Th1 cytokines in pediatric acute lymphoblastic leukemia.

Immune milieus play an important role in various types of cancer. The present study focuses on the effect of Th1 cytokines on pediatric acute lymphoblastic leukemia (ALL). The reaction of ALL cell lines and patient-derived xenografts (PDX) to the most important Th1 cytokines TNF-α (tumor necrosis factor alpha) and IFN-γ (interferon gamma) is analyzed and correlated with the respective cytokine receptors and the intracellular signaling molecules. ALL cell lines and ALL PDX display a great heterogeneity in cell death after incubation with TNF-α and IFN-γ. Several samples show a dose-dependent and additive induction of cell death by both cytokines; others do not react at all or even display an increased viability. Apoptosis is the main type of cell death induced by Th1 cytokines in ALL cells. Over all leukemia cells analyzed, IFN-γ receptor (IFNGR) shows a higher expression than both TNF-receptors, resulting in higher phosphorylation of STAT1 (signal transducer and activator of transcription) compared to phosphorylation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B-cells) in the TNF pathway. The activation of STAT1 correlates with the amount of cell death after stimulation with Th1 cytokines. TNF-α and IFN-γ lead to heterogeneous reactions in ALL cell lines and ALL PDX but are able to induce cell death by apoptosis in the majority of ALL blasts. The correlation of a high expression of IFNGR and following activation of STAT1 with cell death indicates an important role for IFN-γ signaling in this setting.

Epigallocatechin Gallate Inhibits Cell Growth and Hedgehog Signalling in Human Rhabdomyosarcoma Cell Lines.

BACKGROUND/AIM: Epigallocatechin gallate (EGCG), a major polyphenol of green tea, has been shown to inhibit cancer cell proliferation. In this study the effect of EGCG on cell metabolism and the human hedgehog signalling pathway (HH) in human rhabdomyosarcoma (RMS) cells was investigated. MATERIALS AND METHODS: Two human RMS cell lines (RD and RH30) were incubated with EGCG. To evaluate the effects of EGCG on RMS cells, cell viability, colony formation, cell migration, and alteration of genes related to the HH signalling pathway were investigated. RESULTS: EGCG showed cytostatic effects on RMS cells in a dose-dependent manner. Incubation with 25 µM EGCG resulted in a significant reduction of cell migration by 70% and downregulation of the HH pathway transcription factor GLI1. CONCLUSION: EGCG inhibits RMS cells in vitro by reducing cell proliferation and downregulating the HH signalling pathway. It may therefore be a promising agent in chemoresistant or advanced RMS in children.

Combination therapy of doxorubicin and Sildenafil inhibits the growth of pediatric rhabdomyosarcoma.

BACKGROUND: Overexpression of phosphodiesterase type 5 (PDE5) has been detected in many types of malignant tumors. Sildenafil, a potent and selective inhibitor of a cGMP-specific PDE5, has been found to enhance the cytotoxic activity of different chemotherapeutic agents including doxorubicin. The combined therapy of doxorubicin with Sildenafil might reduce the possible side effects of chemotherapy while maintaining synergistic anticancer activity. The present study explored for the first time the effects of Sildenafil, alone and in combination with doxorubicin, on pediatric RMS cells. METHODS: Human RMS cells lines RH30 and RD were used. Western blotting and RT-PCR were performed to analyze PDE5 expression in RMS cells. Cell viability was determined using MTT assay. Cell migration was analyzed via transwell chambers, clonal growth and mitotic cell death were analyzed using the clonogenic assay. FACS analysis was performed to evaluate reactive oxygen species (ROS) and apoptosis. RESULTS: Sildenafil significantly decreased cell viability and migration of RD and RH30 cells. The exposure of RMS cells to doxorubicin resulted in a dose-dependent decrease in their viability. Simultaneous administration of Sildenafil enhanced this effect. The incubation of the RMS cells with Sildenafil in the presence of doxorubicin significantly increased the proportion of apoptotic cells and ROS production compared to the treatment with Sildenafil alone. CONCLUSIONS: The results of our study indicate a link between PDE5 inhibition by Sildenafil and decreased calcium signalling leading to significantly impaired viability, migration, and colony forming of RMS cells. Increased ROS production and apoptosis are mechanisms relevantly contributing to this observation.

Anticancer bioactivity of zerumbone on pediatric rhabdomyosarcoma cells.

PURPOSE: Natural products are generally regarded as safe and have been shown to mediate anticancer activities against a variety of cell types. Zerumbone is a natural cyclic sesquiterpene derived from the rhizome of Zingiber zerumbet, which has attracted extensive attention in the recent decade for anticancer activities. The present study investigates the in vitro effect of zerumbone on rhabdomyosarcoma cells. METHODS: Two rhabdomyosarcoma cell lines (RD and RH30) were used as the model system. The growth inhibition of zerumbone was measured by MTT-assay, apoptosis via flow cytometry, gene expression by real-time PCR, the migration by transwell assay, and intracellular signaling by Western blotting. RESULTS: Zerumbone shows anticancer effects on RD and RH30 cells in a dose-dependent manner via cell growth inhibition and induction of apoptosis. Exposure of RD and RH30 cells on zerumbone also resulted in a decrease of migration and downregulation of the hedgehog pathway. CONCLUSIONS: Taken together, our study provided the first evidence that zerumbone imparted strong inhibitory and apoptotic effects on pediatric rhabdomyosarcoma cell lines and merit further investigation as a promising candidate for the anticancer therapy.

Epitope Detection in Monocytes (EDIM) As a New Method of Liquid Biopsy in Pediatric Rhabdomyosarcoma.

Biomarkers allowing characterization of pediatric rhabdomyosarcoma (RMS) are lacking. Epitope detection in monocytes (EDIM) is a novel method focused on detection of the biomarkers TKTL1 (transketolase-like protein 1) and Apo10 (epitope of DNaseX) in activated monocytes (CD14+/CD16+) from patient's blood. We investigated the expression of these biomarkers in RMS cell lines, tumor material, and peripheral blood from RMS patients. Expression levels of TKTL1 and DNaseX/Apo10 in RMS cell lines (RH30, RD) and tumor samples were analyzed by RT-PCR and flow cytometry. Blood samples of 29 RMS patients were measured and compared to 27 healthy individuals. The percentages of activated CD14+/CD16+ monocytes harboring TKTL1 and Apo10 were determined. EDIM-TKTL1 and EDIM-Apo10 expression scores were calculated. The relationship between TKTL1 expression and DNA-hypomethylation was evaluated. Both RMS cell lines and tumor samples showed significantly higher expression levels of TKTL1 and DNaseX/Apo10 compared to skeletal muscle cells (SkMC). EDIM-TKTL1 and EDIM-Apo10 scores were positive in 96.5% of patients with RMS. All healthy controls had negative corresponding scores. RMS cell lines show increased expression levels of the biomarkers TKTL1 and DNaseX/Apo10. The sensitivity of the EDIM blood test indicates that this assay might serve as an additional tool in pediatric RMS.

Epitope detection in monocytes (EDIM) for liquid biopsy including identification of GD2 in childhood neuroblastoma-a pilot study.

BACKGROUND: Neuroblastoma (NB) is the most common paediatric extracranial solid malignancy. We analysed the role of the epitope detection in monocytes (EDIM) technique for liquid biopsy in NB patients. METHODS: Tumour epitopes transketolase-like 1 (TKTL1), Apo10 (DNaseX) and GD2 were assessed: expression levels in seven NB tumour samples and five NB cell lines were analysed using RT-PCR and flow cytometry. LAN-1 cells were co-cultured with blood and assessed using EDIM. Peripheral blood macrophages of patients with neuroblastoma (n = 38) and healthy individuals (control group, n = 37) were labelled (CD14+/CD16+) and assessed for TKTL1, Apo10 and GD2 using the EDIM technology. RESULTS: mRNA expression of TKTL1 and DNaseX/Apo10 was elevated in 6/7 NB samples. Spike experiments showed upregulation of TKTL1, Apo10 and GD2 in LAN-1 cells following co-culturing with blood. TKTL1 and Apo10 were present in macrophages of 36/38 patients, and GD2 in 15/19 patients. The 37 control samples were all negative. EDIM expression scores of the three epitopes allowed differentiation between NB patients and healthy individuals. CONCLUSIONS: The EDIM test might serve as a non-invasive tool for liquid biopsy in children suffering from NB. Future studies are necessary for assessing risk stratification, tumour biology, treatment monitoring, and early detection of tumour relapses.

D,L-Methadone enhances the cytotoxic activity of standard chemotherapeutic agents on pediatric rhabdomyosarcoma.

PURPOSE: In advanced tumor stages, pediatric rhabdomyosarcoma (RMS) shows an intrinsic resistance to standard chemotherapy, which is associated with a dismal prognosis. Alternative therapeutic approaches and optimization of already existent treatment protocols are urgently needed in these conditions. The µ-opioid receptor (OPRM1) agonist, D,L-methadone is frequently used for analgesia in oncological patients. Recent evidence has shown that D,L-methadone in combination with chemotherapeutic agents may enhance their cytotoxic effect against cancer cells. There are no related data in pediatric rhabdomyosarcoma (RMS). METHODS: Antitumor effects of combined D,L-methadone and doxorubicin, carboplatin, and vincristine on RMS cell lines RD and RH30 were analyzed using following outcome data: expression of the OPRM1 receptor (Western blot), cell growth inhibition (MTT assay), cell migration (wound-healing assay), apoptosis induction (caspase-3/7 assay), and reactive oxygen species (ROS) production (flow cytometry). RESULTS: In both cell lines, OPRM1 expression was significantly increased after combined treatment of D,L-methadone with all three cytotoxic drugs tested, which resulted in suppression of tumor cell growth and increase of apoptosis rates. These effects were mediated by increased ROS production and up-regulation of caspase-3/7 activity. Doxorubicin combined with D,L-methadone significantly reduced cell migration in both cell lines. Carboplatin or vincristine in combination with D,L-methadone had only an impact on cell migration in RH30 cells. CONCLUSIONS: This new therapeutic approach in RMS provides strong antitumor effects in vitro. The combination of standard chemotherapy and D,L-methadone requires further investigation. Especially advanced tumors with a limited effectiveness of conventional treatment regimens seem a potential target of this approach.

Hepatoblastoma: glutamine depletion hinders cell viability in the embryonal subtype but high GLUL expression is associated with better overall survival.

PURPOSE: Glutamine plays an important role in cell viability and growth of various tumors. For the fetal subtype of hepatoblastoma, growth inhibition through glutamine depletion was shown. We studied glutamine depletion in embryonal cell lines of hepatoblastoma carrying different mutations. Since asparagine synthetase was identified as a prognostic factor and potential therapeutic target in adult hepatocellular carcinoma, we investigated the expression of its gene ASNS and of the gene GLUL, encoding for glutamine synthetase, in hepatoblastoma specimens and cell lines and investigated the correlation with overall survival. METHODS: We correlated GLUL and ASNS expression with overall survival using publicly available microarray and clinical data. We examined GLUL and ASNS expression by RT-qPCR and by Western blot analysis in the embryonal cell lines Huh-6 and HepT1, and in five hepatoblastoma specimens. In the same cell lines, we investigated the effects of glutamine depletion. Hepatoblastoma biopsies were examined for histology and CTNNB1 mutations. RESULTS: High GLUL expression was associated with a higher median survival time. Independent of mutations and histology, hepatoblastoma samples showed strong GLUL expression and glutamine synthesis. Glutamine depletion resulted in the inhibition of proliferation and of cell viability in both embryonal hepatoblastoma cell lines. ASNS expression did not correlate with overall survival. CONCLUSION: Growth inhibition resulting from glutamine depletion, as described for the hepatoblastoma fetal subtype, is also detected in established embryonal hepatoblastoma cell lines carrying different mutations. At variance with adult hepatocellular carcinoma, in hepatoblastoma asparagine synthetase has no prognostic significance.

Antitumor effects of curcumin in pediatric rhabdomyosarcoma in combination with chemotherapy and phototherapy in vitro.

Rhabdomyosarcoma (RMS), the most common pediatric soft tissue sarcoma, has an unfavorable outcome in advanced tumor stages with less than 30% failure­free survival. Curcumin (CUR) is a promising drug in complementary oncology with few side effects but proven efficacy in various adult oncological entities. The present study analyzed the effects of CUR on pediatric (RMS) cell lines in vitro. RMS cell lines (RD and RH30), and skeletal muscle cells (SKMC) were treated with different doses of CUR (1.5­30 µM) alone, with phototherapy (PDT, 488 nm) or in combination with vincristine (VCR) or dactinomycin (DAC). MTT assays were used for analysis of RMS tumor cell viability. Clonal cell growth was assessed via colony forming assays and migration of the cells was analyzed with scratch tests. Annexin V staining was used to determine apoptosis in flow cytometry. Possible RMS resistance towards CUR after long­term treatment was analyzed with MTT assays. CUR decreased cell viability in all assessed RMS cell lines in a concentration­dependent manner with IC50=14­20 µM. CUR enhanced the effects of the cytotoxic drugs VCR or DAC, and led to reduced migration and increased cell apoptosis. In combination with PDT, CUR decreased the cell viability in minute quantities with up to a 10­fold lower IC50 than without PDT. CUR effectively inhibited the malignant properties of pediatric RMS cells and should be focused on as a useful additional agent in standard chemotherapy of RMS in children.

Extracellular vesicles released by myeloid-derived suppressor cells from pregnant women modulate adaptive immune responses.

Immunological pregnancy complications are a main challenge in reproductive medicine. Mechanisms regulating the adaptation of the maternal immune system to pregnancy are incompletely understood and therapeutic options limited. Myeloid derived suppressor cells (MDSC) are immune-modulatory cells expanding during healthy pregnancy and seem to play a crucial role for maternal-fetal tolerance. Recent studies showed that exosomes produced by MDSC have immune-modulatory effects corresponding to their parental cells under different pathological conditions. Here, we investigated immunological effects of exosomes of GR-MDSC during pregnancy. Isolated GR-MDSC exosomes from peripheral blood of pregnant women were tested for functionality in different in vitro assays. We show that GR-MDSC exosomes exhibited profound immune-modulatory effects such as suppression of T-cell proliferation, T helper 2 (Th2)-cell polarization, induction of regulatory T-cells and inhibition of lymphocyte cytotoxicity. Our results confirm that MDSC-derived exosomes functionally correspond to their parental cells and identify them as an interesting therapeutic target for immunological pregnancy complications.

The Effect of Direct and Indirect EZH2 Inhibition in Rhabdomyosarcoma Cell Lines.

Enhancer of Zeste homolog 2 (EZH2) is involved in epigenetic regulation of gene transcription by catalyzing trimethylation of histone 3 at lysine 27. In rhabdomyosarcoma (RMS), increased EZH2 protein levels are associated with poor prognosis and increased metastatic potential, suggesting EZH2 as a therapeutic target. The inhibition of EZH2 can be achieved by direct inhibition which targets only the enzyme activity or by indirect inhibition which also affects activities of other methyltransferases and reduces EZH2 protein abundance. We assessed the direct inhibition of EZH2 by EPZ005687 and the indirect inhibition by 3-deazaneplanocin (DZNep) and adenosine dialdehyde (AdOx) in the embryonal RD and the alveolar RH30 RMS cell line. EPZ005687 was more effective in reducing the cell viability and colony formation, in promoting apoptosis induction, and in arresting cells in the G1 phase of the cell cycle than the indirect inhibitors. DZNep was more effective in decreasing spheroid viability and size in both cell lines than EPZ005687 and AdOx. Both types of inhibitors reduced cell migration of RH30 cells but not of RD cells. The results show that direct and indirect inhibition of EZH2 affect cellular functions differently. The alveolar cell line RH30 is more sensitive to epigenetic intervention than the embryonal cell line RD.

Cytotoxic drugs in combination with the CXCR4 antagonist AMD3100 as a potential treatment option for pediatric rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common type of pediatric soft tissue sarcoma. The prognosis of advanced stage RMS remains poor, and metastatic invasion is a major cause of treatment failure. Therefore, there is an urgent need for treatment alternatives focusing on metastatic invasion and drug resistance. The stromal cell­derived factor­1 (SDF­1)/chemokine receptor 4 (CXCR4) axis is a crucial factor for metastatic invasion in RMS. Clinical data has revealed that high CXCR4 expression is associated with a poor outcome and a high metastatic rate in several malignancies, including RMS. Thus, targeting CXCR4 in addition to classical chemotherapy may improve the effectiveness of RMS treatment. In the present study, flow cytometry and reverse transcription­quantitative PCR were used to assess the effects of the combined treatment with a CXCR4 antagonist and chemotherapy on CXCR4 expression in the embryonal RMS (RME) cell line RD and in the alveolar RMS (RMA) cell line RH30. The functional effect of CXCR4 expression on the migratory behavior of RMS cells was analyzed using Transwell assays. Treatment with cytotoxic agents modulated CXCR4 expression in RMS cells in a dose­, drug­ and cell line dependent manner; however, this was not observed in RD cells with vincristine. The expression levels of CXCR4 significantly increased the migratory behavior of RMA and did not affect RME cell migration towards stromal cell­derived factor­1α (SDF­1α). AMD3100 markedly reduced the migration of RH30 cells in the Transwell assays compared with SDF­1α alone, and the cytotoxic agents doxorubicin and vincristine increased this effect. The results of the combined treatment in RMS cells using the CXCR4 antagonist AMD3100 together with cytotoxic drugs demonstrated that this approach may be a promising alternative for the treatment of advanced stage pediatric RMS. The observed effects of circumventing metastatic invasion and drug resistance should be further investigated in vivo.

The histone demethylase KDM2B activates FAK and PI3K that control tumor cell motility.

Recent studies revealed that the histone demethylase KDM2B regulates the epithelial markers E-Cadherin and ZO-1, the RhoA/B/C-small-GTPases and actin cytoskeleton organization, in DU-145 prostate- and HCT-116 colon-tumor cells. Here we addressed the role of KDM2B in the activation of Focal Adhesion Kinase (FAK)-signaling and its involvement in regulating tumor cell motility. We used RT-PCR for gene transcriptional analysis, Western blotting for the assessment of protein expression and activity and wound-healing assay for the study of cell migration. KDM2B overexpression or silencing controls the activity of FAK in DU-145 prostate- and HCT-116 colon-tumor cells without affecting gene transcription and protein expression of this kinase. Upon KDM2B overexpression in DU-145 cells, significantly enhanced migration was observed, which was abolished in cells pretreated by the specific phosphoinositide-3 kinase (PI3 K) inhibitor LY294002, implying involvement of FAK/PI3 K signaling in the migration process. In line with this, the p85-PI3 K-subunit was downregulated upon knockdown of KDM2B in DU-145 cells, while the opposite effect became evident in KDM2B-overexpressing cells. These results revealed a novel functional role of KDM2B in regulating the activation of the FAK/PI3 K signaling in prostate cancer cells that participates in the control of cell motility.

Sildenafil triggers tumor lethality through altered expression of HSP90 and degradation of PKD2.

The repurposing of existing drugs has emerged as an attractive additional strategy to the development of novel compounds in the fight against cancerous diseases. Inhibition of phosphodiesterase 5 (PDE5) has been claimed as a potential approach to target various cancer subtypes in recent years. However, data on the treatment of tumors with PDE5 inhibitors as well as the underlying mechanisms are as yet very scarce. Here, we report that treatment of tumor cells with low concentrations of Sildenafil was associated with decreased cancer cell proliferation and augmented apoptosis in vitro and resulted in impaired tumor growth in vivo. Notably, incubation of cancer cells with Sildenafil was associated with altered expression of HSP90 chaperone followed by degradation of protein kinase D2, a client protein previously reported to be involved in tumor growth. Furthermore, the involvement of low doses of PU-H71, an HSP90 inhibitor currently under clinical evaluation, in combination with low concentrations of Sildenafil, synergistically and negatively impacted on the viability of cancer cells in vivo. Taken together, our study suggests that repurposing of already approved drugs, alone or in combination with oncology-dedicated compounds, may represent a novel cancer therapeutic strategy.

The Epigenetic Factor KDM2B Regulates EMT and Small GTPases in Colon Tumor Cells.

BACKGROUND/AIMS: The epigenetic factor KDM2B is a histone demethylase expressed in various tumors. Recently, we have shown that KDM2B regulates actin cytoskeleton organization, small Rho GTPases signaling, cell-cell adhesion and migration of prostate tumor cells. In the present study, we addressed its role in regulating EMT and small GTPases expression in colon tumor cells. METHODS: We used RT-PCR for the transcriptional analysis of various genes, Western blotting for the assessment of protein expression and immunofluorescence microscopy for visualization of fluorescently labeled proteins. RESULTS: We report here that KDM2B regulates EZH2 and BMI1 in HCT116 colon tumor cells. Knockdown of this epigenetic factor induced potent up-regulation of the protein levels of the epithelial markers E-cadherin and ZO-1, while the mesenchymal marker N-cadherin was downregulated. On the other hand, KDM2B overexpression downregulated the levels of both epithelial markers and upregulated the mesenchymal marker, suggesting control of EMT by KDM2B. In addition, RhoA, RhoB and RhoC protein levels diminished upon KDM2B-knockdown, while all three small GTPases became upregulated in KDM2B-overexpressing HCT116 cell clones. Interestingly, Rac1 GTPase level increased upon KDM2B-knockdown and diminished in KDM2B-overexpressing HCT116 colon tumor- and DU-145 prostate cancer cells. CONCLUSIONS: These results establish a clear functional role of the epigenetic factor KDM2B in the regulation of EMT and small-GTPases expression in colon tumor cells and further support the recently postulated oncogenic role of this histone demethylase in various tumors.

The epigenetic factor KDM2B regulates cell adhesion, small rho GTPases, actin cytoskeleton and migration in prostate cancer cells.

The histone demethylase KDM2B is an epigenetic factor with oncogenic properties that is regulated by the basic fibroblasts growth factor (FGF-2). It has recently been shown that KDM2B co-operates with Polycomb Group proteins to promote cell migration and angiogenesis in tumors. In the present study we addressed the role of KDM2B in regulating actin cytoskeleton signaling, cell-cell adhesion and migration of prostate tumor cells. We report here that KDM2B is functionally expressed in DU-145 prostate cancer cells, activated by FGF-2 and regulates EZH2. KDM2B knockdown induced potent up-regulation of gene transcription and protein expression of the epithelial markers E-cadherin and ZO-1, while KDM2B overexpression down-regulated the levels of both markers, suggesting control of cell adhesion by KDM2B. RhoA and RhoB protein expression and activity were diminished upon KDM2B-knockdown and upregulated in KDM2B-overexpressing cell clones. In accordance, actin reorganization with formation of stress fibers became evident in KDM2B-overexpressing cells and abolished in the presence of the Rho inhibitor C3 transferase. DU-145 cell migration was significantly enhanced in KDM2B overexpressing cells and abolished in C3-pretreated cells. Conversely, the retardation of cell migration observed in KDM2B knockdown cells was enhanced in C3-pretreated cells. These results establish a clear functional link between the epigenetic factor KDM2B and the regulation of cell adhesion and Rho-GTPases signaling that controls actin reorganization and cell migration.

Serum and Glucocorticoid Inducible Kinase 1-Sensitive Survival, Proliferation and Migration of Rhabdomyosarcoma Cells.

BACKGROUND/AIMS: Rhabdomyosarcoma, the most common pediatric soft tissue sarcoma, may show an intrinsic refractoriness to standard chemotherapy in advanced tumor stages, which is associated with poor prognosis. Cellular mechanisms conferring tumor cell survival and therapy resistance in many tumor types include the serum & glucocorticoid inducible kinase (SGK) 1 pathway, a kinase expressed ubiquitously with particularly strong expression in skeletal muscle and some tumor types. The present study explored whether SGK1 is expressed in rhabdomyosarcoma and, if so, whether this kinase impacts on tumor cell survival, proliferation and migration. Multiple in vitro techniques were used to study the role of SGK1 in rhabdomyosarcoma. METHODS: The Gene Chip® Human Genome U133 Plus 2.0 Array were employed to examine SGK1 transcriptional activity in healthy muscle and rhabdomyosarcoma tissue. SGK1 transcript levels were quantified in rhabdomyosarcoma cell lines RD (embryonal subtype) and RH30 (alveolar subtype) by RT-PCR, cell viability was measured using MTT assays. Clonal cell growth was assessed via colony forming assays and migration experiments were performed in a transwell system. RESULTS: SGK1 is expressed in embryonal and alveolar rhabdomyosarcoma tissue samples and in RD and RH30 rhabdomyosarcoma cell lines. Administration of EMD638683 - an inhibitor specific for SGK1 - decreased viability of RD and RH30 cells, enhanced the effects of the cytotoxic drug doxorubicin leading to reduced migration and decreased cell proliferation. CONCLUSIONS: SGK1 is expressed in rhabdomyosarcoma cells where it contributes to survival, therapy resistance, cell proliferation and migration. Thus, SGK1 inhibitors may be considered a therapeutic option for the treatment of therapy-resistant rhabdomyosarcoma.

Istaroxime Inhibits Motility and Down-Regulates Orai1 Expression, SOCE and FAK Phosphorylation in Prostate Cancer Cells.

BACKGROUND/AIMS: Istaroxime is a validated inotropic Na+/K+ ATPase inhibitor currently in development for the treatment of various cardiac conditions. Recent findings established that this steroidal drug exhibits potent apoptotic responses in prostate tumors in vitro and in vivo, by affecting key signaling orchestrating proliferation and apoptosis, such as c-Myc and caspase 3, Rho GTPases and actin cytoskeleton dynamics. In the present study we examined whether istaroxime is affecting cell motility and analyzed the underlying mechanism in prostate tumor cells. METHODS: Migration was assessed by transwell and wound healing assays, Orai1 and Stim1 abundance by RT-PCR and confocal immunofluorescence microscopy, Fura-2 fluorescence was utilized to determine intracellular Ca2+ and Western blotting for FAK/pFAK measurements. RESULTS: We observed strong inhibition of cell migration in istaroxime treated DU-145 prostate cancer cells. Istaroxime further decreased Orai1 and Stim1 transcript levels and downregulated Orai1 protein expression. Moreover, SOCE was significantly decreased upon istaroxime treatment. Furthermore, istaroxime strikingly diminished phosphorylated FAK levels. Interestingly, the efficacy of istaroxime on the inhibition of DU-145 cell migration was further enhanced by blocking Orai1 with 2-APB and FAK with the specific inhibitor PF-00562271. These results provide strong evidence that istaroxime prevents cell migration and motility of DU-145 prostate tumor cells, an effect at least partially attributed to Orai1 downregulation and FAK de-activation. CONCLUSION: Collectively our results indicate that this enzyme inhibitor, besides its pro-apoptotic action, affects motility of cancer cells, supporting its potential role as a strong candidate for further clinical cancer drug development.

C-terminally truncated constitutively active androgen receptor variants and their biologic and clinical significance in castration-resistant prostate cancer.

A mechanism allowing castration resistant prostate cancer cells to escape the effects of conventional anti-hormonal treatments is the synthesis of constitutively active, C-terminally truncated androgen receptor (AR)-variants. Lacking the entire or vast parts of the ligand binding domain, the intended target of traditional endocrine therapies, these AR-variants (termed ARΔLBD) are insensitive to all traditional treatments including second generation compounds like abiraterone, enzalutamide or ARN-509. Although ARΔLBD are predominantly products of alternative splicing, they can also be products of nonsense mutations or proteolytic cleavage. In this review, we will discuss the etiology and function of c-terminally truncated AR-variants and their clinical significance as markers/targets for the treatment of castration resistant prostate cancer.

Chorein Sensitive Orai1 Expression and Store Operated Ca2+ Entry in Rhabdomyosarcoma Cells.

BACKGROUND: Chorein, a protein encoded by VPS13A (vacuolar protein sorting-associated protein 13A), is defective in chorea acanthocytosis, a rare disease characterized by acanthocytosis of red blood cells and neuronal cell death with progressive hyperkinetic movement disorder, cognitive dysfunction, behavioral abnormalities and chronic hyperkalemia. Chorein is highly expressed in ZF rhabdomyosarcoma cells and counteracts apoptosis of those cells. Chorein is effective in part by interacting with and fostering stimulation of phosphoinositide-3-kinase (PI3K)-p85-subunit. PI3K dependent signaling includes the serum and glucocorticoid inducible kinase SGK1. The kinase activates NFκB with subsequent up-regulation of the Ca2+ channel subunit Orai1, which accomplishes store operated Ca2+ entry (SOCE). Orai1 and SOCE have been shown to confer survival of tumor cells. The present study thus explored whether chorein impacts on Orai1 expression and SOCE. METHODS: In rhabdomyosarcoma cells chorein, Orai1, NFκB and SGK1 transcript levels were quantified by RT-PCR, Orai1 protein abundance by Western blotting, FACS analysis and confocal laser microscopy, [Ca2+]i utilizing Fura-2 fluorescence, and SOCE from the increase of [Ca2+]i following store depletion with extracellular Ca2+ removal and inhibition of the sarcoendoplasmatic reticular Ca2+ ATPase with thapsigargin. RESULTS: The mRNA coding for chorein was most abundant in drug resistant, poorly differentiated human ZF rhabdomyosarcoma cells. Chorein silencing significantly decreased Orai1 transcript levels and Orai1 protein expression, as well as SGK1 and NFκB transcript levels. SOCE in ZF rhabdomyosarcoma cells was significantly blunted by chorein silencing, Orai1 inhibitor 2-APB (50 µM), SGK1 inhibitor EMD638683 (50 µM, 10 h) and NFκB inhibitor wogonin (50 µM, 24 h). CONCLUSION: Chorein is a stimulator of Orai1 expression and thus of store operated Ca2+ entry. The effect may involve SGK1 and NFκB.