Hepatocellular carcinoma (HCC) is a common cancer with poor prognosis. Hepatitis B virus (HBV) is one of the leading causes of HCC, but the precise mechanisms by which this infection promotes cancer development are not fully understood. Recently, miR-340-5p, a microRNA (miRNA) that has been identified as a cancer suppressor gene, was found to inhibit the migration and invasion of liver cancer cells. However, the effect of miR-340-5p on cell proliferation and apoptosis in HBV-associated HCC remains unknown. In our study, we show that miR-340-5p plays an important role during HBV infection and hepatocellular carcinoma development. Specifically, this miRNA directly binds to the mRNA encoding activating transcription factor 7 (ATF7), a protein that both promotes cell proliferation and suppresses apoptosis through its interaction with heat shock protein A member 1B (HSPA1B). We further found that miR-340-5p is downregulated by HBV, which enhances ATF7 expression, leading to enhanced cell proliferation and inhibition of apoptosis. Notably, ATF7 is upregulated in HCC tissue, suggesting that HBV may target miR-340-5p in vivo to promote ATF7/HSPA1B-mediated proliferation and apoptosis and regulate liver cancer progression. This work helps to elucidate the complex interactions between HBV and host miRNAs and further suggests that miR-340-5p may represent a promising candidate for the development of improved therapeutic strategies for HCC.

OBJECTIVE: Identification of new molecular markers to enhance early diagnosis, prognosis and/or treatment of hepatocellular carcinoma (HCC) is a need. TOM34 (34 kDa-translocase of the outer mitochondrial membrane) protein expression deregulation has demonstrated to be involved in the growth of many cancers. Here, we aimed at evaluating serum TOM34 and some heat shock proteins (HSPA4, HSPA1B, and HSP90AA1) expressions in hepatitis C virus (HCV)-related cirrhosis and HCV-induced HCC relative to controls and correlating these expressions to the clinicopathological data.
METHODS: Serum specimens were collected from 90 patients with HCV associated complications (30 cirrhotic, 30 early HCC and 30 late HCC) and 60 controls. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed for relative quantification of the four target genes using the Livak method. In silico network analysis was also executed to explore the contribution of the genes in liver cancer.
RESULTS: The serum TOM34 and HSP90AA1 transcripts were significantly upregulated in HCC patients compared to cirrhotic ones with more up-regulation in late HCC patients. Receiver operating characteristic analysis showed the optimum cutoff value of 0.625 corresponding to 71.7% sensitivity and 56.7% specificity, and an area under the curve (AUC) of 0.705 to discriminate HCC from cirrhotic groups (P = .002). In multivariate analysis, ordination plot showed obvious demarcation between the study groups caused by the higher levels of TOM34 among other variables.
CONCLUSIONS: TOM34 and its partner HSP90AA1 might be used as a potential biomarker for monitoring HCV-induced HCC progression in the Egyptian population. Future large-scale validation studies are warranted.

TNFα is a pleiotropic cytokine which fuels tumor cell growth, invasion, and metastasis in some malignancies, while in others it induces cytotoxic cell death. However, the molecular mechanism by which TNFα exerts its diverse effects on breast cancer subtypes remains elusive. Using in vitro assays and mouse xenografts, we show here that TNFα contributes to the aggressive properties of triple negative breast cancer (TNBC) cell lines via upregulation of TNFAIP3(A20). In a striking contrast, TNFα induces a potent cytotoxic cell death in luminal (ER+) breast cancer cell lines which fail to upregulate A20 expression. Overexpression of A20 not only protects luminal breast cancer cell lines from TNFα-induced cell death via inducing HSP70-mediated anti-apoptotic pathway but also promotes a robust EMT/CSC phenotype by activating the pStat3-mediated inflammatory signaling. Furthermore, A20 overexpression in luminal breast cancer cells induces aggressive metastatic properties in mouse xenografts via generating a permissive inflammatory microenvironment constituted by granulocytic-MDSCs. Collectively, our results reveal a mechanism by which A20 mediates pleiotropic effects of TNFα playing role in aggressive behaviors of TNBC subtype while its deficiency results in TNFα-induced apoptotic cell death in luminal breast cancer subtype.

BACKGROUND/AIM: From the standpoint of cancer therapy, it is valuable to enhance the anticancer effects of chemotherapy. Our previous reports revealed that up-regulation of heat-shock protein 27 (HSP27) has been linked to gemcitabine resistance of pancreatic cancer cells. Enzyme-treated asparagus extract (ETAS) is an extract that is produced from asparagus. The purpose of this study was to investigate the effect of ETAS on the expression of HSP27 and other HSPs in the gemcitabine-resistant pancreatic cancer cell line KLM1-R.
MATERIALS AND METHODS: KLM1-R cells were treated with ETAS, and expression levels of HSPs, including HSP27, were investigated by western blotting.
RESULTS: ETAS down-regulated HSP27 and pHSP27 (serine 78) in KLM1-R cells, but, HSP70 and GRP78 levels were not altered.
CONCLUSION: This study suggests the potential therapeutic benefit of ETAS in enhancing anticancer effects by its combination with gemcitabine for patients with pancreatic cancer.

Heat-shock proteins (HSPs), one of the evolutionarily conserved protein families, are widely found in various organisms, and play important physiological functions. Nevertheless, HSPs have not been systematically analyzed in esophageal squamous cell carcinoma (ESCC). In this study, we applied the protein-protein interaction (PPI) network methodology to explore the characteristics of HSPs, and integrate their expression in ESCC. First, differentially expressed HSPs in ESCC were identified from our previous RNA-seq data. By constructing a specific PPI network, we found differentially expressed HSPs interacted with hundreds of neighboring proteins. Subcellular localization analyses demonstrated that HSPs and their interacting proteins distributed in multiple layers, from membrane to nucleus. Functional enrichment annotation analyses revealed known and potential functions for HSPs. KEGG pathway analyses identified four significant enrichment pathways. Moreover, three HSPs (DNAJC5B, HSPA1B, and HSPH1) could serve as promising targets for prognostic prediction in ESCC, suggesting these HSPs might play a significant role in the development of ESCC. These multiple bioinformatics analyses have provided a comprehensive view of the roles of heat-shock proteins in esophageal squamous cell carcinoma.

Non-small cell lung cancer (NSCLC) patients harboring EGFR-activating mutations initially respond to EGFR tyrosine kinase inhibitors (EGFR-TKIs) and have shown favorable outcomes. However, acquired drug resistance to EGFR-TKIs develops in almost all patients mainly due to the EGFR T790 M mutation. Here, we show that treatment with low-dose EGFR-TKI results in the emergence of the EGFR T790 M mutation and in the reduction of HSP70 protein levels in HCC827 cells. Erlotinib treatment inhibits HSP70 phosphorylation at tyrosine 41 and increases HSP70 ubiquitination, resulting in HSP70 degradation. We show that EGFR-TKI treatment causes increased DNA damage and enhanced gene mutation rates, which are secondary to the EGFR-TKI-induced reduction of HSP70 protein. Importantly, HSP70 overexpression delays the occurrence of Erlotinib-induced EGFR T790 M mutation. We further demonstrate that HSP70 interacts with multiple enzymes in the base excision repair (BER) pathway and promotes not only the efficiency but also the fidelity of BER. Collectively, our findings show that EGFR-TKI treatment facilitates gene mutation and the emergence of EGFR T790 M secondary mutation by the attenuation of BER via induction of HSP70 protein degradation.

BACKGROUND: The anticancer effects of histone deacetylase inhibitors (HDACi) vary between patients, and their molecular mechanisms remain poorly understood. Previously, we have identified heat shock 70 kDa protein 1A (HSPA1A, also known as HSP72) as the most overexpressed protein in valproic acid (VPA)-resistant cell lines. KNK437, an inhibitor of heat shock proteins, enhanced the cytotoxic effects of not only VPA but also vorinostat, another HDACi. However, the mechanisms underlying the role of HSP72 in resistance against HDACi remain largely unknown.
OBJECTIVE: The purpose of this study was to identify the mechanisms underlying the role of HSP72 in HDACi resistance.
METHODS: We established an HSP72-overexpressing Jurkat cell line and used it to assess the functional role of HSP72 following treatment with the HDACi vorinostat and VPA.
RESULTS: HDACi-induced apoptosis, assessed using annexin V assays, sub-G1 fraction analysis, and PARP cleavage, was significantly lower in HSP72-overexpressing cells than in control cells. The HDACi-induced upregulation in caspase-3, -8, and -9 activity, as well as the HDACi-induced reduction in mitochondrial membrane potential, were also suppressed following HSP72 overexpression. The basal expression levels of Bcl-2, phosphorylated Bad, and XIAP increased in HSP72-overexpressing cells, whereas HDACi-induced Bid truncation and the suppression of Bad expression. Furthermore, vorinostat-induced histone hyperacetylation was also diminished in HSP72-overexpressing cells.
CONCLUSION: These findings clearly demonstrate that HSP72 inhibits HDACi-induced apoptosis.

AIM: In our previous study, we have built a nine-gene (
METHODS: Logistic regression analysis, discriminant analysis, classification tree analysis, and artificial neural network were used for the multi-parameter gene expression analysis method. One hundred and three patients with early HCC and 54 age-matched healthy normal controls were used to build a diagnostic model. Fifty-two patients with early HCC and 34 healthy people were used for validation. The area under the curve, sensitivity, and specificity were used as diagnostic indicators.
RESULTS: Artificial neural network of the total nine genes had the best diagnostic value, and the AUC, sensitivity, and specificity were 0.943, 98%, and 85%, respectively. At last, 52 HCC patients and 34 healthy normal controls were used for validation. The sensitivity and specificity were 96% and 86%, respectively.
CONCLUSION: Multi-parameter analysis methods may increase the diagnostic value compared to single factor analysis and they may be a trend of the clinical diagnosis in the future.

PURPOSE: The ability to predict individual responsiveness to cancer therapy is urgently needed. This is particularly true for patients with locally advanced rectal cancer (LARC) because a large proportion are resistant to preoperative chemoradiation therapy (CRT). In this study, we sought to identify markers that could predict response by comparing the gene expression profiles of the tumors of patients who received preoperative CRT.
METHODS AND MATERIALS: The basal gene expression profiles of tumors from 22 LARC patients who were responders (n=9) and nonresponders (n=13) to preoperative CRT were analyzed using RNA sequencing (RNA-Seq). To validate the RNA-Seq findings, real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed on tumor samples from an additional 40 LARC patients (n=20 responders; n=20 nonresponders). Candidate genes were stably overexpressed or knocked down in colorectal cancer (CRC) cell lines, and the effect on response to radiation was tested in vitro and also in vivo in a mouse xenograft model.
RESULTS: Eight differentially expressed (>16-fold) genes (B3GALT4, HSPA1B, KRBOX1, PPBP, PPP1R18, PSMB8, SLC39A7, and TAP2) associated with the preoperative CRT response were identified (P<.0005). Among these genes, real-time RT-PCR showed that PSMB8 and SLC39A7 were upregulated in the responsive group of the additional 40 LARC patients. In CRC cell lines, PSMB8 overexpression significantly reduced colony formation and increased the apoptosis-inducing molecules cleaved caspase-3 and cleaved PARP after 6-Gy irradiation. PSMB8 knockdown increased colony formation and decreased caspase-3 activation and cleaved PARP levels after irradiation. SLC39A7 overexpression had no significant effects on irradiated CSC cells. After irradiation of the xenografted mice, tumors that arose from CRC cell line HCT116 overexpressing PSMB8 grew more slowly than did those from HCT116 with vector alone.
CONCLUSION: These results suggest that PSMB8 is a predictive marker of preoperative radiosensitivity in LARC patients. Clinical validation in a larger cohort is now required.

STUDY QUESTION: Is the human blastocyst transcriptome associated with infertility diagnosis, specifically: polycystic ovaries (PCO), male factor (MF) and unexplained (UE)?
SUMMARY ANSWER: The global blastocyst transcriptome was significantly altered in association with a PCO, MF and UE infertility diagnosis.
WHAT IS KNOWN ALREADY: Infertility diagnosis has an impact on the probability for a successful outcome following an IVF cycle. Limited information is known regarding the relationship between a specific infertility diagnosis and blastocyst transcription during preimplantation development.
STUDY DESIGN, SIZE, DURATION: Blastocysts created during infertility treatment from patients with specific infertility diagnoses (PCO, MF and UE) were analyzed for global transcriptome compared to fertile donor oocyte blastocysts (control).
PARTICIPANTS/MATERIALS, SETTING, METHODS: Surplus cryopreserved blastocysts were donated with patient consent and institutional review board approval. Female patients were <38 years old with male patients <40 years old. Blastocysts were grouped according to infertility diagnosis: PCO (n = 50), MF (n = 50), UE (n = 50) and fertile donor oocyte controls (n = 50). Pooled blastocysts were lysed for RNA isolation followed by microarray analysis using the SurePrint G3 Human Gene Expression Microarray. Validation was performed on significant genes of interest using real-time quantitative PCR (RT-qPCR).
MAIN RESULTS AND THE ROLE OF CHANCE: Transcription alterations were observed for all infertility etiologies compared to controls, resulting in differentially expressed genes: PCO = 869, MF = 348 and UE = 473 (P < 0.05; >2-fold). Functional annotation of biological and molecular processes revealed both similarities, as well as differences, across the infertility groups. All infertility etiologies displayed transcriptome alterations in signal transducer activity, receptor binding, reproduction, cell adhesion and response to stimulus. Blastocysts from PCO patients were also enriched for apoptotic genes while MF blastocysts displayed enrichment for genes involved in cancer processes. Blastocysts from couples with unexplained infertility displayed transcription alterations related to various disease states, which included mechanistic target of rapamycin (mTOR) and adipocytokine signaling. RT-qPCR validation confirmed differential gene expression for the following genes: BCL2 like 10 (BCL2L10), heat shock protein family A member 1A (HSPA1A), heat shock protein family A member 1B (HSPA1B), activating transcription factor 3 (ATF3), fibroblast growth factor 9 (FGF9), left-right determination factor 1 (LEFTY1), left-right determination factor 2 (LEFTY2), growth differentiation factor 15 (GDF15), inhibin beta A subunit (INHBA), adherins junctions associated protein 1 (AJAP1), cadherin 9 (CDH9) and laminin subunit alpha 4 (LAMA4) (P < 0.05; >2-fold).
LARGE SCALE DATA: Not available due to participant privacy.
LIMITATIONS, REASONS FOR CAUTION: Blastocyst samples for microarray analysis required pooling. While this allows for an overall average in each infertility etiology group and can reduce noise from sample-to-sample variation, it cannot give a detailed analysis of each blastocyst within the group.
WIDER IMPLICATIONS OF THE FINDINGS: Underlying patient infertility diagnosis has an impact on the blastocyst transcriptome, modifying gene expression associated with developmental competence and implantation potential.
STUDY FUNDING AND COMPETING INTEREST(S): No conflict of interest or outside funding provided.

BACKGROUND: Gliomas are aggressive brain tumors with very high resistance to chemotherapy. Therefore, the aim of the present study was to investigate the effectiveness of sorafenib and Temozolomide in elimination of human glioma cells through apoptosis and autophagy.
METHODS: MOGGCCM (anaplastic astrocytoma) and T98G (glioblastoma multiforme) cell lines incubated with sorafenib and/or Temozolomide were used in the experiments. Cell morphology (ER stress, apoptosis, autophagy, and necrosis) was analyzed microscopically while apoptosis and mitochondrial membrane potential were assessed with flow cytometry. Beclin1, LC3, p62, Hsp27, and Hsp72 levels were analyzed by immunoblotting. The activity of caspase 3, 8, and 9 was evaluated fluorometrically. Expression of Hsps was blocked by transfection with specific siRNA.
RESULTS: In MOGGCCM cells, Temozolomide most frequently induced autophagy, which was accompanied by decreased p62 and increased beclin1 and LC3II levels. Sorafenib initiated mainly apoptosis. Additional incubation with Temozolomide, synergistically potentiated the pro-apoptotic properties of sorafenib, but it was mediated in a caspase-independent way. In T98G cells, the effect of the analyzed drugs on programmed cell death induction was different from that in MOGGCCM cells. Sorafenib induced autophagy, while Temozolomide initiated mainly apoptosis. After simultaneous drug application, apoptosis dominated, suggesting synergistic action of both drugs. Inhibition of Hsp27 and Hsp72 expression increased the sensitivity of both cell lines to ER stress and, to a lesser extent, to induction of apoptosis, but not autophagy.
CONCLUSIONS: Sorafenib and Temozolomide applied in combination are potent apoptosis inducers in T98G and MOGGCCM cells. ER stress precedes the elimination. Blocking of Hsp expression has a greater impact on ER stress rather than apoptosis induction.

To explore a novel combination of chemotherapy, gene therapy, and thermotherapy for osteosarcoma, a targeted heat-sensitive co-delivery system based on bacterial magnetosomes (BMs) was developed. The optimal culture conditions of magnetotactic bacteria (MTB) AMB-1 and characterization of BMs were achieved. A recombinant eukaryotic plasmid heat shock protein 70-polo-like kinase 1-short hairpin RNA (pHSP70-Plk1-shRNA) under transcriptional control of a thermosensitive promoter (human HSP70 promoter) was constructed for gene therapy. Doxorubicin (DOX) and pHSP70-Plk1-shRNA were included in the targeted thermosensitive co-delivery system, and in vitro DOX release activity, targeted gene silencing efficiency and in vitro antitumor efficacy were investigated. The results showed that the optimal culture conditions of MTB AMB-1 are an oxygen concentration of 4.0%, a pH value of 7.0, 20 μmol/L of ferrous sulfate, 800 mg/L of sodium nitrate, and 200 mg/L of succinic acid. The temperature of BMs reached 43°C within 3 minutes and could be maintained for 30 minutes by adjusting the magnitude of the alternating magnetic field (AMF). The diameters of BMs, BM-DOX, BM-recombinant eukaryotic plasmid pHSP70-Plk1-shRNA (shPlk1), and BM-DOX-shPlk1 were 43.7±4.6, 79.2±5.4, 88.9±7.8, and 133.5±11.4 nm, respectively. The zeta potentials of BMs, BM-DOX, BM-shPlk1, and BM-DOX-shPlk1 were -29.4±6.9, -9.5±5.6, -16.7±4.8, and -10.3±3.1 mV, respectively. Besides, the system exhibited good release behavior. DOX release rate from BM-DOX-shPlk1 was 54% after incubation with phosphate-buffered saline at 43°C and 37% after incubation with 50% fetal bovine serum, which was significantly higher than that at 37°C (

Selectively enhance the therapeutic efficacy to malignancy is one of the most important issues for photothermal therapy (PTT). However, most solid tumors, such as triple negative breast cancer (TNBC), do not have identifiable surface markers to distinguish themselves from normal cells, thus it is challenging to selectively identify and eliminate those malignances by PTT. In this report, we hypothesized that, by targeting CD44 (one TNBC-overexpressed surface molecule) and depleting heat shock protein 72 (HSP72, one malignancy-specific-overexpressed thermotolerance-related chaperone) subsequently, the TNBC could be selectively sensitized to PTT and improve the accuracy of treatment. To this end, a rationally designed nanosystem gold nanostar (GNS)/siRNA against HSP72 (siHSP72)/hyaluronic acid (HA) was successfully constructed using a layer-by-layer method. Hydrodynamic diameter and zeta potential analysis demonstrated the formation of GNS/siHSP72/HA having a particle size of 73.2 ± 3.8 nm and a negative surface charge of -18.3 ± 1.6 mV. The CD44-targeting ability of GNS/siHSP72/HA was confirmed by the flow cytometer, confocal microscopic imaging, and competitive binding analysis. The HSP72 silencing efficacy of GNS/siHSP72/HA was ∼95% in complete culture medium. By targeting CD44 and depleting HSP72 sequentially, GNS/siHSP72/HA could selectively sensitize TNBC cells to hyperthermia and enhance the therapeutic efficacy to TNBC with minimal side effect both in vitro and in vivo. Other advantages of GNS/siHSP72/HA included easy synthesis, robust siRNA loading capacity, endosome/lysosome escaping ability, high photothermal conversion efficacy and superior hemo- and biocompatibility.

BACKGROUND: Colorectal cancer (CRC) is the third leading cause of cancer related deaths worldwide both in men and women. Our recent studies have indicated an association of heat shock protein 70-2 (HSP70-2) with bladder urothelial carcinoma. In the present study, we investigated the association of HSP70-2 with various malignant properties of colorectal cancer cells and clinic-pathological features of CRC in clinical specimens.
METHODS: HSP70-2 mRNA and protein was investigated expression by RT-PCR, immunohistochemistry, immunofluorescence, flow cytometry and Western blotting in CRC clinical specimens and COLO205 and HCT116 cell lines. Plasmid-based gene silencing approach was employed to study the association of HSP70-2 with various malignant properties of COLO205 and HCT116 cells in in vitro and with tumor progression in in vivo COLO205 human xenograft mice model.
RESULTS: HSP70-2 expression was detected in 78 % of CRC patients irrespective of various stages and grades by RT-PCR and IHC. Our analysis further revealed that HSP70-2 expression was detected in both COLO205 and HCT116 cell lines. Ablation of HSP70-2 expression resulted in reduced cellular growth, colony forming ability, migratory and invasive ability of CRC cells. In addition, ablation of HSP70-2 expression showed significant reduction in tumor growth in COLO205 human xenograft in in vivo mouse model.
CONCLUSION: Collectively, our results indicate that HSP70-2 is associated with CRC clinical specimens. In addition, down regulation of HSP70-2 expression reduces cellular proliferation and tumor growth indicating that HSP70-2 may be a potential therapeutic target for CRC treatment.

Matrix metalloproteinases (MMPs) are crucial factors in tumor progression, inflammatory/immune responses and tissue development/regeneration. Of note, it has been known that MMPs promote genome instability, epithelial-mesenchymal transition, invasion, and metastasis in tumor progression. We previously reported that human MMP3 could translocate into cellular nuclei and control transcription in human chondrosarcoma-derived cells and in articular cartilage (Eguchi et al. [2008] Mol Cell Biol 28(7):2391-2413); however, further transcriptional target genes and cofactors of intranuclear MMP3 have not been uncovered. In this paper, we used transcriptomics analysis in order to examine novel transcriptional target genes regulated by intracellular MMP3. We found that mRNA levels of HSP family members (HSP70B', HSP72, HSP40/DNAJ, and HSP20/CRYAB) are upregulated by the intracellular MMP3 overload. Bioinformatic analysis predicted several transcription factors that possibly interact with MMP3. Among these factors, heat shock factor 1 (HSF1) cooperated with the MMP3 to activate the HSP70B' gene promoter in reporter gene assays, while a dominant negative HSF1 blocked the role for MMP3 in the trans-activation. The hemopexin-like repeat (PEX) domain of the human MMP3 was essential for transcriptional induction of the HSP70B' gene. In addition, chromobox proteins CBX5/HP1α and CBX3/HP1γ cooperated with the PEX domain in induction of HSP70B' mRNA. Taken together, this study newly clarified that intracellular MMP3 cooperate with CBXs/HP1s in transcriptional promotion of HSP genes. J. Cell. Biochem. 118: 43-51, 2017. © 2016 Wiley Periodicals, Inc.

The stress-inducible chaperone protein HSP70 (HSPA1) is implicated in melanoma development, and HSP70 inhibitors exert tumor-specific cytotoxic activity in cancer. In this study, we documented that a significant proportion of melanoma tumors express high levels of HSP70, particularly at advanced stages, and that phospho-FAK (PTK2) and BRAF are HSP70 client proteins. Treatment of melanoma cells with HSP70 inhibitors decreased levels of phospho-FAK along with impaired migration, invasion, and metastasis in vitro and in vivo Moreover, the HSP70 inhibitor PET-16 reduced levels of mutant BRAF, synergized with the BRAF inhibitor PLX4032 in vitro, and enhanced the durability of response to BRAF inhibition in vivo Collectively, these findings provide strong support for HSP70 inhibition as a therapeutic strategy in melanoma, especially as an adjuvant approach for overcoming the resistance to BRAF inhibitors frequently observed in melanoma patients. Cancer Res; 76(9); 2720-30. ©2016 AACR.

Bacterial infections typically elicit a strong Heat Shock Response (HSR) in host cells. However, the gastric pathogen Helicobacter pylori has the unique ability to repress this response, the mechanism of which has yet to be elucidated. This study sought to characterize the underlying mechanisms by which H. pylori down-modulates host HSP expression upon infection. Examination of isogenic mutant strains of H. pylori defective in components of the type IV secretion system (T4SS), identified the secretion substrate, CagA, to be essential for down-modulation of the HSPs HSPH1 (HSP105), HSPA1A (HSP72), and HSPD1 (HSP60) upon infection of the AGS gastric adenocarcinoma cell line. Ectopic expression of CagA by transient transfection was insufficient to repress HSP expression in AGS or HEK293T cells, suggesting that additional H. pylori factors are required for HSP repression. RT-qPCR analysis of HSP gene expression in AGS cells infected with wild-type H. pylori or isogenic cagA-deletion mutant found no significant change to account for reduced HSP levels. In summary, this study identified CagA to be an essential bacterial factor for H. pylori-mediated suppression of host HSP expression. The novel finding that HSPH1 is down-modulated by H. pylori further highlights the unique ability of H. pylori to repress the HSR within host cells. Elucidation of the mechanism by which H. pylori achieves HSP repression may prove to be beneficial in the identification of novel mechanisms to inhibit the HSR pathway and provide further insight into the interactions between H. pylori and the host gastric epithelium.

BACKGROUND: To investigate polymorphisms in heat shock proteins A1B and A1L (HOM) and associated risk of oesophageal carcinoma in Northeast India.
MATERIALS AND METHODS: The study includes oesophageal cancer (ECA) patients attending general outpatient department (OPD) and endoscopic unit of Gauhati Medical College. Patients were diagnosed based on endoscopic and histopathological findings. Genomic DNA was typed for HSPA1B1267 and HSPA1L2437 SNPs using the polymerase chain reaction with restriction fragment length polymorphisms.
RESULTS: A total of 78 cases and 100 age-sex matched healthy controls were included in the study with a male: female ratio of 5:3 and a mean age of 61.4±8.5 years. Clinico-pathological evaluation showed 84% had squamous cell carcinoma and 16% were adenocarcinoma. Dysphagia grades 4 (43.5%) and 5 (37.1%) were observed by endoscopic and hispathological evaluation. The frequency of genomic variation of A1B from wild type A/A to heterozygous A/G and mutant G/G showed a positive association [chi sq=19.9, p= <0.05] and the allelic frequency also showed a significant correlation [chi sq=10.3, with cases vs. controls, OR=0.32, p≤0.05]. The genomic variation of A1L from wild T/T to heterozygous T/C and mutant C/C were found positively associated [chi sq= 7.02, p<0.05] with development of ECA. While analyzing the allelic frequency, there was no significant association [chi sq= 3.19, OR=0.49, p=0.07]. Among all the risk factors, betel quid [OR =9.79, Chi square= 35.0, p<0.05], tobacco [OR = 2.95, chi square=10.6, p<0.05], smoking [OR=3.23, chi square=10.1, p<0.05] demonstrated significant differences between consumers vs. non consumers regarding EC development. Alcohol did not show any significant association [OR= 1.34, chi square=0.69, p=0.4] independently.
CONCLUSIONS: It can be concluded that the present study provides marked evidence that polymorphisms of HSP70 A1B and HSP70 A1L genes are associated with the development of ECA in a population in Northeast India, A1B having a stronger influence. Betel quid consumption was found to be a highly significant risk factor, followed by smoking and tobacco chewing. Although alcohol was not a potent risk factor independently, alcohol consumption along with tobacco, smoking and betel nut was found to contribute to development of ECA.

BACKGROUND: During the past few years, there has been an increasing interest among the Traditional and Folk medical practitioners of Sri Lanka in the use of a decoction prepared from Flueggea leucopyrus (Willd.) for treating various cancers including breast cancer. In the present study, the cytotoxicity of this decoction and its effects on Heat Shock Protein (HSP) expression and apoptosis were compared in three breast cancer phenotypes, to scientifically evaluate if a decoction prepared from F. leucopyrus (Willd.) is useful for the treatment of breast cancer.
METHODS: Cytotoxic potential of the F. leucopyrus decoction was determined by evaluating its effects in MCF-7, MDA-MB-231 and SKBR-3 breast cancer cell lines, and MCF-10A (non-cancerous) breast cell line, by use of the Sulphorhodamine (SRB) assay. The effect of the decoction on HSP gene expression in the above cells was evaluated by (a) Real time reverse transcription PCR (RT-PCR) and (b) Immunofluorescence analysis of HSP protein expression. Effects of the decoction on apoptosis were evaluated by (a) fluorescent microscopic examination of apoptosis related morphological changes and (b) DNA fragmentation (c) Caspase 3/7 assay.
RESULTS: F. leucopyrus decoction can mediate significant cytotoxic effects in all three breast cancer cells phenotypes (IC50 values: 27.89, 99.43, 121.43 μg/mL at 24 h post incubation periods, for MCF-7, MDA-MB-231, SKBR-3 respectively) with little effect in the non-cancerous breast cell line MCF-10A (IC50: 570.4 μg/mL). Significant (*P <0.05) inhibitions of HSP 90 and HSP 70 expression were mediated by the decoction in MCF-7 and MDA-MB-231, with little effect in the SKBR-3 cells. Clear apoptotic morphological changes on Acridine orange/Ethidium bromide staining and DNA fragmentation were observed in all three breast cancer cell lines. Caspase 3/7 were significantly (*P <0.05) activated only in MDA-MB-231 and SKBR-3 cells indicating caspase dependent apoptosis in these cells and caspase independent apoptosis in MCF-7 cells.
CONCLUSIONS: Modulation of HSP 90 and HSP 70 expressions is a possible mechanism by which the decoction of F. leucopyrus mediates cytotoxic effects MCF-7 and MDA-MB-231 cells. This effect appears to correlate with enhanced apoptosis in these cells. In SKBR-3 cells, mechanisms other than HSP inhibition may be utilized to a greater extent by the decoction to mediate the observed cytotoxic effects. Overall findings suggest that the decoction has the potential to be exploited further for effective treatment of breast cancer.

In the past decade, no significant improvement has been made in chemotherapy for osteosarcoma (OS). To develop improved agents against OS, we screened 70 species of medicinal plants and treated two human OS cell lines with different agent concentrations. We then examined cell viability using the MTT assay. Results showed that a candidate plant, particularly the rhizomes of Anemone altaica Fisch. ex C. A. Mey aqueous extract (AAE), suppressed the viability of HOS and U2OS cells in a concentration-dependent manner. Flow cytometry analysis revealed that AAE significantly increased the amount of cell shrinkage (Sub-G1 fragments) in HOS and U2OS cells. Moreover, AAE increased cytosolic cytochrome c and Bax, but decreased Bcl-2. The amount of cleaved caspase-3 and poly-(ADP-ribose) polymerase-1 (PARP-1) were significantly increased. AAE suppressed the growth of HOS and U2OS through the intrinsic apoptotic pathway. Data suggest that AAE is cytotoxic to HOS and U2OS cells and has no significant influence on human osteoblast hFOB cells. The high mRNA levels of apoptosis-related factors (PPP1R15A, SQSTM1, HSPA1B, and DDIT4) and cellular proliferation markers (SKA2 and BUB1B) were significantly altered by the AAE treatment of HOS and U2OS cells. Results show that the anticancer activity of AAE could up-regulate the expression of a cluster of genes, especially those in the apoptosis-related factor family and caspase family. Thus, AAE has great potential as a useful therapeutic drug for human OS.

BACKGROUND: Heat shock proteins (HSPs) are overexpressed in human hepatocellular carcinoma (HCC) tissue and correlate with aggressiveness and prognosis of HCC.
METHODS: Using the GSE14520 microarray expression profile from Gene Expression Omnibus, we compared HSP gene expression between tumour and non-tumour tissues and correlated this with outcomes in HCC patients.
RESULTS: We analysed 220 hepatitis B virus (HBV)-related HCC patients and 25 HSPs in this study. With the exception of HSPA4L, HSPA12A and HSPB8, members of the HSP family, including HSPH1, HSPBP1, HSPA1A, HSPA1B, HSPA1L, HSPA2, HSPA4, HSPA5, HSPA8, HSPA9, HSPAA1, HSPAB1, HSPA14, HSPB11, HSPA13, HSP90B1 and HSPBAP1, were all overexpressed in tumour tissues (all P < 0.001). In contrast, HSPB6, HSPB7, HSPA6, HSPB2 and HSPB3 were upregulated in non-tumour tissues (all P < 0.001). Multivariate analysis showed that cirrhosis (HR = 5.282, 95% CI = 1.294-21.555, P = 0.02), Barcelona Clinic liver cancer (BCLC) staging (HR = 2.151, 95% CI = 1.682-2.750, P < 0.001), HSPA12A (HR = 1.042, 95% CI = 1.003-1.082, P = 0.033) and HSP90B1 (HR = 1.001, 95% CI = 1.000-1.001, P = 0.011) were negatively associated with survival of HBV-related HCC patients. Furthermore, advanced BCLC staging (HR = 1.797, 95% CI = 1.439-2.244, P < 0.001) was also associated with earlier recurrence of HCC. The high expression of HSPA4 (HR = 1.002, 95% CI = 1.000-1.004, P = 0.019), HSPA5 (HR = 1.0, 95% CI = 1.0-1.0, P = 0.046) and HSPA6 (HR = 1.008, 95% CI = 1.001-1.015, P = 0.021) was similarly associated with HCC recurrence.
CONCLUSIONS: The expression of most HSPs was higher in tumour tissues than in non-tumour tissues. High BCLC staging scores, advanced cirrhosis and the overexpression of HSPA12A and HSP90B1 might be associated with poor survival from HCC, whereas high levels of HSPA4, HSPA5 and HSPA6 might be associated with earlier recurrence of HCC.

Previously, we demonstrated that inhibition of proteasomal chymotrypsin-like (CT-like) activity in human prostate cancer (PCa) PC-3 cultures and PC-3 xenografts results in accumulation of ubiquitinated proteins, followed by induction of cell death. Studies have shown that plasma CT-like proteasomal activity may be a powerful biomarker for risk stratification in hematologic malignancies. We hypothesized that circulating proteasomes could also be used to stratify risk for patients with PCa. A total of 109 patients with suspected PCa underwent prostatic biopsies were enrolled. Subjects were divided into non-cancer, low-risk PCa, and high-risk PCa groups. Three different proteasomal activity markers (CT-like, caspase-like, and trypsin-like) were measured and compared among the three groups. The proteasomal target proteins, Ub-prs, Hsp70, Bax, and P27 in plasma and prostate tissues were also evaluated. Multivariate analysis was used to assess whether CT-like activity was a predictor of PCa progression. Only proteasomal CT-like activity in the high-risk group was statistically higher than in the non-cancer group (P < 0.05). The expression of Ub-prs, Hsp70, Bax, and P27 protein was decreased in both plasma and PCa tissue of high-risk patients. CT-like activity was found to be an independent predictor of high-risk PCa. Subjects with CT-like activity ≥55 had a 2.15-fold higher risk of having high-risk PCa as compared to those with a CT-like activity of <55 (P = 0.021). We found CT-like activity to be an independent predictor of high-risk PCa, and as such, it may be a good candidate as a biomarker for high-risk PCa detection and stratification.

Proteomics-based clinical studies represent promising resources for the discovery of novel biomarkers or for unraveling molecular mechanisms underlying particular diseases. Here, we present a discovery study of hepatocellular carcinoma developed on nonfibrotic liver (nfHCC) that combines complementary quantitative iTRAQ-based proteomics and phosphoproteomics approaches. Using both approaches, we compared a set of 24 samples (18 nfHCC versus six nontumor liver tissue). We identified 43 proteins (67 peptides) differentially expressed and 32 peptides differentially phosphorylated between the experimental groups. The functional analysis of the two data sets pointed toward the deregulation of a protein homeostasis (proteostasis) network including the up-regulation of the Endoplasmic Reticulum (ER) resident HSPA5, HSP90B1, PDIA6, and P4HB and of the cytosolic HSPA1B, HSP90AA1, HSPA9, UBC, CNDP2, TXN, and VCP as well as the increased phosphorylation of the ER resident calnexin at Ser583. Antibody-based validation approaches (immunohistochemistry, immunoblot, Alphascreen(®), and AMMP(®)) on independent nfHCC tumor sets (up to 77 samples) confirmed these observations, thereby indicating a common mechanism occurring in nfHCC tumors. Based on these results we propose that adaptation to proteostasis imbalance in nfHCC tumors might confer selective advantages to those tumors. As such, this model could provide an additional therapeutic opportunity for those tumors arising on normal liver by targeting the tumor proteostasis network. Data are available via ProteomeXchange with identifier PXD001253.

Renal cell carcinoma (RCC) represents one of the most resistant tumors to radiotherapy and chemotherapy. Current therapies for the RCC patients are limited owing to lack of diagnosis and therapeutic treatments. Testis-specific heat-shock protein 70-2 (HSP70-2), a member of HSP70 chaperone family, has been shown to be associated with various cancers. In the present study, we investigated the putative role of HSP70-2 in various malignant properties of the RCC cells. HSP70-2 messenger RNA (mRNA) and protein expression was investigated in A704, ACHN, and Caki-1 cells derived from the RCC patients. We assessed the expression of HSP70-2 gene and protein by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. The expression of HSP70-2 protein was further validated by performing indirect immunofluorescence (IIF) and flow cytometry. The malignant properties of high-grade invasive A704 and Caki-1 cells, such as cellular proliferation, colony formation, migration, invasion, and wound healing, were evaluated by silencing the expression of HSP70-2 gene in these cells. Statistical significance was defined using Student's t test. Our RT-PCR and Western blotting data showed the expression of HSP70-2 in all RCC cells. Our results showed that HSP70-2 was predominantly expressed in cytoplasm and found to be colocalized with endoplasmic reticulum, mitochondria, Golgi body, and plasma membrane but not the nuclear envelope. Knockdown of HSP70-2 expression with specific short hairpin RNA (shRNA) demonstrated significant reduction in cell growth and colony formation. Further, a marked reduction in cell migration and invasion was also observed, indicating the potential role of HSP70-2 in metastasis. Collectively, our data suggest that HSP70-2 plays a key role in cancerous growth and invasive potential of RCC cells. Thus, HSP70-2 could serve as a novel potential therapeutic target for the RCC.

BACKGROUND: Previous epidemiological studies have suggested a potential role of the HSPA1B±1267A/G polymorphism in risk of developing cancer. However, the results were inconsistent. Therefore, we performed this meta-analysis to summarize the possible association with cancer risk.
MATERIALS AND METHODS: We retrieved relevant articles from PubMed, EMBASE, ISI Web of Science, Chinese Biomedical Literature and Chinese National Knowledge Infrastructure. Studies were selected using specific criteria. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to assess those associations. All analyses were performed using STATA software.
RESULTS: Fourteen case-control studies, including 1, 834 cancer cases and 2, 028 controls were included in this meta-analysis. Overall, the results indicated that the G allele of HSPA1B gene ±1267A/G was significantly associated with an increased cancer risk in all genetic models (G vs A: OR=1.51, 95%CI 1.17-1.95, p=0.001; GG vs AA: OR=2.93, 95%CI 1.50-5.74, p=0.002; AG vs AA: OR=1.48, 95%CI 1.10-1.98, p=0.009; GG/AG vs AA: OR=1.69, 95%CI 1.22-2.33, p=0.001; GG vs
AG/AA: OR=2.31, 95%CI 1.24-4.32, p=0.009). In the subgroup analysis stratified by ethnicity, a significant association was identified in Caucasians (G vs A: OR=1.35, 95%CI 1.08-1.69, p=0.008; GG/AG vs AA: OR=1.36, 95%CI 1.09-1.70, p=0.007), but not in Asians. In the stratified analysis by cancer types, individuals with the G allele showed an increased risk of hepatocellular carcinoma compared with carriers of the A allele (OR=2.40, 95%CI 1.47-3.91, p< 0.001). Inversely, individuals with the GG genotype showed a decreased risk of gastric cancer compared with carriers of the AG/GG genotypes (GG vs
AG/AA: OR=0.39, 95%CI 0.20-0.70, p=0.007).
CONCLUSIONS: This meta-analysis suggests associations between the HSPA1B ±1267A/G polymorphism and risk of cancer. However, this association might be Caucasian-specific and the G allele of this polymorphism probably increases risk of hepatocellular carcinoma while decreasing risk of gastric cancer. Further well-designed studies based on larger sample sizes are needed to validate these findings.

The polymorphisms in the three main heat shock protein 70 (HSP70-1, HSP70-2, and HSP70-hom) genes were identified to be associated with cancer risk. However, the results are inconsistent. We perform a meta-analysis to evaluate the association between the three HSP70 polymorphisms and cancer risk. Relevant studies were identified using PubMed, Web of Science, Chinese National Knowledge Infrastructure (CNKI), and Wanfang databases up to March 29, 2014. The cancer risk associated with the HSP70 polymorphisms was estimated for each study by odds ratios (OR) together with its 95% confidence interval (CI), respectively. Twenty case-control studies from eighteen publications were included; a significant association was observed for HSP70-2 polymorphism (dominant model: OR = 1.53, 95% CI: 1.11-2.09; recessive model: OR = 1.91, 95% CI: 1.06-3.45; AG versus AA: OR = 1.38, 95% CI: 1.03-1.84; GG versus AA: OR = 2.34, 95% CI: 1.21-4.54), while there was no significant association for HSP70-1 and HSP70-hom polymorphisms. Besides, in stratification analyses by ethnicity, cancer type, and source of control, significant association was detected for HSP70-2 polymorphism, while for HSP70-hom polymorphism, we found a significant association in hospital-based population under homozygote comparison model. This meta-analysis suggests that the HSP70-2 polymorphism rather than HSP70-hom and HSP70-1 polymorphisms was associated with the risk of cancer.

BACKGROUND: High expression of HSP27 and HSP72 in glioma cells has been closely associated with chemoresistance and decreased sensitivity to programmed cell death induction. Therefore, it is important to devise therapies that effectively target invasive cancer cells by inducing cell death. The aim of our study was to assess the effect of quercetin and imperatorin applied separately and in combinations on the apoptosis and autophagy induction in human T98G cells cultured in vitro.
METHODS: Cell death induction was analyzed by the staining method. The Western blotting technique and fluorimetric measurements of activity were used to assess the expression of marker proteins of apoptosis and autophagy. The specific siRNA transfected method was used for blocking of the expression of HSP27 and HSP72 genes.
RESULTS: The experiments revealed the highest percentage of apoptotic cells after using a 50?M concentration of both compounds. Simultaneous quercetin and imperatorin administration induced apoptosis more effectively than incubation with single drugs. These results were accompanied with decreased HSP27 and HSP72 expression, and a high level of caspase-3 and caspase-9 activity. Autophagy was not observed. Additional experiments were performed on a cell line with blocked Hsp27 and Hsp72 expression and significant increase the sensitivity to apoptosis induction upon quercetin and imperatorin treatment was noticed.
CONCLUSIONS: The present study indicates that quercetin and imperatorin are potent apoptosis inducers, especially when they act synergistically, which may be a promising combination useful in glioma therapy. Our results also demonstrated that blocking the HSP27 and HSP72 gene expression might serve as a therapeutic target for the human brain cancer.

Osteosarcoma (OS) is a type of bone cancer. Eighty percent of this tumor will metastasize to the lungs or liver, and as a result, patients generally need chemotherapy to improve survival possibility. Recently, antitumor activity has been reported in Ocimum gratissimum aqueous extract (OGE), which has been the focus of recent extensive studies on therapeutic strategies due to its antioxidant properties. We performed pharmacogenomics analyses for the effect of OGE on human osteosarcoma U2-OS and HOS cell growth. Cell viability, Western blot and flow cytometry analysis were performed before performing pharmacogenomics analyses for the effect of OGE on human osteosarcoma U2-OS and HOS cell growth, including cDNA microarray and RT-PCR assays. Cell viability assays revealed that OGE significantly and dose-dependently decreased the viability of U2-OS and HOS cells. Increases in cell shrinkage, Sub-G1 fragments and the activation of caspase 3 indicated that OGE induced cell apoptosis in U2-OS and HOS cells. There was no change in human osteoblast hFOS cells. cDNA microarray assay demonstrated that the expression of cell cycle regulators, apoptosis-related factors and cell proliferation markers were all modified by OGE treatment. RT-PCR analysis also confirmed the down-regulation of SKA2 and BUB1B, and the up-regulation of PPP1R15A, SQSTM1, HSPA1B, and DDIT4 by OGE treatment. The finding of anticancer activity in OGE and the identification of some potential target genes raise the expectation that OGE may become a useful therapeutic drug for human OS.

The specific and efficient delivery of small interfering RNA (siRNA) into cancer cells in vivo remains a major obstacle. In this study, we investigated whether ultrasound-targeted microbubble destruction (UTMD) combined with dual targeting of HSP72 and HSC70 in prostate cancer cell lines improve the specific and efficient cell uptake of siRNA, inhibit HSP90 function and induce extensive tumor-specific apoptosis. VCaP cells were transfected with siRNA oligonucleotides. Cell viability assays were used to evaluate the safety of UTMD. The expression of HSP70, HSP90, caspase-8, caspase-3, PARP-1 and cleaved caspase-3 were determined by quantitative PCR and western blotting. Apoptosis and transfection efficiency were detected by flow cytometry. We found that HSP72, HSC70 and HSP90 expression was absent or weak in normal prostate epithelial cells (RWPE-1), and became uniformly and strongly expressed in prostate cancer cells (VCaP). VCaP and RWPE-1 cells expressed very low levels of caspase-8, caspase-3, PARP-1 and cleaved caspase-3. UTMD combined with dual targeting of HSP72 and HSC70 siRNA impoved the efficiency of transfection, cell uptake of siRNA, downregulated HSP70 and HSP90 expression in VCaP cells on the mRNA and protein levels, and upregulated major apoptotic markers (PARP-1, caspase-8, caspase-3 and cleaved caspase-3), thus, inducing extensive tumor-specific apoptosis. The Cell Counting Kit-8 assay showed decreased cellular viability in the HSP72/HSC70-siRNA silenced group. These results suggest that the combination of UTMD with dual targeting of HSP72 and HSC70 may improve the specific and efficient cell uptake of siRNA, inhibit HSP90 function and induce extensive tumor-specific apoptosis, indicating a novel, potential means for targeting therapeutic strategy to prostate cancer cells.

The role of holocarboxylase synthetase (HLCS) in catalyzing the covalent binding of biotin to the five biotin-dependent carboxylases in humans is well established, as are the essential roles of these carboxylases in the metabolism of fatty acids, the catabolism of leucine, and gluconeogenesis. This review examines recent discoveries regarding the roles of HLCS in assembling a multiprotein gene repression complex in chromatin. In addition, emerging evidence suggests that the number of biotinylated proteins is far larger than previously assumed and includes members of the heat-shock superfamily of proteins and proteins coded by the ENO1 gene. Evidence is presented linking biotinylation of heat-shock proteins HSP60 and HSP72 with redox biology and immune function, respectively, and biotinylation of the two ENO1 gene products MBP-1 and ENO1 with tumor suppression and glycolysis, respectively.