Survivin-mediated Therapeutic Efficacy of Gemcitabine through Glucose-regulated Protein 78 in Hepatocellular Carcinoma

Background

Survivin is an antiapoptotic molecule that is widely expressed in cancers, including hepatocellular carcinoma (HCC). Survivin has become a general therapeutic target for cancers because of its selective overexpression in a majority of tumors. However, little is known regarding the effect of survivin expression in combination with gemcitabine on HCC.

Methods

We generated survivin knockdown cells (survivin-KD) via a short interfering RNA (siRNA) technique. The antiproliferation effects of gemcitabine were determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assay, and cell cycle evaluation.

Results

According to the MTT assay, we found that survivin-KD cells were more sensitive than parental cells and scrambled control cells to gemcitabine treatment. The apoptotic cell population increased in survivin-KD cells that were treated with gemcitabine in comparison to scrambled control cells, as observed by the cell cycle distribution and TUNEL assays. We found that survivin knockdown resulted in a reduction of glucose-regulated protein 78 (GRP78), which may be responsible for the observed increased survivin-KD cell sensitivity to gemcitabine.

Conclusions

We conclude that survivin knockdown may contribute to a therapeutic effect of gemcitabine through GRP78 on HCC cells.     

Knockdown Survivin Expression Reduces the Efficacy of Curcumin Treatment in Hepatocellular Carcinoma Cells

Background

Survivin is a potential therapeutic target for cancer. Increased survivin expression promotes cell survival and therapeutic resistance. However, there is little information regarding whether the expression level of survivin affects curcumin treatment in hepatocellular carcinoma (HCC).

Methods

Survivin expression was suppressed in HCC cells using a short interfering RNA (siRNA) technique. The anticancer effects of curcumin were examined using a biosensor system, MTT assay, TUNEL assay, and cell cycle analysis.

Results

Curcumin resistance developed in cells with suppressed survivin, in contrast to the parental cells, as determined by survival assays. Cell cycle analysis and TUNEL assays revealed that the apoptotic cell population was increased in the scrambled-siRNA cells treated with curcumin compared with the survivin-siRNA cells. Suppression of survivin expression resulted in curcumin resistance via the modulation of Bcl-2 and Bax expression.

Conclusions

We conclude that the expression levels of survivin may mediate the therapeutic efficacy of curcumin in HCC cells.     

Silencing of Glucose-Regulated Protein 78 (GRP78) Enhances Cell Migration Through the Upregulation of Vimentin in Hepatocellular Carcinoma Cells

Background

Glucose-regulated protein 78 (GRP78) plays an important role in embryonic development and cancer progression. However, there is little information regarding the regulation of GRP78 in hepatocellular carcinoma (HCC) metastasis.

Methods

We used RNA silencing and cDNA expression vectors to manipulate target gene expression in HCC cells. The transwell migration assay and xCelligence biosensor system were applied to determine the proliferatory and migratory ability of the HCC cells.

Results

In this study, we found that GRP78 silencing enhanced cell migration in both HepJ5 and Mahlavu cells. Overexpressed GRP78 in skHep1 cells suppressed the migratory ability. In the insight mechanism dissection for GRP78-mediated cancer migration, we found that downregulation of GRP78 caused the increase of vimentin expression on HCC cells. Suppressed vimentin expression also decreased the migratory ability on HCC, indicating that vimentin expression levels modulated the cell migratory ability.

Conclusion

We found that silencing GRP78 in HCC cells may enhance cell migration through the increase of vimentin expression.

     

Normoxically Overexpressed Hypoxia Inducible Factor 1-Alpha is Involved in Arsenic Trioxide Resistance Acquisition in Hepatocellular Carcinoma

Background

The aim of this study was to examine the underlying signaling mechanisms of arsenic trioxide (ATO)-mediated anticancer effects and the responsible biomarker(s) for the acquired resistance in human heptatocellular carcinoma (HCC).

Materials and Methods

The therapeutic effects of ATO were examined using 2 characteristically distinct HCC cell lines, Hep-J5 (overexpressing HIF-1α/GRP78) and SK-Hep-1 (the matched control). ATO-mediated proliferation inhibition, oxidative stress, and apoptosis were analyzed using flowcytometric analysis and western blotting. The role of HIF-1α and GRP78 in HCC resistance to ATO treatment was determined using RNA silencing and inhibitor approaches.

Results

SK-Hep-1 cells, lacking both HIF-1α and GRP78 expressions were responsive to ATO-induced apoptosis via an oxidative-nitrosative mechanism. Intracellular glutathione depletion and lipid peroxidation have been identified as the early cascade of events preceding apoptosis via cytochrome c release and the severe drop of mitochondrial membrane potential (MMP). Conversely, Hep-J5 cells, with normoxic coexpression of HIF-1α and GRP78, were resistant to ATO-induced apoptosis. GRP78-silenced Hep-J5 cells remained resistant to ATO treatment. In contrast, ATO resistance in Hep-J5 cells was overcome by the addition of YC-1, a HIF-1α inhibitor.

Conclusions

HIF-1α was identified as the major positive modifier for ATO resistance acquisition in HCC, and it represents a prime molecular target for overcoming ATO resistance.     

The oncofetal gene survivin is re-expressed in osteoarthritis and is required for chondrocyte proliferation <Emphasis Type="Italic">in vitro</Emphasis>

Background

Regulation of cell death and cell division are key processes during chondrogenesis and in cartilage homeostasis and pathology. The oncogene survivin is considered to be critical for the coordination of mitosis and maintenance of cell viability during embryonic development and in cancer, and is not detectable in most adult differentiated tissues and cells. We analyzed survivin expression in osteoarthritic cartilage and its function in primary human chondrocytes in vitro.

Methods

Survivin expression was analyzed by immunoblotting and quantitative real-time PCR. The localization was visualized by immunofluorescence. Survivin functions in vitro were investigated by transfection of a specific siRNA.

Results

Survivin was expressed in human osteoarthritic cartilage, but was not detectable in macroscopically and microscopically unaffected cartilage of osteoarthritic knee joints. In primary human chondrocyte cultures, survivin was localized to heterogeneous subcellular compartments. Suppression of survivin resulted in inhibition of cell cycle progression and sensitization toward apoptotic stimuli in vitro.

Conclusions

The present study indicates a role for survivin in osteoarthritic cartilage and human chondrocytes. In vitro experiments indicated its involvement in cellular division and viability. Learning more about the functions of survivin in chondrocyte biology might further help toward understanding and modulating the complex processes of cartilage pathology and regeneration.

     

Clinical experience with survivin as a biomarker for urothelial bladder cancer

Objectives

This study was carried out as a prospective pilot study to evaluate the potential of survivin mRNA measurement in patients suspicious for urothelial bladder cancer (BC). Data were also analyzed for possible influences of secondary urological findings on survivin measurements.

Methods

Survivin was measured by an mRNA assay in voided urine samples of 50 patients with suspicion of new or recurrent BC prior to transurethral resection. Sample evaluation was possible in 49 cases. Histopathology revealed no malignancy in 17 (35%) and BC in 32 (65%) patients. Survivin mRNA was quantitated by real-time PCR from frozen cell pellets of centrifuged urine samples. A ROC analysis of the survivin data was performed.

Results

ROC analysis identified the best cut-off level at 10,000 mRNA copies, resulting in a sensitivity of 53% and a specificity of 88%. Seven of the 20 pTa tumors (35%), all four pT1 (100%) and all four muscle-invasive tumors (100%) were detected. Of four patients with carcinoma in situ (Cis), 50% could be identified. Only two patients (4%) were assessed as false positive. Histologically confirmed cystitis and concomitant urological findings (inflammatory cells in urine, microhematuria and others) had no detectable influence on survivin measurements.

Conclusion

In present group of patients, survivin was a reliable biomarker for high-grade urothelial BC (sensitivity 83%), but not for low grade (sensitivity 35%) urothelial BC with a high specificity (88%). No confounders influencing the results of survivin measurements could be identified.     

Role of Survivin Gene Promoter Polymorphism (−31G>C) in Susceptibility and Survival of Esophageal Cancer in Northern India

Background and Aim

Survivin is an upregulated inhibitor of apoptosis protein in esophageal cancer (EC), and a promoter region polymorphism (?31G>C) in the survivin gene has been reported as a modulator of gene expression. We aim to explore the role of survivin ?31G>C polymorphism in susceptibility and survival of EC patients in northern Indian population.

Materials and Methods

A case–control study was performed in 500 subjects (250 EC patients and 250 controls), and genotyping was done by polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) method.

Results

Survivin CC genotype was found to be significantly associated with EC susceptibility [odds ratio (OR) = 2.29; 95% confidence interval (CI) = 1.27–4.14; P = 0.006], particularly in males (OR = 4.91; 95% CI = 2.19–11.02; P = 0.0001) having squamous cell carcinoma (SCC) histopathology (OR = 2.4; 95% CI = 1.36–4.21; P = 0.002) at middle third esophagus location (OR = 2.60; 95% CI = 1.40–4.82; P = 0.002). Patients carrying CC genotype were found to have higher susceptibility to lymph node metastasis (OR = 2.82; 95% CI = 1.46–5.48; P = 0.002). However, on survival analysis, no prognostic role of survivin ?31G>C polymorphism was detected. In case-only analysis, no gene–environment interaction was observed.

Conclusion

Survivin promoter region polymorphism (?31G>C) is associated with susceptibility and clinical characteristics but not prognosis of esophageal cancer in northern Indian population.     

Glucose-regulated protein 78 silencing down-regulates vascular endothelial growth factor/vascular endothelial growth factor receptor 2 pathway to suppress human colon cancer tumor growth

Background

Up to 20% of colorectal cancer (CRC) is diagnosed with distant metastasis. The combination of chemotherapy with anti-vascular endothelial growth factor (VEGF) antibody can improve patient survival. Glucose-regulated protein 78 (GRP78) has an important role in cancer progression, but little is known about its role in VEGF production in CRC. The aim of this study was to explore the mechanism of GRP78 in two human colon cancer cell lines.

Methods

We first checked the expression of GRP78 in human normal and colon cancer tissues and two colon cancer cell lines. Glucose-regulated protein 78 was knocked down using GRP78 small interfering RNA (siRNA) in HT29 and DLD-1 cells. We examined knockdown cells by the cell growth kinetics in vitro and tumor growth rate in vivo, respectively. We also investigated the effect of GRP78 siRNA on the expression of hypoxia inducible factor (HIF-1α), VEGF, and VEGF receptor 2 (VEGFR2).

Results

Compared with their adjacent normal tissue, we detected high expression levels of GRP78 of surgically removed colon cancer tissues. Using GRP78 siRNA, we reduced the expression of GRP78 in HT29 and DLD-1 cells. The GRP78 knockdown cells had a lower proliferation rate with fewer colony-forming units in vitro and produced smaller tumors in vivo. In dissecting the mechanism underlying the reduced cell growth, we found that the down-regulation of GRP78 decreased the production of HIF-1α, VEGF, and VEGFR2 and suppressed angiogenesis.

Conclusions

Silencing GRP78 not only inhibits tumor, but also decreases the expression of VEGF and VEGFR2. Collectively, therapy targeting for GRP78 may inhibit the formation of colon cancer tumors via the HIF-1α/VEGF/VEGFR2 pathway.     

Decreased Expression and Prognostic Role of Mitogen-Activated Protein Kinase Phosphatase 4 in Hepatocellular Carcinoma

Purpose

This study aimed to investigate the potential role and prognostic significance of mitogen-activated protein kinase phosphatase 4 (MKP-4) in the pathology of hepatocellular carcinoma (HCC).

Methods

Western blot analysis and quantitative real-time polymerase chain reaction were performed to detect MKP-4 expression in HCC tissues, pericarcinomatous liver (PCL) tissues, and proliferating HCC cells. The detailed role of MKP-4 was further explored by MKP-4 downregulation in HepG2 cells using small interfering RNA (siRNA). Specimens of 134 HCC patients who had undergone hepatic resection were immunohistochemically evaluated for MKP-4 expression.

Results

MKP-4 protein and mRNA levels were significantly lower in HCC tissues than in PCL tissues. In vitro, its expression was gradually reduced following release of HepG2 cells from serum starvation. The cell counting kit-8 assay and Annexin-V-Fluos staining indicated that MKP-4 knockdown by siRNA in HCC cells enhanced cell survival and inhibited apoptosis. Univariate and multivariate analyses revealed that MKP-4 was a significant predictor for overall survival (OS) and time to recurrence (TTR). High MKP-4 expression was well correlated with prognosis independent of Edmondson grade and microvascular invasion (P?<?0.001).

Conclusions

MKP-4 expression was downregulated in HCC tissues and proliferating HCC cells and correlated with OS and TTR, which suggested that MKP-4 is a candidate prognostic marker for HCC.     

RhoE is Associated with Relapse and Prognosis of Patients with Colorectal Cancer

Background

RhoE, an atypical Rho protein, is differently deregulated in some solid tumors, and there are conflicting data describing the role of RhoE in tumor cell migration and invasion. This study aimed to investigate RhoE expression in human colorectal cancer and its relationship with clinicopathological features and prognosis.

Methods

Colorectal cancer and adjacent normal tissues from 202 patients were examined by immunohistochemistry. Staining evaluation results were analyzed statistically in relation to various clinicopathological parameters, disease-free survival, and overall survival. RhoE expression was also investigated by immunohistochemistry in 80 node metastases and the corresponding primary lesions, and by Western blot test in six cancer and adjacent normal tissues. The relationship between RhoE and invasion was examined by transwell assay and Western blot test.

Results

The positive rate for RhoE in colorectal cancer was significantly higher than that of normal colorectal tissues. In colorectal cancer, RhoE expression was significantly correlated with depth of invasion, lymph node metastasis, and distant metastasis. Consistently, overexpression of RhoE in SW620 cells up-regulated vimentin, down-regulated E-cadherin, increased the expression of matrix metalloproteinase (MMP) 9, and enhanced cell invasion in vitro; in contrast, silencing of RhoE by a specific siRNA caused opposite effects. Most importantly, disease-free and overall survivals were significantly poorer for patients with RhoE-positive tumors than for those with RhoE-negative tumors.

Conclusions

These findings emphasize the positive role of RhoE in invasion and metastasis in human colorectal cancer. It could also serve as an independent prognostic marker in addition to the tumor, node, metastasis staging system.     

MK2206 inhibits hepatocellular carcinoma cellular proliferation via induction of apoptosis and cell cycle arrest

Background

Hepatocellular carcinoma (HCC) is commonly diagnosed at an advanced stage and has limited effective treatment options. The aberrant regulation of the phosphoinositide 3-kinase/Akt pathway in HCC makes it an attractive therapeutic target. The effect of MK2206, a novel, allosteric Akt inhibitor, on HCC cells is not yet fully understood. We hypothesized that inhibition of Akt by MK2206 would impact cellular viability.

Materials and methods

Human Huh7, Hep3B, and HepG2 cell lines were treated with 0–2 μM of MK2206 for 96 h. Cell viability was determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Western blot analysis was used to examine the expression level of various protein markers to assess the mechanism of drug action and proliferation inhibition.

Results

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed a reduction in cellular viability by ≥55% for all cell lines (control versus 2 μM MK2206; P <0.001). Western blot analysis revealed reduction in the level of phosphorylated AKT-Ser473 with no change in AKT-thr308 expression confirming the specificity of MK2206. There was an observed reduction in caspase-9 and survivin. Importantly, there were increases in p21 and p27 along with decreased cyclinD1 expression after treatment.

Conclusions

This study demonstrates the anti-tumor activity of MK2206 in HCC cells. The observed reduction in survivin and pro-caspase 9 suggests that MK2206 induces apoptosis. However, HCC proliferation is also halted via induction of cell cycle arrest as indicated by the increase in p21 and p27 expression and decrease in cyclinD1. Importantly, the concentration needed to achieve growth inhibition in HCC is lower than that needed for other cancer types.     

Salinomycin Selectively Targets ‘CD133+’ Cell Subpopulations and Decreases Malignant Traits in Colorectal Cancer Lines

Background

Cancer stem-like cells (CSCs) in colorectal cancers (CRC) may account for the failure of treatments because they are resistant to many current anticancer therapies. Salinomycin, a potassium ionophore, was recently identified as a selective inhibitor of breast CSCs.

Methods

The human CRC cell lines HT29 and SW480 were treated with salinomycin and oxaliplatin. Cell viability was determined with cell counting kit 8. Fraction of CD133+ cell subpopulations was assessed by Flow Cytometric analysis. Clonogenecity and migration were determined with soft agar and Boyden chamber assays. Molecular changes were assessed by immunofluorescence staining, RT-PCR, and Western blot analysis.

Results

We report that salinomycin reduces the proportion of CD133+ subpopulations in human CRC HT29 and SW480 cells. Furthermore, salinomycin treatment decreases colony-forming ability and cell motility in HT29 cells. Moreover, salinomycin downregulates the expression of vimentin and induces the E-cadherin expression in HT29 cells.

Conclusions

This study demonstrates the ability of salinomycin to selectively target “CD133+” cell subpopulations and decrease the malignant traits in colorectal cancer lines.     

Low Expression of PAI-2 as a Novel Marker of Portal Vein Tumor Thrombosis and Poor Prognosis in Hepatocellular Carcinoma

Background

Plasminogen activator inhibitor (PAI)-2 was previously shown to be less frequently expressed in hepatocellular carcinoma (HCC). The present study was designed to investigate the clinical, pathological, and prognostic significance of PAI-2 expression in HCC.

Methods

Expression of PAI-2 was detected immunohistochemically for specimens from 78 patients with HCC after hepatic resection and correlated with clinicopathological features and patient survival. Risk factors of portal vein tumor thrombosis (PVTT) were also analyzed.

Results

Positive PAI-2 staining was observed in tumor and non-tumor tissues from 21 (26.9 %) and 56 (71.8 %) patients, respectively. Plasminogen activator inhibitor-2 negativity in tumor tissues was significantly associated with PVTT, with a high sensitivity not only in univariate analysis but also in multivariate analysis. In addition, positive PAI-2 staining was related to smaller tumor size and prolonged patient survival. The Cox regression model identified intratumoral PAI-2 staining as an independent prognosticator in patients with HCC after resection.

Conclusions

Our data demonstrated that low expression of PAI-2 serves as a novel marker of PVTT and poor prognosis in HCC.     

SIRT1 Promotes Tumorigenesis and Resistance to Chemotherapy in Hepatocellular Carcinoma and its Expression Predicts Poor Prognosis

Background

SIRT1 is a NAD+-dependent deacetylase that plays crucial roles in many biological processes, including stress response, apoptosis, cellular metabolism, adaptation to calorie restriction, aging, and tumorigenesis. The purpose of this study is to elucidate the clinicopathological and functional significance of SIRT1 expression in hepatocellular carcinoma (HCC).

Methods

SIRT1 expression in HCC was determined by immunohistochemical staining. The results were correlated with clinicopathological parameters. SIRT1 was overexpressed in HCC cell line SK-Hep1 to study its role in tumorigenesis and resistance to chemotherapy.

Results

SIRT1 was overexpressed in 95 of 172 HCCs (55%). SIRT1 overexpression was associated with higher ??-fetoprotein level, higher tumor grade, and absence of ??-catenin mutation. SIRT1 expression predicted poor long-term survival for patients with resected HCC. The elevated SIRT1 protein level in HCC was not attributable to the elevation of mRNA level. The half-life of SIRT1 protein was longer in cell lines with higher expression of SIRT1. We further demonstrated that SIRT1 was degraded by the 26S proteasome in an ubiquitin-dependent manner. Overexpression of SIRT1 promoted tumorigenesis and resistance to chemotherapeutical agent and sorafenib.

Conclusions

SIRT1 is an oncogenic protein for HCC and is a predictor of worse outcome after surgical resection of HCC.     

Strong Smad4 Expression Correlates with Poor Prognosis After Surgery in Patients with Hepatocellular Carcinoma

Background

As a pleiotropic cytokine, transforming growth factor-β (TGF-β) controls the functions of proliferation, adhesion, and differentiation, and contributes to cancer promotion and suppression. Moreover, it is related to the epithelial–mesenchymal transition process and T cell differentiation associated with inflammation. The Smad4 protein is the downstream mediator of TGF-β. In this study, we examined the relationship between Smad4 expression and clinicopathological features in patients with hepatocellular carcinoma (HCC).

Methods

Expression of Smad4 was assessed in five HCC cell lines and in paired cancerous and noncancerous tissues in three patients with HCC, using Western blotting analysis. Moreover, Smad4 expression in 121 HCC patients was evaluated by using immunohistochemistry.

Results

Only the Li7 and HT17 cell lines expressed the Smad4 protein. All human samples expressed the protein. Immunohistochemistry showed that Smad4 expression tended to be strong in small HCC nodules less than 45 mm in diameter (P = 0.06) and in the infiltrated part of the tumor capsule. Postoperative survival analysis indicated that HCC patients with strong Smad4 expression had shorter disease-specific survival than those with weak expression (P = 0.04). Multivariate analysis also showed that Smad4 expression could be one predictor of prognosis, but the correlation was not significant (P = 0.07).

Conclusions

Although TGF-β/Smad4 signaling may have various biological effects on human malignancies, strong Smad4 expression in HCC is likely to suggest poor prognosis. The information has implications for predicting HCC prognosis and developing targeted therapeutics.