Inhibitory effects of cold atmospheric plasma on the growth, virulence factors and HSP90 gene expression in Candida albicans.

In spite of the abundance of antifungal therapies, 75% of women in the world suffer from the second most common cause of vaginal infection named vulvovaginal candidiasis. This complication is characterized with overgrowth of Candida albicans. The low efficacy and side effects of current antifungal therapies have convinced the researchers to look for a non-antibiotic based treatment such as cold atmospheric plasmas (CAP). The aim of this research was to evaluate the effects of CAP on C. albicans growth, ergosterol and biofilm formation. In addition, antibiotic resistance, phospholipase and proteinase activity, and structural properties were examined with different exposure duration. Putative critical effect of CAP on the expression of HSP90 as a target of anti-fungal therapy was investigated. ROS production in C. albicans exposed to CAP was assessed. For this purpose, C. albicans subjected to 0, 90, 120, 150, 180 and 210 s of He/O2 (2%), and non-treated cells as control were examined in terms of the mentioned virulence factors. The results showed that CAP had a significant effect on inhibition of C. albicans growth, Inhibition of biofilm formation, ergosterol content, and fluconazole and amphotericin B antibiotic sensitivity were significant in 210 s treatment group. This effect was validated based on changes of the cell architecture and morphology given the microscopy imaging results. The expression of HSP90 in both C. albicans ATCC 10231 and C. albicans PFCC 9362 was inhibited in 210 s of exposition. CAP exposition induced intracellular ROS, which may cause membrane damage and cell death in C. albicans. Taken together, the potential of CAP for therapeutic purposes in C. albicans-induced fungal infections is supported.

Highly effective methods for expression/purification of recombinant human HSP90 and its four distinct (N-LR-M-C) domains.

Heat shock protein 90 (HSP90) plays essential roles in the normal physiology and comprises four distinct domains, including NH2-terminal (N), charged linker region (LR), middle (M), and COOH-terminal (C) domains, all of which regulate HSP90 biological functions. We reported herein detailed protocols to produce recombinant full-length (FL) and all these four domains of human HSP90 from Escherichia coli. cDNAs encoding FL, N, LR, M and C domains of human HSP90α were amplified and cloned into pET-32b(+) expression vector. All HSP90 constructs were expressed as soluble Trx-His-S tagged proteins after induction with 0.25 mM isopropyl-ß-d-thiogalactopyranoside (IPTG) at 18 °C overnight and further purified by affinity chromatography using nickel-nitrilotriacetic acid (Ni-NTA) resin. The enterokinase (EK) digestion was optimized for efficient cleavage of the Trx-His-S tag from each HSP90 construct by varying concentrations of EK (0.5-1 U) and urea (0-3 M). Each HSP90 construct was highly purified and approximately 0.1-1 mg proteins were obtained from 100 ml of bacterial culture. All the purified HSP90 constructs were successfully confirmed by tandem mass spectrometry (nanoLC-ESI-ETD MS/MS) and their secondary structure was quantified using attenuated total reflection - Fourier-transform infrared (ATR-FTIR) spectroscopy. Our expression and purification protocols would facilitate further structural and functional studies of human HSP90.

Heat shock proteins and their expression in primary murine cardiac cell populations during ischemia and reperfusion.

A tight quality control system over protein folding, turnover and synthesis, involving molecular chaperones and co-chaperones, maintains the balance of cardiac proteins. Various cardiac pathologies, including myocardial infarction, increase stresses and post-translational modifications favoring misfolding due to an overwhelmed quality control system. The toxic nature of accumulated misfolded proteins further worsens the condition. The important molecular chaperones which act as quality control proteins are involved in protecting the heart, these include heat shock protein70 (HSP70) and HSP90. Here, we review the emerging roles of heat shock proteins in the maintenance of cardiac cell populations in experimental models of ischemia/reperfusion (I/R) injury. Furthermore, we discuss the expression of HSP70 and HSP90 with therapeutic and diagnostic considerations. Although there is only a partial understanding of these important HSPs in I/R injury, there is an immense therapeutic potential of modulating these HSPs to counteract the imbalance between misfolding and endogenous protein quality control systems.

Characterization of a putative ribosome binding site at the 5' untranslated region of bovine heat shock protein 90.

Untranslated regions (UTRs) of the transcripts play significant roles in translation regulation and continue to raise many intriguing questions in our understanding of cellular stress physiology. Internal ribosome entry site (IRES) mediated alternative translation initiations are emerging as unique mechanisms. Present study is aimed to indentify a functional short 92 base pair length putative sequence located at the 5' untranslated region of bovine heat shock protein 90 AA1 (Hsp90AA1) may interact with ribosomal as well as eukaryotic initiation factor binding site. Here we have predicted both the two and three dimensional structures of bovine Hsp90AA1 IRES (MF400854) element with their respective free energy. Molecular interactions between bovine RPS5 and IRES have been determined after the preparation of docking complex of IRES bound RPS5. Structure of bovine ribosomal translational initiation factor (TIF) has also been determined and docked with IRES. Molecular interaction between bovine TIF and IRES was analyzed from the complex structure. We further detected the relative expression efficiency of the viral (original) in relation with Hsp90AA1 IRES-driven GFP expression, which revealed that efficiency under the control of identified bovine Hsp90AA1 IRES was slightly lower than viral origin. It was also noted that identified bovine HSP90 IRES may increase the expression level of GFP under in vitro heat stressed condition.

PML/RARA inhibits expression of HSP90 and its target AKT.

Essential for cell survival, the 90 kD Heat Shock Proteins (HSP90) are molecular chaperons required for conformational stabilization and trafficking of numerous client proteins. Functional HSP90 is required for the stability of AKT, a serine-threonine kinase phosphorylated in response to growth factor stimulation. AKT plays a crucial regulatory role in differentiation, cell cycle, transcription, translation, metabolism and apoptosis. Acute promyelocytic leukaemia (APL) is characterized by the presence of the promyelocytic leukaemia/retinoic acid receptor alpha (PML/RARA) fusion protein, which deregulates expression of several genes involved in differentiation and apoptosis. Here, we report inhibition of HSP90AA1 and HSP90AB1 isomer transcription in blasts isolated from patients with APL, associated with reduction of HSP90 protein expression and loss of control on AKT protein phosphorylation. We show that in vitro treatment of PML/RARA expressing cells with all-trans retinoic acid (ATRA) up-regulates HSP90 expression and stabilizes AKT. Addition of the HSP90-inhibitor 17-(allylamino)-17-demethoxygeldanamycin in combination with ATRA, blocks upregulation of AKT protein, indicating that HSP90 is necessary for ATRA action on AKT. This is the first report proving that expression of HSP90 isomers are directly and differentially repressed by PML/RARA, with critical results on cellular homeostasis of target proteins, such as AKT, in APL blasts.

Heat shock protein 90 does not contribute to cutaneous vasodilatation in older adults during heat stress.

OBJECTIVES: Heat shock protein 90 (HSP90) contributes to cutaneous vasodilatation during exercise in the heat through nitric oxide (NO) synthase (NOS)-dependent mechanisms in young adults. We hypothesized that similar responses would be observed in older middle-aged adults. METHODS: In nineteen habitually active older middle-aged (56 ± 5 years) men (n = 9) and women (n = 10), cutaneous vascular conductance (CVC) was measured at four forearm skin sites continuously treated with (a) lactated Ringers solution (Control), (b) 10 mmol/L L-NAME (NOS inhibitor), (c) 178 µmol/L geldanamycin (HSP90 inhibitor), or (d) 10 mmol/L L-NAME and 178 µmol/L geldanamycin combined. Participants rested in an upright semi-recumbent position in the heat (35°C) for 70 minutes, followed by a 50-minute bout of moderate-intensity cycling (~55% peak oxygen uptake) and a 30-minute recovery period in the heat. RESULTS: In both men and women, we observed no significant effects of HSP90 inhibition on CVC throughout rest, exercise, and recovery in the heat (all P > 0.27). Conversely, NOS inhibition and dual NOS and HSP90 inhibition attenuated CVC relative to Control throughout the protocol (all P ≤ 0.05). CONCLUSIONS: While NOS mediates cutaneous vasodilatation during rest, exercise, and recovery in the heat, HSP90 does not measurably influence this response in habitually active older middle-aged men or women under these conditions.

Elevated HSP90 associates with expression of HIF-1α and p-AKT and is predictive of poor prognosis in nasopharyngeal carcinoma.

AIMS: HSP90, as a molecular chaperone, has numerous substrate proteins, including HIF-1α and p-AKT, but the relationships among HSP90, HIF-1α and p-AKT have not been investigated in NPC. We examined and analysed the correlation between expression of HSP90, HIF-1α and p-AKT and clinicopathological features of NPC. METHODS: We collected 445 cases of NPC and 54 cases of non-cancerous nasopharyngeal epithelia tissues, detected expression of HSP90, HIF-1α and p-AKT proteins in these tissues by immunohistochemistry. RESULTS: The results indicated that overexpression of HSP90, HIF-1α and p-AKT in NPC was significantly higher than that in non-cancerous nasopharyngeal epithelia (P < 0.05). The overexpression of HIF-1α in primary NPC was significantly lower than that in matched lymph node metastatic NPC (P = 0.024) or recurrent NPC (P = 0.039). The overexpression of HSP90 (P < 0.001) and HIF-1α (P = 0.031) was evidently higher in late stage NPC. NPC patients with lymph node metastasis (LNM) had a higher overexpression rate of HSP90 (P < 0.001) than those without LNM. Increased HSP90 expression was positively associated with HIF-1α expression (r = 0.367, P < 0.001) and p-AKT (r = 0.142, P = 0.003) expression in NPC. Furthermore, HIF-1α was also related to p-AKT expression (r = 0.114, P = 0.017). The overall survival rate for NPC patients with up-regulated HSP90 was significantly lower than those with down-regulated HSP90 (P < 0.001), as was found with raised HIF-1α (P = 0.036) and increased p-AKT (P = 0.044). Multivariate Cox regression analysis further identified that HSP90 and HIF-1α were independent poor prognostic factors for NPC. CONCLUSIONS: Taken together, elevated HSP90 was associated with expression of HIF-1α and p-AKT in NPC. Furthermore, high expression of HSP90 and HIF-1α could be used as a novel independent poor prognostic biomarker for patients with NPC.

Structural and Functional Recovery of Sensory Cilia in C. elegans IFT Mutants upon Aging.

The majority of cilia are formed and maintained by the highly conserved process of intraflagellar transport (IFT). Mutations in IFT genes lead to ciliary structural defects and systemic disorders termed ciliopathies. Here we show that the severely truncated sensory cilia of hypomorphic IFT mutants in C. elegans transiently elongate during a discrete period of adult aging leading to markedly improved sensory behaviors. Age-dependent restoration of cilia morphology occurs in structurally diverse cilia types and requires IFT. We demonstrate that while DAF-16/FOXO is dispensable, the age-dependent suppression of cilia phenotypes in IFT mutants requires cell-autonomous functions of the HSF1 heat shock factor and the Hsp90 chaperone. Our results describe an unexpected role of early aging and protein quality control mechanisms in suppressing ciliary phenotypes of IFT mutants, and suggest possible strategies for targeting subsets of ciliopathies.

Uncaria rhynchophylla Ameliorates Parkinson's Disease by Inhibiting HSP90 Expression: Insights from Quantitative Proteomics.

BACKGROUND/AIMS: Uncaria rhynchophylla, known as "Gou-teng", is a traditional Chinese medicine (TCM) used to extinguish wind, clear heat, arrest convulsions, and pacify the liver. Although U. rhynchophylla has a long history of being often used to treat central nervous system (CNS) diseases, its efficacy and potential mechanism are still uncertain. This study investigated neuroprotective effect and the underlying mechanism of U. rhynchophylla extract (URE) in MPP+-induced SH-SY5Y cells and MPTP-induced mice. METHODS: MPP+-induced SH-SY5Y cells and MPTP-induced mice were used to established Parkinson's disease (PD) models. Quantitative proteomics and bioinformatics were used to uncover proteomics changes of URE. Western blotting was used to validate main differentially expressed proteins and test HSP90 client proteins (apoptosis-related, autophagy-related, MAPKs, PI3K, and AKT proteins). Flow cytometry and JC-1 staining assay were further used to confirm the effect of URE on MPP+-induced apoptosis in SH-SY5Y cells. Gait analysis was used to detect the behavioral changes in MPTP-induced mice. The levels of dopamine (DA) and their metabolites were examined in striatum (STR) by HPLC-EC. The positive expression of tyrosine hydroxylase (TH) was detected by immunohischemical staining and Western blotting. RESULTS: URE dose-dependently increased the cell viability in MPP+-induced SH-SY5Y cells. Quantitative proteomics and bioinformatics results confirmed that HSP90 was an important differentially expressed protein of URE. URE inhibited the expression of HSP90, which further reversed MPP+-induced cell apoptosis and autophagy by increasing the expressions of Bcl-2, Cyclin D1, p-ERK, p-PI3K p85, PI3K p110α, p-AKT, and LC3-I and decreasing cleaved caspase 3, Bax, p-JNK, p-p38, and LC3-II. URE also markedly decreased the apoptotic ratio and elevated mitochondrial transmembrane potential (DΨm). Furthermore, URE treatment ameliorated behavioral impairments, increased the contents of DA and its metabolites and elevated the positive expressions of TH in SN and STR as well as the TH protein. CONCLUSIONS: URE possessed the neuroprotective effect in vivo and in vitro, regulated MAPK and PI3K-AKT signal pathways, and inhibited the expression of HSP90. U. rhynchophylla has potentials as therapeutic agent in PD treatment.

PET imaging of Hsp90 expression in pancreatic cancer using a new 64Cu-labeled dimeric Sansalvamide A decapeptide.

Heat shock protein 90 (Hsp90) plays a vital role in the progress of malignant disease and elevated Hsp90 expression has been reported in pancreatic cancer. In this study, we radiolabeled a dimeric Sansalvamide A derivative (Di-San A1) with 64Cu, and evaluated the feasibility of using 64Cu-Di-San A1 for PET imaging of Hsp90 expression in a mouse model of pancreatic cancer. A macrocyclic chelator NOTA (1,4,7-triazacyclononane-1,4,7-trisacetic acid) was conjugated to Di-San A1. 64Cu-Di-San A1 was successfully prepared in a radiochemical yield > 97% with a radiochemical purity > 98%. 64Cu-Di-San A1 is stable in PBS and mouse serum with > 92% of parent probe intact after 4 h incubation. The cell binding and uptake revealed that 64Cu-Di-San A1 binds to Hsp90-positive PL45 pancreatic cancer cells, and the binding can be effectively blocked by an Hsp90 inhibitor (17AAG). For microPET study, 64Cu-Di-San A1 shows good in vivo performance in terms of tumor uptake in nude mice bearing PL45 tumors. The Hsp90-specific tumor activity accumulation of 64Cu-Di-San A1 was further demonstrated by significant reduction of PL45 tumor uptake with a pre-injected blocking dose of 17AAG. The ex vivo PET imaging and biodistribution results were consistent with the quantitative analysis of PET imaging, demonstrating good tumor-to-muscle ratio (5.35 ± 0.46) of 64Cu-Di-San A1 at 4 h post-injection in PL45 tumor mouse xenografts. 64Cu-Di-San A1 allows PET imaging of Hsp90 expression in PL45 tumors, which may provide a non-invasive method to quantitatively characterize Hsp90 expression in pancreatic cancer.

Metformin reverses TRAP1 mutation-associated alterations in mitochondrial function in Parkinson's disease.

The mitochondrial proteins TRAP1 and HTRA2 have previously been shown to be phosphorylated in the presence of the Parkinson's disease kinase PINK1 but the downstream signalling is unknown. HTRA2 and PINK1 loss of function causes parkinsonism in humans and animals. Here, we identified TRAP1 as an interactor of HTRA2 using an unbiased mass spectrometry approach. In our human cell models, TRAP1 overexpression is protective, rescuing HTRA2 and PINK1-associated mitochondrial dysfunction and suggesting that TRAP1 acts downstream of HTRA2 and PINK1. HTRA2 regulates TRAP1 protein levels, but TRAP1 is not a direct target of HTRA2 protease activity. Following genetic screening of Parkinson's disease patients and healthy controls, we also report the first TRAP1 mutation leading to complete loss of functional protein in a patient with late onset Parkinson's disease. Analysis of fibroblasts derived from the patient reveal that oxygen consumption, ATP output and reactive oxygen species are increased compared to healthy individuals. This is coupled with an increased pool of free NADH, increased mitochondrial biogenesis, triggering of the mitochondrial unfolded protein response, loss of mitochondrial membrane potential and sensitivity to mitochondrial removal and apoptosis. These data highlight the role of TRAP1 in the regulation of energy metabolism and mitochondrial quality control. Interestingly, the diabetes drug metformin reverses mutation-associated alterations on energy metabolism, mitochondrial biogenesis and restores mitochondrial membrane potential. In summary, our data show that TRAP1 acts downstream of PINK1 and HTRA2 for mitochondrial fine tuning, whereas TRAP1 loss of function leads to reduced control of energy metabolism, ultimately impacting mitochondrial membrane potential. These findings offer new insight into mitochondrial pathologies in Parkinson's disease and provide new prospects for targeted therapies.

Repression of Proteases and Hsp90 Chaperone Expression Induced by an Antiretroviral in Virulent Environmental Strains of Cryptococcus neoformans.

This study evaluated the effect of the antiretroviral ritonavir on protease secretion in different strains of Cryptococcus neoformans isolated from the environment and investigated the expression of heat shock protein (Hsp90), classically described virulence factors in other yeast in the presence of the same antiretroviral. The presence of the enzyme was detected by the formation of a degradation of the halo around the colonies. The results were classified as follows: level 1 (without proteases), level 2 (positive for proteases), and level 3 (strongly positive for proteases). Total protein extract isolated from the cell walls of the 12 strains incubated in the absence and presence of ritonavir (0.3125 mg mL-1) were resolved by SDS-PAGE and analyzed by Western blot assays using an antiserum against Hsp90 from Blastocladiella emersonii. All strains tested showed inhibition of proteinase activity in the presence of ritonavir at 0.3125 to 1.25 mg mL-1. High levels of Hsp90 were observed in the absence of ritonavir (0.3125 mg mL-1), except for the non-virulent control cells. In contrast, in the presence of the antiretroviral, a drastic reduction in the expression of the chaperone was observed. The data suggest that ritonavir, in addition to containing viral replication, could inhibit the expression of virulence factors in opportunistic yeast, as proteases and Hsp90. According to our current knowledge, this is the first time that the inhibition of Hsp90 by an antiretroviral was reported for environmental isolates of C. neoformans.

Dexamethasone treatment decreases replication of viral hemorrhagic septicemia virus in Epithelioma papulosum cyprini cells.

The expression of Mx1 in EPC cells after treatment with poly(I:C) or infection with viral hemorrhagic septicemia virus (VHSV) was significantly suppressed by treatment with dexamethasone. However, the titer of VHSV did not increase but instead decreased after dexamethasone treatment. This suggests that dexamethasone not only downregulates type I IFN but also affects certain factors that are necessary for VHSV replication. An important effect of HSP90 on replication of RNA viruses and downregulation of HSP90 by glucocorticoids have been reported. In this study, dexamethasone downregulated HSP90α expression in EPC cells that were stimulated with poly(I:C) or infected with VHSV. Furthermore, cells treated with an HSP90 inhibitor, geldanamycin, showed significantly decreased titers of VHSV, suggesting that HSP90 may be an important host component involved in VHSV replication, and HSP90 inhibition might be one of the causes for the observed reduction in viral titer caused by dexamethasone treatment.

Predicting mutation outcome from early stochastic variation in genetic interaction partners.

Many mutations, including those that cause disease, only have a detrimental effect in a subset of individuals. The reasons for this are usually unknown, but may include additional genetic variation and environmental risk factors. However, phenotypic discordance remains even in the absence of genetic variation, for example between monozygotic twins, and incomplete penetrance of mutations is frequent in isogenic model organisms in homogeneous environments. Here we propose a model for incomplete penetrance based on genetic interaction networks. Using Caenorhabditis elegans as a model system, we identify two compensation mechanisms that vary among individuals and influence mutation outcome. First, feedback induction of an ancestral gene duplicate differs across individuals, with high expression masking the effects of a mutation. This supports the hypothesis that redundancy is maintained in genomes to buffer stochastic developmental failure. Second, during normal embryonic development we find that there is substantial variation in the induction of molecular chaperones such as Hsp90 (DAF-21). Chaperones act as promiscuous buffers of genetic variation, and embryos with stronger induction of Hsp90 are less likely to be affected by an inherited mutation. Simultaneously quantifying the variation in these two independent responses allows the phenotypic outcome of a mutation to be more accurately predicted in individuals. Our model and methodology provide a framework for dissecting the causes of incomplete penetrance. Further, the results establish that inter-individual variation in both specific and more general buffering systems combine to determine the outcome inherited mutations in each individual.

Erythrocyte heat shock protein responses to chronic (in vivo) and acute (in vitro) temperature challenge in diploid and triploid salmonids.

This research investigated how ploidy level (diploid versus triploid) affects the heat shock protein (HSP) response in erythrocytes under different thermal stress regimes, both in vivo and in vitro, in Atlantic salmon (Salmo salar) and brook charr (Salvelinus fontinalis) in order to address the question of why triploids typically have reduced thermal tolerance. A preliminary study confirmed that identical volumes of diploid and triploid erythrocytes (which equates to a smaller number of larger cells for triploids compared to diploids) did not differ in total protein synthesis rates. After chronic (100d) acclimation of fish to 5, 15 and 25°C, triploid erythrocytes had lower HSP70, HSP90, heat shock factor 1 (HSF1) and ubiquitin (free and total) levels than diploids in both species. Furthermore, Atlantic salmon erythrocytes showed significantly higher protein breakdown (based on conjugated ubiquitin levels) in triploids than diploids after acute heat stress in vitro, but no significant difference was detected between ploidies after acute cold stress. These results indicate that: 1) triploid erythrocytes synthesize more total protein per cell than diploids as a result of increased cell size; 2) triploids have sufficient total HSP levels for survival under low stress conditions; and 3) the lower basal titres of HSPs in triploids may be a handicap when combating acute stress. Taken together, this suggests that triploids are limited in their ability to withstand thermal stress because of a reduced ability to maintain proteostasis under stressful conditions.

Restraining Pathogenicity in Candida albicans by Taxifolin as an Inhibitor of Ras1-pka Pathway.

Candida albicans is one of the most virulent and opportunistic fungal strains. In the present scenario, majority metabolic imbalances and unsuccessful treatments of some severe diseases including cancer, diabetes, HIV, psoriasis are because of invasive Candida emergence. Being a beneficial integral part of human biome, its elimination is not possible. The major pathogenicity characteristics in Candida involve hyphal growth, biofilm formation, HSP90 down regulation and genetic modifications. Ras1-pka pathway initiated by HSP90 down regulation is important for hyphal growth and has been focused in the present study. The principle transcriptional factors that induce hyphal growth causing invasiveness and virulence through this pathway have been identified as Tec1 and Rfg1. In the present study, taxifolin, a naturally occurring polyphenol, has been identified as inhibitor for both the transcriptional factors in parallel.

Subtherapeutic tetracycline concentrations aggravate Salmonella Typhimurium infection by increasing bacterial virulence.

OBJECTIVES: Antibiotics are among the most frequently prescribed drugs in human and animal medicine. With antibiotic resistance being a serious threat to veterinary and public health, the prudent use of antibiotics receives much attention. Less well known is that incorrect use of antimicrobial agents may also lead to increased bacterial virulence with the potential of a more severe clinical course of infection. Therefore, the aim of this study was to investigate the effect of subtherapeutic doses of tetracyclines on htpG virulence gene expression in Salmonella Typhimurium and on the course of salmonellosis. METHODS: Salmonella strains containing an htpG-luxCDABE transcriptional fusion were constructed. Phenotype microarrays and tetracycline treatment were used to investigate their htpG expression. A Salmonella transposon mutant bank was used to identify genes involved in the induction of htpG gene expression. Finally, the in vitro results were linked to the in vivo situation using a Salmonella mouse model. RESULTS: We demonstrate that subtherapeutic antimicrobial concentrations can exacerbate bacterial infections through direct up-regulation of bacterial virulence factors using Salmonella Typhimurium 112910a phage type 120/ad as a model organism. Phenotype microarrays showed that expression of the Salmonella Typhimurium virulence gene htpG is increased by several tetracycline antimicrobials at values below their MIC, a process that requires intact Salmonella LPS genes. Exposure of experimentally infected DBA/2J mice to subtherapeutic doxycycline concentrations resulted in htpG-mediated exacerbation of Salmonella Typhimurium infection. CONCLUSIONS: These findings show that the Salmonella isolate used in this study can respond to subtherapeutic tetracycline pressure by increasing its virulence and disease severity.

Down-regulation of Ras-related protein Rab 5C-dependent endocytosis and glycolysis in cisplatin-resistant ovarian cancer cell lines.

Drug resistance poses a major challenge to ovarian cancer treatment. Understanding mechanisms of drug resistance is important for finding new therapeutic targets. In the present work, a cisplatin-resistant ovarian cancer cell line A2780-DR was established with a resistance index of 6.64. The cellular accumulation of cisplatin was significantly reduced in A2780-DR cells as compared with A2780 cells consistent with the general character of drug resistance. Quantitative proteomic analysis identified 340 differentially expressed proteins between A2780 and A2780-DR cells, which involve in diverse cellular processes, including metabolic process, cellular component biogenesis, cellular processes, and stress responses. Expression levels of Ras-related proteins Rab 5C and Rab 11B in A2780-DR cells were lower than those in A2780 cells as confirmed by real-time quantitative PCR and Western blotting. The short hairpin (sh)RNA-mediated knockdown of Rab 5C in A2780 cells resulted in markedly increased resistance to cisplatin whereas overexpression of Rab 5C in A2780-DR cells increases sensitivity to cisplatin, demonstrating that Rab 5C-dependent endocytosis plays an important role in cisplatin resistance. Our results also showed that expressions of glycolytic enzymes pyruvate kinase, glucose-6-phosphate isomerase, fructose-bisphosphate aldolase, lactate dehydrogenase, and phosphoglycerate kinase 1 were down-regulated in drug resistant cells, indicating drug resistance in ovarian cancer is directly associated with a decrease in glycolysis. Furthermore, it was found that glutathione reductase were up-regulated in A2780-DR, whereas vimentin, HSP90, and Annexin A1 and A2 were down-regulated. Taken together, our results suggest that drug resistance in ovarian cancer cell line A2780 is caused by multifactorial traits, including the down-regulation of Rab 5C-dependent endocytosis of cisplatin, glycolytic enzymes, and vimentin, and up-regulation of antioxidant proteins, suggesting Rab 5C is a potential target for treatment of drug-resistant ovarian cancer. This constitutes a further step toward a comprehensive understanding of drug resistance in ovarian cancer.

Calycosin attenuates osteoporosis and regulates the expression of OPG/RANKL in ovariectomized rats via MAPK signaling.

We aimed at exploring the effect of calycosin (CA) on osteoporosis in ovariectomized (OVX) rats. Sprague-Dawley (SD) rats were divided into five groups: Sham group, OVX group, OVX group treated with estradiol valerate (EV), CAL group treated with 15 mg/kg/d of CA and CAH group treated with 30 mg/kg/d of CA for 12 weeks. Bone mineral density (BMD), histopathology, body weight, parameters in serum and urine were observed. Gene expression and protein level of OPG/RANKL were also studied by real-time PCR and western blot, respectively. We further identified the effect of CA on mitogen-activated protein kinase (MAPK) signaling. In comparison with OVX rats, CAL and CAH significantly increased the BMD by 8.3% and 19.0%. Treatment with CA notably inhibited the excretion of Ca, P and Cr. CAH also significantly increased the level of alkaline phosphatase (ALP) and decreased the level of tartrate-resistant acid phosphatase (TRAP) in serum of OVX rats. CA could improve the trabecular bone area, and increased the trabecular number and the trabecular connection after 12-week. CA also increased the expression of osteoprotegerin (OPG) and decreased the Receptor Activator for Nuclear Factor-κB Ligand (RANKL) mRNA expression compared with the OVX rats. In addition, CA could effectively decrease the phosphorylation of MAPKs induced by ovariectomy. In conclusion, CA had remarkable antiosteoporotic activity and therefore can be a promising candidate for the treatment of postmenopausal osteoporosis.

[The Role of Membrane-Bound Heat Shock Proteins Hsp90 in Migration of Tumor Cells in vitro and Involvement of Cell Surface Heparan Sulfate Proteoglycans in Protein Binding to Plasma Membrane].

Heat shock protein Hsp90, detected in the extracellular space and on the membrane of cells, plays an important role in cell motility, migration, invasion and metastasis of tumor cells. At present, the functional role and molecular mechanisms of Hsp90 binding to plasma membrane are not elucidated. Using isoform-specific antibodies against Hsp90, Hsp9α and Hsp90ß, we showed that membrane-bound Hsp90α and Hsp90ß play a significant role in migration of human fibrosarcoma (HT1080) and glioblastoma (A-172) cells in vitro. Disorders of sulfonation of cell heparan sulfates, cleavage of cell heparan. sulfates by heparinase I/III as well as treatment of cells with heparin lead to an abrupt reduction in the expression level of Hsp90 isoforms. Furthermore, heparin significantly inhibits tumor cell migration. The results obtained demonstrate that two isoforms of membrane-bound Hsp90 are involved in migration of tumor cells in vitro and that cell surface heparan sulfate proteoglycans play a pivotal role in the "anchoring" of Hsp90α and Hsp90ß to the plasma membrane.