Phytochemicals resveratrol and sulforaphane as potential agents for enhancing the anti-tumor activities of conventional cancer therapies.

Even though conventional cancer therapies, comprising surgery and chemo- and radiotherapy, play an important role in the treatment of most solid tumours, successful therapeutic outcome is often limited due to high toxicity and related side-effects, as well as the development of multi-drug resistances. Therefore, there is need for new therapeutic strategies not only to obtain higher treatment efficacy, but also for the reduction of toxicity and adverse effects. Emerging evidence suggests that natural compounds with distinct anticarcinogenic activity may be considered as potential agents for enhancing the therapeutic effects of common cancer treatments. By using the examples of resveratrol and sulforaphane this review will summarize the findings of recent investigations focusing this topic so far and the current knowledge of the molecular mechanisms by which these selected phytochemicals may potentiate the anti-tumor effects of different cancer therapies.

Comparison of an established simple office-based immunological FOBT with fecal tumor pyruvate kinase type M2 (M2-PK) for colorectal cancer screening: prospective multicenter study.

OBJECTIVES: The immunological fecal occult blood test (IFOBT) has established itself as a more precise marker for colorectal cancer (CRC) screening than traditional guaiac-based FOBT. The simpler, cheaper, and more convenient newer office-based IFOBTs have been validated for diagnosing CRC. Dimeric isoenzyme of pyruvate kinase, M2-PK, expressed by tumor cells, has as well been proposed as a screening tool for CRC. This is the first study comparing fecal M2-PK as a screening biomarker for CRC against previously evaluated office-based IFOBT and colonoscopy. METHODS: Six hundred forty consecutive subjects (symptomatic, as well as for CRC screening) referred for colonoscopy for various indications across five centers in Germany provided the stool samples for performing M2-PK and an immunochemical FOB strip test. The IFOBT used was a rapid immunochromatographic assay for detection of fecal hemoglobin. For M2-PK, a commercially available sandwich enzyme-linked immunosorbent assay (ELISA) was used. The M2-PK test needs 6 h, while the office-based test can be read in just 10 min and is five times cheaper. RESULTS: Office-based IFOBT had sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and positive and negative likelihood ratios (LR) of 64.5, 96.3, 72.0, 94.9, 17.5, and 0.4 for diagnosing colorectal neoplasia (CRN), while the above performance characteristics for M2-PK at a cutoff value of 4 U/mL were 72.4, 73.8, 29.0, 94.8, 2.8, and 0.8 respectively. CONCLUSIONS: This office-based IFOBT was found to have significantly higher specificity, PPV, and positive LR as compared with M2-PK. IFOBT proved to be a convenient, noncumbersome, quick, and cheap tool in patients with above-average risk for detection of CRN.

Immune modulatory treatment of trinitrobenzene sulfonic acid colitis with calcitriol is associated with a change of a T helper (Th) 1/Th17 to a Th2 and regulatory T cell profile.

A number of recent studies testify that calcitriol alone or in combination with corticosteroids exerts strong immune modulatory activity. As a new approach, we evaluated the protolerogenic potential of calcitriol and dexamethasone in acute T helper (Th)1-mediated colitis in mice. A rectal enema of trinitrobenzene sulfonic acid (TNBS) (100 mg/kg) was applied to BALB/c mice. Calcitriol and/or dexamethasone were administered i.p. from days 0 to 3 or 3 to 5 following the instillation of the haptenating agent. Assessment of colitis severity was performed daily. Colon tissue was analyzed macroscopically and microscopically, and myeloperoxidase activity, as well as cytokine levels [tumor necrosis factor-alpha, interferon-gamma, interleukin (IL)-12p70, IL-1beta, IL-10, IL-4] were determined by enzyme-linked immunosorbent assay, T-bet, GATA family of transcription factors 3, a Th2 master regulator (GATA3), Foxp3, cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), IL-23p19 and IL-17 expression by immunoblot analysis. The combination of the steroids most effectively reduced the clinical and histopathologic severity of TNBS colitis. Th1-related parameters were down-regulated, whereas Th2 markers like IL-4 and GATA3 were up-regulated. Apart from known steroid effects, calcitriol in particular promoted regulatory T cell profiles as indicated by a marked increase of IL-10, TGFbeta, FoxP3, and CTLA4. Furthermore, analysis of dendritic cell mediators responsible for a proinflammatory differentiation of T cells revealed a significant reduction of IL-12p70 and IL23p19 as well as IL-6 and IL-17. Thus, our data support a rationale for a steroid-sparing, clinical application of calcitriol derivatives in inflammatory bowel disease. Furthermore they suggest that early markers of inflammatory dendritic cell and Th17 differentiation qualify as new target molecules for both calcitriol and highly selective immune-modulating vitamin D analogs.

Dietary omega-3-polyunsaturated fatty acids prevent the development of metastases of colon carcinoma in rat liver.

BACKGROUND: Fish oil consisting of omega-3 polyunsaturated fatty acids (PUFA) seems to reduce the incidence of colon cancer. The effect of PUFAs on metastasis of colon carcinoma is still unclear. AIM: The study was designed to examine the effects of a diet rich in omega-3-PUFAs on a model of colorectal metastasis. METHODS: Thirty animals (WAG/Rij) were randomly assigned to receive an omega-3 diet or a control diet to evaluate their effect on tumor growth. The target male rats (WAG/Rij) were fed a diet containing 15% omega-3-fatty acids three days before and 28 days after intervention and the control rats received 15% coconut oil at the same time points. CC 531 cells, a moderately differentiated colon adenocarcinoma, were injected into the spleen of each rat. After 28 days of diet, animals were sacrificed. The tumor growth was evaluated macroscopically and microscopically in liver tissue. The tissue was examined after immunostaining and the use of monoclonal antibodies. RESULTS: PUFAs decreased the index of tumor load from 1.54 in the controls to 0.79 in the treatment group (P = 0.036). While 69.2% of the control animals were tumor positive, only 21.4% of the target animals showed tumor after omega-3-fatty acid (P < 0.05). CONCLUSION: We could show that omega-3-fatty acids may decrease malignant metastatic tumor growth in the liver.

De novo ceramide biosynthesis is associated with resveratrol-induced inhibition of ornithine decarboxylase activity.

Previous studies could demonstrate, that the naturally occuring polyphenol resveratrol inhibits cell growth of colon carcinoma cells at least in part by inhibition of protooncogene ornithine decarboxylase (ODC). The objective of this study was to provide several lines of evidence suggesting that the induction of ceramide synthesis is involved in this regulatory mechanisms. Cell growth was determined by BrdU incorporation and crystal violet staining. Ceramide concentrations were detected by HPLC-coupled mass-spectrometry. Protein levels were examined by Western blot analysis. ODC activity was assayed radiometrically measuring [(14)CO(2)]-liberation. A dominant-negative PPARgamma mutant was transfected in Caco-2 cells to suppress PPARgamma-mediated functions. Antiproliferative effects of resveratrol closely correlate with a dose-dependent increase of endogenous ceramides (p<0.001). Compared to controls the cell-permeable ceramide analogues C2- and C6-ceramide significantly inhibit ODC-activity (p<0.001) in colorectal cancer cells. C6-ceramide further diminished protein levels of protooncogenes c-myc (p<0.05) and ODC (p<0.01), which is strictly related to the ability of ceramides to inhibit cell growth in a time- and dose-dependent manner. These results were further confirmed using inhibitors of sphingolipid metabolism, where only co-incubation with a serine palmitoyltransferase (SPT) inhibitor could significantly counteract resveratrol-mediated actions. These data suggest that the induction of ceramide de novo biosynthesis but not hydrolysis of sphingomyelin is involved in resveratrol-mediated inhibition of ODC. In contrast to the regulation of catabolic spermidine/spermine acetyltransferase by resveratrol, inhibitory effects on ODC occur PPARgamma-independently, indicating independent pathways of resveratrol-action. Due to our findings resveratrol could show great chemopreventive and therapeutic potential in the treatment of colorectal cancers.

The TGFbeta/Smad 3-signaling pathway is involved in butyrate-mediated vitamin D receptor (VDR)-expression.

Previously, we demonstrated the pivotal role of the vitamin D receptor (VDR) in mediating the butyrate-induced differentiation in colon cancer cells. Smad 3, a downstream component of transforming growth factor-beta (TGFbeta) signaling, has been shown to act as a coactivator of VDR and to possibly regulate the vitamin D signaling pathway. In this study, we demonstrate a distinct impact of the TGFbeta/Smad 3-signaling pathway in the butyrate-mediated VDR expression and induction of differentiation. Butyrate treatment resulted in a significant induction of the phosphorylation level of Smad 3, while the combination of butyrate and a specific TGFbeta1-antibody or a TGFbeta-receptor inhibitor considerably diminished the butyrate-induced upregulation of VDR expression. Using a specific inhibitor, we were also able to demonstrate an involvement of the p38 MAPK in the increase of Smad 3 phosphorylation following butyrate treatment, thus opening the view to further elucidate possible mechanisms mediating the upregulation of VDR expression following butyrate treatment in colon cancer cells.

FTY720 ameliorates oxazolone colitis in mice by directly affecting T helper type 2 functions.

The sphingosine-1-phosphate analogue FTY720 is known to alter migration and homing of lymphocytes via sphingosine-1-phosphate receptors. However, several studies indicate that its mode of action is more complex and that FTY720 may also directly influence cytokine effector functions. Therefore, we studied the effect of FTY720 in T helper type (Th2)-mediated oxazolone-induced colitis in mice. Following rectal oxazolone instillation, Th2 cells producing IL-13 induce a progressive colitis resembling human ulcerative colitis. A rectal enema of oxazolone [90 mg/kg body weight] was applied to BALB/c mice. FTY720 was administered i.p. from day 0 to 3 or from day 3 to 5 following the instillation of the haptenating agent. Assessment of severity of colitis was performed daily. FTY720 plasma levels were detected using LC-MS/MS-analysis. Colon tissue was analyzed macroscopically and microscopically, myeloperoxidase activity as well as cytokine levels of lamina propria CD4(+) T-cells and T1/ST2 expression were determined. Treatment with FTY720 prominently reduced the clinical and histopathologic severity of oxazolone-induced colitis, abrogating body weight loss, diarrhea, and macroscopic and microscopic intestinal inflammation. The therapeutic effects of FTY720 were associated with a prominent reduction of the key effector Th2 cytokines IL-13, IL-4 and IL-5. Strikingly, FTY720 inhibited GATA3 and T1/ST2 expression which represent highly relevant markers for Th2 differentiation and Th2 effector function, respectively. Our data provide the first evidence that FTY720 exhibits beneficial prophylactic as well as therapeutic effects in Th2-mediated experimental colitis by directly affecting Th2 cytokine profiles probably by reducing T1/ST2, thus offering a new auspicious therapeutic instrument for the treatment of human ulcerative colitis.

Prospective multicenter evaluation of fecal tumor pyruvate kinase type M2 (M2-PK) as a screening biomarker for colorectal neoplasia.

Proliferating cells, particularly the tumor cells, express a dimeric isoenzyme of pyruvate kinase, termed M2-PK. It's a direct target of several oncoproteins; the determination of fecal tumor pyruvate kinase type M2 (M2-PK) might be another promising tool for colorectal cancer (CRC) screening. In this study, we have evaluated fecal M2-PK as a screening biomarker for colorectal neoplasia. It was compared against fecal occult blood (FOB) and colonoscopy. Three hundred and seventeen consecutive subjects from 4 different centers were included. Stool specimens were collected before purgation, processed appropriately and were tested for FOB and quantitatively analyzed for M2-PK. Colonoscopies were performed by experienced endoscopists who were unaware of fecal assay results. At cutoff value of 4 U/ml, fecal M2-PK assay had a sensitivity, specificity, PPV and NPV of 81.1, 86.7, 71.1 and 61.9% respectively for diagnosing CRC whereas FOBT showed a sensitivity of 36.5%, specificity of 92.2%, PPV of 72.9% and NPV of 71.5% for CRC. Such low specificity of fecal M2-PK will lead to unacceptably high number of false positives if it is used for mass CRC screening, leading to unindicated colonoscopies with its associated inconveniences, risks and costs. CRC screening test must have high specificity; a high sensitivity is not as vital. To conclude, M2-PK was found to be a poor screening biomarker for CR neoplasia in a subject population at above average risk based on its prospective comparison with colonoscopy. These marginal performance characteristics do not permit its use as a screening tool for CR neoplasia in present clinical settings.

Peroxisome proliferator-activated receptor gamma as a molecular target of resveratrol-induced modulation of polyamine metabolism.

Previous results indicate that the polyphenol resveratrol inhibits cell growth of colon carcinoma cells via modulation of polyamine metabolic key enzymes. The aim of this work was to specify the underlying molecular mechanisms and to identify a possible role of transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma). Cell growth was determined by bromodeoxyuridine incorporation and crystal violet staining. Protein levels were examined by Western blot analysis. Spermine/spermidine acetyltransferase (SSAT) activity was determined by a radiochemical assay. PPARgamma ligand-dependent transcriptional activity was measured by a luciferase assay. A dominant-negative PPARgamma mutant was transfected in Caco-2 cells to suppress PPARgamma-mediated functions. Resveratrol inhibits cell growth of both Caco-2 and HCT-116 cells in a dose- and time-dependent manner (P < 0.001). In contrast to Caco-2-wild type cells (P < 0.05), resveratrol failed to increase SSAT activity in dominant-negative PPARgamma cells. PPARgamma involvement was further confirmed via ligand-dependent activation (P < 0.01) as well as by induction of cytokeratin 20 (P < 0.001) after resveratrol treatment. Coincubation with SB203580 abolished SSAT activation significantly in Caco-2 (P < 0.05) and HCT-116 (P < 0.01) cells. The involvement of p38 mitogen-activated protein kinase (MAPK) was further confirmed by a resveratrol-mediated phosphorylation of p38 protein in both cell lines. Resveratrol further increased the expression of PPARgamma coactivator PGC-1alpha (P < 0.05) as well as SIRT1 (P < 0.01) in a dose-dependent manner after 24 hours of incubation. Based on our findings, p38 MAPK and transcription factor PPARgamma can be considered as molecular targets of resveratrol in the regulation of cell proliferation and SSAT activity, respectively, in a cell culture model of colon cancer.

Molecular mechanisms of the chemopreventive effects of resveratrol and its analogs in carcinogenesis.

Resveratrol (3,4',5-trihydroxy-trans-stilbene), a phytoalexin found in grape skins, peanuts, and red wine, has been reported to exhibit a wide range of biological and pharmacological properties. It has been speculated that dietary resveratrol could be an explanation for the so-called 'French paradox' as it may act as an antioxidant, promote nitric oxide production, inhibit platelet aggregation, and increase high-density lipoprotein cholesterol, and thereby serve as a cardioprotective agent. Recently, it has been demonstrated that resveratrol can function as a cancer chemopreventive agent, and there has been a great deal of experimental effort directed toward defining this effect. It has been shown that resveratrol and some of its analogs interfere with signal transduction pathways, modulate cell cycle-regulating proteins, and is a potent inducer of apoptosis in multiple carcinoma cell lines. This review summarizes the recent advances that have provided new insights into the molecular mechanisms underlying the promising properties of resveratrol.