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.

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.

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.

In vitro cytotoxicity of cyclodextrin-bonded birch bark extract.

Triterpenoids from birch bark, like betulin, seem to have an anticancer potential which needs to be further investigated. Aim of this study was first to explore whether a cyclodextrin-solubilised triterpenoid extract (STE) from birch bark induces selective cytotoxic effects in primary liver cancer cells compared to healthy human hepatocytes. Second, selective cytotoxicity against several tumour cell lines should be analysed. For this purpose, human liver cancer cells derived from mouse xenografts (LIXF 575), healthy human hepatocytes, and 42 different human tumour cell lines were incubated with different concentrations of STE corresponding to 4.3 µM - 137.5 µM betulin (BE). Cytotoxicity was tested with the WST-1 cell proliferation assay, apoptosis with caspase 3/7-activity, and necrosis was determined by the propidiumiodid uptake assay. The pathway of cytotoxic effects was further investigated by immunoblotting of apoptosis inducing factor (AIF) and p53. The monolayer assay was used to analyse selectivity of STE towards different tumour cell lines. STE significantly (p < 0.001) reduced viability and induced apoptosis of LIXF cells in low concentrations corresponding to 8.6 µM BE, while human hepatocytes were affected only in concentrations ≥ 68.8 µM. Cell death occurred in a p53 independent manner, and AIF was not involved. The mean IC50 in the 42 tumour cell lines corresponded to 4.3 µM BE and ranged from 2.05 µM to 8.95 µM BE content. Selectivity was, therefore, rather low. In conclusion, STE exhibits in low concentrations cytotoxicity in a broad spectrum of primary cancer cells and cancer cell lines, which is, at least in LIXF cells, induced by caspase 3/7 mediated apoptosis. STE is far less toxic in hepatocytes. The anticancer potential of STE should be further characterised and also investigated in animal models.

Potential role of P-gp for flavone-induced diminished apoptosis and increased adenoma size in the small intestine of APC(min/+) mice.

APC(min/+) mice, carrying a nonsense mutation in the adenomatous polyposis coli (APC) gene, appear as a perfect model to study development or therapy of intestinal neoplasia. We tested whether the flavonoid flavone is able to affect adenoma development in APC(min/+) mice. Tumor sizes were significantly increased by flavone selectively in small intestine. This was associated with reduced cell numbers displaying cleaved caspase-3 and enhanced expression of phosphoglycoprotein (P-gp). However, according to great variability in P-gp expression in all parts of mice intestines, an association between expression of P-gp and inhibition of apoptosis was demonstrated in human Caco-2 colorectal cancer cells.

An aqueous birch leaf extract of Betula pendula inhibits the growth and cell division of inflammatory lymphocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Leaf extracts of Betula pendula have been traditionally used for the treatment of patients with rheumatoid arthritis (RA) or osteoarthritis. AIM OF THE STUDY: We investigated the anti-proliferative capacity of an aqueous leaf extract of Betula pendula (BPE) on human primary lymphocytes in vitro, because activated lymphocytes play a major role in the initiation and maintenance of RA. MATERIALS AND METHODS: Lymphocyte proliferation and cell division was measured by the activity of mitochondrial dehydrogenases and by using the membrane-permeable dye carboxyfluorescein diacetate succinimidyl ester (CFSE), respectively. Apoptosis was analyzed by surface staining of phosphatidylserine and intracellular activation of effector caspases 3 and 7 in comparison to the drug methotrexate using flow cytometric and photometrical analysis. In addition, the impact of the extract on cell cycle distribution was investigated by propidium iodide staining of DNA. For the bioassays BPE concentrations of 10-160 µg/mL were investigated. A phytochemical analysis, using LC-MS and HPLC, was conducted to identify the polyphenolic constituents of the birch leaf extract. RESULTS: Leaf extracts of Betula pendula inhibited the growth and cell division (CD8(+): 40 µg/mL: 45%; 80 µg/mL: 60%; 160 µg/mL: 87%) (CD4(+): 40 µg/mL: 33%; 80 µg/mL: 54%; 160 µg/mL: 79%) of activated, but not of resting T lymphocytes in a significant dose-dependent manner. The inhibition of lymphocyte proliferation due to apoptosis induction (compared to untreated control: 40 µg/mL: 163%; 80 µg/mL: 240%; 160 µg/mL: 348%) and cell cycle arrest was comparable to that of methotrexate. LC-MS analyses showed that the extract contains different quercetin-glycosides. CONCLUSION: Our results give a rational basis for the use of Betula pendula leaf extract for the treatment of immune disorders, like rheumatoid arthritis, by diminishing proliferating inflammatory lymphocytes.

Chrysin blocks topotecan-induced apoptosis in Caco-2 cells in spite of inhibition of ABC-transporters.

ATP-driven efflux pumps such as phosphoglycoprotein-170 (P-gp), multidrug-resistance-associated protein-2 (MRP-2), or breast cancer resistance protein (BCRP) play a crucial role in limiting the efficacy of tumor pharmacotherapy. Selected flavonoids have been suggested to inhibit individual efflux-transporters and to act therefore as multidrug-resistance reversing agents. In the present study it is shown that the flavonoid chrysin acts as a potent inhibitor of P-gp, MRP-2, and BCRP in Caco-2 colon carcinoma cells. As a consequence, cells accumulated higher rates of the apoptosis-inducing chemotherapeutic topotecan in the presence of chrysin, even though under these conditions the expression of the transporters was markedly increased. Interestingly, in spite of the enhanced cellular drug accumulation the topotecan-induced apoptosis, assessed according to DNA-fragmentation, chromatin condensation, and by determination of sub-G1 peaks using fluorescence-assisted-cell sorting (FACS), was potently inhibited by chrysin. Suggested transport-independent apoptosis inhibiting activities of ATP-binding cassette (ABC)-transporters, such as the inhibition of caspases, were shown to be necessary for the inhibition of topotecan-induced apoptosis and were found to be associated with stabilization of beta-catenin especially in the cytosol. Inhibition of topotecan-induced intracellular acidification, however, was proven not to prevent caspase-activation and apoptosis. In conclusion, our studies show that chrysin in spite of raising the cellular concentrations of topotecan potently inhibits the apoptosis-inducing activities of the anti-tumor drug. Inhibition of caspase-activation was identified as the underlying mechanism and is suggested to be caused by transport-independent functions of ABC-transporters.