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Background Glioblastoma multiforme is the most common form of primary brain tumor, often characterized by poor survival. Glioblastoma initiating cells (GICs) regulate self-renewal, differentiation, and tumor initiation properties and are involved in tumor growth, recurrence, and resistance to conventional treatments. The sonic hedgehog (SHH) signaling pathway is essential for normal development and embryonic morphogenesis. The objectives of this study were to examine the molecular mechanisms by which GIC characteristics are regulated by NPV-LDE-225 (Smoothened inhibitor; (2,2'-[[dihydro-2-(4-pyridinyl)-1,3(2H,4H)-pyrimidinediyl]bis(methylene)]bis[N,N-dimethylbenzenamine). Methods Cell viability and apoptosis were measured by XTT and annexin V-propidium iodide assay, respectively. Gli translocation and transcriptional activities were measured by immunofluorescence and luciferase assay, respectively. Gene and protein expressions were measured by quantitative real-time PCR and Western blot analyses, respectively. Results and conclusion NPV-LDE-225 inhibited cell viability, neurosphere formation, and Gli transcriptional activity and induced apoptosis by activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase. NPV-LDE-225 increased the expression of tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-R1/DR4, TRAIL-R2/DR5, and Fas and decreased the expression of platelet derived growth factor receptor-α and Bcl2, and these effects were abrogated by Gli1 plus Gli2 short hairpin RNAs. NPV-LDE-225 enhanced the therapeutic potential of FasL and TRAIL by upregulating Fas and DR4/5, respectively. Interestingly, NPV-LDE-225 induced expression of programmed cell death 4 and apoptosis and inhibited cell viability by suppressing micro RNA (miR)-21. Furthermore, NPV-LDE-225 inhibited pluripotency-maintaining factors Nanog, Oct4, Sox2, and cMyc. The inhibition of Bmi1 by NPV-LDE-225 was regulated by induction of miR-128. Finally, NPV-LDE-225 suppressed epithelial-mesenchymal transition by upregulating E-cadherin and inhibiting N-cadherin, Snail, Slug, and Zeb1 through modulating the miR-200 family. Our data highlight the importance of the SHH pathway for self-renewal and early metastasis of GICs.

Multiple lines of evidence suggest that the Sonic Hedgehog (Shh) signaling pathway is aberrantly reactivated in pancreatic cancer stem cells (CSCs). The objectives of this study were to examine the molecular mechanisms by which GANT-61 (Gli transcription factor inhibitor) regulates stem cell characteristics and tumor growth. Effects of GANT-61 on CSC's viability, spheroid formation, apoptosis, DNA-binding and transcriptional activities, and epithelial-mesenchymal transition (EMT) were measured. Humanized NOD/SCID/IL2R gamma(null) mice were used to examine the effects of GANT-61 on CSC's tumor growth. GANT-61 inhibited cell viability, spheroid formation, and Gli-DNA binding and transcriptional activities, and induced apoptosis by activation of caspase-3 and cleavage of Poly-ADP ribose Polymerase (PARP). GANT-61 increased the expression of TRAIL-R1/DR4, TRAIL-R2/DR5 and Fas, and decreased expression of PDGFRα and Bcl-2. GANT-61 also suppressed EMT by up-regulating E-cadherin and inhibiting N-cadherin and transcription factors Snail, Slug and Zeb1. In addition, GANT-61 inhibited pluripotency maintaining factors Nanog, Oct4, Sox-2 and cMyc. Suppression of both Gli1 plus Gli2 by shRNA mimicked the changes in cell viability, spheroid formation, apoptosis and gene expression observed in GANT-61-treated pancreatic CSCs. Furthermore, GANT-61 inhibited CSC tumor growth which was associated with up-regulation of DR4 and DR5 expression, and suppression of Gli1, Gli2, Bcl-2, CCND2 and Zeb1 expression in tumor tissues derived from NOD/SCID IL2Rγ null mice. Our data highlight the importance of Shh pathway for self-renewal and metastasis of pancreatic CSCs, and also suggest Gli as a therapeutic target for pancreatic cancer in eliminating CSCs.

Dysregulation of the sonic hedgehog (Shh) signaling pathway has been associated with cancer stem cells (CSC) and implicated in the initiation of pancreatic cancer. Pancreatic CSCs are rare tumor cells characterized by their ability to self-renew, and are responsible for tumor recurrence accompanied by resistance to current therapies. The lethality of these incurable, aggressive and invasive pancreatic tumors remains a daunting clinical challenge. Thus, the objective of this study was to investigate the role of Shh pathway in pancreatic cancer and to examine the molecular mechanisms by which sulforaphane (SFN), an active compound in cruciferous vegetables, inhibits self-renewal capacity of human pancreatic CSCs. Interestingly, we demonstrate here that Shh pathway is highly activated in pancreatic CSCs and plays important role in maintaining stemness by regulating the expression of stemness genes. Given the requirement for Hedgehog in pancreatic cancer, we investigated whether hedgehog blockade by SFN could target the stem cell population in pancreatic cancer. In an in vitro model, human pancreatic CSCs derived spheres were significantly inhibited on treatment with SFN, suggesting the clonogenic depletion of the CSCs. Interestingly, SFN inhibited the components of Shh pathway and Gli transcriptional activity. Interference of Shh-Gli signaling significantly blocked SFN-induced inhibitory effects demonstrating the requirement of an active pathway for the growth of pancreatic CSCs. SFN also inhibited downstream targets of Gli transcription by suppressing the expression of pluripotency maintaining factors (Nanog and Oct-4) as well as PDGFRα and Cyclin D1. Furthermore, SFN induced apoptosis by inhibition of BCL-2 and activation of caspases. Our data reveal the essential role of Shh-Gli signaling in controlling the characteristics of pancreatic CSCs. We propose that pancreatic cancer preventative effects of SFN may result from inhibition of the Shh pathway. Thus Sulforaphane potentially represents an inexpensive, safe and effective alternative for the management of pancreatic cancer.

Activation of the sonic hedgehog (Shh) pathway is required for the growth of numerous tissues and organs and recent evidence indicates that this pathway is often recruited to stimulate growth of cancer stem cells (CSCs) and to orchestrate the reprogramming of cancer cells via epithelial mesenchymal transition (EMT). The objectives of this study were to examine the molecular mechanisms by which (-)-epigallocatechin-3-gallate (EGCG), an active compound in green tea, inhibits self-renewal capacity of pancreatic CSCs, and synergizes with quercetin, a major polyphenol and flavonoid commonly detected in many fruits and vegetables. Our data demonstrated that EGCG inhibited the expression of pluripotency maintaining transcription factors (Nanog, c-Myc and Oct-4), and self-renewal capacity of pancreatic CSCs. Inhibition of Nanog by shRNA enhanced the inhibitory effects of EGCG on self-renewal capacity of CSCs. EGCG inhibited cell proliferation and induced apoptosis by inhibiting the expression of Bcl-2 and XIAP, and activating caspase-3. Interestingly, EGCG also inhibited the components of Shh pathway (smoothened, patched, Gli1 and Gli2) and Gli transcriptional activity. Furthermore, EGCG inhibited EMT by inhibiting the expression of Snail, Slug and ZEB1, and TCF/LEF transcriptional activity, which correlated with significantly reduced CSC’s migration and invasion, suggesting the blockade of signaling involved in early metastasis. Furthermore, combination of quercetin with EGCG had synergistic inhibitory effects on self-renewal capacity of CSCs through attenuation of TCF/LEF and Gli activities. Since aberrant Shh signaling occurs in pancreatic tumorigenesis, therapeutics that target Shh pathway may improve the outcomes of patients with pancreatic cancer by targeting CSCs.

Parkinson's disease (PD) is associated with perturbed mitochondrial function. Studies of cytoplasmic hybrid (cybrid) cell lines containing mitochondria from PD subjects suggest complex I dysfunction in particular is a relatively upstream biochemical defect. To evaluate potential downstream consequences of PD mitochondrial dysfunction, we used a cybrid approach to model PD mitochondrial dysfunction; our cybrid cell lines were generated via transfer of PD or control subject platelet mitochondria to mtDNA-depleted NT2 cells. To confirm our PD cybrid mitochondria did indeed differ from control cybrid mitochondria we measured complex I V(max) activities. Consistent with other PD cybrid reports, relative to control cybrid cell lines the PD cybrid cell line mean complex I V(max) activity was reduced. In this validated model, we used an oxygen electrode to characterize PD cybrid mitochondrial respiration. Although whole cell basal oxygen consumption was comparable between the PD and control cybrid groups, the proton leak was increased and maximum respiratory capacity was decreased in the PD cybrids. PD cybrids also had reduced SIRT1 phosphorylation, reduced peroxisome proliferator-activated receptor-gamma coactivator-1alpha levels, and increased NF-kB activation. We conclude mitochondrial respiration and pathways influenced by aerobic metabolism are altered in NT2 cybrid cell lines generated through transfer of PD subject platelet mitochondria.

Cytochrome oxidase (COX) activity varies between individuals and low activities associate with Alzheimer's disease. Whether genetic heterogeneity influences function of this multimeric enzyme is unknown. To explore this we sequenced three mitochondrial DNA (mtDNA) and ten nuclear COX subunit genes from at least 50 individuals. 20% had non-synonymous mtDNA COX gene polymorphisms, 12% had a COX4I1 non-synonymous G to A transition, and other genes rarely contained non-synonymous polymorphisms. Frequent untranslated region (UTR) polymorphisms were seen in COX6A1, COX6B1, COX6C, and COX7A1; heterogeneity in a COX7A1 5' UTR Sp1 site was extensive. Synonymous polymorphisms were common and less frequent in the more conserved COX1 than the less conserved COX3, suggesting at least in mtDNA synonymous polymorphisms experience selection pressure and are not functionally silent. Compound gene variations occurred within individuals. To test whether variations could have functional consequences, we studied the COX4I1 G to A transition and an AGCCCC deletion in the COX7A1 5' UTR Sp1 site. Cells expressing the COX4I1 polymorphism had reduced COX Vmax activity. In reporter construct-transduced cells where green fluorescent protein expression depended on the COX7A1 Sp1 site, AGCCCC deletion reduced fluorescence. Our findings indicate COX subunit gene heterogeneity is pervasive and may mediate COX functional variation.

Mitochondrial dysfunction has severe cellular consequences, and is linked to aging and neurological disorders in humans. Impaired energy supply or Ca2+ buffering, increased ROS production, or control of apoptosis by mitochondria may contribute to the progressive decline of long-lived postmitotic cells. Mitochondrial biogenesis refers to the process via which cells increase their individual mitochondrial mass. Mitochondrial biogenesis may represent an attempt by cells to increase their aerobic set point, or an attempt to maintain a pre-existing aerobic set point in the face of declining mitochondrial function. Neuronal mitochondrial biogenesis itself has been poorly studied, but investigations from other tissues and model systems suggest a series of transcription factors, transcription co-activators, and signal transduction proteins should function to regulate mitochondrial number and mass within neurons. We review data pertinent to the mitochondrial biogenesis field, and discuss implications for brain aging and neurodegenerative disease research efforts.

Prostate cancer stem cells (CSCs) are defined by their extensive self-renewal, differentiation, and tumor initiation properties. It is now clear that CSCs are involved in tumor growth and recurrence, and resistance to conventional treatments. Thus, the strategy that suppresses stemness and consequently tumorigenic potential of CSCs could be considered for the management of prostate cancer. The objectives of this study were to examine the molecular mechanisms by which NPV-LDE-225 / Erismodegib (smoothened inhibitor) regulates stem cell characteristics in prostate cancer. Effects of NPV-LDE-225 on CSC's viability, sphere formation, apoptosis, transcriptional activity, and epithelial-mesenchymal transition (EMT) were measured. NPV-LDE-225 inhibited cell viability and spheroid formation, and induced apoptosis by activation of caspase-3 and cleavage of PARP. NPV-LDE-225 induced expression of Bax and Bak, and inhibited the expression of Bcl-2, Bcl-XL, XIAP, cIAP1, cIAP2 and survivin. NPV-LDE-225 inhibited Gli transcriptional activity, Gli-DNA interaction, and the expression of Gli1, Gli2, Patched1 and Patched 2 in prostate CSCs. Interestingly, NPV-LDE-225 induced PDCD4 and apoptosis and inhibited cell viability by suppressing miR-21. Furthermore, NPV-LDE-225 inhibited pluripotency maintaining factors Nanog, Oct4, cMyc and Sox-2. The inhibition of Bmi-1 by NPV-LDE-225 was regulated by up-regulation of miR-128. NPV-LDE-225 suppressed EMT by up-regulating E-cadherin and inhibiting N-cadherin, Snail, Slug and Zeb1 through regulating miR-200 family. Finally, NPV-LDE-225 inhibited CSC tumor growth which was associated with the suppression of Gli1, Gli2, Patched-1, Patched-2, Cyclin D1 and PCNA, and cleaved caspase-3 and PARP in tumor tissues derived from NOD/SCID IL2Rϒnull mice. Overall, our findings suggest that inhibition of the Shh signaling pathway in CSCs is a potential therapeutic strategy for prostate cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A261. Citation Format: Rajesh Nanta, Daniel Meeker, Mariana Rodova, Peter J. Van Veldhuizen, Sharmila Shankar, Rakesh K. Srivastava. NPV-LDE-225 (Erismodegib) inhibits human prostate cancer stem cell growth in NOD/SCID IL2γnull mice by regulating Bmi-1 and microRNA-128. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A261.

Multiple lines of evidence suggest that the Sonic Hedgehog (Shh) signaling pathway is aberrantly reactivated in pancreatic cancer stem cells (CSCs). The objectives of this study were to examine the molecular mechanisms by which GANT-61 (Gli transcription factor inhibitor) regulates stem cell characteristics and tumor growth. Effects of GANT-61 on CSC's viability, spheroid formation, apoptosis, DNA-binding and transcriptional activities, and epithelial-mesenchymal transition (EMT) were measured. Humanized NOD/SCID/IL2Rgammanull mice were used to examine the effects of GANT-61 on CSC's tumor growth. GANT-61 inhibited cell viability, spheroid formation, and Gli-DNA binding and transcriptional activities, and induced apoptosis by activation of caspase-3 and cleavage of Poly-ADP ribose Polymerase (PARP). GANT-61 increased the expression of TRAIL-R1/DR4, TRAIL-R2/DR5 and Fas, and decreased expression of PDGFRα and Bcl-2. GANT-61 also suppressed EMT by up-regulating E-cadherin and inhibiting N-cadherin and transcription factors Snail, Slug and Zeb1. In addition, GANT-61 inhibited pluripotency maintaining factors Nanog, Oct4, Sox-2 and cMyc. Suppression of both Gli1 plus Gli2 by shRNA mimicked the changes in cell viability, spheroid formation, apoptosis and gene expression observed in GANT-61-treated pancreatic CSCs. Furthermore, GANT-61 inhibited CSC tumor growth which was associated with up-regulation of DR4 and DR5 expression, and suppression of Gli1, Gli2, Bcl-2, CCND2 and Zeb1 expression in tumor tissues derived from NOD/SCID IL2Rγ null mice. Our data highlight the importance of Shh pathway for self-renewal and metastasis of pancreatic CSCs, and also suggest Gli as a therapeutic target for pancreatic cancer in eliminating CSCs. Citation Format: Junsheng Fu, Mariana Rodova, Sanjit K. Roy, Sharmila Shankar, Rakesh K. Srivastava. GANT-61 inhibits pancreatic cancer stem cell growth in vitro and in NOD/SCID/IL2R gamma null mice xenograft. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4012. doi:10.1158/1538-7445.AM2013-4012

The existence of cancer stem cells (CSCs) in prostate cancer has profound implications for cancer prevention and/or treatment. The CSC population resides within the prostate tumor mass and they may be responsible for drug resistance, and for the recurrence of prostate cancer. Therefore, development of novel strategies to target CSCs by nontoxic agent resveratrol is an innovative and novel idea for the management of prostate cancer. Activation of Wnt, sonic hedgehog (Shh) and Notch pathways are required for the growth of numerous tissues and organs and recent evidence indicates that these pathways are often recruited to regulate self-renewal and metastasis of CSCs. The objectives of this study were to examine the molecular mechanisms by which resveratrol, an active compound in grapes and red vine inhibits self-renewal capacity of prostate CSCs. Our data demonstrated that resveratrol inhibited the expression of pluripotency maintaining transcription factors (Nanog, c-Myc and Oct-4), and spheroid formation by prostate CSCs. Inhibition of Nanog by shRNA enhanced the inhibitory effects of resveratrol on self-renewal capacity of CSCs. Resveratrol suppressed cell proliferation and induced apoptosis by inhibiting the expression of Bcl-2 and IAPs, and activating caspase-3. Interestingly, resveratrol also inhibited the transcriptional activities TCF/LEF, Gli and RBP-Jk. Furthermore, resveratrol inhibited epithelial-mesenchymal transition (EMT) by suppressing the expression of Snail, Slug and ZEB1 transcription factors, which correlated with significantly reduced CSC's migration and invasion, suggesting the blockade of signaling involved in early metastasis. In conclusion, resveratrol can inhibit prostate cancer CSC characteristics by suppressing various signal transduction pathways, and can be used for the management of prostate cancer. Citation Format: Rakesh K. Srivastava, Mariana Rodova, Junsheng Fu, Dara Nall, Sharmila Shankar. Resveratrol inhibits prostate cancer stem cell characteristics by suppressing Wnt, sonic hedgehog, and Notch pathways [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A45.