GPD1 Enhances the Anticancer Effects of Metformin by Synergistically Increasing Total Cellular Glycerol-3-Phosphate.

Metformin is an oral drug widely used for the treatment of type 2 diabetes mellitus. Numerous studies have demonstrated the value of metformin in cancer treatment. However, for metformin to elicit effects on cancer often requires a high dosage, and any underlying mechanism for how to improve its inhibitory effects remains unknown. Here, we found that low mRNA expression of glycerol-3-phosphate dehydrogenase 1 (GPD1) may predict a poor response to metformin treatment in 15 cancer cell lines. In vitro and in vivo, metformin treatment alone significantly suppressed cancer cell proliferation, a phenotype enhanced by GPD1 overexpression. Total cellular glycerol-3-phosphate concentration was significantly increased by the combination of GPD1 overexpression and metformin treatment, which suppressed cancer growth via inhibition of mitochondrial function. Eventually, increased reactive oxygen species and mitochondrial structural damage was observed in GPD1-overexpressing cell lines treated with metformin, which may contribute to cell death. In summary, this study demonstrates that GPD1 overexpression enhances the anticancer activity of metformin and that patients with increased GPD1 expression in tumor cells may respond better to metformin therapy. SIGNIFICANCE: GPD1 overexpression enhances the anticancer effect of metformin through synergistic inhibition of mitochondrial function, thereby providing new insight into metformin-mediated cancer therapy.

Profiling and targeting of cellular bioenergetics: inhibition of pancreatic cancer cell proliferation.

BACKGROUND: Targeting both mitochondrial bioenergetics and glycolysis pathway is an effective way to inhibit proliferation of tumour cells, including those that are resistant to conventional chemotherapeutics. METHODS: In this study, using the Seahorse 96-well Extracellular Flux Analyzer, we mapped the two intrinsic cellular bioenergetic parameters, oxygen consumption rate and proton production rate in six different pancreatic cancer cell lines and determined their differential sensitivity to mitochondrial and glycolytic inhibitors. RESULTS: There exists a very close relationship among intracellular bioenergetic parameters, depletion of ATP and anti-proliferative effects (inhibition of colony-forming ability) in pancreatic cancer cells derived from different genetic backgrounds treated with the glycolytic inhibitor, 2-deoxyglucose (2-DG). The most glycolytic pancreatic cancer cell line was exquisitely sensitive to 2-DG, whereas the least glycolytic pancreatic cancer cell was resistant to 2-DG. However, when combined with metformin, inhibitor of mitochondrial respiration and activator of AMP-activated protein kinase, 2-DG synergistically enhanced ATP depletion and inhibited cell proliferation even in poorly glycolytic, 2-DG-resistant pancreatic cancer cell line. Furthermore, treatment with conventional chemotherapeutic drugs (e.g., gemcitabine and doxorubicin) or COX-2 inhibitor, celecoxib, sensitised the cells to 2-DG treatment. CONCLUSIONS: Detailed profiling of cellular bioenergetics can provide new insight into the design of therapeutic strategies for inhibiting pancreatic cancer cell metabolism and proliferation.

Asparagine depletion potentiates the cytotoxic effect of chemotherapy against brain tumors.

UNLABELLED: Targeting amino acid metabolism has therapeutic implications for aggressive brain tumors. Asparagine is an amino acid that is synthesized by normal cells. However, some cancer cells lack asparagine synthetase (ASNS), the key enzyme for asparagine synthesis. Asparaginase (ASNase) contributes to eradication of acute leukemia by decreasing asparagine levels in serum and cerebrospinal fluid. However, leukemic cells may become ASNase-resistant by upregulating ASNS. High expression of ASNS has also been associated with biologic aggressiveness of other cancers, including gliomas. Here, the impact of enzymatic depletion of asparagine on proliferation of brain tumor cells was determined. ASNase was used as monotherapy or in combination with conventional chemotherapeutic agents. Viability assays for ASNase-treated cells demonstrated significant growth reduction in multiple cell lines. This effect was reversed by glutamine in a dose-dependent manner--as expected, because glutamine is the main amino group donor for asparagine synthesis. ASNase treatment also reduced sphere formation by medulloblastoma and primary glioblastoma cells. ASNase-resistant glioblastoma cells exhibited elevated levels of ASNS mRNA. ASNase cotreatment significantly enhanced gemcitabine or etoposide cytotoxicity against glioblastoma cells. Xenograft tumors in vivo showed no significant response to ASNase monotherapy and little response to temozolomide alone. However, combinatorial therapy with ASNase and temozolomide resulted in significant growth suppression for an extended duration of time. Taken together, these findings indicate that amino acid depletion warrants further investigation as adjunctive therapy for brain tumors. IMPLICATIONS: Findings have potential impact for providing adjuvant means to enhance brain tumor chemotherapy.

Metabolism modifications and apoptosis induction after Cellfood™ administration to leukemia cell lines.

BACKGROUND: Cellfood™ (CF) is a nutritional supplement containing deuterium sulphate, minerals, amino acids, and enzymes, with well documented antioxidant properties. Its organic and inorganic components are extracted from the red algae Lithothamnion calcareum, whose mineral extract has shown growth-inhibitory effect both on in vitro and in vivo models. The purpose of this study was to evaluate the antiproliferative effects of CF on leukemic cells. In fact, according to its capacity to modulate O2 availability and to improve mitochondrial respiratory metabolism, we wondered if CF could affect cancer cell metabolism making cells susceptible to apoptosis. METHODS: Three leukemic cell lines, Jurkat, U937, and K562, were treated with CF 5 µl/ml up to 72 hours. Cell viability, apoptosis (i.e. caspase-3 activity and DNA fragmentation), hypoxia inducible factor 1 alpha (HIF-1α) concentration, glucose transporter 1 (GLUT-1) expression, lactate dehydrogenase (LDH) activity and lactate release in the culture medium were detected and compared with untreated cells. RESULTS: CF significantly inhibited leukemic cell viability by promoting cell apoptosis, as revealed by caspase-3 activation and DNA laddering. In particular, CF treated cells showed lower HIF-1α levels and lower GLUT-1 expression as compared to untreated cells. At the same time, CF was able to reduce LDH activity and, consequently, the amount of lactate released in the extracellular environment. CONCLUSIONS: We supplied evidence for an antiproliferative effect of CF on leukemia cell lines by inducing cell death through an apoptotic mechanism and by altering cancer cell metabolism through HIF-1α and GLUT-1 regulation. Thanks to its antioxidative and proapoptotic properties, CF might be a good candidate for cancer prevention.

Pigment epithelium-derived factor enhances differentiation and mineral deposition of human mesenchymal stem cells.

Pigment epithelium-derived factor (PEDF) is a potent antiangiogenic factor found in a wide variety of tissues. Recent findings indicated that lack of PEDF leads to osteogenesis imperfecta type VI whose hallmark is a defect in mineralization. We investigated the effects of PEDF on human mesenchymal stem cells (hMSCs) and signaling pathways through which PEDF displays its activities in hMSCs. hMSCs incubated in a medium supplemented with PEDF induced expression of osteoblastic-related genes. In addition, PEDF induced alkaline phosphatase (ALP) activity in MSCs at 14 days of incubation in maintenance medium; hMSCs incubated in osteogenic medium in presence of PEDF expressed 19% more ALP activity (35.655 ± 1.827 U/mg protein, p = .041 than cells incubated in the same medium without PEDF supplementation (29.956 ± 2.100 U/µg protein). hMSCs incubated in osteogenic medium in presence of PEDF deposited 50% more mineral (2.108 ± 0.306 OD/ml per well per 1 × 10(4) cells per square centimeter, p = .017) than MSCs incubated in absence of the protein (1.398 ± 0.098 OD/ml per well per 1 × 10(4) cells per square centimeter) as determined by Alizarin Red quantitation. Reduction in PEDF expression in MSCs by siRNA led to decreased ALP activity (33.552 ± 2.009 U/ng protein of knockdown group vs. 39.269 ± 3.533 U/ng protein of scrambled siRNA group, p = .039) and significant reduction in mineral deposition (0.654 ± 0.050 OD/ml per well per 1 × 10(4) cells per square centimeter of knockdown group vs. 1.152 ± 0.132 OD/ml per well per 1 × 10(4) cells per square centimeter of wild-type group, p = .010). Decreased ALP activity and mineral deposition were restored by supplementation with exogenous PEDF protein. PEDF activated ERK and AKT signaling pathways in MSCs to induce expression of osteoblastic-related genes. These data suggest that PEDF is involved in MSCs osteoblastic differentiation.

Concise review: the yin and yang of intestinal (cancer) stem cells and their progenitors.

The intestine has developed over the last few years into a prime model system for adult stem cell research. Intestinal cells have an average lifetime of 5 days, moving within this time from the bottom of intestinal crypts to the top of villi. This rapid self-renewal capacity combined with an easy to follow (mostly) unidirectional movement of cells offers an ideal site to conduct adult stem cell research. The delineation of the active pathways in the intestinal epithelium together with the development of molecular techniques to prove stemness laid the grounds for the identification of the intestinal stem cell. In vitro systems and transgenic mouse models broaden our knowledge on the role of the stem cell niche and those cells that reestablish homeostasis after perturbation of the system. These insights expedited also research on the role of normal adult stem cells in cancer initiation and the factors influencing the maintenance of cancer stem cells.

Characterization of vitamin C-induced cell sheets formed from primary and immortalized human corneal stromal cells for tissue engineering applications.

The purpose of this study was to compare the ability of primary human corneal stromal cells (HuFib cells) and SV40-immortalized human corneal keratocytes (HCK cells) to synthesize their own extracellular matrix induced by vitamin C supplementation. Therefore, the amount of collagen secreted and resulting biomechanical properties based on the culture duration were assessed. Cells were cultivated for several weeks with or without vitamin C. The amount of collagen secreted by the cells was quantified based on the culture duration. Cell viability was simultaneously determined via the MTT assay. Collagen secretion was increased as a result of vitamin C supplementation. The effect was stronger in primary cells. In addition, vitamin C supplementation had a positive effect on HuFib cell viability. Vitamin C supplementation induced the formation of detachable cell sheets in both primary and immortalized cells. The biomechanical properties of the sheets were evaluated using a static material testing machine, and the ultrastructure of the cell sheets was examined using scanning electron microscopy. The cell sheets formed from HuFib cells had a higher percentage of light transmission between 400 and 800 nm and were superior in terms of E-modulus and ultimate strength testing. Indirect immunofluorescence and Western blot confirmed the presence of collagen type I in the HuFib and HCK cell cultures. Stimulating secretion of the extracellular matrix in corneal stromal cells is a promising approach for corneal stroma reconstruction for tissue engineering applications.

E-cadherin and, in its absence, N-cadherin promotes Nanog expression in mouse embryonic stem cells via STAT3 phosphorylation.

We have recently shown that loss of E-cadherin in mouse embryonic stem cells (mESCs) results in significant alterations to both the transcriptome and hierarchy of pluripotency-associated signaling pathways. Here, we show that E-cadherin promotes kruppel-like factor 4 (Klf4) and Nanog transcript and protein expression in mESCs via STAT3 phosphorylation and that ß-catenin, and its binding region in E-cadherin, is required for this function. To further investigate the role of E-cadherin in leukemia inhibitory factor (LIF)-dependent pluripotency, E-cadherin null (Ecad(-/-)) mESCs were cultured in LIF/bone morphogenetic protein supplemented medium. Under these conditions, Ecad(-/-) mESCs exhibited partial restoration of cell-cell contact and STAT3 phosphorylation and upregulated Klf4, Nanog, and N-cadherin transcripts and protein. Abrogation of N-cadherin using an inhibitory peptide caused loss of phospho STAT3, Klf4, and Nanog in these cells, demonstrating that N-cadherin supports LIF-dependent pluripotency in this context. We therefore identify a novel molecular mechanism linking E- and N-cadherin to the core circuitry of pluripotency in mESCs. This mechanism may explain the recently documented role of E-cadherin in efficient induced pluripotent stem cell reprogramming.

Molecular cloning and functional expression of chitinase-encoding cDNA from the cabbage moth, Mamestra brassicae.

Chitinase is a rate-limiting and endo-splitting enzyme involved in the bio-degradation of chitin, an important component of the cuticular exoskeleton and peritrophic matrix in insects. We isolated a cDNA-encoding chitinase from the last larval integument of the cabbage moth, Mamestra brassicae (Lepidoptera; Noctuidae), cloned the ORF cDNA into E. coli to confirm its functionality, and analyzed the deduced amino acid sequence in comparison with previously described lepidopteran chitinases. M. brassicae chitinase expressed in the transformed E. coli cells with the chitinase-encoding cDNA enhanced cell proliferation to about 1.6 times of the untransformed wild type strain in a colloidal chitin-including medium with only a very limited amount of other nutrients. Compared with the wild type strain, the intracellular levels of chitin degradation derivatives, glucosamine and N-acetylglucosamine were about 7.2 and 2.3 times higher, respectively, while the extracellular chitinase activity was about 2.2 times higher in the transformed strain. The ORF of M. brassicae chitinaseencoding cDNA consisted of 1686 nucleotides (562 amino acid residues) except for the stop codon, and its deduced amino acid composition revealed a calculated molecular weight of 62.7 and theoretical pI of 5.3. The ORF was composed of N-terminal leading signal peptide (AA 1-20), catalytic domain (AA 21-392), linker region (AA 393-498), and C-terminal chitin-binding domain (AA 499-562) showing its characteristic structure as a molting fluid chitinase. In phylogenetic analysis, the enzymes from 6 noctuid species were grouped together, separately from a group of 3 bombycid and 1 tortricid enzymes, corresponding to their taxonomic relationships at both the family and genus levels.

Cancer cell dependence on unsaturated fatty acids implicates stearoyl-CoA desaturase as a target for cancer therapy.

Emerging literature suggests that metabolic pathways play an important role in the maintenance and progression of human cancers. In particular, recent studies have implicated lipid biosynthesis and desaturation as a requirement for tumor cell survival. In the studies reported here, we aimed to understand whether tumor cells require the activity of either human isoform of stearoyl-CoA-desaturase (SCD1 or SCD5) for survival. Inhibition of SCD1 by siRNA or a small molecule antagonist results in strong induction of apoptosis and growth inhibition, when tumor cells are cultured in reduced (2%) serum conditions, but has little impact on cells cultured in 10% serum. Depletion of SCD5 had minimal effects on cell growth or apoptosis. Consistent with the observed dependence on SCD1, but not SCD5, levels of SCD1 protein increased in response to decreasing serum levels. Both induction of SCD1 protein and sensitivity to growth inhibition by SCD1 inhibition could be reversed by supplementing growth media with unsaturated fatty acids, the product of the enzymatic reaction catalyzed by SCD1. Transcription profiling of cells treated with an SCD inhibitor revealed strong induction of markers of endoplasmic reticulum stress. Underscoring its importance in cancer, SCD1 protein was found to be highly expressed in a large percentage of human cancer specimens. SCD inhibition resulted in tumor growth delay in a human gastric cancer xenograft model. Altogether, these results suggest that desaturated fatty acids are required for tumor cell survival and that SCD may represent a viable target for the development of novel agents for cancer therapy.

Bcl-x(L) overexpression delays the onset of autophagy and apoptosis in hyperosmotic recombinant Chinese hamster ovary cell cultures.

Hyperosmolality in recombinant Chinese hamster ovary (rCHO) cell cultures induces autophagy and apoptosis. To investigate the effect of Bcl-x(L) overexpression on autophagy and apoptosis in hyperosmotic rCHO cell cultures, an erythropoietin (EPO)-producing rCHO cell line with regulated Bcl-x(L) overexpression was subjected to hyperosmolality resulting from NaCl addition in a batch culture and nutrient supplementation in a fed-batch culture. In the batch culture, Bcl-x(L) overexpression suppressed apoptosis, as evidenced by a decreased amount of cleaved caspase-7 and PARP. Concurrently, Bcl-x(L) overexpression also delayed autophagy, as indicated by reduced LC3 conversion, from LC3-I to LC3-II. As a result, the cell viability and EPO production were improved by Bcl-x(L) overexpression. In the fed-batch culture, the simultaneous application of Bcl-x(L) overexpression and nutrient feeding increased the culture longevity and maximum EPO concentration. Taken together, Bcl-x(L) overexpression delayed autophagy and apoptosis in hyperosmotic rCHO cell cultures, resulting in increased EPO production.

Low-intensity laser irradiation stimulates wound healing in diabetic wounded fibroblast cells (WS1).

BACKGROUND: Patients with diabetes suffer from slow-to-heal wounds, which often necessitate amputation. Low-intensity laser irradiation (LILI) has been shown to reduce the healing time in such patients. This study aimed to determine the effect of different wavelengths of LILI on cellular migration, viability, and proliferation in a wounded diabetic cell model. METHODS: Diabetic wounded and unwounded human skin fibroblast cells (WS1) were irradiated at 632.8, 830, or 1,064 nm with 5 J/cm(2). Cellular morphology and migration were determined microscopically, while cellular viability was determined by ATP luminescence, and proliferation was determined by basic fibroblast growth factor expression and alkaline phosphatase activity. RESULTS: Diabetic wounded cells irradiated at 1,064 nm showed a lesser degree of migration, viability, and proliferation compared to cells irradiated at 632.8 or 830 nm. Cells irradiated at 632.8 nm showed a higher degree of haptotaxis and migration as well as ATP luminescence compared to cells irradiated at 830 nm. CONCLUSIONS: This study showed that LILI of diabetic wounded cells in the visible range (632.8 nm) was more beneficial to wound healing than irradiating the same cells to wavelengths in the infrared range. Cells irradiated at a longer wavelength of 1,064 nm performed worse.

Dietary hempseed meal intake increases body growth and shortens the larval stage via the upregulation of cell growth and sterol levels in Drosophila melanogaster.

Hempseed, a rich source of polyunsaturated fatty acids (PUFAs) and phytosterols, has been recognized as a potential therapeutic food used for cardioprotection, preventing platelet aggregation, and improving atopic dermatitis. Although several studies have revealed the physiological benefits of hempseed on a variety of animals, the effects of dietary hempseed intake on animal development are currently unknown. In this study, we evaluated the developmental effects of the addition of hempseed meal (HSM) to the diet of Drosophila. Interestingly, dietary HSM intake was shown to increase the body size of flies by increasing cell numbers, and also truncated the larval period without affecting survival rate or longevity. The oviposition of female flies was also increased by dietary HSM supplementation. Interestingly, the levels of sterols, which are precursors of ecdysone, a molting hormone, were found to be elevated in the larvae fed on HSM. Additionally, the hexane extracts of hempseed mimicked the effects of HSM on growth, developmental timing, and reproduction. Moreover, among the major nonpolar components of HSM, feeding on cholesterol but not PUFA mix or campesterol accelerated pupariation and increased body size. These results indicate that the dietary intake of HSM accelerates both body growth and developmental rates in Drosophila via the stimulation of cell growth and ecdysone synthesis. Additionally, nonpolar components of hempseed, such as cholesterol, might be responsible for the effects of HSM on development and reproduction.

Vitamin D levels, lung function, and steroid response in adult asthma.

RATIONALE: Patients with asthma exhibit variable response to inhaled corticosteroids (ICS). Vitamin D is hypothesized to exert effects on phenotype and glucocorticoid (GC) response in asthma. OBJECTIVES: To determine the effect of vitamin D levels on phenotype and GC response in asthma. METHODS: Nonsmoking adults with asthma were enrolled in a study assessing the relationship between serum 25(OH)D (vitamin D) concentrations and lung function, airway hyperresponsiveness (AHR), and GC response, as measured by dexamethasone-induced expression of mitogen-activated protein kinase phosphatase (MKP)-1 by peripheral blood mononuclear cells. MEASUREMENTS AND MAIN RESULTS: A total of 54 adults with asthma (FEV(1), 82.9 +/- 15.7% predicted [mean +/- SD], serum vitamin D levels of 28.1 +/- 10.2 ng/ml) were enrolled. Higher vitamin D levels were associated with greater lung function, with a 22.7 (+/-9.3) ml (mean +/- SE) increase in FEV(1) for each nanogram per milliliter increase in vitamin D (P = 0.02). Participants with vitamin D insufficiency (<30 ng/ml) demonstrated increased AHR, with a provocative concentration of methacholine inducing a 20% fall in FEV(1) of 1.03 (+/-0.2) mg/ml versus 1.92 (+/-0.2) mg/ml in those with vitamin D of 30 ng/ml or higher (P = 0.01). In ICS-untreated participants, dexamethasone-induced MKP-1 expression increased with higher vitamin D levels, with a 0.05 (+/-0.02)-fold increase (P = 0.02) in MKP-1 expression observed for each nanogram per milliliter increase in vitamin D, a finding that occurred in the absence of a significant increase in IL-10 expression. CONCLUSIONS: In asthma, reduced vitamin D levels are associated with impaired lung function, increased AHR, and reduced GC response, suggesting that supplementation of vitamin D levels in patients with asthma may improve multiple parameters of asthma severity and treatment response. Clinical trials registered with www.clinicaltrials.gov (NCT00495157, NCT00565266, and NCT00557180).

Dehydroepiandrosterone increased oxidative stress in a human cell line during differentiation.

Dehydroepiandrosterone (DHEA), a reversible inhibitor of glucose-6-phosphate dehydrogenase (G6PD), is increasingly taken as an antioxidative and anti-ageing supplement. This study investigated the effects of DHEA on the expression of G6PD and on the state of oxidative stress in a human promyelocytic leukaemia cell line, HL60, during the differentiation to neutrophil-like cell. This study differentiated HL60 with dimethyl sulfoxide (DMSO) in the presence (DMSO-HL60/DHEA) or absence (DMSO-HL60) of DHEA. During the differentiation, activity, mRNA and protein levels of G6PD were increased. DHEA increased these levels further. DHEA by itself suppressed the production of superoxide from DMSO-HL60 upon stimulation with phorbol myristate acetate (PMA). However, DMSO-HL60/DHEA stimulated with PMA in the absence of DHEA produced superoxide and 8-oxo-deoxyguanosine more than PMA-stimulated DMSO-HL60. After addition of H(2)O(2), the ratio of reduced glutathione to oxidized glutathione was lower in DMSO-HL60/DHEA than in DMSO-HL60. These findings indicate that DHEA acts both as an antioxidant and as a pro-oxidant.

Cell death induced by nutritional starvation in mouse macrophage-like RAW264.7 cells.

Amino acid utilization of mouse macrophage-like RAW264.7 cells was investigated. During the logarithmic growth stage, RAW264.7 cells grew very fast, with an approximate doubling time of 11 hours, in DMEM supplemented with 10% heat-inactivated fetal bovine serum. RAW264.7 cells consumed glutamine at the fastest rate, followed by serine, leucine, isoleucine, arginine, lysine, valine and other amino acids. When the cell density reached a critical threshold level, cells began to suffer non-apoptotic cell death characterized by mitochondrial damage (revealed by transmission electron microscopy) and a smear pattern of DNA fragmentation (revealed by agarose gel electrophoresis). At this point, glutamine, serine and glucose in the medium were almost completely exhausted, whereas other amino acids remained at more than 40% of their initial concentrations. Based on these data, it is recommended that glutamine, serine and glucose should be supplemented for the long culture of RAW264.7 cells.

Circadian stage-dependent inhibition of human breast cancer metabolism and growth by the nocturnal melatonin signal: consequences of its disruption by light at night in rats and women.

The circadian production of melatonin by the pineal gland during the night provides an inhibitory signal to tissue-isolated steroid receptor SR+ and - MCF-7 human breast cancer xenografts in female nude rats. A pivotal mechanism for melatonin's anticancer effects in vivo involves a melatonin receptor-mediated inhibition of linoleic acid (LA) uptake and its metabolism to mitogenically active 13-hydroxyoctadecadienoic acid (13-HODE). Exposure of (SR-) xenograft-bearing rats to increasing intensities of polychromatic white light at night suppresses melatonin while increasing tumor growth rates, DNA content, [3H]thymidine incorporation into DNA, LA uptake, 13-HODE formation, cAMP levels and ERK1/2 activation a dose-dependent manner. Similar effects occur in SR- human breast cancer xenografts perfused in situ with melatonin-depleted blood from healthy female subjects after their exposure to a single bright intensity (2800 lux) of polychromatic light at night. Additionally, SR- human breast cancer xenografts exhibit robust circadian rhythms of LA uptake, 13-HODE formation and proliferative activity. Exposure of xenograft-bearing rats to dim light at night results in the complete elimination of these rhythms which culminates in unfettered, high rates of tumor metabolism and growth. The organization of tumor metabolism and growth within circadian time structure by the oncostatic melatonin signal helps create a balance between the cancer and its host that is disrupted by host exposure to light at night. This biological mechanism may partially explain the higher risk of breast and other cancers in women working rotating night shifts and possibly others who also experience prolonged exposure to light at night.

Recruitment of HDAC4 by transcription factor YY1 represses HOXB13 to affect cell growth in AR-negative prostate cancers.

HOXB13 is a homeodomain protein implicated to play a role in growth arrest in AR (androgen receptor)-negative prostate cancer cells. Expression of HOXB13 is restricted to the AR-expressing prostate cells. In this report, we demonstrate that the HDAC inhibitor NaB (sodium butyrate) was able to induce cell growth arrest and to increase HOXB13 expression in AR-negative prostate cancer cells. We also show that both HDAC4 and YY1 participated in the repression of HOXB13 expression through an epigenetic mechanism involving histone acetylation modification. Specifically, co-immunoprecipitation assays revealed that HDAC4 and YY1 formed a complex. The chromatin immunoprecipitation (ChIP) assays verified that HDAC4 was recruited to HOXB13 promoter by YY1. Moreover, promoter truncation and point mutation studies determined that the two proximal YY1 binding sites on the HOXB13 promoter were essential for the recruitments of YY1 and HDAC4. Data presented in this report suggest that YY1 and HDAC4 affected cell growth by repressing transcriptional regulation of HOXB13 through an epigenetic modification of histones.

Glutathione Peroxidase 2 Inhibits Cyclooxygenase-2-Mediated Migration and Invasion of HT-29 Adenocarcinoma Cells but Supports Their Growth as Tumors in Nude Mice.

The selenoprotein gastrointestinal glutathione peroxidase 2 (GPx2) is up-regulated in a variety of cancer cells with thus far unknown consequences. Therefore, two clones of a human colon cancer cell line (HT-29) in which GPx2 was stably knocked down by small interfering RNA (siRNA; siGPx2) were used to test whether cancer-relevant processes are affected by GPx2. The capacity to grow anchorage independently in soft agar was significantly reduced in siGPx2 cells when compared with controls (i.e., HT-29 cells stably transfected with a scramble siRNA). The weight of tumors derived from siGPx2 cells injected into nude mice was lower in 9 of 10 animals. In contrast, in a wound-healing assay, wound closure was around 50% in controls and 80% in siGPx2 cells, indicating an enhanced capacity of the knockdown cells to migrate. Similarly, invasion of siGPx2 cells in a Transwell assay was significantly increased. Migration and invasion of siGPx2 cells were inhibited by celecoxib, a cyclooxygenase-2 (COX-2)-specific inhibitor, but not by alpha-tocopherol. Selenium supplementation of cell culture medium did not influence the results obtained with siGPx2 cells, showing that none of the other selenoproteins could replace GPx2 regarding the described effects. The data show that GPx2 inhibits malignant characteristics of tumor cells, such as migration and invasion, obviously by counteracting COX-2 expression but is required for the growth of transformed intestinal cells and may, therefore, facilitate tumor cell growth. The data also shed new light on the use of selenium as a chemopreventive trace element: a beneficial effect may depend on the stage of tumor development.

Deletion of Yin Yang 1 protein in osteosarcoma cells on cell invasion and CXCR4/angiogenesis and metastasis.

We know that the Yin Yang 1 protein (YY1) overexpression is positively and strongly correlated with the degree of malignancy of bone tumors. Therefore, we questioned whether we could influence cell invasiveness by deleting YY1 in human osteosarcoma cells (SaOs-2), as tested in Matrigel-coated filters and metastasis implantation of such osteosarcoma cells in vivo, by serial analysis with nuclear magnetic resonance. Moreover, we focused our work on the chemokine receptor CXCR4 and its inhibition by T22 antibody, as well as on systemic (direct in vivo assay) and computer-assisted imaging of angiogenesis-related metastasis. Results showed that cell invasiveness and metastasis implantation by wild-type SaOs-2 cells, as evaluated by histology and immunohistochemistry, are associated with up-regulation of CXCR4 expression, which in turn was significantly reduced by T22. In addition, deletion of YY1 (siRNAYY1-SaOs-2) induced a significant decrease of cell invasion and metastasis growth. This phenomenon was associated with decreased vascular endothelial growth factor (VEGF)/angiogenesis and a complex rearrangement of the gene expression profile as evaluated by microarray analysis. In conclusion, YY1 and VEGF/CXCR4 seem to intervene in the pathogenesis of the malignant phenotype of osteosarcoma by acting on cell invasiveness and metastasis growth.