Matthias Ocker

Reactive oxygen species mediate thymoquinone-induced apoptosis and activate ERK and JNK signaling.

Thymoquinone (TQ), a component of black seed essential oil, is known to induce apoptotic cell death and oxidative stress, however, the direct involvement of oxidants in TQ-induced cell death has not been established yet. Here, we show that TQ inhibited the proliferation of a panel of human colon cancer cells (Caco-2, HCT-116, LoVo, DLD-1 and HT-29), without exhibiting cytotoxicity to normal human intestinal FHs74Int cells. Further investigation in DLD-1 revealed that apoptotic cell death is the mechanism for TQ-induced growth inhibition as confirmed by flow cytometry, M30 cytodeath and caspase-3/7 activation. Apoptosis was induced via the generation of reactive oxygen species (ROS) as evidenced by the abrogation of TQ apoptotic effect in cells preincubated with the strong antioxidant N-acetyl cysteine (NAC). TQ increased the phosphorylation states of the mitogen-activated protein kinases (MAPK) JNK and ERK, but not of p38. Their activation was completely abolished in the presence of NAC. Using PD98059 and SP600125, specific ERK and JNK inhibitors, the two kinases were found to possess pro-survival activities in TQ-induced cell death. These data present evidence linking the pro-oxidant effects of TQ with its apoptotic effects in colon cancer and prove a protective role of MAPK.

The histone-deacetylase inhibitor Trichostatin A blocks proliferation and triggers apoptotic programs in hepatoma cells

BACKGROUND/AIMS Effective treatment for hepatocellular carcinoma is urgently needed. The histone-deacetylase inhibitor Trichostatin A (TSA) was shown to induce apoptosis in non-hepatic cells at submicromolar concentrations. However, the effect of TSA on hepatoma cells is unknown. METHODS The hepatoma cells HepG2, MH1C1, Hepa1-6 and Hep1B as well as human fibroblasts (control cells) were exposed to TSA (10(-6) to 10(-9)M). Cell proliferation was assessed by measuring DNA-synthesis and cell numbers. Apoptosis was quantified by flow cytometry and by the TdT-mediated dUTP nick-end labeling method. Expression patterns of cell cycle- and/or apoptosis-associated p27, p21(cip/waf), bax, bcl-2, cyclin A and (pro)-caspase 3 were studied using quantitative Western blotting. Activation of caspase 3 was analyzed via a colorimetric assay. RESULTS 10(-6)M TSA inhibited DNA-synthesis by 46% (HepG2) to 64% (MH1C1) after 24h, inducing a G(2)/M-phase arrest and apoptosis. TSA increased activation of caspase 3 and expression of cyclin A, p2l(cip/waf), bax and (pro)-caspase 3, while bcl-2 was downregulated. Human fibroblasts remained unaffected. CONCLUSIONS TSA inhibits hepatoma cell growth in vitro, which are otherwise particularly resistant to chemotherapy. Its anti-proliferative activity is paralleled by a comparable rate of apoptosis. TSA may be a promising agent for treatment of hepatocellular carcinoma in vivo.

Thymoquinone Triggers Inactivation of the Stress Response Pathway Sensor CHEK1 and Contributes to Apoptosis in Colorectal Cancer Cells

There are few reports describing the role of p53-dependent gene repression in apoptotic cell death. To identify such apoptosis-associated p53 target genes, we used the pro-oxidant plant-derived drug thymoquinone and compared p53+/+ and p53-/- colon cancer cells HCT116. The p53 wild-type (wt) status correlated with more pronounced DNA damage and higher apoptosis after thymoquinone treatment. A significant up-regulation of the survival gene CHEK1 was observed in p53-/- cells in response to thymoquinone due to the lack of transcriptional repression of p53. In p53-/- cells, transfection with p53-wt vector and CHEK1 small interfering RNA treatment decreased CHEK1 mRNA and protein levels and restored apoptosis to the levels of the p53+/+ cells. p53-/- cells transplanted to nude mice treated with thymoquinone up-regulated CHEK1 expression and did not undergo apoptosis unlike p53+/+ cells. Immunofluorescence analysis revealed that the apoptosis resistance in p53-/- cells after thymoquinone treatment might be conveyed by shuttling of CHEK1 into the nucleus. We confirmed the in vivo existence of this CHEK1/p53 link in human colorectal cancer, showing that tumors lacking p53 had higher levels of CHEK1, which was accompanied by poorer apoptosis. CHEK1 overexpression was correlated with advanced tumor stages (P = 0.03), proximal tumor localization (P = 0.02), and worse prognosis (1.9-fold risk, univariate Cox regression; Kaplan-Meier, P = 0.04). We suggest that the inhibition of the stress response sensor CHEK1 might contribute to the antineoplastic activity of specific DNA-damaging drugs.

Thymoquinone reduces mouse colon tumor cell invasion and inhibits tumor growth in murine colon cancer models

We have shown that thymoquinone (TQ) is a potent antitumor agent in human colorectal cancer cells. In this study, we evaluated TQs therapeutic potential in two different mice colon cancer models [ 1,2 ‐dimethyl hydrazine (DMH) and xenografts]. We also examined TQ effects on the growth of C26 mouse colorectal carcinoma spheroids and assessed tumor invasion in vitro. Mice were treated with saline, TQ, DMH, or combinations once per week for 30 weeks and the multiplicity, size and distribution of aberrant crypt foci (ACF) and tumors were determined at weeks 10, 20 and 30. TQ injected intraperitoneally (i.p.) significantly reduced the numbers and sizes of ACF at week 10; ACF numbers were reduced by 86%. Tumor multiplicity was reduced at week 20 from 17.8 in the DMH group to 4.2 in mice injected with TQ. This suppression was observed at week 30 and was long‐term; tumors did not re‐grow even when TQ injection was discontinued for 10 weeks. In a xenograft model of HCT116 colon cancer cells, TQ significantly (P < 0.05) delayed the growth of the tumor cells. Using a matrigel artificial basement membrane invasion assay, we demonstrated that sub‐cyto‐toxic doses of TQ (40μM) decreased C26 cell invasion by 50% and suppressed growth in three‐dimensional spheroids. Apoptotic signs seen morphologically were increased significantly in TQ‐treated spheroids. TUNEL staining of xenografts and immunostaining for caspase 3 cleavage in DMH tumors confirmed increased apoptosis in mouse tumors in response to TQ. These data should encourage further in vivo testing and support the potential use of TQ as a therapeutic agent in human colorectal cancer.

The heme oxygenase 1 product biliverdin interferes with hepatitis C virus replication by increasing antiviral interferon response.

The anti‐inflammatory and antiapoptotic heme degrading enzyme heme oxygenase‐1 (HO‐1) has been shown recently to interfere with replication of hepatitis C virus (HCV). We investigated the effect of HO‐1 products carbon monoxide (CO), iron and biliverdin on HCV replication using the replicon cell lines Huh‐5‐15 and LucUbiNeo‐ET, stably expressing HCV proteins NS3 through NS5B. Incubation of these cell lines in the presence of the CO donor methylene chloride transiently reduced HCV replication, whereas an increase of iron in cell culture by administration of FeCl3 or iron‐saturated lactoferrin did not interfere with HCV replication. Likewise, depletion of iron by deferoxamine during induction of HO‐1 by cobalt‐protoporphyrin IX did not restore HCV replication. The most prominent effect was observed after incubation of replicon cell lines in the presence of biliverdin. Biliverdin seems to interfere with HCV replication–mediated oxidative stress by inducing expression of antiviral interferons, such as interferon alpha2 and alpha17. Conclusion: The antioxidant biliverdin reduces HCV replication in vitro by triggering the antiviral interferon response and might improve HCV therapy in the future. (HEPATOLOGY 2009.)

PIM1 kinase as a target for cancer therapy

Introduction : Inhibition of protein kinases has become a standard of modern clinical oncology. PIM1 belongs to a novel class of serine/threonine kinases with distinct molecular and biochemical features regulating various oncogenic pathways, for example hypoxia response, cell cycle progression and apoptosis resistance. PIM1 is overexpressed in human cancer diseases and has been associated with metastasis and overall treatment response; in experimental models, inhibition of PIM1 suppressed cell proliferation and migration, induced apoptotic cell death and synergized with other chemotherapeutic agents. Areas covered : A PubMed literature search was performed to review the currently available data on PIM1 expression, regulation and targets; its implication in different types of cancer and its impact on prognosis are described. We present ATP-competitive PIM1 inhibitors and the state of the art of PIM1 inhibitor design. Finally, we highlight the development of the unusual class of highly selective and potent organometallic PIM1 inhibitors. Expert opinion : As PIM1 possesses oncogenic functions and is overexpressed in various kinds of cancer diseases, its inhibition provides a new option in cancer therapy. Based on the ability of highly selective organometallic PIM1 inhibitors, promising in vivo applicability is expected.

Apoptosis, proliferation and differentiation patterns are influenced by Zebularine and SAHA in pancreatic cancer models

Objective. Pancreatic cancer continues to be an urgent clinical problem. We used the novel DNA methyltransferase inhibitor Zebularine and the histone deacetylase inhibitor SAHA to investigate the epigenetic influence on viability and differentiation of the pancreatic cancer cell lines YAP C, DAN G and Panc-89 in vitro and in vivo. Material and methods. Cell vitality, proliferation and expression of PDX-1, cytokeratin 7 and 20, chromogranin A, vimentin, bax and bcl-2 were determined on the protein and mRNA level in vitro and in a subcutaneous xenograft model. Results. A time- and dose-dependent increase of apoptosis, paralleled by decreased proliferation, was observed after incubation with single agents or a combination therapy with lower concentrations. This was associated with up-regulation of pro-apoptotic bax and a phenotypic stabilization by the enhanced expression of cytokeratin 7. In vivo, growth of xenografts was delayed with the most pronounced effect in Panc-89 after 1 week of daily intraperitoneal injections of Zebularine paralleled with CK7 up-regulation and down-regulation of dedifferentiation markers. Conclusions. Epigenetic modulation via inhibition of DNA methyltransferase and histone deacetylase induces apoptosis in human pancreatic cancer cells in vitro and delays xenograft growth in vivo, which is associated with a morphological/molecular phenotypic stabilization. These compounds may therefore be suitable as adjunctive therapeutic agents in the treatment of pancreatic cancer.

Inhibition of heme oxygenase 1 expression by small interfering RNA decreases orthotopic tumor growth in livers of mice

Endogenous overexpression of the antiapoptotic protein heme oxygenase 1 (HO‐1) has been shown to occur in various cancer diseases and might contribute to cancer progression. We compared the expression levels of HO‐1 in human liver to expression levels in hepatocellular carcinoma (HCC), as well as the effect of HO‐1 inhibition by small interfering RNA (siRNA) on cellular survival and apoptosis in the mouse hepatoma cell lines Hepa129 and Hepa1‐6 and on orthotopic tumor growth in immune‐competent C3H/HeN mice. Our results show that HO‐1 is frequently overexpressed in human HCC. Downmodulation of HO‐1 by siRNA resulted in increased cellular damage and apoptosis, reduced proliferation, reduced growth of orthotopic HCC and reduced angiogenesis. Livers and kidneys of treated animals did not reveal signs of damage by this treatment. In conclusion, a specific knockdown of HO‐1 might represent a novel therapeutic approach in HCC therapy.

Clinical significance of histone deacetylases 1, 2, 3, and 7: HDAC2 is an independent predictor of survival in HCC

Histone deacetylases (HDAC) are responsible for the transcriptional control of genes through chromatin remodeling and control tumor suppressor genes. In several tumors, their expression has been linked to clinicopathological factors and patient survival. This study investigates HDACs 1, 2, 3, and 7 expressions in hepatocellular carcinoma (HCC) and their correlation with clinical data and patient survival. Tissue microarrays of 170 surgically resected primary HCCs and adjacent uninvolved tissue were evaluated immunohistochemically for the expression of HDACs 1, 2, 3, 7, and Ki-67 and were analyzed with respect to clinicopathological data and patient survival. HDACs 1, 2, 3, and Ki-67 were expressed significantly higher in cancer cells compared to normal tissue (HDAC1: p = 0.034, HDACs 2 and 3 and Ki-67: p < 0.001), while HDAC7 expression did not differ between HCC and non-cancerous liver tissue. In tumor tissue HDACs 1–3 expression levels showed high concordance with each other, Ki-67 and tumor grade (p < 0.001). High HDAC2 expression was associated with poor survival in low-grade and early-stage tumors (p < 0.05). The expression of the HDACs 1, 2, and 3 (but not HDAC7) isoenzymes correlates with clinicopathological factors, and HDAC2 expression has an impact on patient survival.

Deacetylase inhibitors - focus on non-histone targets and effects.

Inhibitors of protein deacetylases have recently been established as a novel therapeutic principle for several human diseases, including cancer. The original notion of the mechanism of action of these compounds focused on the epigenetic control of transcriptional processes, especially of tumor suppressor genes, by interfering with the acetylation status of nuclear histone proteins, hence the name histone deacetylase inhibitors was coined. Yet, this view could not explain the high specificity for tumor cells and recent evidence now suggests that non-histone proteins represent major targets for protein deacetylase inhibitors and that the post-translational modification of the acetylome is involved in various cellular processes of differentiation, survival and cell death induction.