Changes in the expression of proteins associated with aerobic glycolysis and cell migration are involved in tumorigenic ability of two glioma cell lines

ABSTRACT: BACKGROUND: The most frequent and malignant brain cancer is glioblastoma multiforme (GBM). In gliomas, tumor progression and poor prognosis are associated with the tumorigenic ability of the cells. U87MG cells (wild-type p53) are known to be tumorigenic in nude mice, but T98G cells (mutant p53) are not tumorigenic. We investigated the proteomic profiling of these two cell lines in order to gain new insights into the mechanisms that may be involved in tumorigenesis. RESULTS: We found 24 differentially expressed proteins between T98G and U87MG cells. Gene Ontology supports the notion that over-representation of differentially expressed proteins is involved in glycolysis, cell migration and stress oxidative response. Among those associated with the glycolysis pathway, TPIS and LDHB are up-regulated in U87MG cells. Measurement of glucose consumption and lactate production suggests that glycolysis is more effective in U87MG cells. On the other hand, G6PD expression was 3-fold higher in T98G cells and this may indicate a shift to the pentose-phosphate pathway. Moreover, GRP78 expression was also three-fold higher in T98G than in U87MG cells. Under thapsigargin treatment both cell lines showed increased GRP78 expression and the effect of this agent was inversely correlated to cell migration. Quantitative RT-PCR and immunohistochemistry of GRP78 in patient samples indicated a higher level of expression of GRP78 in grade IV tumors compared to grade I and non-neoplastic tissues, respectively. CONCLUSIONS: Taken together, these results suggest an important role of proteins involved in key functions such as glycolysis and cell migration that may explain the difference in tumorigenic ability between these two glioma cell lines and that may be extrapolated to the differential aggressiveness of glioma tumors.     

The role of A-kinase interacting protein 1 in regulating progression and stemness as well as indicating the prognosis in glioblastoma

BackgroundA-kinase interacting protein 1 (AKIP1) is recently implicated in the pathogenesis of several solid tumors, while its role in glioblastoma multiforme (GBM) is largely unknown. Therefore, the current study aimed to investigate the effect of AKIP1 on GBM cell malignant behaviors, stemness, and its underlying molecular mechanisms.MethodsU-87 MG and A172 cells were transfected with control or AKIP1 overexpression plasmid; control or AKIP1 siRNA plasmid. Then cell proliferation, apoptosis, invasion, CD133⁺ cell proportion, and sphere formation assays were performed. Furthermore, RNA-Seq was performed in U-87 MG cells. Besides, AKIP1 expression was detected in 25 GBM and 25 low-grade glioma (LGG) tumor samples.ResultsAKIP1 was increased in several GBM cell lines compared to the control cell line. After transfections, it was found that AKIP1 overexpression increased cell invasion, CD133⁺ cell proportion, and sphere formation ability while less affecting cell proliferation or cell apoptosis in U-87 MG and A172 cells. Moreover, AKIP1 siRNA achieved the opposite effect in these cells, except that it inhibited cell proliferation but induced cell apoptosis to some extent. Subsequent RNA-Seq assay showed several critical carcinogenetic pathways, such as PI3K/AKT, Notch, EGFR tyrosine kinase inhibitor resistance, Ras, ErbB, mTOR pathways, etc. were potentially related to the function of AKIP1 in U-87 MG cells. Clinically, AKIP1 expression was higher in GBM tissues than in LGG tissues, which was also correlated with the poor prognosis of GBM to some degree.ConclusionsAKIP1 regulates the malignant behaviors and stemness of GBM via regulating multiple carcinogenetic pathways.     

Saponin 1 Induces Apoptosis and Suppresses NF-κB-Mediated Survival Signaling in Glioblastoma Multiforme (GBM)

Saponin 1 is a triterpeniod saponin extracted from Anemone taipaiensis, a traditional Chinese medicine against rheumatism and phlebitis. It has also been shown to exhibit significant anti-tumor activity against human leukemia (HL-60 cells) and human hepatocellular carcinoma (Hep-G2 cells). Herein we investigated the effect of saponin 1 in human glioblastoma multiforme (GBM) U251MG and U87MG cells. Saponin 1 induced significant growth inhibition in both glioblastoma cell lines, with a 50% inhibitory concentration at 24 h of 7.4 µg/ml in U251MG cells and 8.6 µg/ml in U87MG cells, respectively. Nuclear fluorescent staining and electron microscopy showed that saponin 1 caused characteristic apoptotic morphological changes in the GBM cell lines. Saponin 1-induced apoptosis was also verified by DNA ladder electrophoresis and flow cytometry. Additionally, immunocytochemistry and western blotting analyses revealed a time-dependent decrease in the expression and nuclear location of NF-κB following saponin 1 treatment. Western blotting data indicated a significant decreased expression of inhibitors of apoptosis (IAP) family members,(e.g., survivin and XIAP) by saponin 1. Moreover, saponin 1 caused a decrease in the Bcl-2/Bax ratio and initiated apoptosis by activating caspase-9 and caspase-3 in the GBM cell lines. These findings indicate that saponin 1 inhibits cell growth of GBM cells at least partially by inducing apoptosis and inhibiting survival signaling mediated by NF-κB. In addition, in vivo study also demonstrated an obvious inhibition of saponin 1 treatment on the tumor growth of U251MG and U87MG cells-produced xenograft tumors in nude mice. Given the minimal toxicities of saponin 1 in non-neoplastic astrocytes, our results suggest that saponin 1 exhibits significant in vitro and in vivo anti-tumor efficacy and merits further investigation as a potential therapeutic agent for GBM.     

Opposing Effects of PI3K/Akt and Smad-Dependent Signaling Pathways in NAG-1-Induced Glioblastoma Cell Apoptosis

Nonsteroidal anti-inflammatory drug (NSAID) activated gene-1 (NAG-1) is a divergent member of the transforming growth factor-beta (TGF-β) superfamily. NAG-1 plays remarkable multifunctional roles in controlling diverse physiological and pathological processes including cancer. Like other TGF-β family members, NAG-1 can play dual roles during cancer development and progression by negatively or positively modulating cancer cell behaviors. In glioblastoma brain tumors, NAG-1 appears to act as a tumor suppressor gene; however, the precise underlying mechanisms have not been well elucidated. In the present study, we discovered that overexpression of NAG-1 induced apoptosis in U87 MG, U118 MG, U251 MG, and T98G cell lines via the intrinsic mitochondrial pathway, but not in A172 and LN-229 cell lines. NAG-1 could induce the phosphorylation of PI3K/Akt and Smad2/3 in all six tested glioblastoma cell lines, except Smad3 phosphorylation in A172 and LN-229 cell lines. In fact, Smad3 expression and its phosphorylation were almost undetectable in A172 and LN-229 cells. The PI3K inhibitors promoted NAG-1-induced glioblastoma cell apoptosis, while siRNAs to Smad2 and Smad3 decreased the apoptosis rate. NAG-1 also stimulated the direct interaction between Akt and Smad3 in glioblastoma cells. Elevating the level of Smad3 restored the sensitivity to NAG-1-induced apoptosis in A172 and LN-229 cells. In conclusion, our results suggest that PI3K/Akt and Smad-dependent signaling pathways display opposing effects in NAG-1-induced glioblastoma cell apoptosis.     

DHA induces apoptosis by altering the expression and cellular location of GRP78 in colon cancer cell lines

n-3 polyunsaturated fatty acids exert growth-inhibitory and pro-apoptotic effects in colon cancer cells. We hypothesized that the anti-apoptotic glucose related protein of 78kDa (GRP78), originally described as a component of the unfolded protein response in endoplasmic reticulum (ER), could be a molecular target for docosahexaenoic acid (DHA) in these cells. GRP78 total and surface overexpression was previously associated with a poor prognosis in several cancers, whereas its down-regulation with decreased cancer growth in animal models. DHA treatment induced apoptosis in three colon cancer cell lines (HT-29, HCT116 and SW480), and inhibited their total and surface GRP78 expression. The cell ability to undergo DHA-induced apoptosis was inversely related to their level of GRP78 expression. The transfection of the low GRP78-expressing SW480 cells with GRP78-GFP cDNA significantly induced cell growth and inhibited the DHA-driven apoptosis, thus supporting the essential role of GRP78 in DHA pro-apoptotic effect. We suggest that pERK1/2 could be the first upstream target for DHA, and demonstrate that, downstream of GRP78, DHA may exert its proapoptotic role by augmenting the expression of the ER resident factors ERdj5 and inhibiting the phosphorylation of PKR-like ER kinase (PERK), known to be both physically associated with GRP78, and by activating caspase-4. Overall, the regulation of cellular GRP78 expression and location is suggested as a possible route through which DHA can exert pro-apoptotic and antitumoral effects in colon cancer cells.     

Modulation of Sonic hedgehog signaling and WW domain containing oxidoreductase WOX1 expression enhances radiosensitivity of human glioblastoma cells

WW domain containing oxidoreductase, designated WWOX, FOR or WOX1, is a known pro-apoptotic factor when ectopically expressed in various types of cancer cells, including glioblastoma multiforme (GBM). The activation of sonic hedgehog (Shh) signaling, especially paracrine Shh secretion in response to radiation, is associated with impairing the effective irradiation of cancer cells. Here, we examined the role of Shh signaling and WOX1 overexpression in the radiosensitivity of human GBM cells. Our results showed that ionizing irradiation (IR) increased the cytoplasmic Shh and nuclear Gli-1 content in GBM U373MG and U87MG cells. GBM cells with exogenous Shh treatment exhibited similar results. Pretreatment with Shh peptides protected U373MG and U87MG cells against IR in a dose-dependent manner. Cyclopamine, a Hedgehog/Smoothened (SMO) inhibitor, reversed the protective effect of Shh in U87MG cells. Cyclopamine increased Shh plus IR-induced H2AX, a marker of DNA double-strand breaks, in these cells. To verify the role of Shh signaling in the radiosensitivity of GBM cells, we tested the effect of the Gli family zinc finger 1 (Gli-1) inhibitor zerumbone and found that it could sensitize GBM cells to IR. We next examined the role of WOX1 in radiosensitivity. Overexpression of WOX1 enhanced the radiosensitivity of U87MG (possessing wild type p53 or WTp53) but not U373MG (harboring mutant p53 or MTp53) cells. Pretreatment with Shh peptides protected both WOX1-overexpressed U373MG and U87MG cells against IR and increased the cytoplasmic Shh and nuclear Gli-1 content. Zerumbone enhanced the radiosensitivity of WOX1-overexpressed U373MG and U87MG cells. In conclusion, overexpression of WOX1 preferentially sensitized human GBM cells possessing wild type p53 to radiation therapy. Blocking of Shh signaling may enhance radiosensitivity independently of the expression of p53 and WOX1. The crosstalk between Shh signaling and WOX1 expression in human glioblastoma warrants further investigation.     

Phosphodiesterase inhibitors control A172 human glioblastoma cell death through cAMP-mediated activation of protein kinase A and Epac1/Rap1 pathways

AimsWe investigated whether cAMP-mediated protein kinase A(PKA) and Epac1/Rap1 pathways differentially affect brain tumor cell death using 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone(rolipram), specific phosphodiesterase type IV(PDE IV) inhibitor.Main methodsA172 and U87MG human glioblastoma cells were used. Percentage of cell survival was determined by MTT assay. PKA and Epac1/Rap1 activation was determined by western blotting and pull-down assay, respectively. Cell cycle and hypodiploid cell formation were assessed by flow cytometry analysis.Key findingsNon-specific PDE inhibitors, isobutylmethylxanthine(IBMX) and theophylline reduce survival percentage of A172 and U87MG cells. The expression of PDE4A and PDE4B was detected in A172 and U87MG cells. Rolipram-treated A172 or U87MG cell survival was lower in the presence of forskolin, adenylate cyclase activator, than that in its absence. Co-treatment with rolipram and forskolin also enhanced CREB phosphorylation on serine 133 that was inhibited by H-89, PKA inhibitor and cAMP-responsive guanine nucleotide exchange factor 1(Epac1), a Rap GDP exchange factor-mediated Rap1 activity in A172 cells. When A172 cells were treated with cell-permeable dibutyryl-cAMP(dbcAMP), PKA activator or 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate(CPT), Epac1 activator, basal level of cell death was increased and cell cycle was arrested at the phase of G2/M. Rolipram-induced A172 cell death was also increased by the co-treatment with dbcAMP or CPT, but it was inhibited by the pre-treatment with H-89.SignificanceThese findings demonstrate that PKA and Epac1/Rap1 pathways could cooperatively play a role in rolipram-induced brain tumor cell death. It suggests that rolipram might regulate glioblastoma cell density through dual pathways of PKA- and Epac1/Rap1-mediated cell death and cell cycle arrest.     

The Synergistic Effect of Combination Progesterone and Temozolomide on Human Glioblastoma Cells

Glioblastoma multiforme (GBM) is the most common and most aggressive malignant brain tumor. Despite optimal treatment and evolving standard of care, the median survival of patients diagnosed with GBM is only 12–15 months. In this study, we combined progesterone (PROG) and temozolomide (TMZ), a standard chemotherapeutic agent for human GBM, to test whether PROG enhances the antitumor effects of TMZ and reduces its side effects. Two WHO grade IV human GBM cells lines (U87MG and U118MG) and primary human dermal fibroblasts (HDFs) were repeatedly exposed to PROG and TMZ either alone or in combination for 3 and 6 days. Cell death was measured by MTT reduction assay. PROG and TMZ individually induced tumor cell death in a dose-dependent manner. PROG at high doses produced more cell death than TMZ alone. When combined, PROG enhanced the cell death-inducing effect of TMZ. In HDFs, PROG did not reduce viability even at the same high cytotoxic doses, but TMZ did so in a dose-dependent manner. In combination, PROG reduced TMZ toxicity in HDFs. PROG alone and in combination with TMZ suppressed the EGFR/PI3K/Akt/mTOR signaling pathway and MGMT expression in U87MG cells, thus suppressing cell proliferation. PROG and TMZ individually reduced cell migration in U87MG cells but did so more effectively in combination. PROG enhances the cytotoxic effects of TMZ in GBM cells and reduces its toxic side effects in healthy primary cells.     

The curry spice curcumin selectively inhibits cancer cells growth in vitro and in preclinical model of glioblastoma

Previous studies suggested that curcumin is a potential agent against glioblastomas (GBMs). However, the in vivo efficacy of curcumin in gliomas remains not established. In this work, we examined the mechanisms underlying apoptosis, selectivity, efficacy and safety of curcumin from in vitro (U138MG, U87, U373 and C6 cell lines) and in vivo (C6 implants) models of GBM. In vitro, curcumin markedly inhibited proliferation and migration and induced cell death in liquid and soft agar models of GBM growth. Curcumin effects occurred irrespective of the p53 and PTEN mutational status of the cells. Interestingly, curcumin did not affect viability of primary astrocytes, suggesting that curcumin selectivity targeted transformed cells. In U138MG and C6 cells, curcumin decreased the constitutive activation of PI3K/Akt and NFkappaB survival pathways, down-regulated the antiapoptotic NFkappaB-regulated protein bcl-xl and induced mitochondrial dysfunction as a prelude to apoptosis. Cells developed an early G2/M cell cycle arrest followed by sub-G1 apoptosis and apoptotic bodies formation. Caspase-3 activation occurred in the p53-normal cell type C6, but not in the p53-mutant U138MG. Besides its apoptotic effect, curcumin also synergized with the chemotherapeutics cisplatin and doxorubicin to enhance GBM cells death. In C6-implanted rats, intraperitoneal curcumin (50 mg kg(-1) d(-1)) decreased brain tumors in 9/11 (81.8%) animals against 0/11 (0%) in the vehicle-treated group. Importantly, no evidence of tissue (transaminases, creatinine and alkaline phosphatase), metabolic (cholesterol and glucose), oxidative or hematological toxicity was observed. In summary, data presented here suggest curcumin as a potential agent for therapy of GBMs.     

Down-regulation of glucose-regulated protein (GRP) 78 potentiates cytotoxic effect of celecoxib in human urothelial carcinoma cells

Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor that has been reported to elicit anti-proliferative response in various tumors. In this study, we aim to investigate the antitumor effect of celecoxib on urothelial carcinoma (UC) cells and the role endoplasmic reticulum (ER) stress plays in celecoxib-induced cytotoxicity. The cytotoxic effects were measured by MTT assay and flow cytometry. The cell cycle progression and ER stress-associated molecules were examined by Western blot and flow cytometry. Moreover, the cytotoxic effects of celecoxib combined with glucose-regulated protein (GRP) 78 knockdown (siRNA), (-)-epigallocatechin gallate (EGCG) or MG132 were assessed. We demonstrated that celecoxib markedly reduces the cell viability and causes apoptosis in human UC cells through cell cycle G1 arrest. Celecoxib possessed the ability to activate ER stress-related chaperones (IRE-1α and GRP78), caspase-4, and CCAAT/enhancer binding protein homologous protein (CHOP), which were involved in UC cell apoptosis. Down-regulation of GRP78 by siRNA, co-treatment with EGCG (a GRP78 inhibitor) or with MG132 (a proteasome inhibitor) could enhance celecoxib-induced apoptosis. We concluded that celecoxib induces cell cycle G1 arrest, ER stress, and eventually apoptosis in human UC cells. The down-regulation of ER chaperone GRP78 by siRNA, EGCG, or proteosome inhibitor potentiated the cytotoxicity of celecoxib in UC cells. These findings provide a new treatment strategy against UC.     

CFTR activation suppresses glioblastoma cell proliferation,migration and invasion

The aim of this study was to investigate the function of Cystic fibrosis transmembrane conductance regulator (CFTR) in human glioblastoma (GBM) cells. Data dining results of the Human Protein Atlas showed that low CFTR expression was associated with poor prognosis for GBM patients. We found that CFTR protein expression was lower in U87 and U251 GBM cells than that in normal humane astrocyte cells. CFTR activation significantly reduced GBM cell proliferation. In addition, CFTR activation significantly abrogated migration and invasion of GBM cells. Besides, CFTR activator Forskolin treatment markedly reduced MMP-2 protein expression. These effects of CFTR activation were significantly inhibited by CFTR inhibitor CFTRinh-172 pretreatment. Our findings suggested that JAK2/STAT3 signaling was involved in the anti-glioblastoma effects of CFTR activation. Moreover, CFTR overexpression in combination with Forskolin induced a synergistic anti-proliferative response in U87?cells. Overall, our findings demonstrated that CFTR activation suppressed GBM cell proliferation, migration and invasion likely through the inhibition of JAK2/STAT3 signaling.     

Glioblastoma cell secretome: analysis of three glioblastoma cell lines reveal 148 non-redundant proteins

Glioblastoma multiforme (GBM) is the most aggressive among human gliomas with poor prognosis. Study of tumor cell secretome - proteins secreted by cancer cell lines, is a powerful approach to discover potential diagnostic or prognostic biomarkers. Here we report, for the first time, proteins secreted by three GBM cell lines, HNGC2, LN229 and U87MG. Analysis of the conditioned media of these cell lines by LC-MS/MS using ESI-IT mass spectrometer (LTQ) resulted in the confident identification of 102, 119 and 64 proteins, respectively. Integration of the results from all the three cell lines lead to a dataset of 148 non-redundant proteins. Subcellular classification using Genome Ontology indicated that 42% of the proteins identified belonged to extracellular or membrane proteins, viz. Vinculin, Tenascin XB, SERPIN F1 and TIMP-1. 52 proteins matched with the secretomes of 11 major cancer types reported earlier whereas remaining 96 are unique to our study. 25 protein identifications from the dataset represent proteins related to GBM or other cancer tissues as per Human Protein Atlas; at least 22 are detectable in plasma, 11 of them being reported even in cerebrospinal fluid. Our study thus provides a valuable resource of GBM cell secretome with potential for further investigation as GBM biomarkers.     

A curcumin derivative hydrazinobenzoylcurcumin suppresses stem-like features of glioblastoma cells by targeting Ca2+/calmodulin-dependent protein kinase II

Glioblastoma multiforme (GBM) is the most aggressive and common type of human primary brain tumor. Glioblastoma stem-like cells (GSCs) have been proposed to contribute to tumor initiation, progression, recurrence, and therapeutic resistance of GBM. Therefore, targeting GSCs could be a promising strategy to treat this refractory cancer. Calmodulin (CaM), a major regulator of Ca²⁺-dependent signaling, controls various cellular functions via interaction with multiple target proteins. Here, we investigated the anticancer effect of hydrazinobenzoylcurcumin (HBC), a Ca ²⁺/CaM antagonist, against GSCs derived from U87MG and U373MG cells. HBC significantly inhibited not only the self-renewal capacity, such as cell growth and neurosphere formation but also the metastasis-promoting ability, such as migration and invasion of GSCs. HBC induced apoptosis of GSCs in a caspase-dependent manner. Notably, HBC repressed the phosphorylation of Ca ²⁺/CaM-dependent protein kinase II (CaMKII), c-Met, and its downstream signal transduction mediators, thereby reducing the expression levels of GSC markers, such as CD133, Nanog, Sox2, and Oct4. In addition, the knockdown of CaMKIIγ remarkably decreased the cancer stem cell-like phenotypes as well as the expression of stemness markers by blocking c-Met signaling pathway in U87MG GSCs. These results suggest that HBC suppresses the stem-like features of GBM cells via downregulation of CaM/CaMKII/c-Met axis and therefore CaMKII may be a novel therapeutic target to eliminate GSCs.     

Suppressing H19 Modulates Tumorigenicity and Stemness in U251 and U87MG Glioma Cells

Glioblastoma multiforme (GBM) is a type of malignant carcinoma found in the brain. Its high frequency of occurrence and poor survival rate have garnered much research attention in recent years. Long non-coding RNAs (lncRNAs) are known to be related to the formation and progression of several cancer types by both promoting and suppressing tumor transformation. H19 is one such lncRNA and has been shown to be upregulated in a few types of cancer. In this study, we discovered that the expression of H19 increased in GBM cell lines. H19 knocked down GBM cells also displayed decreased cellular proliferation and a higher apoptosis rate when induced by temozolomide. Interestingly, the GBM cell lines U87MG and U251 were found to express cancer stem cell markers CD133, NANOG, Oct4 and Sox2. Expression of these markers was downregulated in H19-deficient cells. Collectively, these data suggest a role for H19 in contributing to GBM malignancy and the maintenance of its stem cell properties.     

Acute inducible ablation of GRP78 reveals its role in hematopoietic stem cell survival, lymphogenesis and regulation of stress signaling

GRP78, a master regulator of the unfolded protein response (UPR) and cell signaling, is required for inner cell mass survival during early embryonic development. However, little is known about its role in adult hematopoietic stem cells (HSCs) and hematopoiesis. Here we generated a conditional knockout mouse model that acutely deletes Grp78 in the adult hematopoietic system. Acute GRP78 ablation resulted in a significant reduction of HSCs, common lymphoid and myeloid progenitors, and lymphoid cell populations in the mutant mice. The GRP78-null induced reduction of the HSC pool could be attributed to increased apoptosis. Chimeric mice with Grp78 deletion only in the hematopoietic cells also showed a loss of HSCs and lymphopenia, suggesting a cell intrinsic effect. Analysis of GRP78 deficient bone marrow (BM) cells showed constitutive activation of all the major UPR signaling pathways, including activation of eIF2α, ATF6, xbp-1 splicing, as well as caspase activation. A multiplex cytokine assay further revealed alteration in select cytokine and chemokine serum levels in the mutant mice. Collectively, these studies demonstrate that GRP78 plays a pleiotropic role in BM cells and contributes to HSC survival and the maintenance of the lymphoid lineage.     

Glucose-regulated protein 78 modulates cell growth,epithelial–mesenchymal transition,and oxidative stress in the hyperplastic prostate

Benign prostatic hyperplasia (BPH) is a chronic condition which mainly affects elderly males. Existing scientific evidences have not completely revealed the pathogenesis of BPH. Glucose-regulated protein 78 (GRP78) is a member of the heat shock protein 70 superfamily, which serves as an important regulator in many diseases. This study aims at elucidating the role of GRP78 in the BPH process. Human prostate tissues, cultured human prostate cell lines (BPH-1 and WPMY-1) and clinical data from BPH patients were utilized. The expression and localization of GRP78 were determined with quantitative real time PCR (qRT-PCR), Western blotting and immunofluorescence staining. GRP78 knockdown and overexpression cell models were created with GRP78 siRNA and GRP78 plasmid transfection. With these models, cell viability, apoptosis rate, as well as marker levels for epithelial-mesenchymal transition (EMT) and oxidative stress (OS) were detected by CCK8 assay, flow cytometry analysis and Western blotting respectively. AKT/mTOR and MAPK/ERK pathways were also evaluated. Results showed GRP78 was localized in the epithelium and stroma of the prostate, with higher expression in BPH tissues. There was no significant difference in GRP78 expression between BPH-1 and WPMY-1 cell lines. In addition, GRP78 knockdown (KD) slowed cell growth and induced apoptosis, without effects on the cell cycle stage of both cell lines. Lack of GRP78 affected expression levels of markers for EMT and OS. Consistently, overexpression of GRP78 completely reversed all effects of knocking down GRP78. We further found that GRP78 modulated cell growth and OS via AKT/mTOR signaling, rather than the MAPK/ERK pathway. Overall, our novel data demonstrates that GRP78 plays a significant role in the development of BPH and suggests that GRP78 might be rediscovered as a new target for treatment of BPH.Subject terms: Prostatic diseases, Preclinical research