF-7、BT-549和 SK-BR-3端粒酶 hTERT mRNA 均为阳性表达,而正常成纤维细胞株 MRC-5和 BJ 端粒酶hTERT mRNA 为阴性。CNHK300在乳腺癌细胞 MCF-7、BT-549和 SK-BR-3中48 h 复制倍数分别为40 625、1265和20000倍,与wtAd5的增殖能力相似,较 ONYX-015增殖能力强,在 MCF-7和 BT-549细胞中复制能力甚至强于野生型腺病毒。然而,在正常成纤维细胞 MRC-5和 BJ 中 CNHK300病毒增殖能力减弱,48 h 增殖倍数为63～192倍,而 wtAd5增殖仍可高达3160～4846倍CNHK300 MOI 10 PFU/cell 作用7天,可有效杀伤半数乳腺癌细胞,与 ONYX-015相比,CNHK300具有更强的肿瘤杀伤能力CNHK300对正常成纤维细胞的杀伤力较 wtAd5明显减弱,CNHK300在 MOI 100 PFU/cell 时 BJ 细胞存活率50%以上。正常成纤维细胞株中未检测到 CNHK300 E1A 基因表达,在293细胞和感染 CNHK300的乳腺癌细胞株中能够检测到 E1A 基因表达。结论 hTERT 启动子可成功地调控腺病毒 CNHK300选择性在端粒酶阳性的乳腺癌细胞中复制,并产生溶瘤作用。可望成为治疗乳癌的一种新的治疗策略。