Tyrosine phosphorylation of HSC70 and its interaction with RFC mediates methotrexate resistance in murine L1210 leukemia cells.

We previously identified and characterized a 66-68 kDa membrane-associated, tyrosine phosphorylated protein in murine leukemia L1210 cells as HSC70 which is a methotrexate (MTX)-binding protein. In order to further characterize the functional role of HSC70 in regulating MTX resistance in L1210 cells, we first showed that HSC70 colocalizes and interacts with reduced folate carrier (RFC) in L1210 cells by confocal laser scanning microscopy and Duolink in situ proximity ligation assay. The tyrosine phosphorylation status of HSC70 found in the membrane fraction was different from the parental L1210/0 and cisplatin (CDDP)-MTX cross resistant L1210/DDP cells. In MTX-binding assays, HSC70 from L1210/DDP cells showed less affinity for MTX-agarose beads than that of L1210/0 cells. In addition, genistein (a tyrosine phosphorylation inhibitor) significantly enhanced the resistance of L1210/0 cells to MTX. Moreover, site-directed mutation studies indicated the importance of tyrosine phosphorylation of HSC70 in regulating its binding to MTX. These findings suggest that tyrosine phosphorylation of HSC70 regulates the transportation of MTX into the cells via the HSC70-RFC system and contributes to MTX resistance in L1210 cells.

Biochanin A reduces pancreatic cancer survival and progression.

Pancreatic cancer has dismally low mean survival rates worldwide. Only a few chemotherapeutic agents including gemcitabine have been shown to improve the survival of pancreatic cancer patients. Biochanin A, an isoflavone, is known to exert an anticancer effect on various cancer types. In this study, we examined the anticancer properties of biochanin A on pancreatic cancer cells. The effect of biochanin A on cellular survival, apoptosis, and proliferation was analyzed using MTT, flow cytometry, and colony formation assay. The effect of biochanin A on pancreatic cancer's mitogenic signaling was determined using western blot analysis. Migration assay and zymography were used to determine biochanin A's effect on pancreatic cancer progression. Biochanin A induced dose-dependent toxicity on pancreatic cancer cells (Panc1 and AsPC-1). It reduced colony formation ability of Panc1 cells and induced dose-dependent apoptosis. Activation of Akt and MAPK was inhibited. Furthermore, the migratory and invasive potential of the cancer cells was also reduced. The results suggest that biochanin A is effective in reducing pancreatic cancer cell survival by inhibiting their proliferation and inducing apoptosis. It affects mitogenic, migratory, and invasive processes involved in cancer progression. These findings may lead to novel approaches to treat pancreatic cancer using isoflavones in combination with other therapeutic drugs.

Interleukin-6 and JAK2/STAT3 signaling mediate the reversion of dexamethasone resistance after dexamethasone withdrawal in 7TD1 multiple myeloma cells.

We previously reported the establishment and characteristics of a DXM-resistant cell line (7TD1-DXM) generated from the IL6-dependent mouse B cell hybridoma, 7TD1 cell line. After withdrawing DXM from 7TD1-DXM cells over 90 days, DXM significantly inhibited the cell growth and induced apoptosis in the cells (7TD1-WD) compared with 7TD1-DXM cells. Additionally, IL-6 reversed while IL-6 antibody and AG490 enhanced the effects of growth inhibition and apoptosis induced by DXM in 7TD1-WD cells. Our study demonstrates that 7TD1-DXM cells become resensitized to DXM after DXM withdrawal, and IL-6 and JAK2/STAT3 pathways may regulate the phenomenon.

Identification and characterization of a 66-68-kDa protein as a methotrexate-binding protein in murine leukemia L1210 cells.

We previously observed an unidentified, tyrosine-phosphorylated, membrane-associated, 66-68-kDa protein which was present in the L1210 murine leukemia cells but not present, at least in the tyrosine-phosphorylated form, in cisplatin-methotrexate (CDDP-MTX) cross-resistant L1210/DDP cells. We purified and characterized this 66-68-kDa protein by affinity chromatography purification using its two identified properties, tyrosine phosphorylation and MTX-binding, and yielded a single band of 66-68 kDa. The purified protein was subjected to trypsin digestion and the isolated peptide fragments were sequenced and yielded two partial peptide sequences: VEIIANDQ and VTNAVVTVPAYFNDSQRQA. The two peptide sequences were used to search for the mouse genome at the national center for biotechnology information (NCBI) database for Open Reading Frame Sequence (ORFs) containing these peptides using the TBLASTN function. A single gene was identified containing both sequences, the HSPa8 gene, which codes for the heat shock family protein, HSC70. We further demonstrated that HSC70 is a MTX-binding protein using a binding assay with MTX-agarose beads followed by Western blotting. The HSC70 also existed in various cancer cell lines and showed binding to MTX. Additionally, the HSC70 protein, cloned from the L1210 murine leukemia cells, was expressed and purified from E. coli cells using a polyhistidine-tag purification system and it also showed the binding properties with MTX. DnaK, the HSC70 homologue in E. coli, also binds to MTX. By using the purified truncated HSC70 domains, we identified the adenosine triphosphatase (ATPase) domain of HSC70 that can bind to MTX. Thus, we have tentatively characterized a new, novel property of HSC70 as a MTX-binding protein.

Comprehensive review on the HSC70 functions, interactions with related molecules and involvement in clinical diseases and therapeutic potential.

Heat shock cognate protein 70 (HSC70) is a constitutively expressed molecular chaperone which belongs to the heat shock protein 70 (HSP70) family. HSC70 shares some of the structural and functional similarity with HSP70. HSC70 also has different properties compared with HSP70 and other heat shock family members. HSC70 performs its full functions by the cooperation of co-chaperones. It interacts with many other molecules as well and regulates various cellular functions. It is also involved in various diseases and may become a biomarker for diagnosis and potential therapeutic targets for design, discovery, and development of novel drugs to treat various diseases. In this article, we provide a comprehensive review on HSC70 from the literatures including the basic general information such as classification, structure and cellular location, genetics and function, as well as its protein association and interaction with other proteins. In addition, we also discussed the relationship of HSC70 and related clinical diseases such as cancer, cardiovascular, neurological, hepatic and many other diseases and possible therapeutic potential and highlight the progress and prospects of research in this field. Understanding the functions of HSC70 and its interaction with other molecules will help us to reveal other novel properties of this protein. Scientists may be able to utilize this protein as a biomarker and therapeutic target to make significant advancement in scientific research and clinical setting in the future.

Treatment of human astrocytoma U87 cells with silicon dioxide nanoparticles lowers their survival and alters their expression of mitochondrial and cell signaling proteins.

Recent evidence suggests silicon dioxide micro- and nanoparticles induce cytotoxic effects on lung cells. Thus, there is an increasing concern regarding their potential health hazard. Nevertheless, the putative toxicity of nanoparticles in mammalian cells has not yet been systematically investigated. We previously noted that several metallic oxide nanoparticles exert differential cytotoxic effects on human neural and nonneural cells. Therefore, we hypothesized that silicon dioxide nanoparticles induce cytotoxicity in U87 cells by lowering their survival by decreasing cell survival signaling and disturbing mitochondrial function. To investigate this hypothesis, we determined the activities of the key mitochondrial enzymes, citrate synthase and malate dehydrogenase, in astrocytoma U87 cells treated with silicon dioxide nanoparticles. In addition, we studied the expression of the mitochondrial DNA-encoded proteins, cytochrome C oxidase II and nicotinamide adenine dinucleotide (NADPH) dehydrogenase subunit 6, and cell signaling pathway protein extracellular signal-regulated kinase (ERK) and phosphorylated ERK in treated U87 cells. The activated form of ERK controls cell growth, differentiation, and proliferation. In parallel, we determined survival of U87 cells after treating them with various concentrations of silicon dioxide nanoparticles. Our results indicated that treatment with silicon dioxide nanoparticles induced decreases in U87 cell survival in a dose-related manner. The activities of citrate synthase and malate dehydrogenase in treated U87 cells were increased, possibly due to an energetic compensation in surviving cells. However, the expression of mitochondrial DNA-encoded cytochrome C oxidase subunit II and NADH dehydrogenase subunit 6 and the cell signaling protein ERK and phosphorylated ERK were altered in the treated U87 cells, suggesting that silicon dioxide nanoparticles induced disruption of mitochondrial DNA-encoded protein expression, leading to decreased mitochondrial energy production and decreased cell survival/proliferation signaling. Thus, our results strongly suggest that the cytotoxicity of silicon dioxide nanoparticles in human neural cells implicates altered mitochondrial function and cell survival/proliferation signaling.

Apoptotic resistance exhibited by dexamethasone-resistant murine 7TD1 cells is controlled independently of interleukin-6 triggered signaling.

Interleukin-6 (IL6)-mediated signaling is known to play a role in pathogenesis and resistance in several cancers like multiple myeloma (MM). In this report we used the IL6-dependent 7TD1 murine B-cell hybridoma as an in vitro model to study the interactions between IL6-signaling pathways and the development of dexamethasone resistance. Though in initial stages, 7TD1 cells grew IL6-dependent and were sensitive to dexamethasone-induced apoptosis, chronic exposure to dexamethasone led to a dexamethasone-resistant phenotype (7TD1-Dxm) that grew independent of exogenous IL6. While IL6-mediated JAK/STAT3 and PI3K/AKT signaling was important for proliferation of both cell lines, as shown in proliferation assays using the respective pathway inhibitors, AG490 and LY294002, the resistant cells were insensitive to induction of apoptosis using the same. STAT3 was constitutively phosphorylated in resistant cells and inhibition of its dimerization induced apoptosis but did not alter their insensitivity to dexamethasone. Our results suggest a role of entities downstream of IL6-mediated JAK/STAT3 signaling in development of dexamethasone resistance by 7TD1-Dxm cells.

Exposure to titanium dioxide and other metallic oxide nanoparticles induces cytotoxicity on human neural cells and fibroblasts.

The use of titanium dioxide (TiO(2)) in various industrial applications (eg, production of paper, plastics, cosmetics, and paints) has been expanding thereby increasing the occupational and other environmental exposure of these nanoparticles to humans and other species. However, the health effects of exposure to TiO(2) nanoparticles have not been systematically assessed even though recent studies suggest that such exposure induces inflammatory responses in lung tissue and cells. Because the effects of such nanoparticles on human neural cells are unknown, we have determined the putative cytotoxic effects of these nanoparticles on human astrocytes-like astrocytoma U87 cells and compared their effects on normal human fibroblasts. We found that TiO(2) micro- and nanoparticles induced cell death on both human cell types in a concentration-related manner. We further noted that zinc oxide (ZnO) nanoparticles were the most effective, TiO(2) nanoparticles the second most effective, and magnesium oxide (MgO) nanoparticles the least effective in inducing cell death in U87 cells. The cell death mechanisms underlying the effects of TiO(2) micro- and nanoparticles on U87 cells include apoptosis, necrosis, and possibly apoptosis-like and necrosis-like cell death types. Thus, our findings may have toxicological and other pathophysiological implications on exposure of humans and other mammalian species to metallic oxide nanoparticles.

Signaling pathways mediating manganese-induced toxicity in human glioblastoma cells (u87).

Although essential, manganese (Mn) intake in excess leads to neurotoxicity. Mn neurotoxicity induces impairment of energy metabolism and ultimately cell death. Nevertheless, the signaling mechanisms underlying Mn toxicity are unknown. Employing human glioblastoma (U87) cells, we investigated several signaling pathways (ones promoting cellular proliferation and invasion) underlying Mn toxicity. Mn-treatment of U87 cells induced a down-regulation of MAPK pathway but the AKT pathway was not markedly affected. Mn-treatment of these cells induced decreases in their levels of c-Jun and c-Fos transcription factors and extracellular matrix degrading enzymes like MMP-2, which are associated with glioblastoma invasiveness. Mn-treatment also induced apoptosis in U87 cells. Thus, our results indicate that other than inducing apoptosis in U87 cells, Mn exerts differential effects on several signaling pathways promoting glioblastoma proliferation and invasion. Consequently, Mn may have pathophysiological roles in inducing apoptosis and in blocking glioblastoma invasion. Our results may thus have therapeutic implications.

Manganese-induced neurotoxicity is differentially enhanced by glutathione depletion in astrocytoma and neuroblastoma cells.

Manganese (Mn) is neurotoxic: the underlying mechanisms have not been fully elucidated. L: -Buthionine-(S,R)-sulfoximine (BSO) is an irreversible inhibitor of gamma-glutamylcysteine synthetase, an important enzyme in glutathione (GSH) synthesis. To test the hypothesis that BSO modulates Mn toxicity, we investigated the effects of treatment of U-87 or SK-N-SH cells with MnCl(2), BSO, or MnCl(2) plus BSO. We monitored cell viability using MTT assay, staining with HO-33342 to assess live and/or apoptotic cells, and staining with propidium iodide (PI) to assess necrotic cells; we also measured cellular glutathione. Our results indicate decreased viability in both cell types when treated with MnCl(2) or BSO: Mn was more toxic to SK-N-SH cells, whereas BSO was more toxic to U-87 cells. Because BSO treatment accentuated Mn toxicity in both cell lines, GSH may act to combat Mn toxicity. Thus, further investigation in oxidative stress mediated by glutathione depletion will unravel new Mn toxicity mechanism(s).

Manganese treatment modulates the expression of peroxisome proliferator-activated receptors in astrocytoma and neuroblastoma cells.

Peroxisome proliferator-activated receptors (PPARs) play roles in neural cells by regulating energy balance, cell proliferation and anti-oxidant responses although the molecular mechanisms underlying such roles are unclear. Chronic exposure to excess manganese (Mn) leads to neurotoxicity, although Mn-induced neurotoxic mechanisms have not been fully elucidated. We hypothesized Mn neurotoxicity differentially alters the expression of PPARs. We investigated the effects of manganese chloride treatment (0.01-4 mM) on protein expression of PPAR isoforms (alpha, beta, and gamma) in human astrocytoma (U87) and neuroblastoma (SK-N-SH) cells. The two cell types expressed the 3 PPAR isoforms differentially: their expression of the PPARs was altered by Mn-treatment. Furthermore, nuclear and cytosolic fractions derived from the 2 cell types, with and without Mn-treatment, exhibited marked differences in the protein content of PPARs. Our results constitute the first demonstration that the PPAR signaling pathway may assume pathophysiological importance in Mn neurotoxicity.

Differential lowering by manganese treatment of activities of glycolytic and tricarboxylic acid (TCA) cycle enzymes investigated in neuroblastoma and astrocytoma cells is associated with manganese-induced cell death.

Manganese (Mn) is a trace metal required for normal growth and development. Manganese neurotoxicity is rare and usually associated with occupational exposures. However, the cellular and molecular mechanisms underlying Mn toxicity are still elusive. In rats chronically exposed to Mn, their brain regional Mn levels increase in a dose-related manner. Brain Mn preferentially accumulates in mitochondria; this accumulation is further enhanced with Mn treatment in vivo. Exposure of mitochondria to Mn in vitro leads to uncoupling of oxidative phosphorylation. These observations prompted us to investigate the hypothesis that Mn induces alterations in energy metabolism in neural cells by interfering with the activities of various glycolytic and TCA cycle enzymes using human neuroblastoma (SK-N-SH) and astrocytoma (U87) cells. Treatments of SK-N-SH and U87 cells with MnCl2 induced cell death in these cells, in a concentration- and time-dependent manner, as determined by MTT assays. In parallel with the Mn-induced, dose-dependent decrease in cell survival, treatment of these cells with 0.01 to 4.0 mM MnCl2 for 48 h also induced dose-related decreases in their activities of hexokinase, pyruvate kinase, lactate dehydrogenase, citrate synthase, and malate dehydrogenase. Hexokinase in SK-N-SH cells was the most affected by Mn treatments, even at the lower range of concentrations. Mn treatment of SK-N-SH cells affected pyruvate kinase and citrate synthase to a lesser extent as compared to its effect on other enzymes investigated. However, citrate synthase and pyruvate kinase in U87 cells were more vulnerable than other enzymes investigated to the effects of Mn. The results suggest the two cell types exhibited differential susceptibility toward the Mn-induced effects. Additionally, the results may have significant implications in flux control because HK is the first and highly regulated enzyme in brain glycolysis. Thus these results are consistent with our hypothesis and may have pathophysiological implications in the mechanisms underlying Mn neurotoxicity.