MICU1 drives glycolysis and chemoresistance in ovarian cancer.

Cancer cells actively promote aerobic glycolysis to sustain their metabolic requirements through mechanisms not always clear. Here, we demonstrate that the gatekeeper of mitochondrial Ca2+ uptake, Mitochondrial Calcium Uptake 1 (MICU1/CBARA1) drives aerobic glycolysis in ovarian cancer. We show that MICU1 is overexpressed in a panel of ovarian cancer cell lines and that MICU1 overexpression correlates with poor overall survival (OS). Silencing MICU1 in vitro increases oxygen consumption, decreases lactate production, inhibits clonal growth, migration and invasion of ovarian cancer cells, whereas silencing in vivo inhibits tumour growth, increases cisplatin efficacy and OS. Mechanistically, silencing MICU1 activates pyruvate dehydrogenase (PDH) by stimulating the PDPhosphatase-phosphoPDH-PDH axis. Forced-expression of MICU1 in normal cells phenocopies the metabolic aberrations of malignant cells. Consistent with the in vitro and in vivo findings we observe a significant correlation between MICU1 and pPDH (inactive form of PDH) expression with poor prognosis. Thus, MICU1 could serve as an important therapeutic target to normalize metabolic aberrations responsible for poor prognosis in ovarian cancer.

Gold Nanoparticle Reprograms Pancreatic Tumor Microenvironment and Inhibits Tumor Growth.

Altered tumor microenvironment (TME) arising from a bidirectional crosstalk between the pancreatic cancer cells (PCCs) and the pancreatic stellate cells (PSCs) is implicated in the dismal prognosis in pancreatic ductal adenocarcinoma (PDAC), yet effective strategies to disrupt the crosstalk is lacking. Here, we demonstrate that gold nanoparticles (AuNPs) inhibit proliferation and migration of both PCCs and PSCs by disrupting the bidirectional communication via alteration of the cell secretome. Analyzing the key proteins identified from a functional network of AuNP-altered secretome in PCCs and PSCs, we demonstrate that AuNPs impair secretions of major hub node proteins in both cell types and transform activated PSCs toward a lipid-rich quiescent phenotype. By reducing activation of PSCs, AuNPs inhibit matrix deposition, enhance angiogenesis, and inhibit tumor growth in an orthotopic co-implantation model in vivo. Auto- and heteroregulations of secretory growth factors/cytokines are disrupted by AuNPs resulting in reprogramming of the TME. By utilizing a kinase dead mutant of IRE1-α, we demonstrate that AuNPs alter the cellular secretome through the ER-stress-regulated IRE1-dependent decay pathway (RIDD) and identify endostatin and matrix metalloproteinase 9 as putative RIDD targets. Thus, AuNPs could potentially be utilized as a tool to effectively interrogate bidirectional communications in the tumor microenvironment, reprogram it, and inhibit tumor growth by its therapeutic function.

Cystathionine ß-synthase regulates endothelial function via protein S-sulfhydration.

Deficiencies of the human cystathionine ß-synthase (CBS) enzyme are characterized by a plethora of vascular disorders and hyperhomocysteinemia. However, several clinical trials demonstrated that despite reduction in homocysteine levels, disease outcome remained unaffected, thus the mechanism of endothelial dysfunction is poorly defined. Here, we show that the loss of CBS function in endothelial cells (ECs) leads to a significant down-regulation of cellular hydrogen sulfide (H2S) by 50% and of glutathione (GSH) by 40%. Silencing CBS in ECs compromised phenotypic and signaling responses to the VEGF that were potentiated by decreased transcription of VEGF receptor (VEGFR)-2 and neuropilin (NRP)-1, the primary receptors regulating endothelial function. Transcriptional down-regulation of VEGFR-2 and NRP-1 was mediated by a lack in stability of the transcription factor specificity protein 1 (Sp1), which is a sulfhydration target of H2S at residues Cys68 and Cys755. Reinstating H2S but not GSH in CBS-silenced ECs restored Sp1 levels and its binding to the VEGFR-2 promoter and VEGFR-2, NRP-1 expression, VEGF-dependent proliferation, and migration phenotypes. Thus, our study emphasizes the importance of CBS-mediated protein S-sulfhydration in maintaining vascular health and function.-Saha, S., Chakraborty, P. K., Xiong, X., Dwivedi, S. K. D., Mustafi, S. B., Leigh, N. R., Ramchandran, R., Mukherjee, P., Bhattacharya, R. Cystathionine ß-synthase regulates endothelial function via protein S-sulfhydration.

Role of cystathionine beta synthase in lipid metabolism in ovarian cancer.

Elevated lipid metabolism is implicated in poor survival in ovarian cancer (OC) and other cancers; however, current lipogenesis-targeting strategies lack cancer cell specificity. Here, we identify a novel role of cystathionine beta-synthase (CBS), a sulphur amino acid metabolizing enzyme highly expressed in several ovarian cancer cell lines, in driving deregulated lipid metabolism in OC. We examined the role of CBS in regulation of triglycerides, cholesterol and lipogenic enzymes via the lipogenic transcription factors SREBP1 and SREBP2. CBS silencing attenuated the expression of number of key enzymes involved in lipid synthesis (FASN and ACC1). Additionally CBS abrogates lipid uptake in OC cells. Gene silencing of CBS or SREBPs abrogated cellular migration and invasion in OC, while ectopic expression of SREBPs can rescue phenotypic effects of CBS silencing by restoring cell migration and invasion. Mechanistically, CBS represses SREBP1 and SREBP2 at the transcription levels by modulating the transcription factor Sp1. We further established the roles of both CBS and SREBPs in regulating ovarian tumor growth in vivo. In orthotopic tumor models, CBS or SREBP silencing resulted in reduced tumor cells proliferation, blood vessels formation and lipid content. Hence, cancer-selective disruption of the lipid metabolism pathway is possible by targeting CBS and, at least for OC, promises a profound benefit.

Understanding protein-nanoparticle interaction: a new gateway to disease therapeutics.

Molecular identification of protein molecules surrounding nanoparticles (NPs) may provide useful information that influences NP clearance, biodistribution, and toxicity. Hence, nanoproteomics provides specific information about the environment that NPs interact with and can therefore report on the changes in protein distribution that occurs during tumorigenesis. Therefore, we hypothesized that characterization and identification of protein molecules that interact with 20 nm AuNPs from cancer and noncancer cells may provide mechanistic insights into the biology of tumor growth and metastasis and identify new therapeutic targets in ovarian cancer. Hence, in the present study, we systematically examined the interaction of the protein molecules with 20 nm AuNPs from cancer and noncancerous cell lysates. Time-resolved proteomic profiles of NP-protein complexes demonstrated electrostatic interaction to be the governing factor in the initial time-points which are dominated by further stabilization interaction at longer time-points as determined by ultraviolet-visible spectroscopy (UV-vis), dynamic light scattering (DLS), ζ-potential measurements, transmission electron microscopy (TEM), and tandem mass spectrometry (MS/MS). Reduction in size, charge, and number of bound proteins were observed as the protein-NP complex stabilized over time. Interestingly, proteins related to mRNA processing were overwhelmingly represented on the NP-protein complex at all times. More importantly, comparative proteomic analyses revealed enrichment of a number of cancer-specific proteins on the AuNP surface. Network analyses of these proteins highlighted important hub nodes that could potentially be targeted for maximal therapeutic advantage in the treatment of ovarian cancer. The importance of this methodology and the biological significance of the network proteins were validated by a functional study of three hubs that exhibited variable connectivity, namely, PPA1, SMNDC1, and PI15. Western blot analysis revealed overexpression of these proteins in ovarian cancer cells when compared to normal cells. Silencing of PPA1, SMNDC1, and PI15 by the siRNA approach significantly inhibited proliferation of ovarian cancer cells and the effect correlated with the connectivity pattern obtained from our network analyses.

Pituitary homeobox 2 (PITX2) protects renal cancer cell lines against doxorubicin toxicity by transcriptional activation of the multidrug transporter ABCB1.

The multidrug resistance (MDR) P-glycoprotein ABCB1 plays a major role in MDR of malignant cells and is regulated by various transcription factors, including Wnt/ß-catenin/TCF4. The transcription factor PITX2 (Pituitary homeobox-2) is essential for embryonic development. PITX2 operates by recruiting and interacting with ß-catenin to increase the expression of growth-regulating genes, such as cyclin D1/2 and c-Myc. The importance of PITX2 in malignancy is not yet known. Here we demonstrate that in the renal cancer cell lines ACHN and A498, the level of ABCB1 expression and function correlate with nuclear PITX2 localization and PITX2-luciferase reporter gene activity (A498 > ACHN). In A498 cells, doxorubicin toxicity is augmented by the ABCB1 inhibitor, PSC833. PITX2 overexpression increases ABCB1 expression and cell survival in ACHN cells. Silencing of PITX2 by siRNA downregulates ABCB1 and induces a greater chemotherapeutic response to doxorubicin in A498 cells, as determined by MTT cell viability and clonogenic survival assays. Two PITX2 binding sequences were identified in the ABCB1 promoter sequence. PITX2 binding was confirmed by chromatin immunoprecipitation. ß-Catenin is not required for PITX2 upregulation of ABCB1 because ABCB1 mRNA increased and doxorubicin toxicity decreased upon PITX2 overexpression in ß-catenin(-/-) cells. The data show for the first time that ABCB1 is a target gene of PITX2 transcriptional activity, promoting MDR and cell survival of cancer cells.

ERK1/2-dependent bestrophin-3 expression prevents ER-stress-induced cell death in renal epithelial cells by reducing CHOP.

Upon endoplasmic reticulum (ER) stress induction, cells endeavor to survive by engaging the adaptive stress response known as the unfolded protein response or by removing aggregated proteins via autophagy. Chronic ER stress culminates in apoptotic cell death, which involves induction of pro-apoptotic CHOP. Here, we show that bestrophin-3 (Best-3), a protein previously associated with Ca(2+)-activated Cl(-) channel activity, is upregulated by the ER stressors, thapsigargin (TG), tunicamycin (TUN) and the toxic metal Cd(2+). In cultured rat kidney proximal tubule cells, ER stress, CHOP and cell death were induced after 6h by Cd(2+) (25µM), TG (3µM) and TUN (6µM), were associated with increased cytosolic Ca(2+) and downstream formation of reactive oxygen species and attenuated by the Ca(2+) chelator BAPTA-AM (10µM), the antioxidant α-tocopherol (100µM), or overexpression of catalase (CAT). Immunofluorescence staining showed Best-3 expression in the plasma membrane, nuclei and intracellular compartments, though not in the ER, in cultured cells and rat kidney cortex sections. Best-3 mRNA was augmented by ER stress and signaled through increased Ca(2+), oxidative stress and ERK1/2 phosphorylation, because it was attenuated by α-tocopherol, CAT expression, BAPTA-AM, calmodulin kinase inhibitor calmidazolium (40µM), ERK1/2 inhibitor U0126 (10µM), and ERK1/2 RNAi. Knockdown of Best-3 resulted in decreased cell number consequentially of cell death, as determined by nuclear staining and PARP-1 cleavage. Furthermore, reduced ER stress-cell death by Best-3 overexpression is attributed to diminished CHOP. Since Best-3 overexpression did not affect upstream signaling pathways, we hypothesize that Best-3 possibly interferes with CHOP transcription. From our novel observations, we conclude that ERK1/2-dependent Best-3 activation regulates cell fate decisions during ER stress by suppressing CHOP induction and death.

Chronic cadmium exposure induces transcriptional activation of the Wnt pathway and upregulation of epithelial-to-mesenchymal transition markers in mouse kidney.

The transition metal cadmium (Cd) is an environmental pollutant which damages the kidneys. Chronic Cd exposure may induce renal fibrosis and/or cancer, but the signaling pathways involved are not understood. The Wnt pathway is a key signaling cascade responsible for renal development, fibrosis and cancer. Hence the effect of chronic in vivo Cd exposure (100 mg/l drinking water for 12 weeks) on transcriptional activation of the Wnt pathway and markers of epithelial-to-mesenchymal transition (EMT) was investigated in mouse kidneys. Cd exposure increased kidney Cd content from 0.023+/-0.001 microg/g to 61+/-7 microg/g wet weight (means+/-S.D. of 6-7 animals). This was accompanied by increased expression of Wnt ligands (Wnt3a/6/7a/7b/9a/9b/10a/11), as determined by RT-PCR. The Wnt receptors Frizzled (Fz1/2/4,5,7-10) were also upregulated, as were the co-receptors low-density lipoprotein receptor-related proteins 5/6. Immunoblots with Wnt10a and Fz7 antibodies also revealed increased protein expression induced by Cd exposure. In contrast, Wnt antagonists were largely unaffected. Upregulation of Wnt signaling components induced by Cd was corroborated by increased expression of Wnt target genes, i.e. cell proliferation and survival genes c-Myc, cyclin D1 and the multidrug transporter P-glycoprotein Abcb1b, which promote malignancy. Lastly the EMT markers Twist, fibronectin and collagen I, but not alpha-smooth muscle actin, were also upregulated, suggesting that Cd-induced changes of renal epithelial tissue characteristics towards fibrosis and cancer may be mediated by Wnt signaling.

Cadmium induces Wnt signaling to upregulate proliferation and survival genes in sub-confluent kidney proximal tubule cells.

BACKGROUND: The class 1 carcinogen cadmium (Cd2+) disrupts the E-cadherin/beta-catenin complex of epithelial adherens junctions (AJs) and causes renal cancer. Deregulation of E-cadherin adhesion and changes in Wnt/beta-catenin signaling are known to contribute to carcinogenesis. RESULTS: We investigated Wnt signaling after Cd2+-induced E-cadherin disruption in sub-confluent cultured kidney proximal tubule cells (PTC). Cd2+ (25 microM, 3-9 h) caused nuclear translocation of beta-catenin and triggered a Wnt response measured by TOPflash reporter assays. Cd2+ reduced the interaction of beta-catenin with AJ components (E-cadherin, alpha-catenin) and increased binding to the transcription factor TCF4 of the Wnt pathway, which was upregulated and translocated to the nucleus. While Wnt target genes (c-Myc, cyclin D1 and ABCB1) were up-regulated by Cd2+, electromobility shift assays showed increased TCF4 binding to cyclin D1 and ABCB1 promoter sequences with Cd2+. Overexpression of wild-type and mutant TCF4 confirmed Cd2+-induced Wnt signaling. Wnt signaling elicited by Cd2+ was not observed in confluent non-proliferating cells, which showed increased E-cadherin expression. Overexpression of E-cadherin reduced Wnt signaling, PTC proliferation and Cd2+ toxicity. Cd2+ also induced reactive oxygen species dependent expression of the pro-apoptotic ER stress marker and Wnt suppressor CHOP/GADD153 which, however, did not abolish Wnt response and cell viability. CONCLUSIONS: Cd2+ induces Wnt signaling in PTC. Hence, Cd2+ may facilitate carcinogenesis of PTC by promoting Wnt pathway-mediated proliferation and survival of pre-neoplastic cells.

Modulation of Akt and ERK1/2 pathways by resveratrol in chronic myelogenous leukemia (CML) cells results in the downregulation of Hsp70.

BACKGROUND: Resveratrol is known to downregulate the high endogenous level of Heat shock protein 70 (Hsp70) in Chronic Myelogenous Leukemia (CML) K562 cells and induce apoptosis. Since Heat Shock Factor 1 (HSF1) controls transcription of Hsp70, we wanted to probe the signaling pathways responsible for transcriptional activation of HSF1. METHODOLOGY/PRINCIPAL FINDINGS: Cells exposed to 40microM Resveratrol rapidly abolished serine473 phosphorylation of Akt and significantly reduced its kinase activity. Inactivation of Akt pathway by Resveratrol subsequently blocked serine9 phosphorylation of Gsk3beta. Active non-phosphorylated Gsk3beta rendered HSF1 transcriptionally inactive and reduced Hsp70 production. Blocking PI3K/Akt activity also demonstrated similar effects on Hsp70 comparable to Resveratrol. Inactivation of Gsk3beta activity by inhibitors SB261763 or LiCl upregulated Hsp70. Resveratrol significantly modulated ERK1/2 activity as evident from hyper phosphorylation at T302/Y304 residues and simultaneous upregulation in kinase activity. Blocking ERK1/2 activation resulted in induction of Hsp70. Therefore, increase in ERK1/2 activity by Resveratrol provided another negative influence on Hsp70 levels through negative regulation of HSF1 activity. 17-allylamino-17-demethoxygeldanamycin (17AAG), a drug that inhibits Hsp90 chaperone and degrades its client protein Akt concomitantly elevated Hsp70 levels by promoting nuclear translocation of HSF1 from the cytosol. This effect is predominantly due to inhibition of both Akt and ERK1/2 activation by 17AAG. Simultaneously treating K562 with Resveratrol and 17AAG maintained phosho-ERK1/2 levels close to untreated controls demonstrating their opposite effects on ERK1/2 pathway. Resveratrol was found not to interfere with Bcr-Abl activation in K562 cells. CONCLUSION/SIGNIFICANCE: Thus our study comprehensively illustrates that Resveratrol acts downstream of Bcr-Abl and inhibits Akt activity but stimulates ERK1/2 activity. This brings down the transcriptional activity of HSF1 and Hsp70 production in K562 cells. Additionally, Resveratrol can be used in combination with chemotherapeutic agents such as 17AAG, an Hsp90 inhibitor reported to induce Hsp70 and hence compromise its chemotherapeutic potential.

Resveratrol induces apoptosis in K562 (chronic myelogenous leukemia) cells by targeting a key survival protein, heat shock protein 70.

Chronic myelogenous leukemia (CML) is a myeloproliferative disease associated with a characteristic chromosomal translocation called the Philadelphia chromosome. This results in the expression of the Bcr-Abl fusion protein, a constitutively active protein tyrosine kinase. Although there are a few treatment options with Bcr-Abl kinase inhibitors, drug resistance is often encountered. One of the major obstacles in overcoming drug resistance in CML is the high endogenous levels of heat shock protein 70 (Hsp70). Resveratrol is a phytoalexin produced by several plants. We studied the chemotherapeutic effects and mode of action of resveratrol on K562 (CML) cells. Resveratrol induced apoptosis in K562 cells in a time-dependent manner. This was established by increased annexin V binding, corroborated with an enhanced caspase-3 activity and a rise in the sub-G(0)/G(1) population. Resveratrol treatment also caused suppression of Hsp70 both in mRNA and protein levels. The downregulation of Hsp70 by resveratrol exposure was correlated with a diminished presence of heat shock factor 1 (HSF1) in the nucleus, and the downregulation of transcriptional activity of HSF1. High endogenous levels of Hsp70 have been found to be a deterrent for sensitivity to chemotherapy. We show here that resveratrol could considerably enhance the apoptosis induction in K562 cells by 17-allylamino-17-demethoxygeldanamycin, an anticancer agent that inhibits Hsp90 but augments Hsp70 levels. We conclude that resveratrol significantly downregulated Hsp70 levels through inhibition of HSF1 transcriptional activity and appreciably augmented the pro-apoptotic effects of 17-allylamino-17-demethoxygeldanamycin.

Interaction of piroxicam with mitochondrial membrane and cytochrome c.

Modulation of surface properties of biomembranes by any ligand leading to permeabilization, fusion, rupture, etc. is a fundamental requirement for many biological processes. In this work, we present the interaction of piroxicam, a long acting Non-Steroidal Anti-Inflammatory Drug (NSAID) with isolated mitochondria, membrane mimetic systems, intact cells and a mitochondrial protein cytochrome c. Dye permeabilization study on isolated mitochondria indicates that piroxicam can permeabilize mitochondrial membrane. Direct imaging by Scanning Electron Microscope (SEM) shows that piroxicam induces changes in mitochondrial membrane morphology leading to fusion and rupture. Transmission Electron Microscope (TEM) imaging of piroxicam treated DMPC vesicles and mixed micelles formed from CTAB and SDS show that causing membrane fusion is a general property of piroxicam at physiological pH. In intact cells viz., V79 Chinese Hamster lung fibroblast, piroxicam is capable of releasing cytochrome c from mitochondria into the cytosol in a dose dependent manner along with the enhancement of downstream proapoptotic event viz., increase in caspase-3 activity. We have also shown that piroxicam can reduce cytochrome c within a time frame relevant to its lifetime in blood plasma. UV-visible spectroscopy has been used to study the reaction mechanism and kinetics in detail, allowing us to propose and validate a Michaelis-Menten like reaction scheme. CD spectroscopy shows that small but significant changes occur in the structure of cytochrome c when reduced by piroxicam.

Aberrant interchromosomal exchanges are the predominant cause of the 22q11.2 deletion.

Chromosome 22q11.2 deletions are found in almost 90% of patients with DiGeorge/velocardiofacial syndrome (DGS/VCFS). Large, chromosome-specific low copy repeats (LCRs), flanking and within the deletion interval, are presumed to lead to misalignment and aberrant recombination in meiosis resulting in this frequent microdeletion syndrome. We traced the grandparental origin of regions flanking de novo 3 Mb deletions in 20 informative three-generation families. Haplotype reconstruction showed an unexpectedly high number of proximal interchromosomal exchanges between homologs, occurring in 19/20 families. Instead, the normal chromosome 22 in these probands showed interchromosomal exchanges in 2/15 informative meioses, a rate consistent with the genetic distance. Meiotic exchanges, visualized as MLH1 foci, localize to the distal long arm of chromosome 22 in 75% of human spermatocytes tested, also reflecting the genetic map. Additionally, we found no effect of proband gender or parental age on the crossover frequency. Parental origin studies in 65 de novo 3 Mb deletions (including these 20 patients) demonstrated no bias. Unlike Williams syndrome, we found no chromosomal inversions flanked by LCRs in 22 sets of parents of 22q11 deleted patients, or in eight non-deleted patients with a DGS/VCFS phenotype using FISH. Our data are consistent with significant aberrant interchromosomal exchange events during meiosis I in the proximal region of the affected chromosome 22 as the likely etiology for the deletion. This type of exchange occurs more often than is described for deletions of chromosomes 7q11, 15q11, 17p11 and 17q11, implying a difference in the meiotic behavior of chromosome 22.