Heparanase inhibitor OGT 2115 induces prostate cancer cell apoptosis via the downregulation of MCL­1.

Heparanase (HPSE), an endo-ß-D-glucuronidase, cleaves heparan sulfate and serves an important role in the tumor microenvironment and thus in tumorigenesis. HPSE is known to promote tumor cell evasion of apoptosis. However, the underlying mechanism of this requires further study. In the present study, the results demonstrated that myeloid cell leukemia-1 (MCL-1), an antiapoptotic protein, and HPSE were upregulated in prostate cancer tissues compared with adjacent normal tissues. In addition, the HPSE inhibitor, OGT 2115, inhibited PC-3 and DU-145 prostate cancer cell viability in a dose-dependent manner, with IC50 values of 20.2 and 97.2 µM, respectively. Furthermore, annexin V/PI double-staining assays demonstrated that OGT 2115 induced apoptosis in prostate cancer cells. OGT 2115 treatment markedly decreased MCL-1 protein expression levels, whereas RNA interference-mediated downregulation of MCL-1 and OGT 2115 drug treatment synergistically induced apoptosis in PC-3 and DU-145 cells. In vivo, OGT 2115 40 mg/kg (ig) significantly inhibited PC-3 cell xenograft growth in nude mice and increased the positive TUNEL staining rate of xenograft tissues. It was therefore hypothesized that MCL-1 was an important signaling molecule in OGT 2115-induced apoptosis. The results of the present study also demonstrated that the proteasome inhibitor, MG-132, markedly inhibited the downregulation of MCL-1 protein expression levels induced by OGT 2115. However, the protein synthesis inhibitor, cycloheximide, did not affect the role of OGT 2115 in regulating MCL-1. In summary, the results of the present study demonstrated that the proapoptotic activity of OGT 2115 was achieved by downregulating MCL-1.

Modulation of gut microbiota alleviates cerebral ischemia/reperfusion injury in rats by inhibiting M1 polarization of microglia.

Gut microbiota affects the gut-brain axis; hence, the modulation of the microbiota has been proposed as a potential therapeutic strategy for cerebral ischemia/reperfusion injury (CIRI). However, the role and mechanism of the gut microbiota in regulating microglial polarization during CIRI remain poorly understood. Herein, using a middle cerebral artery occlusion and reperfusion (MCAO/R) rat model, we evaluated changes in the gut microbiota after CIRI and the potential effects of fecal microbiota transplant (FMT) on the brain. Rats underwent either MCAO/R or sham surgery, and then they received FMT (started 3 days later; continued for 10 days). 2,3,5-Triphenyltetrazolium chloride staining, neurological outcome scale, and Fluoro-Jade C staining showed that MCAO/R induced cerebral infarction, neurological deficits, and neuronal degeneration. In addition, immunohistochemistry or real-time PCR assay showed increased expression levels of M1-macrophage markers-TNF-α, IL-1ß, IL-6, and iNOS-in the rats following MCAO/R. Our finding suggests that microglial M1 polarization is involved in CIRI. 16 S ribosomal RNA gene sequencing data revealed an imbalance in the gut microbiota of MCAO/R animals. In contrast, FMT reversed this MCAO/R-induced imbalance in the gut microbiota and ameliorated nerve injury. In addition, FMT prevented the upregulation in the ERK and NF-κB pathways, which reversed the M2-to-M1 microglial shift 10 days after MCAO/R injury in rats. Our primary data showed that the modulation of the gut microbiota can attenuate CIRI in rats by inhibiting microglial M1 polarization through the ERK and NF-κB pathways. However, an understanding of the underlying mechanism requires further study.

The Specifically Androgen-Regulated Gene (SARG) Promotes Papillary Thyroid Carcinoma (PTC) Lymphatic Metastasis Through Vascular Endothelial Growth Factor C (VEGF-C) and VEGF Receptor 3 (VEGFR-3) Axis.

The papillary thyroid carcinoma (PTC) metastasizes through lymphatic spread, but the follicular thyroid cancer (FTC) metastasis occurs by following hematogenous spread. To date, the molecular mechanism underlying different metastatic routes between PTC and FTC is still unclear. Here, we showed that specifically androgen-regulated gene (SARG) was significantly up-regulated in PTC, while obviously down-regulated in FTC through analyzing the Gene Expression Omnibus (GEO) database. Immunohistochemistry assay verified that the PTC lymph node metastasis was associated with higher levels of SARG protein in clinical PTC patient samples. SARG-knockdown decreased TPC-1 and CGTH-W3 cells viability and migration significantly. On the contrary, SARG-overexpressed PTC cells possessed more aggressive migratory ability and viability. In vivo, SARG overexpression dramatically promoted popliteal lymph node metastasis of xenografts from TPC-1 cells mouse footpad transplanting. Mechanistically, SARG overexpression and knockdown significantly increased and decreased the expression of vascular endothelial growth factor C (VEGF-C) and VEGF receptor 3 (VEGFR-3), respectively, thereby facilitating or inhibiting the tube formation in HUVECs. The tube formation experiment showed that SARG overexpression and knockdown promoted or inhibited the number of tube formations in HUVEC cells, respectively. Taken together, we showed for the first time the differential expression profile of SARG between PTC and FTC, and SARG promotes PTC lymphatic metastasis via VEGF-C/VEGFR-3 signal. It indicates that SARG may represent a target for clinical intervention in lymphatic metastasis of PTC.

Transmembrane 4 L Six Family Member 1 Suppresses Hormone Receptor--Positive, HER2-Negative Breast Cancer Cell Proliferation.

Background: The prognosis of breast cancer varies according to the molecular subtype. Transmembrane 4 L six family 1 (TM4SF1) exhibits different expression patterns among the molecular subtypes of breast cancer. However, the expression profile of TM4SF1 in hormone receptor HR+HER2- breast cancer remains unclear. Methods: TM4SF1 mRNA levels were examined in major subclasses of breast cancer by analyzing The Cancer Genome Atlas (TCGA) datasets. In addition, TM4SF1 protein and mRNA levels in HR+HER2- breast cancer tissue samples were determined by immunohistochemistry and Western blot assay. The effect of TM4SF1 on cell proliferation was evaluated using MTT, colony formation, 3D organoid, and xenograft models, following the TM4SF1 overexpression or knockdown. Results: TCGA database analysis demonstrated that TM4SF1 was downregulated in breast cancer compared with the healthy adjacent breast tissue. In addition, the expression of TM4SF1 in basal-like one and the mesenchymal TNBC tissue was higher than that of the healthy adjacent breast tissue. Other types, including the luminal androgen receptor-positive TNBC tissue, expressed lower levels of TM4SF1. Immunohistochemistry and real-time quantitative PCR assays demonstrated that the TM4SF1 protein and mRNA levels were downregulated in the HR+HER2- breast cancer tissue compared with the healthy adjacent tissue. Moreover, the TM4SF1 overexpression reduced the viability of MCF-7 and ZR-75-1 breast cancer cells, whilst reducing the number of colonies and 3D-organoids formed by these cell lines. By contrast, TM4SF1 knockdown led to an increased MCF-7 cell proliferation. However, in the TNBC cell line, MDA-MB-231, TM4SF1 silencing reduced cell proliferation. In vivo, the TM4SF1 overexpression inhibited MCF-7 xenograft growth in a nude mouse model, which was associated with the downregulation of the Ki-67 expression, apoptosis induction, and inhibition of the mTOR pathway. Conclusion: TM4SF1 is downregulated in HR + HER2-breast cancer, and the overexpression of TM4SF1 suppresses cell proliferation in this cancer subtype.

KIR2DL4 promotes the proliferation of RCC cell associated with PI3K/Akt signaling activation.

BACKGROUND: Killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4) is a transmembrane glycoprotein that is expressed by natural killer (NK) cells and certain subsets of T cells. However, its expression profiles and functions in solid tumor progression remain poorly defined. METHODS: In the present study, using bioinformatics analysis, immunohistochemistry, immunoblotting, MTT cell viability assay, soft agar colony formation assay and a human renal cell carcinoma (RCC) cell xenograft model in nude mice, we examined whether KIR2DL4 is expressed by RCC and its possible roles in RCC progression. RESULTS: We confirmed that KIR2DL4 is overexpressed by RCC cells. MTT and soft agar cloning assays showed that KIR2DL4 knockdown delayed cell proliferation and viability in RCC cell lines, Caki-1 and 769-P, in vitro. By contrast, KIR2DL4 overexpression promoted Caki-1 cell proliferation both in vitro and in vivo, which was observed in a BALB/c-nu/nu xenograft mouse model. Moreover, RNA sequencing data demonstrated that the differentially expressed genes found between parallel-controlled and Caki-1 cells overexpressing KIR2DL4 were highly associated with cancer development, of which those related to the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway were particularly enriched, immunoblotting data showed that the level of AKT phosphorylation was higher or lower in KIR2DL4 overexpressing or KIR2DL4 knocking-down Caki-1 cells compared with that in the parallel-controlled cells. In addition, PI3K inhibitor wortmannin treatment and KIR2DL4-shRNA transfection further deregulated the levels of phosphorylated AKT and Caki-1 cell proliferation. CONCLUSIONS: Our results indicate that KIR2DL4 is also expressed by RCC cells, which promotes RCC progression associated with PI3K/AKT activation.

XMD-17-51 Inhibits DCLK1 Kinase and Prevents Lung Cancer Progression.

Doublecortin-like kinase 1 (DCLK1) is a cancer stem cell marker that is highly expressed in various types of human cancer, and a protein kinase target for cancer therapy that is attracting increasing interest. However, no drug candidates targeting DCLK1 kinase have been developed in clinical trials to date. XMD-17-51 was found herein to possess DCLK1 kinase inhibitory activities by cell-free enzymatic assay. In non-small cell lung carcinoma (NSCLC) cells, XMD-17-51 inhibited DCLK1 and cell proliferation, while DCLK1 overexpression impaired the anti-proliferative activity of XMD-17-51 in A549 cell lines. Consequently, XMD-17-51 decreased Snail-1 and zinc-finger-enhancer binding protein 1 protein levels, but increased those of E-cadherin, indicating that XMD-17-51 reduces epithelial-mesenchymal transition (EMT). Furthermore, sphere formation efficiency was significantly decreased upon XMD-17-51 treatment, and XMD-17-51 reduced the expression of stemness markers such as ß-catenin, and pluripotency factors such as SOX2, NANOG and OCT4. However, the percentage of ALDH+ cells was increased significantly following treatment with XMD-17-51 in A549 cells, possibly due to EMT inhibition. In combination, the present data indicated that XMD-17-51 inhibited DCLK1 kinase activity in a cell-free assay with an IC50 of 14.64 nM, and decreased DCLK1 protein levels, cell proliferation, EMT and stemness in NSCLC cell lines. XMD-17-51 has the potential to be a candidate drug for lung cancer therapy.

LPAR5 promotes thyroid carcinoma cell proliferation and migration by activating class IA PI3K catalytic subunit p110ß.

Lysophosphatidic acid receptor 5 (LPAR5) is involved in mediating thyroid cancer progression, but the underlying mechanism needs to be further revealed. In this study, we confirmed that LPAR5 is upregulated in papillary thyroid carcinoma (PTC), especially in BRAF-like PTC, by analyzing The Cancer Genome Atlas (TCGA) database and performing immunohistochemistry assay in human thyroid cancer tissues. LPAR5-specific antagonist TC LPA5 4 treatment inhibited CGTH-W3, TPC-1, B-CPAP, and BHT-101 cell proliferation, CGTH-W3 and TPC-1 cell migration significantly. In vivo, TC LPA5 4 treatment could delay CGTH-W3 xenograft growth in nude mice. We also found that LPAR5-specific antagonist TC LPA5 4, PI3K inhibitor wortmannin, or mTOR inhibitor rapamycin pretreatment abrogated phosphorylation of Akt and p70S6K1 stimulated by LPA in CGTH-W3 and TPC-1 cells. Stimulating CGTH-W3 cells transfected with pEGFPC1-Grp1-PH fusion protein with LPA resulted in the generation of phosphatidylinositol (3,4,5)-triphosphate, which indicates that PI3K was activated by LPA directly. The p110ß-siRNA instead of p110α-siRNA transfection abrogated the increase of levels of phosphorylated Akt and S6K1 stimulated by LPA. Furthermore, immunoprecipitation assay confirmed an interaction between LPAR5 and p110ß. Overall, we provide new insights that the downregulation of LPAR5 decreased the proliferation and migration phenotype via the PI3K/Akt pathway. Inhibition of LPAR5 or the PI3K/Akt signal may be a novel therapeutic strategy for treating thyroid cancer.

Sodium-glucose co-transporter-2 (SGLT-2) inhibition reduces glucose uptake to induce breast cancer cell growth arrest through AMPK/mTOR pathway.

OBJECTIVE: The sodium-glucose transporter 2 (SGLT2) inhibitors Canagliflozin and Dapagliflozin are recently approved medications for type 2 diabetes. Recent studies indicate the potential ability of SGLT2 inhibitors to attenuate cancer growth of SGLT2-expressing cancer cells, but there is little known about the effects of SGLT2 inhibitors on breast cancer. The goal in this research was to assess the anticancer activity of SGLT2 inhibitors in breast cancerin vitro and in vivo. METHODS: We test the SGLT2 expression in breast cancer using immunohistochemistry and immunoblot assay. MTT cytotoxicity assay, colony formation assay and human breast cancer cells nude mice xenograft model were performed to detect the effects of SGLT2 inhibitors on cancer cell proliferation and growth. Flow Cytometry assay was performed to determine if the SGLT2 inhibitors induced cell cycle arrest and apoptosis. RESULTS: We proved that SGLT2 expresses in breast cancer cell lines and human breast tumor tissue samples. SGLT2 inhibitors Dapagliflozin and Canagliflozin exhibited a potent anti-proliferative effect in breast cancer cells as demonstrated by MTT, clonogenic survival assay in vitro and xenograft growth model in vivo. Furthermore, we found that SGLT2 inhibitors arrested cell cycle in G1/G0 phase and induced cell apoptosis. Western blot analysis demonstrated that treatment with SGLT2 inhibitors increased the phosphorylation of Amp-activated protein kinase (AMPK) and decreased the phosphorylation of 70 kDa ribosomal protein S6 kinase 1 (p70S6K1) in breast cancer cells. CONCLUSIONS: These findings indicate that SGLT2 inhibitor-therapy induced AMPK-mediated cell cycle arrest and apoptosis, which is a potential novel strategy for the treatment of breast cancer.

CA8 promotes RCC proliferation and migration though its expression level is lower in tumor compared to adjacent normal tissue.

Chemotherapy and radiotherapy are not as successful in the case of renal cell carcinoma (RCC) although some targeted drugs were approved for RCC therapy recently. Analysis of whole genomic data will lead to improvements in understanding RCC and identifying novel anticancer targets. Here, we found the differential mRNA expression and copy number variation (CNV) of Carbonic anhydrase-related protein VIII (CA8) gene in RCC through integrated bioinformatics analysis of TCGA database, which was confirmed in 5 cases of samples collected from RCC patients who underwent radical nephrectomy by analysis of CA8 mRNA and protein levels using RT-PCR immunohistochemical assay. However, we got a completely opposite result that CA8 promoted RCC progression, those are CA8 overexpression promoted the proliferative and migratory ability of Caki-1 and 769-P cells in vitro as determined with MTT and transwell assay, and CA8 overexpression could also promote Caki-1 xenograft growth in BALB/C­nu/nu mice. On the contrary, CA8-knockdown reduced Caki-1 and 769-P cell proliferation and migration. Moreover, knockdown of CA8 decreased pAKT and MMP2 protein levels in Caki-1 cells while overexpressing CA8 increased pAKT and MMP2. In conclusion, we showed that CA8 promoted RCC cell proliferation and migration, but it was down-regulated in RCC, which requires an additional mechanism study.

Everolimus Inhibits the Progression of Ductal Carcinoma In Situ to Invasive Breast Cancer Via Downregulation of MMP9 Expression.

PURPOSE: We evaluated the role of everolimus in the prevention of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) progression. EXPERIMENTAL DESIGN: The effects of everolimus on breast cancer cell invasion, DCIS formation, and DCIS progression to IDC were investigated in a 3D cell culturing model, intraductal DCIS xenograft model, and spontaneous MMTV-Her2/neu mouse model. The effect of everolimus on matrix metalloproteinase 9 (MMP9) expression was determined with Western blotting and IHC in these models and in patients with DCIS before and after a window trial with rapamycin. Whether MMP9 mediates the inhibition of DCIS progression to IDC by everolimus was investigated with knockdown or overexpression of MMP9 in breast cancer cells. RESULTS: Everolimus significantly inhibited the invasion of human breast cancer cells in vitro. Daily intragastric treatment with everolimus for 7 days significantly reduced the number of invasive lesions from intraductal DCIS foci and inhibited DCIS progression to IDC in the MMTV-Her2/neu mouse mammary tumor model. Mechanistically, everolimus treatment decreased the expression of MMP9 in the in vitro and in vivo models, and in breast tissues from patients with DCIS treated with rapamycin for 1 week. Moreover, overexpression of MMP9 stimulated the invasion, whereas knockdown of MMP9 inhibited the invasion of breast cancer cell-formed spheroids in vitro and DCIS in vivo. Knockdown of MMP9 also nullified the invasion inhibition by everolimus in vitro and in vivo. CONCLUSIONS: Targeting mTORC1 can inhibit DCIS progression to IDC via MMP9 and may be a potential strategy for DCIS or early-stage IDC therapy.

Everolimus induces G1 cell cycle arrest through autophagy-mediated protein degradation of cyclin D1 in breast cancer cells.

Everolimus inhibits mammalian target of rapamycin complex 1 (mTORC1) and is known to cause induction of autophagy and G1 cell cycle arrest. However, it remains unknown whether everolimus-induced autophagy plays a critical role in its regulation of the cell cycle. We, for the first time, suggested that everolimus could stimulate autophagy-mediated cyclin D1 degradation in breast cancer cells. Everolimus-induced cyclin D1 degradation through the autophagy pathway was investigated in MCF-10DCIS.COM and MCF-7 cell lines upon autophagy inhibitor treatment using Western blot assay. Everolimus-stimulated autophagy and decrease in cyclin D1 were also tested in explant human breast tissue. Inhibiting mTORC1 with everolimus rapidly increased cyclin D1 degradation, whereas 3-methyladenine, chloroquine, and bafilomycin A1, the classic autophagy inhibitors, could attenuate everolimus-induced cyclin D1 degradation. Similarly, knockdown of autophagy-related 7 (Atg-7) also repressed everolimus-triggered cyclin D1 degradation. In addition, everolimus-induced autophagy occurred earlier than everolimus-induced G1 arrest, and blockade of autophagy attenuated everolimus-induced G1 arrest. We also found that everolimus stimulated autophagy and decreased cyclin D1 levels in explant human breast tissue. These data support the conclusion that the autophagy induced by everolimus in human mammary epithelial cells appears to cause cyclin D1 degradation resulting in G1 cell cycle arrest. Our findings contribute to our knowledge of the interplay between autophagy and cell cycle regulation mediated by mTORC1 signaling and cyclin D1 regulation.

VHL regulates NEK1 via both HIF-2α pathway and ubiquitin-proteasome pathway in renal cancer cell.

Both Von Hippel-Lindau tumor suppressor (VHL) and Never-in-mitosis A (NIMA)-related kinase 1 (NEK1) are involved in primary cilium formation, but whether VHL could regulate NEK1 is unknown. Here, we demonstrated that renal cancer cells Caki-1 and ACHN with wild-type VHL expressed lower level of NEK1 than that of VHL-defective cells including 786-O, 769-P and A498 cells. VHL-overexpression down-regulated NEK1 in 769-P cells, while VHL-knockdown up-regulated NEK1 in Caki-1 cells. In addition, we found the hypoxia response element (HRE) in the promoter sequence of NEK1 and hypoxia induced NEK1 expression both in vitro and in vivo. HIF-2α knockdown blocked hypoxia induced NEK1 upregulation instead of HIF-1α, which indicates that NEK1 may be a new target of HIF-2α. Moreover, we confirmed the association between VHL and NEK1 in Caki-1 cell, then showed VHL promoted NEK1 protein degradation and ubiquitination. In conclusion, our findings showed VHL regulates NEK1 via both HIF-2α pathway and ubiquitin-proteasome pathway in renal cancer cells.

Never-in-mitosis A-related kinase 8, a novel target of von-Hippel-Lindau tumor suppressor protein, promotes gastric cancer cell proliferation.

Previous research has revealed that the von-Hippel-Lindau tumor suppressor protein (pVHL) may downregulate never-in-mitosis A-related kinase 8 (NEK8) via hypoxia-inducible factor-α (HIF-α). The HIF-independent functions of pVHL also serve an important role in its tumor-suppressor action. In the present study, the association between pVHL and NEK8 was demonstrated in the human gastric cancer cell line, SGC-7901, indicating a direct interaction of pVHL with NEK8. Subsequently, it was reported that MG-132, a specific proteasome inhibitor, may attenuate pVHL overexpression-induced reductions in NEK8 protein expression levels. In addition, the present study revealed that pVHL may stimulate the rapid degradation of NEK8 protein and promote its ubiquitination. The association between the expression profile of NEK8 and the survival status of patients with gastric cancer was analyzed from an online database. Kaplan-Meier survival plots indicated that higher expression levels of NEK8 may lead to poor survival, as suggested by the transcriptomic data of 1,065 patients with gastric cancer. It was found that NEK8-knockdown mediated by RNA interference inhibited SGC-7901 and SNU-1 proliferation, colony formation and migration in vitro, and tumor growth in vivo. Collectively, the present study proposed that NEK8 may be a novel target of pVHL as a ubiquitin E3 ligase, and may serve a role as a potential oncoprotein in human gastric cancer.

VHL loss predicts response to Aurora kinase A inhibitor in renal cell carcinoma cells.

The majority of molecular targets of anticancer agents are limited to a subset of patients, and therefore identification of more specific biomarkers that can be used to improve clinical outcomes is of increasing interest. The present study showed that von Hippel­Lindau tumor suppressor (VHL) tumor­suppressor activity may influence the therapeutic response to Aurora kinase A (AURKA) inhibitors in human renal cell carcinoma (RCC). VHL protein (pVHL) expression was evaluated by immunoblotting in the human RCC cell lines CAKI, ACHN, 786­O, 769­P and A498. The anti­tumor activities of alisertib, an AURKA­specific chemical inhibitor, were detected by Cell Counting Kit­8 assay in vitro and mouse xenograft model in vivo. Additionally, the VHL­mediated anti­tumor activity was assessed in 769­P and CAKI cells via the loss or gain of VHL. The results revealed that VHL­deficient 786­O, 769­P and A498 cells were sensitive to alisertib. By contrast, alisertib­resistant CAKI and ACHN cells expressed the wild type VHL gene. In addition, rescue or knockdown of VHL was observed to increase or decrease alisertib anti­proliferation activity, respectively, in RCC cells. The inverse correlation between the VHL gene expression profile and alisertib sensitivity was further confirmed in human cancer xenografts models. Taken together, these results suggested that VHL loss could potentially serve as a biomarker for predicting the efficacy of AURKA inhibitors.

Combinatorial Antitumor Effect of Rapamycin and ß-Elemene in Follicular Thyroid Cancer Cells.

Background. mTOR signaling would be a promising target for thyroid cancer therapy. However, in clinical trials, objective response rate with mTOR inhibitor monotherapy in most cancer types was modest. A new focus on development of combinatorial strategies with rapalogs is increasing. Objective. Investigating the combinatorial antitumor effect of rapamycin and ß-elemene in follicular thyroid cancer cells. Methods. MTT assay was used to determine the FTC-133 cell proliferation after culturing with rapamycin and/or ß-elemene. To analyze their combinatorial effect, immunoblotting was performed to analyze the activation status of AKT. Moreover, ß-elemene attenuated rapamycin-induced immunosuppression was tested in mice. Results. Combination of rapamycin and ß-elemene exerted significant synergistic antiproliferative effects in FTC-133 cell lines in vitro, based on inhibiting the AKT feedback activation induced by rapamycin. In vivo, the ß-elemene could attenuate rapamycin-induced immunosuppression via reversing imbalance of Treg/Th17, with the underlying mechanism needed to be declared. Conclusions. We demonstrate that the novel combination of mTOR inhibitor with ß-elemene synergistically attenuates tumor cell growth in follicular thyroid cancer, which requires additional preclinical validation.

Loss of PIG3 increases HIF-1α level by promoting protein synthesis via mTOR pathway in renal cell carcinoma cells.

PIG3 is a target of the tumor suppressor p53 and is thought to be involved in p53-mediated cell apoptosis. Although PIG3 is similar to oxidoreductases involved in generating ROS, whether PIG3 would regulate HIF-1α was never characterized directly. Here we demonstrated that knockdown of PIG3 by transfecting with specific siRNA could increase the expression of HIF-1α in several human cancer cell lines, including CAKI, FTC-133 and A549. It indicates that PIG3 may be involved in the regulation of HIF-1α. Furthermore, we revealed that PIG3-siliencing increased HIF-1α protein level through promoting its protein biosynthesis via mTOR pathway. In addition, the effect of PIG3 on the production of HIF-1α was further related to VEGF secretion and cell migration. PIG3-downregulation increased the secretion of VEGF and promoted the migration of renal cancer cells obviously. Taken together, these data suggest that PIG3 was involved in HIF-1α regulation, and reveal a novel signaling pathway of PIG3/HIF-1α in the regulation of cell migration in renal cell carcinoma.

Validation of p27KIP1 expression levels as a candidate predictive biomarker of response to rapalogs in patient-derived breast tumor xenografts.

Blockade of mammalian target of rapamycin (mTOR) is a promising area in breast cancer therapy. However, in clinical trials, objective response rate with mTOR inhibitor monotherapy in breast cancer was modest. Biomarker studies designed to identify the responders of rapalogs are of increasing interest. We validated p27KIP1 expression levels as a candidate predictive biomarker of response to rapalogs. We also analyzed the correlation between rapamycin activity and p27KIP1 expression in the primary breast cancer cells and the patient-derived breast tumor xenograft models. The cells isolated from the breast tumor tissues expressing high levels of p27KIP1 were sensitive to rapamycin, whereas the cells from the tissues expressing low levels of p27KIP1 exhibited resistance to rapamycin. The correlation between p27KIP1 expression and rapamycin antitumor activity was also observed in the patient-derived breast tumor xenograft models. Moreover, we also found rapamycin significantly decreased phosphorylated p70S6K1 and phosphorylated 4EBP1 in both samples. It seemed that the different sensitivity of tumor cells to rapamycin did not owe to its different potency against mTOR activity. We further propose p27KIP1 expression level may be also a candidate predictive biomarker of rapalogs for breast cancer therapy, which requires additional clinical validation.

The tumor suppressor pVHL down-regulates never-in-mitosis A-related kinase 8 via hypoxia-inducible factors to maintain cilia in human renal cancer cells.

NEK8 (never in mitosis gene A (NIMA)-related kinase 8) is involved in cytoskeleton, cilia, and DNA damage response/repair. Abnormal expression and/or dysfunction of NEK8 are related to cancer development and progression. However, the mechanisms that regulate NEK8 are not well declared. We demonstrated here that pVHL may be involved in regulating NEK8. We found that CAK-I cells with wild-type vhl expressed a lower level of NEK8 than the cells loss of vhl, such as 786-O, 769-P, and A-498 cells. Moreover, pVHL overexpression down-regulated the NEK8 protein in 786-O cells, whereas pVHL knockdown up-regulated NEK8 in CAK-I cells. In addition, we found that the positive hypoxia response elements (HREs) are located in the promoter of the nek8 sequence and hypoxia could induce nek8 expression in different cell types. Consistent with this, down-regulation of hypoxia-inducible factors α (HIF-1α or HIF-2α) by isoform-specific siRNA reduced the ability of hypoxia inducing nek8 expression. In vivo, NEK8 and HIF-1α expression were increased in kidneys of rats subjected to an experimental hypoxia model of ischemia and reperfusion. Furthermore, NEK8 siRNA transfection significantly blocked pVHL-knockdown-induced cilia disassembling, through impairing the pVHL-knockdown-up-regulated NEK8 expression. These results support that nek8 may be a novel hypoxia-inducible gene. In conclusion, our findings show that nek8 may be a new HIF target gene and pVHL can down-regulate NEK8 via HIFs to maintain the primary cilia structure in human renal cancer cells.

Mobile bearing or fixed bearing? A meta-analysis of outcomes comparing mobile bearing and fixed bearing bilateral total knee replacements.

BACKGROUND: To compare outcomes between mobile-bearing (MB) and fixed-bearing (FB) in bilateral total knee replacements. METHODS: The MEDLINE, EMBASE and Cochrane Library databases were searched. Randomized controlled trials of bilateral total knee arthroplasty with one of each design implanted were identified. Weighted mean differences (WMDs) and pooled risk ratios (RRs) were calculated using fixed- or random-effects models. RESULTS: Twelve studies were identified with a total of 807 patients and 1614 knees. All RCTs were of high quality with a low risk of bias. No statistical difference was found between MB and FB at 2- to 5-year follow-up in terms of America Knee Society score (WMD: -1.29, 95% CI: -5.65 to 3.06), pain score (WMD: -3.26, 95% CI: -10.45 to 3.93), range of motion (WMD: -4.16, 95% CI: -9.97 to 1.66), reoperation (RR: 1.00, 95% CI: 0.28 to 3.60), and radiolucent lines (RR: 1.51, 95% CI: 0.70 to 3.24). The results were similar at 1-, 5- to 8-, or >8-year follow-up. Patient's satisfaction (RR: 0.85, 95% CI: 0.54 to 1.34), and complication (≤2-year, RR: 0.55, 95% CI: 0.29 to 1.04; >2-year, RR: 1.0, 95% CI=0.73 to 1.38) also showed no difference between two groups. CONCLUSIONS: Based on this meta-analysis we are unable to detect the superiority of MB as compared to FB. More randomized trials with a larger sample size and longer follow-up are needed to evaluate these two kinds of prosthesis. LEVEL OF EVIDENCE: Therapeutic Level II.

Characterization of eubacterial and archaeal community diversity in the pit mud of Chinese Luzhou-flavor liquor by nested PCR-DGGE.

The aim of this study was to investigate and compare the microbial community structures of eubacteria and archaea in the pit mud of Chinese Luzhou-flavor liquor from the wall (C(w)) and bottom (C(b)) of cellar through nested PCR-denaturing gradient gel electrophoresis (DGGE). The Shannon-Wiener index (H) calculated from the DGGE profiles showed that the community diversities of eubacteria and archaea in samples from C(b) were almost higher than that from C(w). In addition, cluster analysis of the DGGE profiles revealed that some differences were found in the microbial community structure in samples from different locations. The closely relative microorganisms of all eubacterial 16S rRNA gene sequences fell into four phyla (Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria), including 12 genera and 2 uncultured eubacteria. Moreover, 37.1% eubacteria were affiliated with Clostridium. Particularly, genus Acinetobacter was absent in all samples from C(b) but present in all samples from C(w). The closely relative microorganisms of all archaeal 16S rRNA gene sequences fell into four genera, which included Methanobrevibacter, Methanoculleus, Methanobacterium and Methanosaeta, while the dominant archaea in samples from C(w) and C(b) were similar. Results presented in this study provide further understanding of the spatial differences in microbial community structure in the pit mud, and is of great importance for the production and quality improvement of Luzhou-flavor liquor.