Cell Death Detection ELISA Plus Research Articles

Abstract 40: Methylated resveratrol analogue DMU-212 undergoes metabolic activation to DMU-214 in ovarian A-2780 and SKOV-3 cancer cells

Abstract Resveratrol (3,4',5-trans-trihydroxystilbene) is a naturally occurring phytoalexin which shows a wide spectrum of biological activity. Although the anticancer effects of resveratrol have been revealed, its clinical application is limited because of a low bioavailability and a rapid clearance from the circulation. The structural alteration of the stilbene motif of resveratrol has been found to be a promising strategy for generation of several synthetic analogues with improved pharmacokinetic parameters. The substitution of hydroxyl groups of resveratrol for methoxy groups may increase the molecule stability, making it less susceptible to phase II conjugation reactions in vivo. The structure-activity studies have revealed that introduction of additional methoxy groups resulted in an increased cytotoxic properties of resveratrol analogues. Furthermore, the methoxy groups at positions 3,5- and 3,4,5- of the trihydroxystilbene scaffold have been identified as crucial for pro-apoptotic activity of the compound. To date few reports have demonstrated the antiproliferative effect of DMU-212 (3,4,4',5-tetramethoxystilbene) in transformed fibroblasts, prostate, liver, colon, cervical, breast and ovarian cancer cells. It has been shown that DMU-212 is biotransformed to four metabolites: 3'-hydroxy-3,4,5,4'-tetramethoxystilbene (DMU-214), 4'-hydroxy-3,4,5-trimethoxystilbene (DMU-281), 4-hydroxy-3,5,4'-trimethoxystilbene (DMU-291), and 3-hydroxy-4,5,4'-trimethoxystilbene (DMU-807). However, if these metabolites retain the biological activity of DMU-212 remains unknown. Therefore, we evalueted the cytotoxic activity of these four metabolites of DMU-212 and the induction of apoptosis in ovarian A-2780 and SKOV-3 cancer cell lines. CYP1A1 and CYP1B1 are known to catalyze the conversion of estradiol to 4-hydroxyestradiol. Hence, the compounds such as resveratrol or DMU-212, which are structurally similar to the endogenous oestrogen estradiol, have been suggested to undergo metabolism by these enzymes. Surprisingly, DMU-212 has been shown to entirely inhibit CYP1B1 in ovarian A-2780 cancer cell line. Since CYP1B1 has been suggested to be involved in DMU-212 biotransformation, it can be hypothesised that one or more metabolites of DMU-212 regulate the expression of CYP1B1 by the feedback inhibition. To have an insight into DMU-212 metabolites' role in the expression of isoenzymes of CYP1 family we assessed CYP1A1 and CYP1B1 mRNA and protein levels in metabolites-treated A-2780 and SKOV-3 cancer cells. The inhibitory effect of four metabolites of DMU-212 was investigated in ovarian A-2780 and SKOV-3 cancer cell lines by MTT assays. The induction of apoptosis was assayed by Cell Death Detection ELISAPLUS test, Caspase-Glo® 3/7 luminescent test and flow cytometry analysis. The expression of CYP1A1 and CYP1B1 mRNA and protein levels was investigated by RT-qPCR and Western blott, respectively. From among four metabolites of DMU-212, DMU-214 exhibited the highest cytotoxic activity (IC50 A-2780=13.26±2.11 nM; IC50 SKOV-3=29.21±3.17nM). Furthermore, DMU-214 has been revealed to exert more pronounced inhibitory effects than the prototype compound, DMU-212. In addition, we observed that the stronger cytotoxic activity of DMU-214 evoked its greater pro-apoptotic effect. DMU-214 caused an increase in nucleosomes level in A-2780 and SKOV-3 cells, EF= 15.89 and 7.91, respectively. Exposure of either cell line to DMU-214 resulted in a statistically significant decrease in CYP1A1 and CYP1B1 mRNA and protein levels as compared to control. We showed for the first time that DMU-212 undergoes metabolic activation to DMU-214 in ovarian A-2780 and SKOV-3 cancer cell lines. Since DMU-214 has been observed to decrease expression of CYP1A1 and CYP1B1 in A-2780 and SKOV-3 cells our results provide a new insight into the concept of their feedback inhibition by the compound tested. Citation Format: Sylwia Borys, Malgorzata Kucinska, Dorota Trzybulska, Mariusz Kaczmarek, Marcin Wierzchowski, Marek Murias, Jadwiga Jodynis-Liebert, Hanna Piotrowska. Methylated resveratrol analogue DMU-212 undergoes metabolic activation to DMU-214 in ovarian A-2780 and SKOV-3 cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 40.

Apoptosis and Phagocytosis Activity of Macrophages Infected by Mycobacterium tuberculosis Resistant and Sensitive Isoniazid Clinical Isolates

Mycobacterium tuberculosis (M.tb) is the main causative pathogen that cause the pulmonary tuberculosis.Intracellular M.tb was reported able to induce macrophages apoptosis, which may have crucial role in the regulationof immun response against M.tb infection. As an intracellular bacteria, M.tb able to live and replicate withinmacrophages. Phagocytosis is the first step to achieved this condition. The induction of macrophages apoptosis byINH resistant and sensitive M.tb clinical isolates, and H37Rv was studied. The macrophages apoptosis level weremeasured using an Ag-capture ELISA for histone and fragmented DNA (Cell Death Detection ELISAplus, RocheDiagnostic GmBH). Phagocytosis activity also analyzed, after staining using fluorescence dye (AcriFluorTM, ScientificDevice Lab.). The results showed that there was no significantly different between INH resistant and sensitive M.tbclinical isolates in respect their ability to induce apoptosis. The phagocytosis activity among the clinical isolates wasshown to be strain dependent, and undistinguishable between the Mtb clinical isolates. There was no associationbetween macrophages apoptosis level and the phagocytosis activity. These data suggested that among the virulentMtb clinical isolates, the ability to induce macrophages apoptosis and phagocytosis were consistently in comparablelevel

Apoptosis and Phagocytosis Activity of Macrophages Infected by Mycobacterium tuberculosis Resistant and Sensitive Isoniazid Clinical Isolates

Mycobacterium tuberculosis (M.tb) is the main causative pathogen that cause the pulmonary tuberculosis.Intracellular M.tb was reported able to induce macrophages apoptosis, which may have crucial role in the regulationof immun response against M.tb infection. As an intracellular bacteria, M.tb able to live and replicate withinmacrophages. Phagocytosis is the first step to achieved this condition. The induction of macrophages apoptosis byINH resistant and sensitive M.tb clinical isolates, and H37Rv was studied. The macrophages apoptosis level weremeasured using an Ag-capture ELISA for histone and fragmented DNA (Cell Death Detection ELISAplus, RocheDiagnostic GmBH). Phagocytosis activity also analyzed, after staining using fluorescence dye (AcriFluorTM, ScientificDevice Lab.). The results showed that there was no significantly different between INH resistant and sensitive M.tbclinical isolates in respect their ability to induce apoptosis. The phagocytosis activity among the clinical isolates wasshown to be strain dependent, and undistinguishable between the Mtb clinical isolates. There was no associationbetween macrophages apoptosis level and the phagocytosis activity. These data suggested that among the virulentMtb clinical isolates, the ability to induce macrophages apoptosis and phagocytosis were consistently in comparablelevelKeywords: Mycobacterium tuberculosis, apoptosis, phagocytosis, macrophages, isoniazid

Docetaxel in combination with octreotide shows synergistic apoptotic effect by increasing SSTR2 and SSTR5 expression levels in prostate and breast cancer cell lines.

Docetaxel (DTX) is widely used for the treatment of metastatic prostate and breast cancers. Despite the clinical success of DTX, drug-related cumulative toxicity restricts its clinical use in cancer therapy. Thus, there is an urgent need for new therapeutic options. Octreotide (OCT) is a synthetic somatostatin analog that induces apoptosis in different cancer cell lines in vitro. In this study, we investigated the possible synergistic apoptotic effects of DTX in combination with OCT in prostate and breast cancer cell lines. The XTT cell viability assay was used to determine cytotoxicity. Apoptosis was evaluated by Cell Death Detection ELISA(Plus) Kit. The expression levels of apoptotic proteins were assessed by human apoptosis antibody array. Levels of SSTR2 and SSTR5 proteins were determined by western blot analysis. DTX and OCT combination induced apoptosis in both breast and prostate cancer cells in a concentration- and time-dependent manner. Moreover, combination treatment resulted in inhibition of anti-apoptotic proteins such as Bcl-2 and Bcl-xL and induction of pro-apoptotic proteins Bax, Cytochrome c and IAPs in all of the tested cancer cell lines. SSTR2 and SSTR5 protein levels were induced as compared to any agent alone. These results indicate that this combination treatment is a significant inducer of apoptosis in a synergistic manner in breast and prostate cancer cells. This strong synergism helps to lower the dose of DTX in both types of cancers, thus letting DTX to be used for longer periods by delaying resistance development and lesser side effects.

Apoptosis- and differentiation-inducing activities of jacaric acid, a conjugated linolenic acid isomer, on human eosinophilic leukemia EoL-1 cells.

Conjugated linolenic acids(CLNAs) are a group of naturally occurring positional and geometrical isomers of the C18 polyunsaturated essential fatty acid, linolenic acid(LNA), with three conjugated double bonds(C18:3). Although previous research has demonstrated the growth-inhibitory effects of CLNA on a wide variety of cancer cell lines invitro, their action mechanisms and therapeutic potential on human myeloid leukemia cells remain poorly understood. In the present study, we found that jacaric acid(8Z,10E,12Z-octadecatrienoic acid), a CLNA isomer which is present in jacaranda seed oil, inhibited the invitro growth of human eosinophilic leukemia EoL-1 cells in a time- and concentration-dependent manner. Mechanistic studies showed that jacaric acid triggered cell cycle arrest of EoL-1 cells at the G0/G1 phase and induced apoptosis of the EoL-1 cells, as measured by the Cell Death Detection ELISAPLUS kit, AnnexinV assay and JC-1 dye staining. Notably, the jacaric acid-treated EoL-1 cells also underwent differentiation as revealed by morphological and phenotypic analysis. Collectively, our results demonstrated the capability of jacaric acid to inhibit the growth of EoL-1 cells invitro through triggering cell cycle arrest and by inducing apoptosis and differentiation of the leukemia cells. Therefore, jacaric acid might be developed as a potential candidate for the treatment of certain forms of myeloid leukemia with minimal toxicity and few side effects.

Effect of trastuzumab/AT-101 combination on apoptosis and cytotoxicity in HER2-positive breast cancer cells: A novel combination to effect resistance to anti-HER2 therapy through inhibition of PI3K signaling.

e13512 Background: Trastuzumab is a humanized monoclonal antibody approved for the treatment of breast cancer with HER2 amplification and/or overexpression. AT-101, the (-/-) enantiomer of gossypol...

Paclitaxel in combination with AT-101 induces apoptosis via supressing Bcl-2, bcl-XL, mcl-1 proteins in human breast cancer cells.

e13578 Background: Paclitaxel, a microtubule stabilizing agent, has been a standard of care for breast cancer. AT-101, is an -/- enantiomer of gossypol, inhibits the Bcl-2 family proteins which contain BH3 domain. We reported previously that Paclitaxel in combination with AT-101 showed strong synergistic cytotoxic and apoptotic effects in human breast cancer cells. In this study, to elucidate the molecular mechanisms involved in the apoptotic effect of AT-101/Paclitaxel combination treatment in breast cancer cells, we investigated the possible roles of anti-apoptotic Bcl-2, Bcl-XL and Mcl-1 proteins which contain BH3 domain. Involvement of caspase 3 and 7 activation was also investigated. Methods: Human breast cancer cells were treated with increasing concentrations of drugs alone or with the synergistic combination doses of AT-101 and Paclitaxel. Cell Death Detection Elisa Plus Kit (Roche) was used to detect apoptosis. Caspase 3/7 activity was evaluated by Caspase-Glo 3/7 (Promega, Madison, WI) kit. Changes in the mRNA levels of Bcl-2, Bcl-XL and Mcl-1 genes were evaluated by qRT-PCR. Expression levels of these proteins were also investigated by Western blot analysis. Results: Combined treatment was shown to have strong synergistic apoptotic effects in MCF-7 and MDA- MB-231 human breast cancer cells. mRNA levels of Bcl-2, Bcl-XL and Mcl-1 molecules were reduced by the combination treatment in both cell lines. In parallel with mRNA levels, Bcl-2, Bcl-XL and Mcl-1 protein levels were significantly reduced after this novel drug combination. Combined treatment also induced caspase 3/7 activation in breast cancer cells. Conclusions: These preliminary data suggest that anti-apoptotic proteins such as Bcl-2, Bcl-XL and Mcl-1 may play important role in the underlying mechanistic rationale of apoptotic effect of AT-101/paclitaxel combination, while pro-apoptotic Bcl-2 related genes (Caspase-3 and Caspase-7) also seem to regulate this synergistic interaction.

7-Aminoactinomycin D for apoptosis staining in flow cytometry

7-Aminoactinomycin D (7-AAD) is a DNA dye that distinguishes viable, apoptotic, and late apoptotic/dead cells in flow cytometry. Several staining protocols using 7-AAD have been described, but data on the influence of the 7-AAD concentration on the readout are not available. Therefore, we compared the results obtained by staining with 1, 5, 10, and 20μg/ml 7-AAD for 20min with the PE–Annexin V Apoptosis Detection Kit and Cell Death Detection ELISAPLUS in lymphocytes and CEM human leukemia cells. The results showed that 7-AAD staining with 5, 10, and 20μg/ml, but not with 1μg/ml, is suitable for quantification of apoptosis in flow cytometry.

99P Zoledronic Acid in Combination with Serine/Threonine Phosphatase Inhibitors Induces Cytotoxicity and Apoptosis in Human Breast Cancer Cells via Decreasing the Activities of Pp1 and Pp2A

ABSTRACT Introduction Zoledronic acid (ZA), a nitrogen containing bisphosphonate (N-BP), is able to affect tumor cells by inhibiting the enzyme farnesylpyrophosphate synthase (FPPS) in the mevalonate pathway (MVP). Several studies have investigated its potential role in the treatment of breast cancer. The aim of our study was to investigate the role of the serine/threonine protein phosphatase inhibitors, Okadaic acid (OA) or Calyculin A (CA), in combination with ZA on human breast cancer cells, MCF-7 and MDA-MB-231. Methods Cytotoxicity was assessed by XTT cell viability assay. Apoptosis was shown by using Cell Death Detection ELISA Plus Kit and verified by measuring caspase 3/7 enzyme activity. PP1 and PP2A enzyme activities were evaluated by serine/threonine phosphatase ELISA assay and expression levels of PP1 and PP2A protein levels were then assessed by western blot analysis. Results ZA induced cytotoxicity and apoptosis in both breast cancer cells in a dose and time dependent manner. Combination of ZA with either CA or OA showed synergistic cytotoxicity and apoptosis as compared to any agent alone in both breast cancer cells. The combination of ZA with phosphatase inhibitors resulted in enhanced suppression in PP1 and PP2A enzyme activities and also protein levels within breast cancer cells. Conclusion Results from our study increase the translational potential of our in vitro findings and offer the basic rationale for the potential use of protein phosphatase inhibitors in combination with ZA for breast cancer. Disclosure All authors have declared no conflicts of interest.

Cartilage Destruction in Granulomatosis with Polyangiitis (Wegener's Granulomatosis) Is Mediated by Human Fibroblasts after Transplantation into Immunodeficient Mice

A key feature of granulomatosis with polyangiitis (GPA; or Wegener's granulomatosis) is the granulomatous inflammation of the upper respiratory tract, which leads to the subsequent destruction of adjacent tissues. The aim of our work was to study the histopathological and cellular components of tissue destruction of human GPA tissue transplanted into immunodeficient mice. Biopsy specimens from patients with active GPA (n = 10) or sinusitis (controls, n = 6) were s.c. co-implanted with healthy allogeneic human nasal cartilage into immunodeficient pfp/rag2(-/-) mice. Transplants were examined for their destructive capability of the allografted human cartilage. In addition, nasal fibroblasts from patients with GPA (n = 8) and control healthy nasal fibroblasts (n = 5) were cultured, and cell proliferation and apoptosis were quantified. mRNA and protein levels of matrix metalloproteinases and cytokines were evaluated at baseline and after proinflammatory stimulation. GPA implants showed massive destruction of the co-implanted human cartilage, whereas cartilage destruction was only marginal in control samples. Destruction was mediated by human fibroblasts and could be inhibited by corticoid treatment. The up-regulated production of matrix metalloproteinases 1, 3, and 13 and cytokines IL-6 and IL-8 was found in vivo and in vitro. Although proliferation of isolated fibroblasts was comparable between GPA and controls, GPA samples showed a significant delay of apoptosis. The destruction of nasal cartilage in GPA is mainly mediated by fibroblasts that can be blocked by corticosteroids, and this tissue destruction is not dependent on the influx of leukocytes.

PI3 Kinase δ Is a Key Regulator of Synoviocyte Function in Rheumatoid Arthritis

Class I phosphoinositide 3 kinase (PI3K) δ is a promising therapeutic target for rheumatoid arthritis (RA) because of its contribution to leukocyte biology. However, its contribution in fibroblasts has not been studied as a mechanism that contributes to efficacy. We investigated the expression and function of PI3Kδ in synovium and cultured fibroblast-like synoviocytes (FLS). Immunohistochemistry demonstrated that PI3Kδ is highly expressed in RA synovium, especially in the synovial lining. Using quantitative PCR and Western blot analysis, we found that PI3Kδ mRNA and protein expression is higher in RA than in osteoarthritis (OA) synovium. PI3Kδ was also expressed in cultured FLS, along with PI3Kα and PI3Kβ, whereas PI3Kγ was not detectable. PI3Kδ mRNA expression was selectively induced by inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) but not by growth factors platelet-derived growth factor (PDGF) and transforming growth factor β (TGFβ). The use of inhibitors that block individual PI3K isoforms, including the novel selective PI3Kδ inhibitor INK007, showed that PI3Kδ is required for PDGF- and TNF-induced Akt activation. PI3Kδ inhibition also diminished PDGF-mediated synoviocyte growth and sensitized cells to H(2)O(2)-induced apoptosis. These data are the first documentation of increased PI3Kδ expression in both RA synovium and cultured synoviocytes. Furthermore, these are the first data demonstrating that PI3Kδ is a major regulator of PDGF-mediated fibroblast growth and survival via Akt. Thus, targeting PI3Kδ in RA could modulate synoviocyte function via anti-inflammatory and disease-altering mechanisms.

P3-18-05: Triple-Negative Breast Cancer: Stem Cells, Cancer and New Treatment Strategy.

Abstract Background: Women with triple negative breast cancer (TNBC) have a worse prognosis compared with other breast cancer subtypes. Hormonal or Herceptin-based therapies were found to be ineffective because of the loss of target receptors such as ER, PR and HER-2 amplification. TNBC has increased recurrence and poor survival, despite initial response to neoadjuvant chemotherapy. The aim of this study was to determine whether current drug treatment(s) eliminates bulk of tumor cells, but it has a minimal effect on cancer stem cells (CSCs) leading to tumor recurrence. Materials and Methods: Cancer stem cells were identified by ALDH-positive population by fluorescence-activated cell sorting. CSCs ability to form mammospheres, are generated in nonadherent and nondifferentiated conditions. Apoptosis was assessed using the Cell Death Detection ELISAPLUS kit. Xenograft tumor formation was evaluated using Nod/Scid mice. Cancer stem cell levels in human TNBC and ER+, PR+ and HER2− tumors were obtained by CD44+/ CD24low/− immunohistochemistry. Results: TNBC tumors express relatively higher levels of cancer stem cells compared to ER-positive, PR-positive and HER2−negative breast tumors. We studied the effects of PARP inhibitors (AZD2281 and BSI-201), paclitaxel, docetaxel, cisplatin and cisplatin plus TRAIL on CSCs derived from CRL-2335 and MDA-MB-468 TNBC cells in vitro. The in vitro data indicates that cisplatin plus TRIAL treatment was most effective in eliminating CSCs compared to PARP inhibitors, cisplatin, paclitaxel and docetaxel. Treatment with cisplatin plus TRAIL also inhibits Wnt-1 signaling and its downstream target, β-catenin, phospho β-catenin, cyclin D1, increased apoptosis, reduced proliferation and mammosphere formation. Inhibition of Wnt-1 by siRNA significantly reduced the ability of CSCs to form mammospheres compared to control. However, maximum effect was seen in cisplatin plus TRAIL treated cells. The combination of cisplatin/TRAIL also inhibited the growth of CRL-2335 Xenografts. Discussion: We demonstrate for the first time that cisplatin plus TRAIL is highly effective in reducing CSCs compared to PARP inhibitors, docetaxel or paclitaxel in TNBC cells in vitro. Inhibition of Wnt-1 signaling pathways by siRNA or cisplatin plus TRAIL reduces cell proliferation and increases apoptosis in CSCs. Taken together the data suggests that cisplatin plus TRAIL treatment has the potential of providing a new strategy for improving the therapeutic outcome in TNBC patients. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-18-05.

Abstract 5044: P144, an inhibitory peptide of the TGF-beta signaling pathway, inhibits growth and increases apoptosis in brain tumor cell lines

Abstract Transforming growth factor beta (TGF-beta), a cytokine with potent promalignant properties, is overexpressed in different tumors. Therefore, the development of TGF-beta inhibitory molecules is of paramount importance to improve the efficacy of antitumor therapies. Several strategies using neutralizing antibodies, TGF-beta soluble binding proteins, antisense compounds, or small molecule inhibitors targeting TGF-beta signaling, have shown that TGF-beta inhibition helps in antitumor therapy. Some of these compounds have shown their value by directly inhibiting tumor growth and metastasis, whereas others have been able to enhance antitumor immune responses. The aim of our study was to investigate the effect of two peptide inhibitors of TGF-beta, namely P144 (1) and P17 (2), in 6 human tumor cell lines derived from medulloblastoma (DAOY), rhabdomyosarcoma (TE671), neuroblastoma (SK-NSH and SH-SY5Y), and glioblastoma (A172 and U87-MG). P144 and P17 are derived from human TGF-beta type III receptor sequence (1) and phage display library technology (2) respectively. Both peptides have high affinity for soluble TGF-beta and potent inhibitory effects on TGF-beta binding to its receptors. MTT assay and an apoptosis test (Cell Death Detection ELISA PLUS, Roche) were performed before and after treatment with the peptides, which were added to the media at a final concentration of 100 ug/ml. P17 did not produce any effect on the cell lines, while P144 induced growth inhibition and apoptosis in three brain tumor cell lines (DAOY, A172, and U87-MG). The level of phosphorylated Smad2 (that initiates the activation of the TGF-beta intracelular signaling pathway) was decreased in presence of P144 in all cases, as determined by Western blot, confirming that P144 inhibits TGF-beta signaling. qRT-PCR was performed in order to analyze different genes related with the regulation of the TGF-beta signaling, such as Smad7, Ski, SnoN, FoxG1, which are inhibitors of the pathway, and p21CIP. Our preliminary data show that P144 affects the transcription of Smad dependent genes, e.g. Smad7, SnoN and Ski, although confirmatory experiments are still required. Our data suggest that inhibition of the TGF-beta pathway by P144 inhibits cell growth and increases apoptosis in brain tumor cell lines, possibly in a Smad-dependent manner. Therefore, P144 should be further tested as a therapeutic agent against brain tumors. 1. Ezquerro IJ, et al. A synthetic peptide from transforming growth factor beta type III receptor inhibits liver fibrogenesis in rats with carbon tetrachloride liver injury. Cytokine 22:12-20, 2003. 2. Dotor J, et al. Identification of peptide inhibitors of transforming growth factor beta 1 using a phage-displayed peptide library. Cytokine. 39:106-115, 2007. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5044. doi:10.1158/1538-7445.AM2011-5044

Hormona Refrakter Prostat Kanseri Hücre Dizisi DU-145’in Kemoterapi Duyarlılığını Artırmak İçin Dosetaksel ve Gossipolün Sinerjik Bileşimi

Objective: Docetaxel has become the standard of care for hormone-refractory prostate cancer (HRPC), however drug resistance and toxicity are still challenging in daily cancer practice. The anti-apoptotic pathway centered around the Bcl-2 protein might be one of the responsible pathways. Adding a second drug to docetaxel treatment is one of the most common approaches to solve this problem. Gossypol was reported to have potent anticancer activities in prostate cancer. In this study, we searched for the possible synergistic cytotoxic/apoptotic effects of this combination treatment in hormone- and drug resistant prostate cancer cell line, DU-145 via inhibition of Bcl-2 protein. Material and Methods: XTT cell viability assay was used to assess cytotoxicity of the drugs alone and in combination. For verifying apoptosis, Cell Death Detection Elisa Plus Kit and Caspase-Glo 3/7 assay were used. Western Blot analysis for Bcl-2 protein was carried out. Results: A novel drug combination of docetaxel and gossypol resulted in a significant synergistic cytotoxic activity and apoptosis as compared to any single agent alone, in a dose- and time dependent manner, and also significantly reduced Bcl-2 protein levels in DU-145, in doses that can be used clinically. Conclusion: Adding gossypol to docetaxel as a combination treatment in HRPC patients might be a solution for taxane resistant patients. Inhibition of Bcl-2 might be one of the underlying routes of activity for this combination.

STAT1 Is a Master Regulator of Pancreatic β-Cell Apoptosis and Islet Inflammation

Cytokines produced by islet-infiltrating immune cells induce β-cell apoptosis in type 1 diabetes. The IFN-γ-regulated transcription factors STAT1/IRF-1 have apparently divergent effects on β-cells. Thus, STAT1 promotes apoptosis and inflammation, whereas IRF-1 down-regulates inflammatory mediators. To understand the molecular basis for these differential outcomes within a single signal transduction pathway, we presently characterized the gene networks regulated by STAT1 and IRF-1 in β-cells. This was done by using siRNA approaches coupled to microarray analysis of insulin-producing cells exposed or not to IL-1β and IFN-γ. Relevant microarray findings were further studied in INS-1E cells and primary rat β-cells. STAT1, but not IRF-1, mediates the cytokine-induced loss of the differentiated β-cell phenotype, as indicated by decreased insulin, Pdx1, MafA, and Glut2. Furthermore, STAT1 regulates cytokine-induced apoptosis via up-regulation of the proapoptotic protein DP5. STAT1 and IRF-1 have opposite effects on cytokine-induced chemokine production, with IRF-1 exerting negative feedback inhibition on STAT1 and downstream chemokine expression. The present study elucidates the transcriptional networks through which the IFN-γ/STAT1/IRF-1 axis controls β-cell function/differentiation, demise, and islet inflammation.

Pterostilbene and tamoxifen show an additive effect against breast cancer in vitro

BackgroundTamoxifen is widely used for the treatment of breast cancer. Pterostilbene, a bioavailable stilbenoid found in blueberries, has been found to inhibit breast cancer growth in vitro. It was hypothesized that combining pterostilbene with tamoxifen would produce additive effects on estrogen receptor–positive breast cancer cells. MethodsTwo estrogen receptor–positive breast cancer cell lines, MCF7 and ZR-751, were pretreated with graduated doses of pterostilbene for 24 hours, followed by 5 μmol/L tamoxifen. MTT proliferation assays and Cell Death Detection ELISAPLUS tests evaluated cell viability and apoptosis. ResultsMCF7 cells showed inhibition (10 and 20 μmol/L, P < .001; 30 μmol/L, P < .05) at all time points when combined with tamoxifen. ZR-751 cells showed additive reductions in cell viability (P < .001). Cell Death Detection ELISAPLUS indicated increased apoptosis (P < .01). ConclusionsPterostilbene shows an additive inhibitory effect on breast cancer cells when combined with tamoxifen, most likely from augmented cancer cell apoptosis.

Effects of the pan-DAC inhibitor LBH589 in estrogen-sensitive and -insensitive breast cancer cells.

e13618 Background: The pan-deacetylase inhibitor LBH589 (panobinostat), that is now in phase I-II trials for its antitumor activity in advanced refractory solid tumors and hematologic malignancies, is one of those drugs with both antitumor activity and the ability of modulating the expression of selected molecules involved in different response to therapy. Advanced breast cancer, becoming resistant to conventional treatment, or metastatic disease are potential application of these new approaches. Methods: In the present study, we report the effects of LBH589 in estrogen-sensitive MCF-7 and T47D, and estrogen-insensitive MDA-MB 231 breast cancer cells. Cells were treated with 5-100 nM LBH589 for different periods (untreated cells were used as control). After treatment, we evaluated: 1. cell viability and cytotoxicity with the WST-1 method; 2. apoptosis induction with the Cell Death Detection ELISA PLUS (Roche Applied Science, Germany); 3. cell cycle progression with flow cytometry; 4. estrogen sensitivity gene expression with RT- real time PCR for estrogen receptor (ER), progesterone receptor (PR), and FOXA1. Results: LBH589 caused a significant reduction in cell viability within nanomolar range and the anti-tumour effect was sustained by apoptosis induction and cell cycle arrest in G2/M. The cytotoxic effect was evident in both estrogen-sensitive and insensitive cells. LBH589 at concentrations higher than 50 nM caused also a reduction of ER alpha, PR and FOXA1 expression in MCF7 and T47D cells; no effect was observed in MDA-MB 231 cells where ER alpha, PR and FOXA1 were not detectable before and after treatment. LBH589 is able to smooth down estrogen-sensitivity pathway, almost certainly causing additional modification in cell behavior, that deserves further studies. Conclusions: Our data suggest that LBH589 is a useful compound in the managing of breast cancer due to its potent cytotoxic effect that is detectable in both estrogen-sensitive and insensitive cells (differently by what reported for other DAC-inhibitors). Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Novartis

Transcriptional Changes Induced by Imatinib and Nilotinib in the Chronic Myelogenous Leukemia (CML) Cell Line K562.

Abstract 4250Nilotinib is a selective bcr-abl tyrosine kinase inhibitor that is 30-fold more potent than Imatinib in vitro. To examine the molecular and functional effects of Nilotinib and Imatinib we performed gene expression and functional analyses in K562 cells following in vitro treatment with the two tyrosine kinase inhibitors. Affymetrix U133A 2.0 microarrays covering 21.722 probe sets were used to analyse the gene expression profile of 5×10 7 K562 cells after 24h in vitro treatment with Imatinib (0.5 μM) or Nilotinib (0.05 μM) (half maximal inhibitory concentration). Gene expression data of the treated cells were compared with data of untreated cells. In addition, proliferation (MTS Assay, Promega), apoptosis (Cell Death Detection ELISAPLUS, Roche) and cell cycle (FITC BrdU Flow Kit, BD Pharmingen) assays were performed. Protein levels of STAT5, pSTAT5(Thr694), PIM-1 and PIM-2 were detected with Western blots. Particular emphasis was put on 303 genes which we found to be differentially expressed in primary CD34+ cells from patients with CML in vivo during treatment with Imatinib (Bruennert et al, Leukemia, 2008). In K562 cells, we found that Imatinib led to a significant differential expression of 45 of those genes. In general, the effect of Nilotinib with regard to the number of genes affected and degree of suppression was more pronounced resulting in a significant differential expression of 120 genes of the aforementioned genes. Of note, genes affected by Nilotinib included all genes altered by Imatinib. Downregulation of genes involved in cell cycle was observed in 17 genes following Nilotinib exposure, but only in the PIM-1 gene following Imatinib exposure. This effect of Nilotinib is in line with the results of cell cycle experiments showing that Nilotinib exposed cells had the lowest proportion of actively cycling cells. The proportion of apoptotic K562 cells was 5.5-fold greater following treatment with Nilotinib in comparison to treatment with DMSO, whereas Imatinib treated K562 cells had a 1.3-fold higher apoptosis rate compared to DMSO treated 562 cells after 24 hours. The superiority of Nilotinib is also reflected by the results of protein analysis of STAT5, pSTAT5(Thr694), PIM-1 and PIM-2: Both tyrosine kinase inhibitors completely inhibit the phosphorylation of STAT5, but Nilotinib inhibits PIM-1 and PIM-2 stronger than Imatinib (by 85% vs. 70%) in K562 cells. In summary, on a molecular level Nilotinib is apparently more potent than Imatinib with regard to the number of genes affected and the degree of their suppression. Among the 45 genes that were significant differentially expressed with both drugs, genes of Imatinib treated K562 cells were downregulated 1.68fold (mean) whereas genes of Nilotinib treated K562 cells were downregulated 2.41fold (mean). The genes altered are mainly associated with cell cycle regulation. In addition to this Nilotinib has a stronger inhibitory effect on PIM 1 and 2. Disclosures:Gattermann:Novartis: Honoraria, Participation in Advisory Boards on deferasirox clinical trials.

Rapid simultaneous determination of apoptosis, necrosis, and viability in sulfur mustard exposed HaCaT cell cultures

Sulfur mustard (SM; bis(2-chloroethyl)sulphide; HD) is a blister inducing agent causing DNA damage and subsequently, cell death, mostly by apoptosis in basal keratinocytes. Despite intensive investigations on the cellular mechanism, there are, as of now, no causal therapeutics to prevent or antagonize SM-related damage to cells and tissues. In order to develop treatment strategies against vesication, it is important to distinguish apoptosis from necrosis in SM treated human keratinocytes. DNA fragmentation is a hallmark of apoptosis and regulated by a cascade of enzymes (endonucleases, DNase I, NUC 18), which finally cut the chromatin into specific formations of 180–200 base pairs, the nucleosomes. A feasible way to monitor apoptosis is the detection of nucleosomes by means of the Cell Death Detection ELISAplus® (CDDE). In contrast, during necrosis DNA fragmentation is at random and delivers larger fragments, which therefore are significantly less in number and predominantly occur in cell culture supernatant.To monitor necrosis, we measured the release of intracellular adenylate kinase (AK) into cell culture supernatant by means of the ToxiLight® Bioluminescence Assay (TL). With combination of the Cell Death Detection ELISAplus® and the ToxiLight® Bioluminescence Assay, we acquired more comprehensive information on cell survival and mechanisms of cell death, following an SM exposure. To validate the assay we tested common apoptosis- and necrosis-inducing agents like SM 300μM for 30min, Lewisite (L) 60μM for 5min and Triton X-100 0.1%. The results show that it is possible to differentiate between the two modes of cell death and to quantify their extent.This assay is highly effective in quantifying apoptosis and necrosis caused by cytotoxic agents and in estimating protective effects of potential active pharmaceutical ingredients.

Brucein D induces apoptosis in pancreatic adenocarcinoma cell line PANC-1 through the activation of p38-mitogen activated protein kinase

Pancreatic cancer is a malignant disease with extremely high mortality. Our previous work found that Brucea javanica fruit possesses potent anti-pancreatic cancer activity. In the present study, brucein D (BD), a quassinoid found abundantly in B. javanica fruit, was evaluated for its anti-proliferative and apoptogenic actions. BD inhibited the growth of three pancreatic cancer cell lines, i.e., PANC-1, SW1990 and CAPAN-1, but exerted only modest cytotoxicity on non-tumorigenic Hs68 cells. Hoechst 33342 staining and Cell Death Detection ELISA PLUS assay revealed that BD-induced DNA fragmentation in PANC-1 cells. Moreover, subG1 phase was observed in the BD-treated cells. Western blot experiments indicated that BD exposure augmented caspase 3, 8, 9 and bak protein levels, while attenuating the expression of bcl-2. Furthermore, BD treatment promoted phosphorylation of p38-MAPK. The selective p38-MAPK inhibitor SB203580 effectively mitigated the BD-induced apoptosis in PANC-1 cells, suggesting that p38-MAPK signaling pathway was involved in the BD-induced apoptosis in pancreatic cancer cells. Taken together, our results provide experimental evidence to support the traditional use of B. javanica fruit in cancer treatment, and render BD a promising chemical candidate for further development into anti-pancreatic cancer agent.