Characterizing the toxicological responses to inorganic arsenicals and their metabolites in immortalized human bladder epithelial cells.

Arsenic is highly toxic to the human bladder. In the present study, we established a human bladder epithelial cell line that closely mimics normal human bladder epithelial cells by immortalizing primary uroplakin 1B-positive human bladder epithelial cells with human telomerase reverse transcriptase (HBladEC-T). The uroplakin 1B-positive human bladder epithelial cell line was then used to evaluate the toxicity of seven arsenicals (iAsV, iAsIII, MMAV, MMAIII, DMAV, DMAIII, and DMMTAV). The cellular uptake and metabolism of each arsenical was different. Trivalent arsenicals and DMMTAV exhibited higher cellular uptake than pentavalent arsenicals. Except for MMAV, arsenicals were transported into cells by aquaglyceroporin 9 (AQP9). In addition to AQP9, DMAIII and DMMTAV were also taken up by glucose transporter 5. Microarray analysis demonstrated that arsenical treatment commonly activated the NRF2-mediated oxidative stress response pathway. ROS production increased with all arsenicals, except for MMAV. The activating transcription factor 3 (ATF3) was commonly upregulated in response to oxidative stress in HBladEC-T cells: ATF3 is an important regulator of necroptosis, which is crucial in arsenical-induced bladder carcinogenesis. Inorganic arsenics induced apoptosis while MMAV and DMAIII induced necroptosis. MMAIII, DMAV, and DMMTAV induced both cell death pathways. In summary, MMAIII exhibited the strongest cytotoxicity, followed by DMMTAV, iAsIII, DMAIII, iAsV, DMAV, and MMAV. The cytotoxicity of the tested arsenicals on HBladEC-T cells correlated with their cellular uptake and ROS generation. The ROS/NRF2/ATF3/CHOP signaling pathway emerged as a common mechanism mediating the cytotoxicity and carcinogenicity of arsenicals in HBladEC-T cells.

OTUD1 deubiquitinase regulates NF-κB- and KEAP1-mediated inflammatory responses and reactive oxygen species-associated cell death pathways.

Deubiquitinating enzymes (DUBs) regulate numerous cellular functions by removing ubiquitin modifications. We examined the effects of 88 human DUBs on linear ubiquitin chain assembly complex (LUBAC)-induced NF-κB activation, and identified OTUD1 as a potent suppressor. OTUD1 regulates the canonical NF-κB pathway by hydrolyzing K63-linked ubiquitin chains from NF-κB signaling factors, including LUBAC. OTUD1 negatively regulates the canonical NF-κB activation, apoptosis, and necroptosis, whereas OTUD1 upregulates the interferon (IFN) antiviral pathway. Mass spectrometric analysis showed that OTUD1 binds KEAP1, and the N-terminal intrinsically disordered region of OTUD1, which contains an ETGE motif, is indispensable for the KEAP1-binding. Indeed, OTUD1 is involved in the KEAP1-mediated antioxidant response and reactive oxygen species (ROS)-induced cell death, oxeiptosis. In Otud1-/--mice, inflammation, oxidative damage, and cell death were enhanced in inflammatory bowel disease, acute hepatitis, and sepsis models. Thus, OTUD1 is a crucial regulator for the inflammatory, innate immune, and oxidative stress responses and ROS-associated cell death pathways.

The carbonic anhydrase inhibitor acetazolamide inhibits urinary bladder cancers via suppression of ß-catenin signaling.

Carbonic anhydrases (CAs) play an important role in maintaining pH homeostasis. We previously demonstrated that overexpression of CA2 was associated with invasion and progression of urothelial carcinoma (UC) in humans. The purpose of the present study was to evaluate the effects of the CA inhibitor acetazolamide (Ace) on N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced bladder carcinogenesis in mice and explore the function of CA2 in muscle invasion by UC. Male mice were treated with 0.025% (experiment 1) or 0.05% BBN (experiment 2) in their drinking water for 10 weeks, then treated with cisplatin (Cis), Ace, or Cis plus Ace for 12 weeks. In experiment 1, the overall incidence of BBN-induced UCs was significantly decreased in the BBN→Ace and BBN→Cis+Ace groups. In experiment 2, the overall incidence of BBN-induced UCs was significantly decreased in the BBN→Cis+Ace group, and the incidence of muscle invasive UC was significantly decreased in both the BBN→Ace and the BBN→Cis+Ace groups. We also show that overexpression of CA2 by human UC cells T24 and UMUC3 significantly increased their migration and invasion capabilities, and that Ace significantly inhibited migration and invasion by CA2-overexpressing T24 and UMUC3 cells. These data demonstrate a functional association of CA2 with UC development and progression, confirming the association of CA2 with UC that we had shown previously by immunohistochemical analysis of human UC specimens and proteome analysis of BBN-induced UC in rats. Our finding that inhibition of CA2 inhibits UC development and muscle invasion also directly confirms that CA2 is a potential therapeutic target for bladder cancers.

Suppression of the doxorubicin response by hypoxia-inducible factor-1α is strictly dependent on oxygen concentrations under hypoxic conditions.

Hypoxia-inducible factor-1α (HIF-1α) and p53 are involved in anticancer drug resistance under hypoxic conditions. Here, we found that the cytotoxicity of anticancer drugs (doxorubicin, gemcitabine, and cisplatin) was lower at 1% O2 than at 5% O2. We examined the effects of these drugs on HIF-1α and p53 expression under different hypoxic oxygen concentrations. At 5% O2, the drugs decreased HIF-1α expression and increased p53 levels. At 1% O2, the drugs increased HIF-1α expression but did not alter p53 levels. When the HIF-1α protein was stabilized by DMOG under normoxic conditions, doxorubicin did not increase the level of p53 expression. These results show that the maintenance of HIF-1α expression blocked doxorubicin-dependent increases in p53 expression. We hypothesized the mechanism of HIF-1α protein translation might be different between at 5% and at 1% O2, because many reports indicate that the same mechanism of HIF-1α protein stabilization occurs under hypoxic conditions, such as 5% and 1% O2. The level of phosphorylated-4E-BP1, which causes translation of HIF-1α, was higher at 1% O2 than at 5% O2. Our results suggest that the sensitivity of tumor cells to anticancer drugs is dependent oxygen concentrations under hypoxic conditions, and involves 4E-BP1-dependent stabilization of the HIF-1α protein.

Canopy Homolog 2 as a Novel Molecular Target in Hepatocarcinogenesis.

In the present study, the role of a novel protein involved in neurite development and endoplasmic reticulum (ER) stress, canopy homolog 2 (CNPY2), was investigated in mouse and human hepatocarcinogenesis. Firstly, a sensitive quantitative and qualitative detection of protein expression using QSTAR Elite LC-Ms/Ms was performed for the analysis of lysates of microdissected hepatocellular altered foci (AF), adenomas (HCAs), carcinomas (HCCs) and peri-tumoral livers from C57Bl/6J mice treated with diethylnitrosamine (DEN) and then maintained for 27 or 38 weeks on basal diet. Significant overexpression of 18.5 kDa CNPY2 processed form was demonstrated in AF, HCAs and HCCs, while low expression was observed in the livers of DEN-treated and control mice. Furthermore, CNPY2 elevation in AF and tumors was coordinated with accumulation of numerous cytoskeletal proteins, including cytokeratins 8 and 18, actin, non-muscle myosin and septin 9 and those involved in ER and mitochondrial stresses such as calreticulin, prohibitins 1 and 2 and YME1-like-1. Knockdown of CNPY2 in Huh7 and HepG2 human liver cancer cells resulted in significant suppression of cell survival and invasive potential, inhibition of cyclin D1, induction of p21Waf1/Cip1 and suppression of the apoptosis inhibitor Bcl2. In contrast, transfection of a mouse CNPY2 (mCNPY2-Ds-Red) vector plasmid in Huh7 and HepG2 cancer cells, with subsequent accumulation of CNPY2 in the ER, resulted in significant increase in cancer cells survival. Clinicopathological analysis in 90 HCV-positive HCC patients, revealed significant association of CNPY2 overexpression with poor overall (p = 0.041) survival. Furthermore, CNPY2 increase was associated with vessel invasion (p = 0.038), poor histological differentiation (p = 0.035) and advanced clinical stage (p = 0.016). In conclusion, CNPY2 is a promising molecular target elevated early in hepatocarcinogenesis and prognostic marker for human HCV-associated HCC. CNPY2 is involved in the processes of ER stress, cell cycle progression, proliferation, survival and invasion of liver tumor cells.

ER membrane protein complex 1 interacts with STIM1 and regulates store-operated Ca2+ entry.

Store-operated calcium entry (SOCE) is the process by which the emptying of endoplasmic reticulum (ER) Ca2+ stores causes an influx of Ca2+ across the plasma membrane (PM). It is the major Ca2+ influx pathway in nonexcitable cells and has a wide array of physiological functions. Upon store depletion, stromal interaction molecule 1 (STIM1), an ER calcium sensor relocates into discrete puncta at the ER-PM junction region, which results in the coupling of Ca2+ channels to initiate SOCE. However, the mechanism regulating STIM1 activity remains poorly understood. Here, we performed affinity purification of STIM1 and uncovered ER membrane protein complex 1 (EMC1) as an STIM1 binding partner. We showed that this interaction occurred in the ER through the intraluminal region of STIM1. After store depletion, EMC1 does not cluster adjacent to the PM, which suggests that it is distributed differently from STIM1. EMC1 knockdown with small interfering RNA resulted in a marked decrease in SOCE. Thus, these findings suggest that EMC1 functions as a positive regulator of SOCE.

Upregulation of PTK7 and ß-catenin after vaginal mechanical dilatation: an examination of fibulin-5 knockout mice.

INTRODUCTION AND HYPOTHESIS: Pelvic organ prolapse (POP) in women is associated with deficiency of elastic fibers, and fibulin-5 is known to be a critical protein in the synthesis of elastin. The purpose of this study is to investigate the related pathway for the synthesis of elastin via fibulin-5 using fibulin-5 knockout mice. METHODS: Fibulin-5 knockout mice were generated using the CRISPR/Cas9 system, and vaginal dilatation was used to mimic vaginal delivery. We divided the mice into three groups: Fbln5+/+ mice immediately after dilatation (Fbln5+/+ day0), Fbln5+/+ mice 3 days after dilatation (Fbln5+/+ day3) and Fbln5-/- mice 3 days after dilatation (Fbln5-/- day3). Proteins related to elastogenesis in the vaginal wall were measured by liquid chromatography mass spectrometry (LC-MS/MS) analysis, and differences in the expression of these proteins between the Fbln5-/- mice and the Fbln5+/+ mice were analyzed using western blotting. RESULTS: In the LC-MS/MS analysis, protein tyrosine kinase 7 (PTK7) was not detected in the Fbln5-/- day3 group, although the expression increased by > 1.5 times between the Fbln5+/+ day0 and day3 groups. PTK7 and ß-catenin are known to act in the Wnt/ß-catenin pathway, and both were upregulated after dilatation in the Fbln5+/+ mice, though not in the Fbln5-/- mice. CONCLUSION: Our findings suggest that these proteins are involved in elastogenesis via fibulin-5, and the impairment of these proteins might be the underlying cause of POP manifestation.

Generation of Rat Monoclonal Antibodies Specific for Human Stromal Cell-Derived Factor-2.

Stromal cell-derived factor-2 (SDF-2) is reportedly involved in multiple endoplasmic reticulum (ER) functions, including the misfolded protein catabolic process, protein glycosylation, and ER protein quality control. However, the precise molecular and cellular functions of SDF-2 remain unknown. Previously, we discovered that SDF-2 mediates acquired resistance to oxaliplatin in human gastric cancer cells. In this study, we have generated SDF-2-specific monoclonal antibodies (mAbs), using the rat medial iliac lymph node method, as a tool to explore novel mechanisms of oxaliplatin resistance. The antibodies detected endogenous human SDF-2 in immunoblotting analyses. In addition, immunoprecipitation analyses revealed the availability of these antibodies for human SDF-2. Thus, these mAbs will be available to elucidate molecular and cellular functions of SDF-2 in cancer cells.

Effects of orally active hypoxia inducible factor alpha prolyl hydroxylase inhibitor, FG4592 on renal fibrogenic potential in mouse unilateral ureteral obstruction model.

Orally active hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors that stabilize HIF protein and stimulate the production of erythropoietin have been approved to treat renal anemia. Our previous report suggested that HIF-1α dependent fibrogenic mechanisms are operating at the early onset of renal fibrosis and its contribution declines with the progression in mouse unilateral ureteral obstruction (UUO) model. The aim of the study is to evaluate the renal fibrogenic potential of FG4592, a recently approved orally active HIF prolyl hydroxylase inhibitor in mouse UUO model. Male C57BL/6J mice orally given FG-4592 (12.5 mg/kg/day and 50 mg/kg/day) were subjected to UUO. Neither dose of FG-4592 affected renal fibrosis or macrophage infiltration. FG-4592 had no effects on increased mRNA of collagen I, collagen III or transforming growth factor-ß1. At 3 days after UUO, higher dose of FG-4592 potentiated the increased mRNA expression of profibrogenic molecules, plasminogen activator inhibitor 1 (Pai-1) and connective tissue growth factor (Ctgf) but such potentiation disappeared at 7 days after UUO. It is suggested that FG-4592 used in the present study had little effects on renal fibrosis even though high dose of FG-4592 used in the present study transiently potentiated gene expression of Pai-1 and Ctgf in the UUO kidney.

The Autophagy-Related Protein GABARAP Is Induced during Overwintering in the Bean Bug (Hemiptera: Alydidae).

In most insects dependent on food resources that deplete seasonally, mechanisms exist to protect against starvation. Insects overcome periods of food depletion using diapause-associated physiological mechanisms, such as increased energy resources in fat bodies and suppression of metabolism. Because autophagy supplies energy resources through the degradation of intracellular components, we hypothesized that it might be an additional strategy to combat starvation during overwintering. In this study, we measured the abundance of the proteins involved in the signaling pathway of autophagy during overwintering in adults of the bean bug Riptortus pedestris (Fabricius) (Hemiptera: Alydidae), which must withstand the periodic depletion of its host plants from late fall to early spring. Although the levels of gamma-aminobutyric acid receptor-associated protein (GABARAP) markedly increased after the cessation of food supply, AMP-activated protein kinase (AMPK) and target of rapamycin (TOR) were not found to be associated with food depletion. Thus, food depletion appears to induce autophagy independent of AMPK and TOR. The GABARAP levels significantly increased universally when the food supply ceased, irrespective of the diapause status of adults and low-temperature conditions. In overwintering diapause adults under seminatural conditions, the GABARAP levels significantly increased during early spring. Thus, autophagy appears to assist the survival of the bean bugs under natural conditions of food deficiency.

Controlling the Phenotype of Tumor-Infiltrating Macrophages via the PHD-HIF Axis Inhibits Tumor Growth in a Mouse Model.

The tumor microenvironment (TME) polarizes tumor-infiltrating macrophages toward tumor support. Macrophage-abundant tumors are highly malignant and are the cause of poor prognosis and therapeutic resistance. In this study, we show that the prolyl hydroxylase (PHD) inhibitor FG-4592 (FG) inhibits tumor growth of macrophage-abundant tumors and prolongs mouse survival. FG not only normalizes tumor vessels and improves tumor oxygenation but also directly affects macrophages and activates phagocytosis through the PHD-hypoxia-inducible factor (HIF) axis. Remarkably, FG can promote phagocytic ability of the Ly6Clo subset of tumor-infiltrating macrophages, leading to tumor growth inhibition. Moreover, Ly6Cneg macrophages contributed to blood vessel normalization. Using a malignant tumor mouse model, we characterized macrophage function and subsets. Altogether, our findings suggest that the PHD inhibitor can promote the anti-tumor potential of macrophages to improve cancer therapy.

Therapeutic Silencing of Centromere Protein X Ameliorates Hyperglycemia in Zebrafish and Mouse Models of Type 2 Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) is characterized by persistent hyperglycemia and is influenced by genetic and environmental factors. Optimum T2DM management involves early diagnosis and effective glucose-lowering therapies. Further research is warranted to improve our understanding of T2DM pathophysiology and reveal potential roles of genetic predisposition. We have previously developed an obesity-induced diabetic zebrafish model that shares common pathological pathways with humans and may be used to identify putative pharmacological targets of diabetes. Additionally, we have previously identified several candidate genes with altered expression in T2DM zebrafish. Here, we performed a small-scale zebrafish screening for these genes and discovered a new therapeutic target, centromere protein X (CENPX), which was further validated in a T2DM mouse model. In zebrafish, cenpx knockdown by morpholino or knockout by CRISPR/Cas9 system ameliorated overfeeding-induced hyperglycemia and upregulated insulin level. In T2DM mice, small-interfering RNA-mediated Cenpx knockdown decreased hyperglycemia and upregulated insulin synthesis in the pancreas. Gene expression analysis revealed insulin, mechanistic target of rapamycin, leptin, and insulin-like growth factor 1 pathway activation following Cenpx silencing in pancreas tissues. Thus, CENPX inhibition exerted antidiabetic effects via increased insulin expression and related pathways. Therefore, T2DM zebrafish may serve as a powerful tool in the discovery of new therapeutic gene targets.

A dipeptidyl peptidase-4 (DPP-4) inhibitor, linagliptin, attenuates cardiac dysfunction after myocardial infarction independently of DPP-4.

Dipeptidyl peptidase-4 (DPP-4) inhibitors not only improve impaired glucose tolerance in diabetes, but also have pleiotropic extra-pancreatic effects such as preconditioning effect for myocardial ischemia-reperfusion injury. Here, we investigated the anti-remodeling effects of linagliptin, a DPP-4 inhibitor, by use of DPP-4-deficient rats. After the induction of myocardial infarction (MI), Fischer 344 rats with inactivating mutation of DPP-4 were orally administrated with a DPP-4 inhibitor, linagliptin (5 mg kg-1·day-1), or vehicle in drinking water for 4 weeks. Linagliptin did not affect hemodynamic status, body weight, and infarct size. In echocardiography, linagliptin tended to improve left ventricular (LV) systolic function, and significantly improved LV diastolic function, surprisingly. Interstitial fibrosis in marginal region and macrophage infiltration were significantly lower in the linagliptin group than those in the vehicle group. Fibrosis-related gene expressions, such as collagen I and transforming growth factor-ß1 (TGF-ß1), and inflammation-related expressions, such as macrophage chemotactic protein 1 and matrix metalloproteinase-2 (MMP-2), were significantly suppressed in marginal area of the linagliptin-treated rats compared with the vehicle rats. The TGF-ß1 and MMP-2 protein levels were attenuated by linagliptin in DPP-4-deficient cardiac fibroblasts. Linagliptin can attenuate MI-induced cardiac remodeling via a DPP-4-independent pathway.

Generation of Rat Monoclonal Antibodies Against a Deubiquitinase, Ovarian Tumor Domain-Containing Protein 1.

Ovarian tumor domain-containing protein 1 (OTUD1), an OTU-family deubiquitinating enzyme, has been reported to be involved in cancer progression through the regulation of p53 and SMAD7. However, the precise pathophysiological functions of OTUD1 remain elusive. Here, we report the establishment of OTUD1-specific monoclonal antibodies (mAbs), using the rat medial iliac lymph node method. The generated antibodies recognize the N-terminal portion (aa. 1-290) of human and mouse OTUD1 proteins. In addition, immunofluorescent staining and subcellular fractionation analyses using these antibodies indicated that OTUD1 is predominantly localized in the cytosol. Thus, these mAbs can be further used to elucidate cellular functions of OTUD1 and its involvement in processes such as cancer progression.

Generation of Rat Monoclonal Antibodies Specific for DZIP3.

DAZ interacting zinc finger 3 (DZIP3), an RNA-binding RING-type ubiquitin ligase, has been reported to be involved in multiple physiological functions, including the regulation of chemokine- or estradiol-induced gene expression, self-renewal, and maintaining pluripotency in mouse embryonic stem cells. However, the precise cellular functions of DZIP3 remain elusive. In this study, we report the establishment of DZIP3-specific monoclonal antibodies (MAbs), using the rat medial iliac lymph node method. In immunoblotting analyses, our antibodies detected endogenous human and mouse DZIP3. In addition, immunoprecipitation analyses revealed the availability of these antibodies for human or mouse DZIP3. Thus, these MAbs will be available to elucidate cellular functions of DZIP3.

Role of hypoxia-inducible factor-1 in the development of renal fibrosis in mouse obstructed kidney: Special references to HIF-1 dependent gene expression of profibrogenic molecules.

The aim of the study is to clarify the role of hypoxia-inducible factor-1 (HIF-1) in the development of renal fibrosis in mouse obstructive nephropathy. We used mice with floxed HIF-1α alleles and tamoxifen-inducible Cre/ERT2 recombinase under ubiquitin C promoter to induce global HIF-1α deletion. Following tamoxifen administration, mice were subjected to unilateral ureteral obstruction (UUO). At 3, 7 and 14 days after UUO, renal gene expression profiles and interstitial fibrosis were assessed. HIF-1 dependent up-regulation of prolyl hydroxylase 3 and glucose transporter-1 was observed in the obstructed kidney at 3 and 7 days but not at 14 days after UUO. Various factors promoting fibrosis were up-regulated during the development of fibrosis. HIF-1 dependent gene expression of profibrotic molecules, plasminogen activator inhibitor 1, connective tissue growth factor, lysyl oxidase like 2 and transglutaminase 2 was observed in the obstructed kidney but such HIF-1 dependency was limited to the early onset of renal fibrosis. Global HIF-1 deletion tended to attenuate interstitial collagen I deposition at 3 days but had no effects thereafter. It is suggested that HIF-1 dependent profibrogenic mechanisms are operating at the early onset of renal fibrosis but its contribution declines with the progression in mouse UUO model.

Heat shock protein 70 inhibitors suppress androgen receptor expression in LNCaP95 prostate cancer cells.

Androgen deprivation therapy is initially effective for treating patients with advanced prostate cancer; however, the prostate cancer gradually becomes resistant to androgen deprivation therapy, which is termed castration-resistant prostate cancer (CRPC). Androgen receptor splice variant 7 (AR-V7), one of the causes of CRPC, is correlated with resistance to a new-generation AR antagonist (enzalutamide) and poor prognosis. Heat shock protein 70 (Hsp70) inhibitor is known to decrease the levels of full-length AR (AR-FL), but little is known about its effects against CRPC cells expressing AR-V7. In this study, we investigated the effect of the Hsp70 inhibitors quercetin and VER155008 in the prostate cancer cell line LNCaP95 that expresses AR-V7, and explored the mechanism by which Hsp70 regulates AR-FL and AR-V7 expression. Quercetin and VER155008 decreased cell proliferation, increased the proportion of apoptotic cells, and decreased the protein levels of AR-FL and AR-V7. Furthermore, VER155008 decreased AR-FL and AR-V7 mRNA levels. Immunoprecipitation with Hsp70 antibody and mass spectrometry identified Y-box binding protein 1 (YB-1) as one of the molecules regulating AR-FL and AR-V7 at the transcription level through interaction with Hsp70. VER155008 decreased the phosphorylation of YB-1 and its localization in the nucleus, indicating that the involvement of Hsp70 in AR regulation might be mediated through the activation and nuclear translocation of YB-1. Collectively, these results suggest that Hsp70 inhibitors have potential anti-tumor activity against CRPC by decreasing AR-FL and AR-V7 expression through YB-1 suppression.

Macrophage-derived exosomes induce inflammatory factors in endothelial cells under hypertensive conditions.

Hypertension is one of the most important cardiovascular risk factors and results in macrophage infiltration of blood vessels. However, how macrophages coordinate inflammatory responses with endothelial cells (ECs) remains unclear. In this study, we investigated whether exosomes upregulate the expression of inflammatory factors in ECs under hypertensive conditions. Hypertension was induced in rats by continuous infusion of angiotensin II (Ang II). Exosomes were purified from rat serum by density gradient and ultracentrifugation and used to stimulate human coronary artery ECs (HCAECs). Moreover, the interactions between HCAECs and exosomes from human THP-1-derived macrophages were analyzed. Administration of Ang II enhanced the expression of CD68, a macrophage marker, in rat hearts, suggesting enhanced infiltration of macrophages. In addition, the expression of intracellular adhesion molecule-1 (ICAM1) and plasminogen activator inhibitor-1 (PAI-1), a proinflammatory factor, was increased in hypertensive rat hearts compared with control rats. CD68 protein expression and an increase in the expression of some exosome markers were detected in exosomes from hypertensive rat serum. Moreover, the exosomes upregulated the expression levels of ICAM1 and PAI-1 in HCAECs. The level of miR-17, a negative regulator of ICAM1 expression, was markedly decreased in exosomes from hypertensive rat serum compared with exosomes from control rats. Interestingly, Ang II-stimulated THP-1-derived exosomes also enhanced the expression of ICAM1 and PAI-1 and contained reduced levels of miR-17 compared with exosomes from unstimulated cells. These results suggest that inflammation of ECs under hypertensive conditions is caused, at least in part, by macrophage-derived exosomes.

Protection of stromal cell-derived factor 2 by heat shock protein 72 prevents oxaliplatin-induced cell death in oxaliplatin-resistant human gastric cancer cells.

Heat shock protein 72 (Hsp72) is a molecular chaperone that assists in the folding of nascent polypeptides and in the refolding of denatured proteins. In many cancers, Hsp72 is constitutively expressed at elevated levels, which can result in enhanced stress tolerance. Similarly, following treatment with anticancer drugs, Hsp72 binds to denatured proteins that may be essential for survival. We therefore hypothesized that Hsp72 client proteins may play a crucial role in drug resistance. Here, we aimed to identify proteins that are critical for oxaliplatin (OXA) resistance by analyzing human gastric cancer cell lines, as well as OXA-resistant cells via a mass spectrometry-based proteomic approach combined with affinity purification using anti-Hsp72 antibodies. Stromal cell-derived factor 2 (SDF-2) was identified as an Hsp72 client protein unique to OCUM-2M/OXA cells. SDF-2 was overexpressed in OXA-resistant cells and SDF-2 silencing promoted the apoptotic effects of OXA. Furthermore, Hsp72 prevented SDF-2 degradation in a chaperone activity-dependent manner. Together, our data demonstrate that Hsp72 protected SDF-2 to avoid OXA-induced cell death. We propose that inhibition of SDF-2 may comprise a novel therapeutic strategy to counteract OXA-resistant cancers.