The MAPK Pathway Signals Telomerase Modulation in Response to Isothiocyanate-Induced DNA Damage of Human Liver Cancer Cells

4-methylthiobutyl isothiocyanate (MTBITC), an aliphatic, sulphuric compound from Brassica vegetables, possesses in vitro and in vivo antitumor activity. Recently we demonstrated the potent growth inhibitory potential of the DNA damaging agent MTBITC in human liver cancer cells. Here we now show that MTBITC down regulates telomerase which sensitizes cells to apoptosis induction. This is mediated by MAPK activation but independent from production of reactive oxygen species (ROS). Within one hour, MTBITC induced DNA damage in cancer cells correlating to a transient increase in hTERT mRNA expression which then turned into telomerase suppression, evident at mRNA as well as enzyme activity level. To clarify the role of MAPK for telomerase regulation, liver cancer cells were pre-treated with MAPK-specific inhibitors prior to MTBITC exposure. This clearly showed that transient elevation of hTERT mRNA expression was predominantly mediated by the MAPK family member JNK. In contrast, activated ERK1/2 and P38, but not JNK, signalled to telomerase abrogation and consequent apoptosis induction. DNA damage by MTBITC was also strongly abolished by MAPK inhibition. Oxidative stress, as analysed by DCF fluorescence assay, electron spin resonance spectroscopy and formation of 4-hydroxynonenal was found as not relevant for this process. Furthermore, N-acetylcysteine pre-treatment did not impact MTBITC-induced telomerase suppression or depolarization of the mitochondrial membrane potential as marker for apoptosis. Our data therefore imply that upon DNA damage by MTBITC, MAPK are essential for telomerase regulation and consequent growth impairment in liver tumor cells and this detail probably plays an important role in understanding the potential chemotherapeutic efficacy of ITC.     

Endoplasmic reticulum stress activates telomerase

Telomerase contributes to cell proliferation and survival through both telomere‐dependent and telomere‐independent mechanisms. In this report, we discovered that endoplasmic reticulum (ER) stress transiently activates the catalytic components of telomerase (TERT) expression in human cancer cell lines and murine primary neural cells. Importantly, we show that depletion of hTERT sensitizes cells to undergo apoptosis under ER stress, whereas increased hTERT expression reduces ER stress‐induced cell death independent of catalytically active enzyme or DNA damage signaling. Our findings establish a functional link between ER stress and telomerase, both of which have important implications in the pathologies associated with aging and cancer.     

Curcumin inhibits telomerase and induces telomere shortening and apoptosis in brain tumour cells

Curcumin, a polyphenolic compound isolated from Curcuma longa (Turmeric) is widely used in traditional Ayurvedic medicine. Its potential therapeutic effects on a variety of diseases have long been known. Though anti‐tumour effects of curcumin have been reported earlier, its mode of action and telomerase inhibitory effects are not clearly determined in brain tumour cells. In the present study, we demonstrate that curcumin binds to cell surface membrane and infiltrates into cytoplasm to initiate apoptotic events. Curcumin treatment has resulted in higher cytotoxicity in the cells that express telomerase enzyme, highlighting its potential as an anticancer agent. Curcumin induced growth inhibition and cell cycle arrest at G2/M phase in the glioblastoma and medulloblastoma cells used in the study. Gene and protein expression analyses revealed that curcumin down‐regulated CCNE1, E2F1 and CDK2 and up‐regulated the expression of PTEN genes resulting in growth arrest at G2/M phase. Curcumin‐induced apoptosis is found to be associated with increased caspase‐3/7 activity and overexpression of Bax. In addition, down‐regulation of Bcl2 and survivin was observed in curcumin‐treated cells. Besides these effects, we found curcumin to be inhibiting telomerase activity and down‐regulating hTERT mRNA expression leading to telomere shortening. We conclude that telomerase inhibitory effects of curcumin underscore its use in adjuvant cancer therapy. J. Cell. Biochem. 114: 1257–1270, 2013. © 2012 Wiley Periodicals, Inc.     

Activity of the Human Telomerase Catalytic Subunit (hTERT) Gene Promoter Could Be Increased by the SV40 Enhancer

Telomerase is a ribonucleoprotein complex of which the function is to add telomeric repeats to chromosomal ends. Telomerase consists of two essential components, the telomerase RNA template (hTR) and the catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor and fetal cells. The aim of this study is to test the increased telomerase promoter activity for cancer gene therapy in adenovirus vector. We cloned the hTERT promoter in place of the SV40 promoter in the pGL3-contol vector to be increased by the SV40 enhancer sequences, resulting in strong expression of luc+ only in telomerase positive cancer cells. Then we transfected the constructed plasmid into a normal human cell line and several cancer cell lines. Through these experiments, we identified the selective and increased expression of the luciferase gene controlled by the hTERT promoter and the SV40 enhancer in the telomerase positive cancer cell lines. To investigate the possibility of utilizing the hTERT promoter and the SV40 enhancer in targeted cancer gene therapy, we constructed an adenovirus vector expressing HSV-TK controlled by the hTERT promoter and the SV40 enhancer for the induction of specific telomerase positive cancer cell death. NSCLC cells infected by Ad-hT-TK-enh were more significantly suppressed and induced apoptosis than those infected by Ad-hT-TK. Telomerase is activated in 80～90% of cancers, so adenovirus with increasing telomerase promoter activity might be used for targeted cancer gene therapy using suicide genes. These results show that the hTERT promoter and the SV40 enhancer might be used for targeted cancer gene therapy.     

Overcoming resistance to mitochondrial apoptosis by BZML‐induced mitotic catastrophe is enhanced by inhibition of autophagy in A549/Taxol cells

Objectives

Our previous in vitro study showed that 5‐(3, 4, 5‐trimethoxybenzoyl)‐4‐methyl‐2‐(p‐tolyl) imidazol (BZML) is a novel colchicine binding site inhibitor with potent anti‐cancer activity against apoptosis resistance in A549/Taxol cells through mitotic catastrophe (MC). However, the mechanisms underlying apoptosis resistance in A549/Taxol cells remain unknown. To clarify these mechanisms, in the present study, we investigated the molecular mechanisms of apoptosis and autophagy, which are closely associated with MC in BZML‐treated A549 and A549/Taxol cells.

Methods

Xenograft NSCLC models induced by A549 and A549/Taxol cells were used to evaluate the efficacy of BZML in vivo. The activation of the mitochondrial apoptotic pathway was assessed using JC‐1 staining, Annexin V‐FITC/PI double‐staining, a caspase‐9 fluorescence metric assay kit and western blot. The different functional forms of autophagy were distinguished by determining the impact of autophagy inhibition on drug sensitivity.

Results

Our data showed that BZML also exhibited desirable anti‐cancer activity against drug‐resistant NSCLC in vivo. Moreover, BZML caused ROS generation and MMP loss followed by the release of cytochrome c from mitochondria to cytosol in both A549 and A549/Taxol cells. However, the ROS‐mediated apoptotic pathway involving the mitochondria that is induced by BZML was only fully activated in A549 cells but not in A549/Taxol cells. Importantly, we found that autophagy acted as a non‐protective type of autophagy during BZML‐induced apoptosis in A549 cells, whereas it acted as a type of cytoprotective autophagy against BZML‐induced MC in A549/Taxol cells.

Conclusions

Our data suggest that the anti‐apoptosis property of A549/Taxol cells originates from a defect in activation of the mitochondrial apoptotic pathway, and autophagy inhibitors can potentiate BZML‐induced MC to overcome resistance to mitochondrial apoptosis.

     

Critical roles of tyrosyl‐DNA phosphodiesterases in cell tolerance to carnosol‐induced DNA damage

Carnosol is a natural compound with pharmacological action due to its anti‐cancer properties. However, the precise mechanism for its anti‐carcinogenic effect remains elusive. In this study, we used lymphoblastoid TK6 cell lines to identify the DNA damage and repair mechanisms of carnosol. Our results showed that carnosol induced DNA double‐strand breaks (DSBs). We also found that cells lacking tyrosyl‐DNA phosphodiesterase 1 (TDP1), an enzyme related to topoisomerase 1 (TOP1), and tyrosyl‐DNA phosphodiesterase 2 (TDP2), an enzyme related to topoisomerase 2 (TOP2), were supersensitive to carnosol. Carnosol was found to induce the formation of the TOP1‐DNA cleavage complex (TOP1cc) and TOP2‐DNA cleavage complex (TOP2cc). When comparing the accumulation of γ‐H2AX foci and the number of chromosomal aberrations (CAs) with wild‐type (WT) cells, the susceptivity of the TDP1^?/? and TDP2^?/? cells were associated with an increased DNA damage. Our results provided evidence of carnosol inducing DNA lesions in TK6 cells and demonstrated that the damage induced by carnosol was associated with abnormal topoisomerase activity. We conclude that TDP1 and TDP2 play important roles in the anti‐cancer effect of carnosol.     

Sulforaphene enhances radiosensitivity of hepatocellular carcinoma through suppression of the NF‐κB pathway

Sulforaphene (SFE), a naturally occurring isothiocyanate found in cruciferous vegetables, has attracted increasing attention for its anti‐cancer effect in many cancers, including hepatocellular carcinoma (HCC). However, the precise role of SFE in the radiosensitivity of HCC is still unclear. Here, cell proliferation and apoptosis were detected by MTT and flow cytometry assay, respectively. The activity of NF‐κB was further evaluated by ELISA. We also observed the effect of SFE and/or radiation on tumor growth. The results showed that SFE inhibited cell proliferation and induced apoptosis in HCC cells. Radiation increased NF‐kB activity, while PDTC, a NF‐kB inhibitor, enhanced radiation‐induced cell death. SFE inhibited NF‐kB activity and the downstream gene expressions of the NF‐kB pathway in HCC cells. Moreover, SFE enhanced the inhibitory effect of radiation on tumor growth both in vitro and in vivo. This study indicated that SFE sensitized the radiosensitivity of HCC by blocking the NF‐kB pathway.     

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.     

Psidium guajava L. anti-neoplastic effects: induction of apoptosis and cell differentiation

Objectives: Curative properties of medicinal plants such as Psidium guajava L. (Myrtaceae) have often been indicated by epidemiological studies on populations in which these fruits are consumed daily. However, complete characterization of the active principles responsible for this ability has never been performed. Here, we have characterized P. guajava’s anti‐cancer potential and identified the parts of the fruit involved in its anti‐neoplastic action. Materials and methods: We studied morphology of our cells, cell cycle characteristics and apoptosis and performed immunostaining, differentiation and western blot analyses. Results: We report that the P. guajava extract exerted anti‐cancer control on both haematological and solid neoplasias. P. guajava extract’s anti‐tumour properties were found to be tightly bound to induction of apoptosis and differentiation. Use of ex vivo myeloid leukaemia blasts corroborated that P. guajava was able to induce cell death but did not exhibit anti‐cancer effects on all malignant cells investigated, indicating selective activity against certain types of tumour. Analyses of P. guajava pulp, peel and seeds identified the pulp as being the most relevant component for causing cell cycle arrest and apoptosis, whereas peel was responsible for causing cell differentiation. P. guajava itself and its pulp‐derived extract were found to induce apoptosis accompanied by caspase activation and p16, p21, Fas ligand (FASL TNF super‐family, member 6), Bcl‐2‐associated agonist of cell death (BAD) and tumour necrosis factor receptor super‐family, member 10b (DR5), overexpression. Conclusions: Our findings showed that P. guajava L. extract was able to exert anti‐cancer activity on cultures in vitro and ex vivo, supporting the hypothesis of its anti malignant pro‐apoptotic modulation.     

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.     

Apoptosis‐inducing effect of garcinol is mediated by NF‐κB signaling in breast cancer cells

Garcinol, obtained from Garcinia indica in tropical regions, is used for its numerous biological effects. Its anti‐cancer activity has been suggested but the mechanism of action has not been studied in‐detail, especially there is no report on its action against breast cancer cells. Here we tested our hypothesis that garcinol may act as an anti‐proliferative and apoptosis‐inducing agent against breast cancer cell lines. Using multiple techniques such as MTT, Histone‐DNA ELISA, Annexin V‐PI staining, Western blot for activated caspases and cleaved PARP, homogenous caspase‐3/7 fluorometric assay and EMSA, we investigated the mechanism of apoptosis‐inducing effect of garcinol in ER‐positive MCF‐7 and ER‐negative MDA‐MB‐231 cells. We found that garcinol exhibits dose‐dependent cancer cell‐specific growth inhibition in both the cell lines with a concomitant induction of apoptosis, and has no effect on non‐tumorigenic MCF‐10A cells. Our results suggested induction of caspase‐mediated apoptosis in highly metastatic MDA‐MB‐231 cells by garcinol. Down‐regulation of NF‐κB signaling pathway was observed to be the mechanism of apoptosis‐induction. Garcinol inhibited constitutive NF‐κB activity, which was consistent with down‐regulation of NF‐κB‐regulated genes. This is the first report on anti‐proliferative and apoptosis‐inducing action of garcinol against human breast cancer cells and the results suggest that this natural compound merits investigation as a potential chemo‐preventive/‐therapeutic agent, especially against breast cancer. J. Cell. Biochem. 109: 1134–1141, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Theabrownin triggers DNA damage to suppress human osteosarcoma U2OS cells by activating p53 signalling pathway

Osteosarcoma becomes the second leading cause of cancer death in the younger population. Current outcomes of chemotherapy on osteosarcoma were unsatisfactory to date, demanding development of effective therapies. Tea is a commonly used beverage beneficial to human health. As a major component of tea, theabrownin has been reported to possess anti‐cancer activity. To evaluate its anti‐osteosarcoma effect, we established a xenograft model of zebrafish and employed U2OS cells for in vivo and in vitro assays. The animal data showed that TB significantly inhibited the tumour growth with stronger effect than that of chemotherapy. The cellular data confirmed that TB‐triggered DNA damage and induced apoptosis of U2OS cells by regulation of Mki67, PARP, caspase 3 and H2AX, and Western blot assay showed an activation of p53 signalling pathway. When P53 was knocked down by siRNA, the subsequent downstream signalling was blocked, indicating a p53‐dependent mechanism of TB on U2OS cells (p53 wt). Using osteosarcoma cell lines with p53 mutations (HOS, SAOS‐2 and MG63), we found that TB exerted stronger inhibitory effect on U2OS cells than that on p53‐mut cell lines, but it also exerted obvious effect on SAOS‐2 cells (p53 null), suggesting an activation of p53‐independent pathway in the p53‐null cells. Interestingly, theabrownin was found to have no toxicity on normal tissue in vivo and could even increase the viability of p53‐wt normal cells. In sum, theabrownin could trigger DNA damage and induce apoptosis on U2OS cells via a p53‐dependent mechanism, being a promising candidate for osteosarcoma therapy.