Antiproliferative and apoptotic effect of LY2090314, a GSK-3 inhibitor, in neuroblastoma in vitro.

BACKGROUND: Neuroblastoma (NB) is a devastating disease. Despite recent advances in the treatment of NB, about 60% of high-risk NB will have relapse and therefore long-term event free survival is very minimal. We have reported that targeting glycogen synthase kinase-3 (GSK-3) may be a potential strategy to treat NB. Consequently, investigating LY2090314, a clinically relevant GSK-3 inhibitor, on NB cellular proliferation and may be beneficial for NB treatment. METHODS: The effect of LY2090314 was compared with a previously studied GSK-3 inhibitor, Tideglusib. Colorimetric, clonogenic, and live-cell image confluency assays were used to study the proliferative effect of LY2090314 on NB cell lines (NGP, SK-N-AS, and SH-SY-5Y). Western blotting and caspase glo assay were performed to determine the mechanistic function of LY2090314 in NB cell lines. RESULTS: LY2090314 treatment exhibited significant growth reduction starting at a 20 nM concentration in NGP, SK-N-AS, and SH-SY-5Y cells. Western blot analysis indicated that growth suppression was due to apoptosis as evidenced by an increase in pro-apoptotic markers cleaved PARP and cleaved caspase-3 and a reduction in the anti-apoptotic protein, survivin. Further, treatment significantly reduced the level of cyclin D1, a key regulatory protein of the cell cycle and apoptosis. Functionally, this was confirmed by an increase in caspase activity. LY2090314 treatment reduced the expression levels of phosphorylated GSK-3 proteins and increased the stability of ß-catenin in these cells. CONCLUSIONS: LY2090314 effectively reduces growth of both human MYCN amplified and non-amplified NB cell lines in vitro. To our knowledge, this is the first study to look at the effect of LY2090314 in NB cell lines. These results indicate that GSK-3 may be a therapeutic target for NB and provide rationale for further preclinical analysis using LY2090314.

Inhibition of the AKT pathway in cholangiocarcinoma by MK2206 reduces cellular viability via induction of apoptosis.

INTRODUCTION: Cholangiocarcinoma (CCA) is an aggressive disease with limited effective treatment options. The PI3K/Akt/mTOR pathway represents an attractive therapeutic target due to its frequent dysregulation in CCA. MK2206, an allosteric Akt inhibitor, has been shown to reduce cellular proliferation in other cancers. We hypothesized that MK2206 mediated inhibition of Akt would impact CCA cellular viability. STUDY METHODS: Post treatment with MK2206 (0-2 µM), cellular viability was assessed in two human CCA cell lines-CCLP-1 and SG231-using an MTT assay. Lysates from the MK2206 treated CCA cells were then examined for apoptotic marker expression levels using Western blot analysis. Additionally, the effect on cellular proliferation of MK2206 treatment on survivin depleted cells was determined. RESULTS: CCLP-1 and SG231 viability was significantly reduced at MK2206 concentrations of 0.5, 1, and 2 µM by approximately 44%, 53%, and 64% (CCLP-1; p = 0.01) and 32%, 32%, and 42% (SG231; p < 0.00005) respectively. Western analysis revealed a decrease in AKT(Ser473), while AKT(Thr308) expression was unchanged. In addition, cleaved PARP as well as survivin expression increased while pro-caspase 3 and 9 levels decreased with treatment. Depletion of survivin in CCLP-1 cells resulted in apoptosis as evidenced by increased cleaved PARP. In addition, survivin siRNA further enhanced the antitumor activity of MK2206. CONCLUSIONS: This study demonstrates that by blocking phosphorylation of Akt at serine473, CCA cellular growth is reduced. The growth suppression appears to be mediated via apoptosis. Importantly, combination of survivin siRNA transfection and MK2206 treatment significantly decreased cell viability.

Curcumin-mediated regulation of Notch1/hairy and enhancer of split-1/survivin: molecular targeting in cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma (CCA) is highly malignant and characterized by poor prognosis with chemotherapeutic resistance. Therefore, continued development of novel, effective approaches are needed. Notch expression is markedly upregulated in CCA, but the utility of Notch1 inhibition is not defined. Based on recent findings, we hypothesized that curcumin, a polyphenolic phytochemical, suppresses CCA growth in vitro via inhibition of Notch1 signaling. METHODS: Established CCA cell lines CCLP-1 and SG-231 were treated with varying concentrations of curcumin (0-20 µM). Viability was assessed through 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and clonogenic assays. Evaluation of apoptosis was determined via Western analysis for apoptotic markers and Caspase-Glo 3/7 assay. Cell lysates were further analyzed via Western blotting for Notch1/HES-1/survivin pathway expression, cell cycle progression, and survival. RESULTS: Curcumin-treated CCA cells exhibited reduced viability compared with control treatment. Statistically significant reductions in cell viability were observed with curcumin treatment at concentrations of 7.5, 10, and 15 µM by approximately 10%, 48%, and 56% for CCLP-1 and 13%, 25%, and 50% for SG-231, respectively. On Western analysis, concentrations of ≥10 µM showed reductions in Notch1, HES-1, and survivin. Apoptosis was evidenced by an increase in expression of cleaved poly [ADP] ribose polymerase and an increase in caspase activity. Cyclin D1 (cell cycle progression) expression levels were also reduced with treatment. CONCLUSIONS: Curcumin effectively induces CCA (CCLP-1 and SG-231) growth suppression and apoptosis at relatively low treatment concentrations when compared with the previous research. A concomitant reduction of Notch1, HES-1, and survivin expression in CCA cell lines provides novel evidence for a potential antitumorigenic mechanism-of-action. To our knowledge, this is the first report showing reduction in HES-1 expression via protein analysis after treatment with curcumin. Such findings merit further investigation of curcumin-mediated inhibition of Notch signaling in CCA either alone or in combination with chemotherapeutic agents.

Glycogen synthase kinase-3 inhibitor AR-A014418 suppresses pancreatic cancer cell growth via inhibition of GSK-3-mediated Notch1 expression.

BACKGROUND: Glycogen synthase kinase-3 (GSK-3) can act as either a tumour promoter or suppressor by its inactivation depending on the cell type. There are conflicting reports on the roles of GSK-3 isoforms and their interaction with Notch1 in pancreatic cancer. It was hypothesized that GSK-3α stabilized Notch1 in pancreatic cancer cells thereby promoting cellular proliferation. METHODS: The pancreatic cancer cell lines MiaPaCa2, PANC-1 and BxPC-3, were treated with 0-20 µM of AR-A014418 (AR), a known GSK-3 inhibitor. Cell viability was determined by the MTT assay and Live-Cell Imaging. The levels of Notch pathway members (Notch1, HES-1, survivin and cyclinD1), phosphorylated GSK-3 isoforms, and apoptotic markers were determined by Western blot. Immunoprecipitation was performed to identify the binding of GSK-3 specific isoform to Notch1. RESULTS: AR-A014418 treatment had a significant dose-dependent growth reduction (P < 0.001) in pancreatic cancer cells compared with the control and the cytotoxic effect is as a result of apoptosis. Importantly, a reduction in GSK-3 phosphorylation lead to a reduction in Notch pathway members. Overexpression of active Notch1 in AR-A014418-treated cells resulted in the negation of growth suppression. Immunoprecipitation analysis revealed that GSK-3α binds to Notch1. CONCLUSIONS: This study demonstrates for the first time that the growth suppressive effect of AR-A014418 on pancreatic cancer cells is mainly mediated by a reduction in phosphorylation of GSK-3α with concomitant Notch1 reduction. GSK-3α appears to stabilize Notch1 by binding and may represent a target for therapeutic development. Furthermore, downregulation of GSK-3 and Notch1 may be a viable strategy for possible chemosensitization of pancreatic cancer cells to standard therapeutics.

MK-2206 causes growth suppression and reduces neuroendocrine tumor marker production in medullary thyroid cancer through Akt inhibition.

BACKGROUND: Development of targeted therapies for medullary thyroid cancer (MTC) has focused on inhibition of the rearranged during transfection (RET) proto-oncogene. Akt has been demonstrated to be a downstream target of RET via the key mediator phosphoinositide-3-kinase. MK-2206 is an orally administered allosteric Akt inhibitor that has exhibited minimal toxicity in phase I trials. We explored the antitumor effects of this compound in MTC. METHODS: Human MTC-TT cells were treated with MK-2206 (0-20 µM) for 8 days. Assays for cell viability were performed at multiple time points with MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide). The mechanism of action, mechanism of growth inhibition, and production of neuroendocrine tumor markers were assessed with Western blot analysis. RESULTS: MK-2206 suppressed MTC cell proliferation in a dose-dependent manner (p ≤ 0.001). Levels of Akt phosphorylated at serine 473 declined with increasing doses of MK-2206, indicating successful Akt inhibition. The apoptotic proteins cleaved poly (ADP-ribose) polymerase and cleaved caspase-3 increased in a dose-dependent manner with MK-2206, while the apoptosis inhibitor survivin was markedly reduced. Importantly, the antitumor effects of MK-2206 were independent of RET inhibition, as the levels of RET protein were not blocked. CONCLUSIONS: MK-2206 significantly suppresses MTC proliferation without RET inhibition. Given its high oral bioavailability and low toxicity profile, phase II studies with this drug alone or in combination with RET inhibitors are warranted.

Synergistic effect of pasireotide and teriflunomide in carcinoids in vitro.

BACKGROUND/AIM: Somatostatin (SST) analogs are mainstay for controlling tumor proliferation and hormone secretion in carcinoid patients. Recent data suggest that extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation may potentiate the anti-tumor effects of SST analogs in carcinoids. Additionally, ERK1/2 phosphorylating agents have been shown to suppress biomarker expression in carcinoids. Thus, Raf-1/MEK/ERK1/2 pathway activating drugs may be synergistic with SST analogs such as pasireotide (SOM230), which may be more effective than others in its class given its elevated receptor affinity and broader binding spectrum. Here, we investigate the effects of SOM230 in combination with teriflunomide (TFN), a Raf-1 activator, in a human carcinoid cell line. METHODS: Human pancreatic carcinoid cells (BON) were incubated in TFN, SOM230 or a combination. Cell proliferation was measured using a rapid colorimetric assay. Western analysis was performed to analyze expression levels of achaete-scute complex-like 1 (ASCL1), chromogranin A (CgA), phosphorylated and total ERK1/2, and markers for apoptosis. RESULTS: Combination treatment with SOM230 and TFN reduced cell growth beyond the additive effect of either drug alone. Combination indices (CI) fell below 1, thus quantifiably verifying synergy between both drugs as per the Chou-Talalay CI scale. Combined treatment also reduced ASCL1 and CgA expression beyond the additive effect of either drug alone. Furthermore, it increased levels of phosphorylated ERK1/2, cleaved poly(ADP)-ribose polymerase and caspase-3, and reduced levels of anti-apoptotic biomarkers. Elevated phosphorylated ERK1/2 expression following combination therapy may underlie the synergistic interaction between the two drugs. CONCLUSION: Since efficacy is achieved at lower doses, combination therapy may palliate symptoms at low toxicity levels. Because each drug has already been evaluated in clinical trials, combinatorial drug trials are warranted.

Mitochondria-targeted antioxidant and glycolysis inhibition: synergistic therapy in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. Mito-carboxy proxyl (Mito-CP), a lipophilic cationic nitroxide, accumulates in the mitochondria because of the large negative transmembrane potential. Studies have shown that these agents act by disrupting the energy-producing mechanism, inducing mitochondrial-mediated apoptosis, and also enhancing the action of other chemotherapeutic agents in cancer cells. We hypothesized that the combination of Mito-CP and glycolysis inhibitor, 2-deoxyglucose (2-DG), would synergistically inhibit HCC in vitro. HepG2 cells and primary hepatocytes were treated with various combinations of Mito-CP and 2-DG. Cell cytotoxicity was measured using the methylthiazolyldiphenyl-tetrazolium bromide assay and ATP bioluminescence assay. In addition, caspase 3/7 enzymatic activity was examined after treatment. Mito-CP and 2-DG induced synergistic cytotoxicity in HepG2 cells in a dose-dependent and time-dependent manner, whereas primary cells remained viable and unaffected after treatment. The intracellular ATP levels of HepG2 cells were suppressed within 6 h of combination treatment, whereas primary cells maintained higher levels of ATP. Dose-dependent increases in caspase 3/7 activity occurred in HepG2 cells in a time-dependent manner, showing the initiation of cell death through the apoptotic pathway. These findings indicate that a combination of Mito-CP and 2-DG effectively inhibits HCC growth in vitro. The increase in caspase 3/7 activity supports the occurrence of 2-DG-induced and Mito-CP-induced apoptotic death in HCC. The inability of the compounds to induce cytotoxicity or suppress the production of ATP in primary hepatocytes provides a selective and synergistic approach for the treatment of HCC.

Neuroendocrine phenotype alteration and growth suppression through apoptosis by MK-2206, an allosteric inhibitor of AKT, in carcinoid cell lines in vitro.

Carcinoids are neuroendocrine malignancies characterized by their overproduction of various bioactive hormones that lead to the carcinoid syndrome. We have shown previously that AKT serves as a key regulator of growth and phenotypic expression of tumor markers in carcinoids by the genetic depletion of AKT expression. However, no small-molecule inhibitor of AKT kinase activity has been developed until recently. MK-2206, a novel allosteric inhibitor of AKT, is currently undergoing clinical trials for the treatment of solid tumors. In this study, we explored the effect of MK-2206 on carcinoid cell proliferation and bioactive hormone production in vitro in two carcinoid cell lines - pancreatic carcinoid BON and bronchopulmonary H727. Treatment with MK-2206 effectively suppressed AKT phosphorylation at serine 473 and significantly reduced cell proliferation in a dose-dependent manner. Most importantly, MK-2206 treatment resulted in a significant reduction in ASCL1, CgA, and NSE expression, collectively recognized as markers of neuroendocrine tumor malignancy. Furthermore, MK-2206-treated cells showed an increase in levels of cleaved PARP and cleaved caspase-3, with a concomitant reduction in levels of Mcl-1 and XIAP, indicating that the antiproliferative effect of MK-2206 occurs through the induction of apoptosis. In conclusion, MK-2206 suppresses carcinoid tumor growth, and alters its neuroendocrine phenotype, indicating that this drug may be beneficial for patients with carcinoid syndrome. These studies merit further clinical investigation.

Leflunomide suppresses growth in human medullary thyroid cancer cells.

BACKGROUND: Medullary thyroid cancer (MTC) is a neuroendocrine tumor that arises from the calcitonin-secreting parafollicular cells of the thyroid gland. Leflunomide (LFN) is a disease-modifying antirheumatic drug approved for the treatment of rheumatoid arthritis, and its active metabolite teriflunomide has been identified as a potential anticancer drug. In this study we investigated the ability of LFN to similarly act as an anticancer drug by examining the effects of LFN treatment on MTC cells. METHODS: Human MTC-TT cells were treated with LFN (25-150 µmol/L) and Western blotting was performed to measure levels of neuroendocrine markers. MTT assays were used to assess the effect of LFN treatment on cellular proliferation. RESULTS: LFN treatment downregulated neuroendocrine markers ASCL1 and chromogranin A. Importantly, LFN significantly inhibited the growth of MTC cells in a dose-dependent manner. CONCLUSIONS: Treatment with LFN decreased neuroendocrine tumor marker expression and reduced the cell proliferation in MTC cells. As the safety of LFN in human beings is well established, a clinical trial using this drug to treat patients with advanced MTC may be warranted.

Molecular mechanisms of resistance to kinase inhibitors and redifferentiation in thyroid cancers.

Protein kinases play critical roles in cell survival, proliferation, and motility. Their dysregulation is therefore a common feature in the pathogenesis of a number of solid tumors, including thyroid cancers. Inhibiting activated protein kinases has revolutionized thyroid cancer therapy, offering a promising strategy in treating tumors refractory to radioactive iodine treatment or cytotoxic chemotherapies. However, despite satisfactory early responses, these drugs are not curative and most patients inevitably progress due to drug resistance. This review summarizes up-to-date knowledge on various mechanisms that thyroid cancer cells develop to bypass protein kinase inhibition and outlines strategies that are being explored to overcome drug resistance. Understanding how cancer cells respond to drugs and identifying novel molecular targets for therapy still represents a major challenge for the treatment of these patients.

A preclinical and clinical study of lithium in low-grade neuroendocrine tumors.

BACKGROUND: Low-grade neuroendocrine tumors (NETs) respond poorly to chemotherapy; effective, less toxic therapies are needed. Glycogen synthase kinase (GSK)-3ß has been shown to regulate growth and hormone production in NETs. Use of lithium chloride in murine models suppressed carcinoid cell growth, reduced GSK-3ß levels, and reduced expression of chromogranin A. This study assessed the efficacy of lithium chloride in patients with NETs. DESIGN: Eligible patients had low-grade NETs. A single-arm, open-label phase II design was used. Lithium was dosed at 300 mg orally three times daily, titrated to serum levels of 0.8-1.0 mmol/L. The primary endpoint was objective tumor response by the Response Evaluation Criteria in Solid Tumors. Secondary endpoints included overall survival, progression-free survival, GSK-3ß phosphorylation, and toxicity. RESULTS: Fifteen patients were enrolled between October 3, 2007 and July 17, 2008, six men and nine women. The median age was 58 years. Patient diagnoses were carcinoid tumor for eight patients, islet cell tumor for five patients, and two unknown primary sites. Eastern Cooperative Oncology Group performance status scores were 0 or 1. Two patients came off study because of side effects. The median progression-free survival interval was 4.50 months. There were no radiographic responses. Because of an early stopping rule requiring at least one objective response in the first 13 evaluable patients, the study was closed to further accrual. Patients had pre- and post-therapy biopsies. CONCLUSIONS: Lithium chloride was ineffective at obtaining radiographic responses in our 13 patients who were treated as part of this study. Based on the pre- and post-treatment tumor biopsies, lithium did not potently inhibit GSK-3ß at serum levels used to treat bipolar disorders.

Resveratrol induces Notch2-mediated apoptosis and suppression of neuroendocrine markers in medullary thyroid cancer.

BACKGROUND: Currently, complete surgical resection is the only curative option for medullary thyroid cancer (MTC). Previous work has shown the Notch pathway is a potent tumor suppressor in MTC and that resveratrol activates the Notch pathway in carcinoid cancer, a related neuroedocrine malignancy. In this study, we hypothesized that the effects observed on carcinoid cells could be extended to MTC. METHODS: MTC cells treated with varying doses of resveratrol were assayed for viability by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. Western blot analysis for achaete-scute complex-like 1 (ASCL1), chromogranin A (CgA), full-length and cleaved caspase 3, and poly-ADP ribose polymerase (PARP) was performed. Quantitative real-time polymerase chain reaction (qPCR) was used to measure relative mRNA expression. RESULTS: Treatment with resveratrol resulted in growth suppression and an increase in the cleavage of caspase-3 and PARP. A dose-dependent inhibition of ASCL1, a neuroedocrine transcription factor, was observed at the protein and mRNA levels. Protein levels of CgA, a marker of hormone secretion, were also reduced after treatment with resveratrol. A dose-dependent induction of Notch2 mRNA was observed by qPCR. CONCLUSIONS: Resveratrol suppresses in vitro growth, likely through apoptosis, as demonstrated by cleavage of caspase-3 and PARP. Furthermore, resveratrol decreased neuroedocrine markers ASCL1 and chromogranin A. Induction of Notch2 mRNA suggests that this pathway may be central in the anti-MTC effects observed.

Expression of the active Notch1 decreases MTC tumor growth in vivo.

BACKGROUND: Human medullary thyroid cancer (MTC) is a neuroendocrine (NE) tumor, derived from thyroid C-cells. Besides surgery, there are no curative therapies for MTC. This emphasizes the need for the development of new therapies. In MTC, Notch1 signaling pathway is absent and Notch1 activation in MTC-TT cells has been shown to reduce growth and NE markers in vitro. While the in vitro studies will provide insight into the potential mechanisms by which Notch inhibits growth, only by in vivo model one can recreate the conditions found in patients with MTC and assess effects on metastatic potential and microscopic disease. MATERIALS AND METHODS: Doxycycline inducible TT-NOTCH1 cells were utilized in a murine subcutaneous xenograft model to study tumor development and growth. Doxycycline was used to induce the expression of Notch1 in these tumors. RESULTS: Measurements of tumor volume showed that doxycycline treated mice had slower tumor growth than control mice. Western blot analysis of tumor lysates demonstrated activation of Notch1 protein only in doxycycline treated mice suggesting that active Notch1 slowed tumor growth. Furthermore, this activation led to a significant reduction in the levels of achaete-scute complex-like1 and chromogranin A important NE markers. CONCLUSION: Based on these data, activation of Notch signaling pathway could be a therapeutic strategy to treat patients with MTC.

Antiproliferative effect of chrysin on anaplastic thyroid cancer.

BACKGROUND: Anaplastic thyroid cancer (ATC) is an undifferentiated, aggressive malignancy, for which there are no effective therapies. Though ATCs only make up less than 2% of all thyroid cancer cases, they represent over half of the thyroid cancer-related deaths. Chrysin, a natural flavonoid, has recently been reported as a potential anti-cancer agent. However, the effect of this compound on ATC cells is not known. Thus, in this study, we evaluated the antiproliferative nature of chrysin in ATC cells. METHODS: HTH7 and KAT18 cells, derived from patients with ATC, were treated with chrysin (25-50 µM) for up to 6 d. Cell proliferation was measured every 2 d using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Western blot analysis for molecular makers of apoptosis was carried out to investigate the effect and mechanism of Chrysin on ATC. RESULTS: Chrysin inhibited proliferation of HTH7 and KAT18 in a dose- and time-dependent manner. HTH7 and KAT18 cells with Chrysin treatment showed a significant increase in cleaved caspase-3, cleaved PolyADP Ribose Polymerase (PARP), along with a decrease in cyclin D1, Mcl-1, and XIAP. Furthermore, the ratio of Bax to Bcl-2 expression in ATC cells revealed an increase after the treatment. CONCLUSIONS: Chrysin inhibits growth in ATC cells via apoptosis in vitro. Therefore, the natural flavonoid chrysin warrants further clinical investigation as a new potential drug for the treatment for ATC.

Notch 1 signaling is active in ovarian cancer.

OBJECTIVE.: Despite advances in chemotherapy and radical surgery, most advanced stage ovarian cancer patients die from their disease, highlighting the need for the development of novel treatment strategies. The Notch signaling pathway plays an important role in cellular differentiation, proliferation and apoptosis. We hypothesized that the active form of Notch 1, the Notch 1 intracellular domain (NICD), would be overexpressed in ovarian cancer cells and that depletion of NICD would lead to growth reduction. METHODS.: Following institutional review board approval, NICD expression was analyzed in human ovarian cancer specimens as well as the ovarian cancer cell lines OVCAR3, SKOV3, and CaOV3. In addition, the effects of Notch 1 depletion on ovarian cancer cell growth were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) growth assay for 6 days following transfection with siRNA against Notch 1. RESULTS.: Western blot analysis revealed abundant NICD expression in all 3 ovarian cancer cell lines, as well as in 16 of 21 (76%) human ovarian cancer samples. Following treatment with Notch 1 siRNA, expression of NICD was greatly reduced in all three cell lines. Furthermore, this depletion of NICD was associated with significant growth inhibition of all three ovarian cancer cell lines. CONCLUSIONS.: NICD was frequently expressed in ovarian cancer cell lines and human ovarian cancer specimens. Importantly, depletion of Notch 1 led to growth inhibition of ovarian cancer cells. These findings support the hypothesis that Notch 1 plays a role in ovarian cancer proliferation, encouraging the investigation of this pathway as a therapeutic target.

Inhibition of growth in medullary thyroid cancer cells with histone deacetylase inhibitors and lithium chloride.

BACKGROUND: While representing only 3% of thyroid malignancies, medullary thyroid cancer (MTC) accounts for 14% of thyroid cancer deaths. MTC has a high rate of recurrence and lacks effective treatments. The histone deacetylase (HDAC) inhibitors valproic acid (VPA) and suberoyl bis-hydroxamic acid (SBHA) activate the Notch1 signaling pathway, while lithium chloride inhibits the glycogen synthase kinase-3ss (GSK-3ss) pathway. These compounds have been shown to limit growth and suppress hormonal secretion; thus, targeting different signaling pathways may be an effective treatment. METHODS: MTC cells were treated with varying combinations of up to 20 mM lithium chloride with either 3 mM VPA or 20 muM SBHA for 48 h. Western analysis was used to measure the effects on Notch1, GSK-3ss, and neuroendocrine (NE) markers. Growth was assessed by a methylthiazolyldiphenyl-tetrazolium (MTT) bromide cellular proliferation assay. Western analysis was used to determine the mechanism of growth regulation. RESULTS: Combination therapy increased active Notch1, inhibited the GSK-3ss pathway, and decreased NE markers. Additive inhibition of growth was observed with combination therapy. Lower-dose combination therapy achieved greater decreases on NE markers and growth than treatment with any of the drugs alone. Moreover, an increase in the cleavage of the apoptotic markers caspase-3 and PARP was observed. CONCLUSIONS: Combination therapy with lithium chloride and HDAC inhibitors suppresses NE markers and decreases growth via apoptosis of MTC cells in vitro. With the possibility of increased efficacy and decreased toxicity, combination therapy may represent a new strategy to treat MTC.

ZM336372 induces apoptosis associated with phosphorylation of GSK-3beta in pancreatic adenocarcinoma cell lines.

INTRODUCTION: ZM336372 is small molecule tyrosine kinase modulator. It has been shown to inhibit glycogen synthase kinase-3beta (GSK-3beta) through phosphorylation of GSK-3beta at Ser 9. GSK-3beta has previously been shown to mediate cell survival in pancreatic cancer cells. Here we determine the effects of ZM336372 on GSK-3beta phosphorylation, apoptosis, and growth in pancreatic adenocarcinoma cell lines. METHODS: Panc-1 and MiaPaCa-2 cells were treated with ZM336372 or lithium chloride (LiCl) and compared with solvent control. The effects on proliferation for each cell line were determined using the MTT assay. Western blot analysis was performed to examine the effects of treatment on the phosphorylation of GSK-3beta. In addition, western blot was utilized to examine the cleavage of poly (ADP-ribose) polymerase (PARP), a marker of apoptosis. RESULTS: A dose-dependent increase in phosphorylation of GSK-3beta was observed after treatment with both ZM336372 and LiCl. Growth inhibition due to treatment with ZM336372 and LiCl also occurred in a dose-dependent fashion. An increase in cleaved PARP was demonstrated after treatment with both agents, as was seen previously with GSK-3beta inhibition in pancreatic adenocarcinoma cells. CONCLUSION: This is the first description of growth inhibition and apoptosis in pancreatic cancer cells related to GSK-3beta inhibition through treatment with ZM336372.

MG-132 inhibits carcinoid growth and alters the neuroendocrine phenotype.

BACKGROUND: Carcinoid cancers are the most common neuroendocrine (NE) tumors, and limited treatment options exist. The inhibition of glycogen synthase kinase-3beta (GSK-3beta) has been shown to be a potential therapeutic target for the treatment of carcinoid disease. In this study, we investigate the ability of MG-132, a proteasome inhibitor, to inhibit carcinoid growth, the neuroendocrine phenotype, and its association with GSK-3beta. MATERIALS AND METHODS: Human pulmonary (NCI-H727) and gastrointestinal (BON) carcinoid cells were treated with MG-132 (0-4microM). Cellular growth was measured by the 3-[4,5-dimethylthiazole-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Levels of total and phosphorylated GSK-3beta and the NE markers chromogranin A (CgA), Achaete-Scute complex-like 1 (ASCL1), as well as the apoptotic markers poly (ADP-ribose), polymerase (PARP), and cleaved caspase-3 were determined by Western blot. RESULTS: Treating carcinoid cells with MG-132 resulted in growth inhibition, a dose-dependent inhibition of CgA and ASCL1, as well as an increase in the levels of cleaved PARP and cleaved caspase-3. Additionally, an increase in the level of phosphorylated GSK-3beta was observed. CONCLUSION: MG-132 inhibits cellular growth and the neuroendocrine phenotype. This proteasome inhibitor warrants further preclinical investigation as a possible therapeutic strategy for intractable carcinoid disease.

Tautomycetin and tautomycin suppress the growth of medullary thyroid cancer cells via inhibition of glycogen synthase kinase-3beta.

Medullary thyroid cancer (MTC) is a relatively uncommon neuroendocrine tumor that arises from the calcitonin-secreting parafollicular cells of the thyroid gland. Unfortunately, MTC frequently metastasizes, precluding curative surgical resection and causing significant morbidity. Thus, there is an urgent need for new treatment modalities. Tautomycin and tautomycetin are antifungal antibiotics isolated from Streptomyces spiroverticillatus and Streptomyces griseochromogens, respectively. Glycogen synthase kinase-3beta is a serine/threonine protein kinase that regulates multiple cellular processes and is important in various cancers, including MTC. Treatment with tautomycin and tautomycetin decreased neuroendocrine markers, suppressed hormonal secretion, and inhibited growth through apoptosis in MTC cells. Importantly, we describe a novel action of these compounds: inhibition of glycogen synthase kinase-3beta.

A novel gene panel for prediction of lymph-node metastasis and recurrence in patients with thyroid cancer.

BACKGROUND: Although well-differentiated papillary thyroid cancer may remain indolent, lymph node metastases and the recurrence rates are approximately 50% and 20%, respectively. No current biomarkers are able to predict metastatic lymphadenopathy and recurrence in early stage papillary thyroid cancer. Hence, identifying prognostic biomarkers predicting cervical lymph-node metastases would prove very helpful in determining treatment. METHODS: The database of the Cancer Genome Atlas included 495 papillary thyroid cancer samples. Using this database, we developed a machine learning model to define a gene signature that could predict lymph-node metastasis (N0 or N1). Kruskal-Wallis tests, univariate and multivariate logistic and Cox regression models, and Kaplan-Meier analyses were performed to correlate the gene signature with clinical outcomes. RESULTS: We identified a panel of 25 genes and constructed a risk score that can differentiate N0 and N1 papillary thyroid cancer samples (P < .001) with a sensitivity of 86%, a specificity of 62%, a positive predictive value of 93%, and a negative predictive value of 42%. This panel represents an independent biomarker to predict metastatic lymphadenopathy (OR = 8.06, P < .001) specifically in patients with T1 lesions (OR = 7.65, P = .002) and disease-free survival (HR = 2.64, P = .043). CONCLUSION: This novel 25-gene panel may be used as a potential prognostic marker for accurately predicting lymph-node metastasis and disease-free survival in patients with early-stage papillary thyroid cancer.