Deletion of Clusterin Protects Cochlear Hair Cells against Hair Cell Aging and Ototoxicity.

Hearing loss is a debilitating disease that affects 10% of adults worldwide. Most sensorineural hearing loss is caused by the loss of mechanosensitive hair cells in the cochlea, often due to aging, noise, and ototoxic drugs. The identification of genes that can be targeted to slow aging and reduce the vulnerability of hair cells to insults is critical for the prevention of sensorineural hearing loss. Our previous cell-specific transcriptome analysis of adult cochlear hair cells and supporting cells showed that Clu, encoding a secreted chaperone that is involved in several basic biological events, such as cell death, tumor progression, and neurodegenerative disorders, is expressed in hair cells and supporting cells. We generated Clu-null mice (C57BL/6) to investigate its role in the organ of Corti, the sensory epithelium responsible for hearing in the mammalian cochlea. We showed that the deletion of Clu did not affect the development of hair cells and supporting cells; hair cells and supporting cells appeared normal at 1 month of age. Auditory function tests showed that Clu-null mice had hearing thresholds comparable to those of wild-type littermates before 3 months of age. Interestingly, Clu-null mice displayed less hair cell and hearing loss compared to their wildtype littermates after 3 months. Furthermore, the deletion of Clu is protected against aminoglycoside-induced hair cell loss in both in vivo and in vitro models. Our findings suggested that the inhibition of Clu expression could represent a potential therapeutic strategy for the alleviation of age-related and ototoxic drug-induced hearing loss.

Netrin-1 and clusterin: Innovative potential diagnostic biomarkers for early renal damage in ß-thalassemia major children.

BACKGROUND: Children with ß-thalassemia major (ß-TM) suffer from tubular dysfunction even before the onset of any renal impairment symptoms and/or clinical signs. Therefore, identifying innovative biomarkers allowing early renal damage detection has focused attention. AIM: This study aims to preliminary assess Netrin-1(NTN-1) and clusterin (CLU) in ß-TM children and explore their possible roles as surrogate noninvasive biomarkers of renal tubular dysfunction. SUBJECTS AND METHODS: In this study, 40 ß-TM children and 30 healthy children were enrolled. Routine serum and urinary biochemical variables were determined. Urinary NTN-1 and CLU levels were measured using ELISA and their mRNA expression in PBMCs were assayed using real-time PCR. Serum TNF-α, MDA levels and GST activity were measured. RESULTS: Urinary NTN-1 and CLU concentrations and mRNA relative expression levels in PBMCs were significantly increased in ß-TM children relative to controls. Oxidative stress and inflammatory markers revealed significant elevation in ß-TM children compared to controls. The change in these parameters correlated significantly with other renal parameters. ROC curves analysis showed that urinary NTN-1 and CLU levels are of promising diagnostic performance. CONCLUSION: Our results suggest that NTN-1 and CLU are qualified as new noninvasive biomarker panels for early detection of renal injury in ß-TM children. Moreover, urinary NTN-1 is recommended as a precise one during the clinical practices.

Neuronal clusterin expression is associated with cognitive protection in amyotrophic lateral sclerosis.

AIMS: Clusterin is a topologically dynamic chaperone protein with the ability to participate in both intra- and extacellular proteostasis. Clusterin has been shown to be upregulated in the spinal cord of patients with amyotrophic lateral sclerosis (ALS) and has been shown to protect against TDP-43 protein misfolding in animal and cell models. Previous studies have demonstrated an association between the pathological burden of TDP-43 misfolding and cognitive deficits in ALS, demonstrating high specificity, but correspondingly low sensitivity owing to a subset of individuals with no evidence of cognitive deficits despite a high burden of TDP-43 pathology, called mismatch cases. METHODS: Hypothesizing that differences in the ability to cope with protein misfolding in these cases may be due to differences in expression of protective mechanisms such as clusterin expression, we assessed the spatial expression of clusterin and another chaperone protein, HspB8, in post mortem brain tissue of mismatch cases. We employed a modified in situ hybridization technique called BaseScope, with single cell, single transcript resolution. RESULTS: Mismatch cases demonstrated differential spatial expression of clusterin, with a predominantly neuronal pattern, compared to cases with cognitive manifestations of their TDP-43 pathology who demonstrated a predominantly glial distribution of expression. CONCLUSIONS: Our data suggest that, in individuals with TDP-43 pathology, predominantly neuronal expression of clusterin in extra-motor brain regions may indicate a cell protective mechanism delaying clinical manifestations such as cognitive dysfunction.

Clusterin is highly expressed in tubular complexes during spontaneous pancreatitis of spontaneous hypertensive rats.

Pancreatitis is an inflammatory disorder of pancreas which leads to varying degrees of pancreatic endocrine and exocrine dysfunction and manifests in either acute or chronic forms. Spontaneous pancreatitis in experimental animals has rarely been reported. Here, we found acute to chronic courses of spontaneous pancreatitis in spontaneously hypertensive rats (SHRs), showing the formation of tubular complexes (TCs) and enhanced islet regeneration. We investigated the expression pattern of clusterin in the pancreas of SHRs based on immunohistochemistry (IHC). IHC analysis revealed the strong expression of clusterin in dedifferentiated duct-like cells and regenerative islets of TCs. These results imply that clusterin might be involved in the formation of TCs and parenchymal regeneration during rat pancreatitis.

Clusterin, a Novel DEC1 Target, Modulates DNA Damage-Mediated Cell Death.

Differentiated embryonic chondrocyte expressed gene 1 (DEC1, also known as Sharp2/Stra13/BHLHE40) is a basic helix-loop-helix transcription factor that plays an important role in circadian rhythms, cell proliferation, apoptosis, cellular senescence, hypoxia response, and epithelial-to-mesenchymal transition of tumor cells. Secretory clusterin (sCLU) is a cytoprotective protein that guards against genotoxic stresses. Here, clusterin (CLU) was identified as a novel target gene of DEC1 and suppresses DNA damage-induced cell death in tumor cells. Mechanistically, based on chromatin immunoprecipitation and luciferase assays, DEC1 binds to and activates the promoter of the CLU gene. DEC1 and DNA-damaging agents induce sCLU expression, whereas DEC1 knockdown decreases the expression of sCLU upon DNA damage. Moreover, the data demonstrate that DEC1 inhibits, whereas sCLU knockdown enhances, DNA damage-induced cell death in MCF7 breast cancer cells. Given that DEC1 and sCLU are frequently overexpressed in breast cancers, these data provide mechanistic insight into DEC1 as a prosurvival factor by upregulating sCLU to reduce the DNA damage-induced apoptotic response. Together, this study reveals sCLU as a novel target of DEC1 which modulates the sensitivity of the DNA damage response.Implications: DEC1 and sCLU are frequently overexpressed in breast cancer, and targeting the sCLU-mediated cytoprotective signaling pathway may be a novel therapeutic approach. Mol Cancer Res; 16(11); 1641-51. ©2018 AACR.

Expression of clusterin in colorectal carcinoma in relation to clinicopathological criteria.

BACKGROUND/AIM: Colorectal carcinoma (CRC) carries a high incidence of morbidity and mortality. Prognosis is related to nodal metastasis and stage. Clusterin is a widely distributed glycoprotein with not yet fully understood functions. Clusterin may be overexpressed in some tumours or under expressed in other tumours. The aim behind this study is to examine the relation of clusterin cytoplasmic immunostaining to tumour characteristics, disease relapse, and survival in CRC. MATERIALS AND METHODS: Paraffin blocks of 133 CRCs were retrieved from the Department of Pathology, King Abdulaziz University, Jeddah, Saudi Arabia. Immunostaining was done using antibody to clusterin. Staining expression in 10% of malignant cells was used as a cut-off to determine low immunostaining and high immunostaining. Statistical tests were used to evaluate the association of clusterin immunostaining with clinicopathological parameters. RESULTS: Immunohistochemical results showed clusterin low immunostaining in CRC and nodal metastases. No association was found between clusterin immunostaining and tumour grade, age, tumour invasiveness, distant metastases, vascular invasion, nodal metastases, relapse, and survival. CONCLUSION: Our study showed low clusterin immunostaining in CRC with lack of association with prognostic indicators in CRC. These results raise the controversy of understanding the role of clusterin in CRC. Further molecular studies are required to explore more about possible mechanisms of clusterin association with tumorigenicity, apoptosis, tumour growth progression, local and vascular invasion, and metastasis of CRC.

Pseudoexfoliation and Alzheimer's associated CLU risk variant, rs2279590, lies within an enhancer element and regulates CLU, EPHX2 and PTK2B gene expression.

Genetic variants at PTK2B-CLU locus pose as high-risk factors for many age-related disorders. However, the role of these variants in disease progression is less characterized. In this study, we aimed to investigate the functional significance of a clusterin intronic SNP, rs2279590, that has been associated with pseudoexfoliation, Alzheimer's disease (AD) and diabetes. We have previously shown that the alleles at rs2279590 differentially regulate clusterin (CLU) gene expression in lens capsule tissues. This polymorphism resides in an active regulatory region marked by H3K27Ac and DNase I hypersensitive site and is an eQTL for CLU expression. Here, we report the presence of an enhancer element in surrounding region of rs2279590. Deletion of a 115 bp intronic region flanking the rs2279590 variant through CRISPR-Cas9 genome editing in HEK293 cells demonstrated a decreased clusterin gene expression. Electrophoretic mobility shift and chromatin immunoprecipitation assays show that rs2279590 with allele 'A' constitutes a transcription factor binding site for heat shock factor-1 (HSF1) but not with allele 'G'. By binding to allele 'A', HSF1 abrogates the enhancer effect of the locus as validated by reporter assays. Interestingly, rs2279590 locus has a widespread enhancer effect on two nearby genes, protein tyrosine kinase 2 beta (PTK2B) and epoxide hydrolase-2 (EPHX2); both of which have been previously associated with AD as risk factors. To summarize, our study unveils a mechanistic role of the common variant rs2279590 that can affect a variety of aging disorders by regulating the expression of a specific set of genes.

Clusterin inhibition mediates sensitivity to chemotherapy and radiotherapy in human cancer.

Since its discovery in 1983, the protein clusterin (CLU) has been isolated from almost all human tissues and fluids and linked to the development of different physiopathological processes, including carcinogenesis and tumor progression. During the last few years, several studies have shown the cytoprotective role of secretory CLU in tumor cells, inhibiting their apoptosis and enhancing their resistance to conventional treatments including hormone depletion, chemotherapy, and radiotherapy. In an effort to determine the therapeutic potential that the inhibition of this protein could have on the development of new strategies for cancer treatment, numerous studies have been carried out in this field, with results, in most cases, satisfactory but sometimes contradictory. In this document, we summarize for the first time the current knowledge of the effects that CLU inhibition has on sensitizing tumor cells to conventional cancer treatments and discuss its importance in the development of new strategies against cancer.

Estrogen receptor ß, a regulator of androgen receptor signaling in the mouse ventral prostate.

As estrogen receptor ß-/- (ERß-/-) mice age, the ventral prostate (VP) develops increased numbers of hyperplastic, fibroplastic lesions and inflammatory cells. To identify genes involved in these changes, we used RNA sequencing and immunohistochemistry to compare gene expression profiles in the VP of young (2-mo-old) and aging (18-mo-old) ERß-/- mice and their WT littermates. We also treated young and old WT mice with an ERß-selective agonist and evaluated protein expression. The most significant findings were that ERß down-regulates androgen receptor (AR) signaling and up-regulates the tumor suppressor phosphatase and tensin homolog (PTEN). ERß agonist increased expression of the AR corepressor dachshund family (DACH1/2), T-cadherin, stromal caveolin-1, and nuclear PTEN and decreased expression of RAR-related orphan receptor c, Bcl2, inducible nitric oxide synthase, and IL-6. In the ERß-/- mouse VP, RNA sequencing revealed that the following genes were up-regulated more than fivefold: Bcl2, clusterin, the cytokines CXCL16 and -17, and a marker of basal/intermediate cells (prostate stem cell antigen) and cytokeratins 4, 5, and 17. The most down-regulated genes were the following: the antioxidant gene glutathione peroxidase 3; protease inhibitors WAP four-disulfide core domain 3 (WFDC3); the tumor-suppressive genes T-cadherin and caveolin-1; the regulator of transforming growth factor ß signaling SMAD7; and the PTEN ubiquitin ligase NEDD4. The role of ERß in opposing AR signaling, proliferation, and inflammation suggests that ERß-selective agonists may be used to prevent progression of prostate cancer, prevent fibrosis and development of benign prostatic hyperplasia, and treat prostatitis.

Identification of plexin A4 as a novel clusterin receptor links two Alzheimer's disease risk genes.

Although abundant genetic and biochemical evidence strongly links Clusterin (CLU) to Alzheimer disease (AD) pathogenesis, the receptor for CLU within the adult brain is currently unknown. Using unbiased approaches, we identified Plexin A4 (PLXNA4) as a novel, high-affinity receptor for CLU in the adult brain. PLXNA4 protein expression was high in brain with much lower levels in peripheral organs. CLU protein levels were significantly elevated in the cerebrospinal fluid (CSF) of Plxna4-/- mice and, in humans, CSF levels of CLU were also associated with PLXNA4 genotype. Human AD brains had significantly increased the levels of CLU protein but decreased levels of PLXNA4 by ∼50%. To determine whether PLXNA4 levels influenced cognition, we analyzed the behaviour of Plxna4+/+, Plxna4+/-, and Plxna4-/- mice. In comparison to WT controls, both Plxna4+/- and Plxna4-/- mice were hyperactive in the open field assay while Plxna4-/- mice displayed a hyper-exploratory (low-anxiety phenotype) in the elevated plus maze. Importantly, both Plxna4+/- and Plxna4-/- mice displayed prominent deficits in learning and memory in the contextual fear-conditioning paradigm. Thus, even a 50% reduction in the level of PLXNA4 is sufficient to cause memory impairments, raising the possibility that memory problems seen in AD patients could be due to reductions in the level of PLXNA4. Both CLU and PLXNA4 have been genetically associated with AD risk and our data thus provide a direct relationship between two AD risk genes. Our data suggest that increasing the levels of PLXNA4 or targeting CLU-PLXNA4 interactions may have therapeutic value in AD.

Clusterin/Akt Up-Regulation Is Critical for GATA-4 Mediated Cytoprotection of Mesenchymal Stem Cells against Ischemia Injury.

BACKGROUND: Clusterin (Clu) is a stress-responding protein with multiple biological functions. Our preliminary microarray studies show that clusterin was prominently upregulated in mesenchymal stem cells (MSCs) overexpressing GATA-4 (MSCGATA-4). We hypothesized that the upregulation of clusterin is involved in overexpression of GATA-4 mediated cytoprotection. METHODS: MSCs harvested from bone marrow of rats were transduced with GATA-4. The expression of clusterin in MSCs was further confirmed by real-time PCR and western blotting. Simulation of ischemia was achieved by exposure of MSCs to a hypoxic environment. Lactate dehydrogenase (LDH) released from MSCs was served as a biomarker of cell injury and MTs uptake was used to estimate cell viability. Mitochondrial function was evaluated by measuring mitochondrial membrane potential (ΔΨm) and caspase 3/7 activity. RESULTS: (1) Clusterin expression was up-regulated in MSCGATA-4 compared to control MSCs transfected with empty-vector (MSCNull). MSCGATA-4 were tolerant to 72 h hypoxia exposure as shown by reduced LDH release and higher MTs uptake. This protection was abrogated by transfecting Clu-siRNA into MSCGATA-4. (2) Exogenous clusterin significantly decreased LDH release and increased MSC survival in hypoxic environment. Moreover, ΔΨm was maintained and caspase 3/7 activity was reduced by clusterin in a concentration-dependent manner. (3) p-Akt expression in MSCs was upregulated following pre-treatment with clusterin, with no change in total Akt. Moreover, cytoprotection mediated by clusterin was partially abrogated by Akt inhibitor LY294002. CONCLUSIONS: Clusterin/Akt signaling pathway is involved in GATA-4 mediated cytoprotection against hypoxia stress. It is suggested that clusterin may be therapeutically exploited in MSC based therapy for cardiovascular diseases.

Distinct promoters, subjected to epigenetic regulation, drive the expression of two clusterin mRNAs in prostate cancer cells.

The human clusterin (CLU) gene codes for several mRNAs characterized by different sequences at their 5' end. We investigated the expression of two CLU mRNAs, called CLU 1 and CLU 2, in immortalized (PNT1a) and tumorigenic (PC3 and DU145) prostate epithelial cells, as well as in normal fetal fibroblasts (WI38) following the administration of the epigenetic drugs 5-aza-2'-deoxycytidine (AZDC) and trichostatin A (TSA) given either as single or combined treatment (AZDC-TSA). Our experimental evidences show that: a) CLU 1 is the most abundant transcript variant. b) CLU 2 is expressed at a low level in normal fibroblasts and virtually absent in prostate cancer cells. c) CLU 1, and to a greater extent CLU 2 expression, increased by AZDC-TSA treatment in prostate cancer cells. d) Both CLU 1 and CLU 2 encode for secreted CLU. e) P2, a novel promoter that overlaps the CLU 2 Transcription Start Site (TSS), drives CLU 2 expression. f) A CpG island, methylated in prostate cancer cells and not in normal fibroblasts, is responsible for long-term heritable regulation of CLU 1 expression. g) ChIP assay of histone tail modifications at CLU promoters (P1 and P2) shows that treatment of prostate cancer cells with AZDC-TSA causes enrichment of Histone3(Lys9)acetylated (H3K9ac) and reduction of Histone3(Lys27)trimethylated (H3K27me3), inducing active transcription of both CLU variants. In conclusion, we show for the first time that the expression of CLU 2 mRNA is driven by a novel promoter, P2, whose activity responds to epigenetic drugs treatment through changes in histone modifications.

Clusterin inhibition using OGX-011 synergistically enhances zoledronic acid activity in osteosarcoma.

PURPOSE: Despite recent improvements in therapeutic management of osteosarcoma, ongoing challenges in improving the response to chemotherapy warrants new strategies still needed to improve overall patient survival. Among new therapeutic approaches, zoledronic acid (ZOL) represents a promising adjuvant molecule to chemotherapy to limit the osteolytic component of bone tumors. However, ZOL triggers the elevation of heat shock proteins (Hsp), including Hsp27 and clusterin (CLU), which could enhance tumor cell survival and treatment resistance. We hypothesized that targeting CLU using siRNA or the antisense drug, OGX-011, will suppress treatment-induced CLU induction and enhance ZOL-induced cell death in osteosarcoma (OS) cells. METHODS: The combined effects of OGX-011 and ZOL were investigated in vitro on cell growth, viability, apoptosis and cell cycle repartition of ZOL-sensitive or -resistant human OS cell lines (SaOS2, U2OS, MG63 and MNNG/HOS). RESULTS: In OS cell lines, ZOL increased levels of HSPs, especially CLU, in a dose- and time-dependent manner by mechanism including increased HSF1 transcription activity. The OS resistant cells to ZOL exhibited higher CLU expression level than the sensitive cells. Moreover, CLU overexpression protects OS sensitive cells to ZOL-induced cell death by modulating the MDR1 and farnesyl diphosphate synthase expression. OGX-011 suppressed treatment-induced increases in CLU and synergistically enhanced the activity of ZOL on cell growth and apoptosis. These biologic events were accompanied by decreased expression of HSPs, MDR1 and HSF1 transcriptional activity. In vivo, OGX-011, administered 3 times a week (IP, 20mg/kg), potentiated the effect of ZOL (s.c; 50µg/kg), significantly inhibiting tumor growth by 50% and prolonging survival in MNNG/HOS xenograft model compared to ZOL alone. CONCLUSION: These results indicate that ZOL-mediated induction of CLU can be attenuated by OGX-011, with synergistic effects on delaying progression of osteosarcoma.

Clusterin expression level correlates with increased oxidative stress in asthmatics.

BACKGROUND: Oxidative stress is thought to play a role in the pathogenesis of asthma. Clusterin is a sensitive cellular biosensor of oxidative stress and has antioxidant properties. The function and expression of clusterin in patients with asthma have not been fully investigated. OBJECTIVE: To investigate whether the expression of clusterin in patients with asthma is regulated by increased oxidative burden and whether clusterin expression could be used to assess the response to inhaled corticosteroids. METHODS: Clusterin levels in serum, induced sputum, and peripheral blood mononuclear cells of patients with asthma were measured by enzyme-linked immunosorbent assay and western blotting and compared with pulmonary function and levels of expression of hyperoxidized peroxiredoxins. Serum concentrations of clusterin in treatment-naive patients were compared before and after inhaled corticosteroid use. RESULTS: Serum clusterin concentration was significantly elevated in patients with severe asthma and was inversely correlated with pulmonary function. The expression of hyperoxidized peroxiredoxins was greatly increased in peripheral blood mononuclear cells of patients with asthma and was strongly correlated with clusterin expression. Serum clusterin concentrations in treatment-naive patients with asthma were decreased significantly after initial treatment with inhaled corticosteroids. CONCLUSION: Clusterin may be a biomarker of asthma severity and the burden of oxidative stress in patients with asthma. Moreover, clusterin may be useful for the prompt assessment of airway inflammation.

Far-infrared radiation inhibits proliferation, migration, and angiogenesis of human umbilical vein endothelial cells by suppressing secretory clusterin levels.

Far-infrared (FIR) radiation is known to lessen the risk of angiogenesis-related diseases including cancer. Because deficiency of secretory clusterin (sCLU) has been reported to inhibit angiogenesis of endothelial cells (EC), we investigated using human umbilical vein EC (HUVEC) whether sCLU mediates the inhibitory effects of FIR radiation. Although FIR radiation ranging 3-25µm wavelength at room temperature for 60min did not alter EC viability, further incubation in the culture incubator (at 37°C under 5% CO2) after radiation significantly inhibited EC proliferation, in vitro migration, and tube formation in a time-dependent manner. Under these conditions, we found decreased sCLU mRNA and protein expression in HUVEC and decreased sCLU protein secreted in culture medium. Expectedly, the replacement of control culture medium with the FIR-irradiated conditioned medium significantly decreased wound closure and tube formation of HUVEC, and vice versa. Furthermore, neutralization of sCLU with anti-sCLU antibody also mimicked all observed inhibitory effects of FIR radiation. Moreover, treatment with recombinant human sCLU protein completely reversed the inhibitory effects of FIR radiation on EC migration and angiogenesis. Lastly, vascular endothelial growth factor also increased sCLU secretion in the culture medium, and wound closure and tube formation of HUVEC, which were significantly reduced by FIR radiation. Our results demonstrate a novel mechanism by which FIR radiation inhibits the proliferation, migration, and angiogenesis of HUVEC, via decreasing sCLU.

Analysis of the hippocampal proteome in ME7 prion disease reveals a predominant astrocytic signature and highlights the brain-restricted production of clusterin in chronic neurodegeneration.

Prion diseases are characterized by accumulation of misfolded protein, gliosis, synaptic dysfunction, and ultimately neuronal loss. This sequence, mirroring key features of Alzheimer disease, is modeled well in ME7 prion disease. We used iTRAQ(TM)/mass spectrometry to compare the hippocampal proteome in control and late-stage ME7 animals. The observed changes associated with reactive glia highlighted some specific proteins that dominate the proteome in late-stage disease. Four of the up-regulated proteins (GFAP, high affinity glutamate transporter (EAAT-2), apo-J (Clusterin), and peroxiredoxin-6) are selectively expressed in astrocytes, but astrocyte proliferation does not contribute to their up-regulation. The known functional role of these proteins suggests this response acts against protein misfolding, excitotoxicity, and neurotoxic reactive oxygen species. A recent convergence of genome-wide association studies and the peripheral measurement of circulating levels of acute phase proteins have focused attention on Clusterin as a modifier of late-stage Alzheimer disease and a biomarker for advanced neurodegeneration. Since ME7 animals allow independent measurement of acute phase proteins in the brain and circulation, we extended our investigation to address whether changes in the brain proteome are detectable in blood. We found no difference in the circulating levels of Clusterin in late-stage prion disease when animals will show behavioral decline, accumulation of misfolded protein, and dramatic synaptic and neuronal loss. This does not preclude an important role of Clusterin in late-stage disease, but it cautions against the assumption that brain levels provide a surrogate peripheral measure for the progression of brain degeneration.

Characterization of mechanism involved in acquired resistance to sorafenib in a mouse renal cell cancer RenCa model.

PURPOSE: The objective of this study was to investigate the mechanism mediating the acquisition of a resistant phenotype to sorafenib in renal cell carcinoma (RCC). METHODS: A parental mouse RCC cell line, RenCa (RenCa/P), was continuously exposed to increasing doses of sorafenib, and a cell line resistant to sorafenib (RenCa/R), showing an approximately sixfold higher IC(50) than that of RenCa/P, was established. Changes in the expression of several molecules in these cell lines following sorafenib treatment were evaluated by western blotting, and the effects of sorafenib treatment on the in vivo growth patterns were compared. RESULTS: There were no significant differences in sensitivities to potential agents against RCC between RenCa/P and RenCa/R. Among several apoptosis-related proteins, the expression of clusterin in RenCa/R was significantly greater than that in RenCa/P. Following treatment with sorafenib, the expression level of phosphorylated p44/42 mitogen-activated protein kinase (MAPK) in RenCa/P, but not that in RenCa/R, was significantly decreased. Furthermore, additional treatment with a specific inhibitor of the MAPK signaling pathway significantly increased the sensitivity of RenCa/R to sorafenib, but not that of RenCa/P. There was no significant difference between the in vivo growth patterns of RenCa/P and RenCa/R in mice without sorafenib treatment; however, the growth inhibitory effect of sorafenib on the RenCa/P tumor was significantly greater than that on the RenCa/R tumor. CONCLUSIONS: These findings suggest that the upregulation of clusterin and continuous activation of the MAPK pathway during sorafenib treatment may be involved in the acquisition of a resistance to sorafenib in RCC.

Apocynin, an NADPH oxidase inhibitor, suppresses rat prostate carcinogenesis.

Recent evidence suggests that oxidative stress contributes to the pathogenesis of prostate cancer. The present study focused on the effect of apocynin, an inhibitor of NADPH oxidase, on prostate carcinogenesis using the transgenic rat for adenocarcinoma of prostate (TRAP) model. There were no toxic effects with apocynin treatment. The percentages and numbers of carcinomas in both the ventral and lateral prostate were significantly reduced by apocynin treatment, with dose dependence. Reduction of reactive oxygen species by apocynin was confirmed by immunohistochemistry of 8-OHdG and dihydroethidium staining. Positivity of Ki67 was significantly reduced by apocynin treatment, and downregulation of clusterin expression, as well as inactivation of the MEK-ERK1/2 pathway, was a feature of the apocynin treated groups. In human prostate cancer cell line LNCaP, apocynin also inhibited reactive oxygen species production and blocked cell growth by inducing G0/G1 arrest with downregulation of clusterin and cyclin D1. These data suggest that apocynin possesses chemopreventive potential against prostate cancer.

Anti-apoptotic effect of clusterin on cisplatin-induced cell death of retinoblastoma cells.

Clusterin is a cytoprotective chaperone protein that is known to protect various retinal cells. It was also reported to be overexpressed in several types of malignant tumors, whose chemoresistance correlates with the expression of clusterin. Herein, we investigated the effect of clusterin on cisplatin-induced cell death of retinoblastoma cells. Firstly, evaluation of clusterin expression demonstrated that it was highly expressed in human retinoblastoma tissues and cell lines (SNUOT-Rb1 and Y79) particularly in the area between viable cells around vessels and necrotic zones in the relatively avascular area in human retinoblastoma tissues. Furthermore, the effects of cisplatin on retinoblastoma cells were evaluated. Cisplatin (1 µg/ml) significantly affected cell viability of SNUOT-Rb1 cells by inducing caspase-3-dependent apoptosis. Notably, the cell death due to cisplatin was prevented by 5 µg/ml of clusterin administered 4 h prior to cisplatin treatment by inhibiting cisplatin-induced apoptosis. Furthermore, overexpression of clusterin exerted its anti-apoptotic effect on cisplatin-induced apoptosis, and effectively prevented cisplatin-induced cell death. These data suggest that clusterin, found to be expressed in human retinoblastoma, may exert anti-apoptotic effects on cisplatin-induced apoptosis and prevent cell death. Therefore, clusterin can contribute to cisplatin resistance of retinoblastoma.

Podoplanin and clusterin are reliable markers of nonneoplastic synovium at various sites.

INTRODUCTION: Clusterin (CLU) has been noted to mark synovium adjacent to tenosynovial tumors, and studies suggest that podoplanin (PP) is upregulated in inflammatory arthritis. Characterization of synovial staining with CLU and PP in various nonneoplastic disease states has not been described. METHODS: A microarray was created from paraffin-embedded human synovium, including 19 normal/noninflammatory (10 weight-bearing joints, 8 non-weight-bearing joints), 9 rheumatoid arthritis, 10 synovial cysts, and 3 osteoarthritis and stained with PP (D2-40) and CLU. Staining intensity was graded semiquantitatively (0-3+). RESULTS: PP and CLU stained synovium in 88% and 95% cases, respectively. PP and CLU showed moderate to strong (3+) staining in 26% and 19% of noninflammatory and 44% and 0% of inflammatory synovia, respectively (P < .01). CONCLUSIONS: PP and CLU are reliable markers of human synovium and can confirm its presence in limited specimens. Although CLU was more sensitive, PP may be more useful in the setting of chronic inflammation.