In Vitro Inhibitory Potential of Different Anthocyanin-Rich Berry Extracts in Murine CT26 Colon Cancer Cells.

Anti-oxidant, -inflammatory, and -carcinogenic activities of bioactive plant constituents, such as anthocyanins, have been widely discussed in literature. However, the potential interaction of anthocyanin-rich extracts with routinely used chemotherapeutics is still not fully elucidated. In the present study, anthocyanin-rich polyphenol extracts of blackberry (BB), bilberry (Bil), black currant (BC), elderberry (EB), and their respective main anthocyanins (cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, cyanidin-3-O-rutinoside, and cyanidin-3-O-sambubioside) were investigated concerning their cytotoxic and DNA-damaging properties in murine CT26 cells either alone or in combination with the chemotherapeutic agent SN-38. BB exerted potent cytotoxic effects, while Bil, BC, and EB only had marginal effects on cell viability. Single anthocyanins comprised of the extracts could not induce comparable effects. Even though the BB extract further pronounced SN-38-induced cytotoxicity and inhibited cell adhesion at 100-200 µg/mL, no effect on DNA damage was observed. In conclusion, anti-carcinogenic properties of the extracts on CT26 cells could be ranked BB >> BC ≥ Bil ≈ EB. Mechanisms underlying the potent cytotoxic effects are still to be elucidated since the induction of DNA damage does not play a role.

Biological activity of PtIV prodrugs triggered by riboflavin-mediated bioorthogonal photocatalysis.

We have recently demonstrated that riboflavin (Rf) functions as unconventional bioorthogonal photocatalyst for the activation of PtIV prodrugs. In this study, we show how the combination of light and Rf with two PtIV prodrugs is a feasible strategy for light-mediated pancreatic cancer cell death induction. In Capan-1 cells, which have high tolerance against photodynamic therapy, Rf-mediated activation of the cisplatin and carboplatin prodrugs cis,cis,trans-[Pt(NH3)2(Cl)2(O2CCH2CH2CO2H)2] (1) and cis,cis,trans-[Pt(NH3)2(CBDCA)(O2CCH2CH2CO2H)2] (2, where CBDCA = cyclobutane dicarboxylate) resulted in pronounced reduction of the cell viability, including under hypoxia conditions. Such photoactivation mode occurs to a considerable extent intracellularly, as demonstrated for 1 by uptake and cell viability experiments. 195Pt NMR, DNA binding studies using circular dichroism, mass spectrometry and immunofluorescence microscopy were performed using the Rf-1 catalyst-substrate pair and indicated that cell death is associated with the efficient light-induced formation of cisplatin. Accordingly, Western blot analysis revealed signs of DNA damage and activation of cell death pathways through Rf-mediated photochemical activation. Phosphorylation of H2AX as indicator for DNA damage, was detected for Rf-1 in a strictly light-dependent fashion while in case of free cisplatin also in the dark. Photochemical induction of nuclear pH2AX foci by Rf-1 was confirmed in fluorescence microscopy again proving efficient light-induced cisplatin release from the prodrug system.

Distinct activity of the bone-targeted gallium compound KP46 against osteosarcoma cells - synergism with autophagy inhibition.

BACKGROUND: Osteosarcoma is the most frequent primary malignant bone tumor. Although survival has distinctly increased due to neoadjuvant chemotherapy in the past, patients with metastatic disease and poor response to chemotherapy still have an adverse prognosis. Hence, development of new therapeutic strategies is still of utmost importance. METHODS: Anticancer activity of KP46 against osteosarcoma cell models was evaluated as single agent and in combination approaches with chemotherapeutics and Bcl-2 inhibitors using MTT assay. Underlying mechanisms were tested by cell cycle, apoptosis and autophagy assays. RESULTS: KP46 exerted exceptional anticancer activity at the nanomolar to low micromolar range, depending on the assay format, against all osteosarcoma cell models with minor but significant differences in IC50 values. KP46 treatment of osteosarcoma cells caused rapid loss of cell adhesion, weak cell cycle accumulation in S-phase and later signs of apoptotic cell death. Furthermore, already at sub-cytotoxic concentrations KP46 reduced the migratory potential of osteosarcoma cells and exerted synergistic effects with cisplatin, a standard osteosarcoma chemotherapeutic. Moreover, the gallium compound induced signs of autophagy in osteosarcoma cells. Accordingly, blockade of autophagy by chloroquine but also by the Bcl-2 inhibitor obatoclax increased the cytotoxic activity of KP46 treatment significantly, suggesting autophagy induction as a protective mechanism against KP46. CONCLUSION: Together, our results identify KP46 as a new promising agent to supplement standard chemotherapy and possible future targeted therapy in osteosarcoma.

Active vitamin D potentiates the anti-neoplastic effects of calcium in the colon: A cross talk through the calcium-sensing receptor.

Epidemiological studies suggest an inverse correlation between dietary calcium (Ca(2+)) and vitamin D intake and the risk of colorectal cancer (CRC). It has been shown in vitro that the active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25-D3) can upregulate expression of the calcium-sensing receptor (CaSR). In the colon, CaSR has been suggested to regulate proliferation of colonocytes. However, during tumorigenesis colonic CaSR expression is downregulated and we hypothesized that the loss of CaSR could influence the anti-tumorigenic effects of Ca(2+) and vitamin D. Our aim was to assess the impact of CaSR expression and function on the anti-neoplastic effects of 1,25-D3 in colon cancer cell lines. We demonstrated that in the healthy colon of mice, high vitamin D diet (2500 IU/kg diet) increased expression of differentiation and apoptosis markers, decreased expression of proliferation markers and significantly upregulated CaSR mRNA expression, compared with low vitamin D diet (100 IU/kg diet). To determine the role of CaSR in this process, we transfected Caco2-15 and HT29 CRC cells with wild type CaSR (CaSR-WT) or a dominant negative CaSR mutant (CaSR-DN) and treated them with 1,25-D3 alone, or in combination with CaSR activators (Ca(2+) and NPS R-568). 1,25-D3 enhanced the anti-proliferative effects of Ca(2+) and induced differentiation and apoptosis only in cells with a functional CaSR, which were further enhanced in the presence of NPS R-568, a positive allosteric modulator of CaSR. The mutant CaSR inhibited the anti-tumorigenic effects of 1,25-D3 suggesting that the anti-neoplastic effects of 1,25-D3 are, at least in part, mediated by the CaSR. Taken together, our data provides molecular evidence to support the epidemiological observation that both, vitamin D and calcium are needed for protection against malignant transformation of the colon and that their effect is modulated by the presence of a functional CaSR. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

2-deprenyl-rheediaxanthone B isolated from Metaxya rostrata induces active cell death in colorectal tumor cells.

Metaxya rostrata C. Presl (Metaxyaceae) is a common tree fern in Central and South America that is used for the treatment of intestinal ulcers and tumours in ethnic medicine. Using a bioactivity-guided strategy 2-deprenyl-rheediaxanthone B (XB) has been isolated as one of the active principles in this plant. XB induced loss of cell viability in colorectal cancer cell lines at IC50 concentrations of 11-23 µM. This was caused by both accumulation of cells in the G2- and S-phase as well as by induction of active cell death in a time and concentration-dependent manner. Cells exposed to XB were incapable of undergoing regular mitosis due to down-regulation of FoxM1 and absence of chromosome condensation. The apoptosis-related proteins Bcl2 and Bclxl were up-regulated so that Caspase 3 was not activated and classical apoptosis was not observed. However, XB triggered damage pathways down-stream of ATR and activated Caspase 2 causing cell death by a mechanism similar to mitotic catastrophe. Our observations are the first to show the cytotoxic activity of 2-deprenyl-rheediaxanthone B and indicate that XB is an interesting new lead compound for cancer therapy that merits further development.

Synergistic anticancer activity of arsenic trioxide with erlotinib is based on inhibition of EGFR-mediated DNA double-strand break repair.

Arsenic trioxide (ATO), one of the oldest remedies used in traditional medicine, was recently rediscovered as an anticancer drug and approved for treatment of relapsed acute promyelocytic leukemia. However, its activity against nonhematologic cancers is rather limited so far. Here, we show that inhibition of ATO-mediated EGF receptor (EGFR) activation can be used to potently sensitize diverse solid cancer types against ATO. Thus, combination of ATO and the EGFR inhibitor erlotinib exerted synergistic activity against multiple cancer cell lines. Subsequent analyses revealed that this effect was based on the blockade of ATO-induced EGFR phosphorylation leading to more pronounced G2-M arrest as well as enhanced and more rapid induction of apoptosis. Comparable ATO-sensitizing effects were also found with PI3K/AKT and mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitors, suggesting an essential role of the EGFR-mediated downstream signaling pathway in cancer cell protection against ATO. H2AX staining and comet assay revealed that erlotinib significantly increases ATO-induced DNA double-strand breaks (DSB) well in accordance with a role of the EGFR signaling axis in DNA damage repair. Indeed, EGFR inhibition led to downregulation of several DNA DSB repair proteins such as Rad51 and Rad50 as well as reduced phosphorylation of BRCA1. Finally, the combination treatment of ATO and erlotinib was also distinctly superior to both monotreatments against the notoriously therapy-resistant human A549 lung cancer and the orthotopic p31 mesothelioma xenograft model in vivo. In conclusion, this study suggests that combination of ATO and EGFR inhibitors is a promising therapeutic strategy against various solid tumors harboring wild-type EGFR.

Trabectedin has promising antineoplastic activity in high-grade meningioma.

BACKGROUND: Meningiomas are common intracranial tumors arising from the meninges and usually are benign. However, a few meningiomas have aggressive behavior and, for such patients, effective treatment options are needed. Trabectedin is a novel, marine-derived, antineoplastic agent that has been approved and is used routinely as therapy for advanced soft tissue sarcoma and ovarian cancer. METHODS: The authors investigated the in vitro effects of trabectedin alone and in combination with hydroxyurea, cisplatin, and doxorubicin in primary cell cultures of benign (n = 9), atypical (n = 6), and anaplastic (n = 4) meningiomas using chemosensitivity assays (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide [MTT]), Western blot analysis, cell cycle analysis, and immunofluorescent staining. RESULTS: Strong antimeningioma activity of trabectedin was observed and was characterized by distinct cell cycle arrest, down-regulation of multiple cyclins, deregulated expression of cell death-regulatory genes, and massive apoptosis induction. Cytotoxic activity was especially intense in higher grade meningiomas with a half-maximal inhibitory concentration <10 nM. Combination with trabectedin synergistically enhanced the antimeningioma activity of hydroxyurea but also enhanced the activity of doxorubicin and cisplatin. On the basis of these findings, trabectedin was given to 1 patient who had heavily pretreated, anaplastic meningioma, and a favorable response was observed with radiologic disease stabilization, marked reductions in brain edema and requirement for corticosteroids, and improvement of clinical symptoms. However, treatment had to be discontinued after 5 cycles because of adverse drug effects. CONCLUSIONS: The current results indicated that trabectedin may represent a promising new therapeutic option for patients with aggressive meningioma and should be evaluated in prospective clinical studies.

Fibroblast growth factor receptors as therapeutic targets in human melanoma: synergism with BRAF inhibition.

Cutaneous melanoma is a tumor with rising incidence and a very poor prognosis at the disseminated stage. Melanomas are characterized by frequent mutations in BRAF and also by overexpression of fibroblast growth factor 2 (FGF2), offering opportunities for therapeutic intervention. We investigated inhibition of FGF signaling and its combination with dacarbazine or BRAF inhibitors as an antitumor strategy in melanoma. The majority of melanoma cell lines displayed overexpression of FGF2 but also FGF5 and FGF18 together with different isoforms of FGF receptors (FGFRs) 1-4. Blockade of FGF signals with dominant-negative receptor constructs (dnFGFR1, 3, or 4) or small-molecule inhibitors (SU5402 and PD166866) reduced melanoma cell proliferation, colony formation, as well as anchorage-independent growth, and increased apoptosis. DnFGFR constructs also significantly inhibited tumor growth in vivo. Combination of FGF inhibitors with dacarbazine showed additive or antagonistic effects, whereas synergistic drug interaction was observed when combining FGFR inhibition with the multikinase/BRAF inhibitor sorafenib or the V600E mutant-specific BRAF inhibitor RG7204. In conclusion, FGFR inhibition has antitumor effects against melanoma cells in vitro and in vivo. Combination with BRAF inhibition offers a potential for synergistic antimelanoma effects and represents a promising therapeutic strategy against advanced melanoma.

EGCG-meditated cyto- and genotoxicity in HaCat keratinocytes is impaired by cell-mediated clearance of auto-oxidation-derived H2O2: an algorithm for experimental setting correction.

Several lines of evidence suggest that besides antioxidant also prooxidant properties are crucially involved in cytotoxic and protective activities of the major green tea catechin epigallocatechin-3-gallate (EGCG) in vitro (Elbling et al., 2011). Furthermore recent data suggest that EGCG induces oxidative stress also in vivo (Li et al., 2010). Here we set out to identify factors modulating cellular effects of EGCG in vitro. Using the HaCat keratinocytes model, we demonstrate that the cytotoxic, genotoxic and signal-activating effects of EGCG are significantly dependent on the ratio of cell number to working volume. Treatment with identical EGCG concentrations at altered experimental settings resulted in IC(50) values differing up to orders of magnitude and could even exert contradictory effects. This effect was based on cell-mediated clearance of autooxidation-derived H(2)O(2) from the supernatant. In order to estimate EGCG/H(2)O(2) concentrations equally effective under different settings, we have rationally derived and experimentally verified a simple algorithm relating concentration, working volume, cell number and - indirectly - exposure time. Algorithm application resulted in similar H(2)O(2) clearance curves from cell supernatants as well as comparable EGCG/H(2)O(2) effects at different settings. Our results demonstrate the importance of standardized experimental settings when investigating cytotoxic and/or beneficial effects of autooxidizing compounds.

Hydrogen peroxide mediates EGCG-induced antioxidant protection in human keratinocytes.

The beneficial health effects of (-)-epigallocatechin-3-gallate (EGCG), the main catechin of green tea, have been attributed to complex interactions with a focus on antioxidative properties. Susceptibility to autoxidation and production of cytotoxic reactive oxygen species (ROS), mostly H(2)O(2), have been suggested to occur in vitro but also in vivo. In this study, we address whether autoxidation-derived H(2)O(2) may be involved in the cytoprotective effects of EGCG. To that end we investigated keratinocyte-derived HaCat and HL-60 promyelocytic leukemia cells with significantly different sensitivities to H(2)O(2) (IC(50) 117.3 versus 58.3 µM, respectively) and EGCG (134.1 versus 84.1 µM). HaCat cells significantly resisted cytotoxicity and DNA damage based on enhanced H(2)O(2) clearance, improved DNA repair, and reduced intracellular ROS generation. Cumulative versus bolus EGCG and H(2)O(2) treatment and H(2)O(2) pretreatment before subsequent high-dose EGCG and vice versa significantly reduced DNA damage and cytotoxicity in HaCat cells only. Addition of catalase abolished the protective activities of low-dose H(2)O(2) and EGCG. In summary, our data suggest that autoxidative generation of low-dose H(2)O(2) is a significant player in the cell-type-specific cytoprotection mediated by EGCG and support the hypothesis that regular green tea consumption can contribute as a pro-oxidant to increased resistance against high-dose oxidative stressors.

Green tea extract and (-)-epigallocatechin-3-gallate, the major tea catechin, exert oxidant but lack antioxidant activities.

Green tea is the most widely consumed beverage. It has attained high reputation as a health-promoting dietary component ascribed to the antioxidant activity of (-)-epigallocatechin-3-gallate (EGCG), its main polyphenolic constituent. Evidence is increasing that tea constituents can be cell damaging and pro-oxidant themselves. These effects were suggested to be due to spontaneous H2O2 generation by polyphenols in solution. In the present study, we investigated the oxidant and antioxidant properties of green tea extracts (GTE) and of EGCG by means of the rodent macrophage-like RAW 264.7 and human promyelocytic leukemic HL60 cell lines. The results obtained show that both under cell-free conditions and in the presence of cells the oxidant activities of GTE and EGCG exceeded those of spontaneously generated H2O2 (FOX assay). Increase of intracellular oxidative stress was indicated by 2',7'-dichlorofluorescin probing, and the enhanced genotoxicity was demonstrated by the alkaline comet assay and by the micronucleus assay (cytokinesis block). Time- and dose-dependent induction of cell death was monitored by trypan blue exclusion, MTT assay, and Hoechst staining. Furthermore, in our systems in vitro, EGCG neither directly scavenges H2O2 nor mediates other antioxidant activities but rather increased H2O2-induced oxidative stress and DNA damage. In conclusion, our data suggest that detailed mechanistic studies on the effects of GTE and EGCG should be performed in vivo before excessive intake and/or topical application of green tea products can be recommended to healthy and/or diseased persons.