The isothiocyanate erucin abrogates telomerase in hepatocellular carcinoma cells in vitro and in an orthotopic xenograft tumour model of HCC

In contrast to cancer cells, most normal human cells have no or low telomerase levels which makes it an attractive target for anti‐cancer drugs. The small molecule sulforaphane from broccoli is known for its cancer therapeutic potential in vitro and in vivo. In animals and humans it was found to be quickly metabolized into 4‐methylthiobutyl isothiocyanate (MTBITC, erucin) which we recently identified as strong selective apoptosis inducer in hepatocellular carcinoma (HCC) cells. Here, we investigated the relevance of telomerase abrogation for cytotoxic efficacy of MTBITC against HCC. The drug was effective against telomerase, independent from TP53 and MTBITC also blocked telomerase in chemoresistant subpopulations. By using an orthotopic human liver cancer xenograft model, we give first evidence that MTBITC at 50 mg/KG b.w./d significantly decreased telomerase activity in vivo without affecting enzyme activity of adjacent normal tissue. Upon drug exposure, telomerase decrease was consistent with a dose‐dependent switch to anti‐survival, cell arrest and apoptosis in our in vitro HCC models. Blocking telomerase by the specific inhibitor TMPyP4 further sensitized cancer cells to MTBITC‐mediated cytotoxicity. Overexpression of hTERT, but not enzyme activity deficient DNhTERT, protected against apoptosis; neither DNA damage nor cytostasis induction by MTBITC was prevented by hTERT overexpression. These findings imply that telomerase enzyme activity does not protect against MTBITC‐induced DNA damage but impacts signalling processes upstream of apoptosis execution level.     

hTERT: Another brick in the wall of cancer cells

In human cancer, expression of telomerase is positively correlated with tumour aggressiveness and metastatic potential. There is accumulating evidence that hTERT (the catalytic subunit of telomerase) favours an immortal phenotype by blocking programmed cell death (apoptosis) independently of its protective function at the telomere ends. This review summarized existing evidence for the anti-apoptotic role of hTERT in the context of tumour-cell resistance against DNA damage and aims to put hTERT in the context of cell-signal-transduction pathways leading either to survival or cell death. We found evidence that telomerase is cross-linked with many different signalling pathways that regulate cell proliferation, DNA damage repair, and also cell death. Thereby, hTERT survival function seems to occur at early stages of DNA damage recognition. We found some discrepancies in the published data though. Based on our findings, we suggest further exploration is needed of the interplay of the DNA damage response signalling network, including MAPK and p53 family activation, on telomerase regulation. This interaction is probably an important factor for fine tuning of the sensitivity of the cell to genotoxic stress. Using anti-neoplastic agents, further dose relationships on timing and extent of DNA damage, cellular repair and death should be established and correlated with hTERT expression/telomerase activation. Closing the data gaps identified here could profoundly improve our understanding of the relevance of telomerase for protecting the cell against anti-cancer agents and would contribute to developing new strategies for cancer therapy.     

Preclinical Evaluation of 4-Methylthiobutyl Isothiocyanate on Liver Cancer and Cancer Stem Cells with Different p53 Status

Isothiocyanates from plants of the order Brassicales are considered promising cancer chemotherapeutic phytochemicals. However, their selective cytotoxicity on liver cancer has been barely researched. Therefore, in the present study, we systematically studied the chemotherapeutic potency of 4-methylthiobutyl isothiocyanate (MTBITC). Selective toxicity was investigated by comparing its effect on liver cancer cells and their chemoresistant subpopulations to normal primary hepatocytes and liver tissue slices. Additionally, in a first assessment, the in vivo tolerability of MTBITC was investigated in mice. Growth arrest at G2/M and apoptosis induction was evident in all in vitro cancer models treated with MTBITC, including populations with cancer initiating characteristics. This was found independent from TP53; however cell death was delayed in p53 compromised cells as compared to wt-p53 cells which was probably due to differential BH3 only gene regulation i. e. Noxa and its antagonist A1. In normal hepatocytes, no apoptosis or necrosis could be detected after repeated administration of up to 50 µM MTBITC. In mice, orally applied MTBITC was well tolerated over 18 days of treatment for up to 50 mg/kg/day, the highest dose tested. In conclusion, we could show here that the killing effect of MTBITC has a definite selectivity for cancer cells over normal liver cells and its cytotoxicity even applies for chemoresistant cancer initiating cells. Our study could serve for a better understanding of the chemotherapeutic properties of isothiocyanates on human liver-derived cancer cells.     

Inhibition of telomerase activity and human telomerase reverse transcriptase gene expression by histone deacetylase inhibitor in human brain cancer cells

The aim of the present study is to investigate the effect of histone deacetylase inhibitor, trichostatin A (TSA) on the cell growth, apoptosis, genomic DNA damage and the expression of telomerase and associated factors in human normal and brain cancer cells. Here, human normal un-transformed fibroblasts (MRC-5), human normal hTERT-immortalised fibroblasts (hTERT-BJ1) and human brain cancer cell lines (glioblastoma cell line, A-172 and medulloblastoma cell line, ONS-76) were treated with 0.5–3.0 μM TSA for 24 h. Exposure to TSA resulted in apoptosis in a dose-dependent manner in the brain cancer cells. Glioblastoma cell line (A-172) displayed higher sensitivity to TSA-induced cell killing effect and apoptosis than the medulloblastoma cell line (ONS-76). The brain cancer cell lines and hTERT-BJ1 cell line displayed significant inhibition in telomerase activity and hTERT mRNA level after 2 μM TSA treatment. Elevated expressions of p53 and p21 with a decrease in cyclin-D level supported the observation on cell cycle arrest following TSA treatment. Upregulation of Bax and cytochrome c correlated with the apoptotic events in TSA-treated cells. This study suggests that telomerase and hTERT might be the primary targets of TSA which may have the potential to be used as a telomerase inhibitor in cancer therapy.     

Expression of human telomerase (hTERT) does not prevent stress-induced senescence in normal human fibroblasts but protects the cells from stress-induced apoptosis and necrosis

Cells subjected to sub-lethal doses of stress such as irradiation or oxidative damage enter a state that closely resembles replicative senescence. What triggers stress-induced premature senescence (SIPS) and how similar this mechanism is to replicative senescence are not well understood. It has been suggested that stress-induced senescence is caused by rapid telomere shortening resulting from DNA damage. In order to test this hypothesis directly, we examined whether overexpression of the catalytic subunit of human telomerase (hTERT) can protect cells from SIPS. We therefore analyzed the response of four different lines of normal human fibroblasts with and without hTERT to stress induced by UV, gamma-irradiation, and H(2)O(2). SIPS was induced with the same efficiency in normal and hTERT-immortalized cells. This suggests that SIPS is not triggered by telomere shortening and that nonspecific DNA damage serves as a signal for induction of SIPS. Although telomerase did not protect cells from SIPS, fibroblasts expressing hTERT were more resistant to stress-induced apoptosis and necrosis. We hypothesize that healing of DNA breaks by telomerase inhibits the induction of cell death, but because healing does not provide legitimate DNA repair, it does not protect cells from SIPS.     

The 5'-end of hTERT mRNA is a good target for hammerhead ribozyme to suppress telomerase activity

Because the expression level of hTERT, a catalytic subunit of human telomerase, is a rate-limiting determinant of telomerase activity, hTERT mRNA would be an excellent target of hammerhead ribozymes for the regulation of telomerase activity. We studied the efficiency of several hammerhead ribozymes targeting hTERT mRNA by transient and stable transfection procedures. To screen the potency of the ribozymes, transient ribozyme transfection and telomerase determination were performed. The ribozyme targeting 13 nucleotides downstream from the 5'-end of hTERT mRNA (13-ribozyme) exhibited the strongest telomerase-inhibitory activity, and the ribozyme to target 59 nucleotides upstream from the poly(A) tail showed clear activity. A stable transfection study confirmed that the 13-ribozyme suppressed telomerase. These observations suggest that the 13-ribozyme can regulate telomerase activity and may possess potential for cancer therapy.     

EPA诱导人肝癌细胞SMMC-7721凋亡及端粒酶活性调控机制研究

目的:探究二十碳五烯酸(Eicosa Pentaenoic Acid.EPA)对SMMC-7721人肝癌细胞的凋亡、端粒逆转录酶h TERT的调控作用及端粒酶表达活性的影响。方法:体外培养SMMC-7721人肝癌细胞,用不同浓度的EPA(0μM、25μM、50μM、100μM、200μM)作用于SMMC-7721肝癌细胞(24 h、48 h、72 h)后,显微镜下观察其形态学变化;应用MTT法检测SMMC-7721肝癌细胞细胞增殖变化情况;Western-blot法检测h TERT、Bax、Bcl-2蛋白表达水平变化;Real Time-PCR检测h TERTm RNA的表达变化;ELISA法检测SMMC-7721肝癌细胞端粒酶活性的表达水平。结果:EPA可诱导肝癌细胞SMMC-7721发生细胞凋亡,具有明显的时间计量依赖关系。在此过程中Bcl-2蛋白表达的降低和Bax蛋白表达上调,同时端粒酶逆转录酶h TERT蛋白及其m RNA的表达水平和端粒酶活性均明显降低。结论:抑制端粒酶逆转录酶基因(h TERTm RNA)表达而抑制端粒酶的活性、诱导癌细胞凋亡,可能是EPA的抗癌作用机制之一。     

Estrogen receptor-alpha mediates the effects of estradiol on telomerase activity in human mesenchymal stem cells

Sex steroid hormone receptors play a central role in modulating telomerase activity, especially in cancer cells. However, information on the regulation of steroid hormone receptors and their distinct functions on telomerase activity within the mesenchymal stem cell are largely unavailable due to low telomerase activity in the cell. In this study, the effects of estrogen (E2) treatment and function of estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) on telomerase activity were investigated in human mesenchymal stem cells (hMSCs). Telomerase activity and mRNA expression of the catalytic subunit of telomerase (hTERT) were upregulated by treatment of the cells with E2. The protein concentration of ERalpha was also increased by E2 treatment, and enhancement of ERalpha accumulation in the nucleus was clearly detected with immunocytochemistry. When ERalpha expression was reduced by siRNA transfection into hMSCs, the effect of E2 on the induction of hTERT expression and telomerase activity was diminished. In contrast, the transient overexpression of ERalpha increased the effect of E2 on the expression of hTERT mRNA. These findings indicate that the activation of hTERT expression and telomerase activity by E2 in hMSCs depends on ERalpha, but not on ERbeta.     

人端粒酶逆转录酶核酶抑制端粒酶活性

为有效切割端粒酶逆转录酶mRNA以降低端粒酶活性 ,从而使肿瘤细胞生长变慢 ,凋亡增加。设计并合成了针对端粒酶逆转录酶mRNA的锤头状核酶基因 ,构建了该核酶基因的体外转录和真核表达质粒。检测了该核酶对端粒酶逆转录酶mRNA的体外切割效力。并将该核酶基因转染至肿瘤细胞中 ,检测其对肿瘤细胞端粒酶活性和生物学性状的影响。结果表明 ,该核酶在体外和细胞内均能有效切割端粒酶逆转录酶mRNA ;在细胞内能明显抑制端粒酶活性 ,使细胞生长变慢 ,倍增时间延长。因而 ,该核酶可望成为有效的端粒酶抑制剂 ,在抑制肿瘤生长中发挥作用     

Knock-Down of the 37kDa/67kDa Laminin Receptor LRP/LR Impedes Telomerase Activity

Cancer has become a major problem worldwide due to its increasing incidence and mortality rates. Both the 37kDa/67kDa laminin receptor (LRP/LR) and telomerase are overexpressed in cancer cells. LRP/LR enhances the invasiveness of cancer cells thereby promoting metastasis, supporting angiogenesis and hampering apoptosis. An essential component of telomerase, hTERT is overexpressed in 85–90% of most cancers. hTERT expression and increased telomerase activity are associated with tumor progression. As LRP/LR and hTERT both play a role in cancer progression, we investigated a possible correlation between LRP/LR and telomerase. LRP/LR and hTERT co-localized in the perinuclear compartment of tumorigenic breast cancer (MDA_MB231) cells and non-tumorigenic human embryonic kidney (HEK293) cells. FLAG^® Co-immunoprecipitation assays confirmed an interaction between LRP/LR and hTERT. In addition, flow cytometry revealed that both cell lines displayed high cell surface and intracellular LRP/LR and hTERT levels. Knock-down of LRP/LR by RNAi technology significantly reduced telomerase activity. These results suggest for the first time a novel function of LRP/LR in contributing to telomerase activity. siRNAs targeting LRP/LR may act as a potential alternative therapeutic tool for cancer treatment by (i) blocking metastasis (ii) promoting angiogenesis (iii) inducing apoptosis and (iv) impeding telomerase activity.