Zoledronic acid reverses cisplatin resistance in nasopharyngeal carcinoma cells by activating the mitochondrial apoptotic pathway.

Despite improvements to radiotherapeutic strategies, resistance to adjuvant chemotherapy remains the main problem underlying the low 5-year survival rate in patients with nasopharyngeal carcinoma (NPC). In the present study, the human NPC cell line HNE1 was exposed to gradually increasing concentrations of cisplatin (CDDP) in order to establish a drug-resistant sub-cell line, HNE1/CDDP. HNE1/CDDP cells exhibited multidrug resistance and a prolonged doubling time, as compared with the parent HNE1 cells. Furthermore, pretreatment with zoledronic acid (ZOL) appeared to resensitize the CDDP-resistant cells by inducing S-phase cell cycle arrest and the mitochondrial apoptotic pathway by upregulating the expression of B-cell lymphoma-2 (BCL-2)-associated X protein and caspase-9 and downregulating the expression of BCL-2. The results of the present study suggested that HNE1/CDDP cells are a stable, multidrug-resistant NPC cell line that may serve as an important tool for research in drug resistance. In addition, the application of ZOL may hold clinical therapeutic potential for the treatment of drug resistance in NPC.

Down-regulated ECRG4 expression in breast cancer and its correlation with tumor progression and poor prognosis--A short Report.

BACKGROUND: Recently, we identified the esophageal carcinoma related gene 4 (ECRG4) as a novel candidate tumor suppressor gene and a promising therapeutic target in nasopharyngeal carcinoma (NPC). In addition, we found that reduced ECRG4 expression in NPC was associated with promoter hypermethylation. The aim of the current study was to assess the expression status of the ECRG4 protein in breast cancer and to clarify its clinicopathological significance and potential prognostic implications. METHODS: Western blotting was used to examine ECRG4 protein levels in 20 paired breast cancer tissues and adjacent noncancerous tissues. In addition, we performed ECRG4 immunohistochemistry on 113 clinicopathologically well-characterized breast cancer samples and assessed putative associations between its expression and overall patient survival rates. RESULTS: We found that ECRG4 protein expression was significantly reduced in the breast cancer tissues compared to the noncancerous tissues. Clinicopathological analyses revealed that loss of ECRG4 protein expression, observed in 41.6 % (47/113) of the primary breast cancer tissues tested, was significantly correlated with lymph node metastasis (P = 0.026), advanced tumor stage (P = 0.042) and unfavorable overall survival (P = 0.004). Additional multivariate analyses revealed that ECRG4 protein expression may serve as an independent prognostic factor for the prediction of patient survival (P = 0.033). CONCLUSION: Our data suggest that loss of ECRG4 protein expression may be involved in tumor progression and may serve as a prognostic biomarker for breast cancer.

Decreased CDK10 expression correlates with lymph node metastasis and predicts poor outcome in breast cancer patients - a short report.

BACKGROUND: Cyclin-dependent kinase 10 (CDK10) has recently been identified as a tumor suppressor and, concordantly, its encoding gene has frequently been found to be inactivated in various human cancers. Here, we examined the expression status of CDK10 in a panel of primary human breast cancers and evaluated its correlation with clinicopathological parameters and clinical outcome. METHODS: Western blotting was used to assess CDK10 protein levels in 20 paired breast cancer tissues and adjacent noncancerous tissues. In addition, immunohistochemistry was performed in 128 formalin-fixed, paraffin-embedded tumor tissues. Associations of CDK10 expression with various clinicopathological parameters were evaluated and Kaplan-Meier survival analyses and Cox proportional hazards models were used to estimate its effect on patient survival. RESULTS: We found that CDK10 protein expression was markedly decreased in cancer tissues compared to adjacent noncancerous tissues. Immunohistochemistry revealed decreased CDK10 levels in 65/128 (50.8 %) of the primary breast cancer tissues tested. These decreased levels were found to be significantly associated with lymph node metastasis (P = 0.003), advanced tumor stage (P < 0.001) and unfavorable overall survival (P < 0.001). Furthermore, multivariate analyses indicated that CDK10 expression may serve as an independent prognostic factor for survival (P = 0.001). CONCLUSION: Down-regulated CDK10 expression frequently occurs in breast cancers and correlates with disease progression and poor survival. CDK10 may serve as a prognostic biomarker for breast cancer.

ECRG4 acts as a tumor suppressor and as a determinant of chemotherapy resistance in human nasopharyngeal carcinoma.

BACKGROUND: Human nasopharyngeal carcinoma (NPC) is a malignant type of cancer with an increasing incidence. As yet, however, molecular biomarkers with a strong diagnostic impact and a major therapeutic promise have remained elusive. Here, we identified the esophageal carcinoma related gene 4 (ECRG4) as a novel candidate tumor suppressor gene and a promising therapeutic target for NPC. METHODS: RT-PCR, Western blotting, methylation-specific PCR and bisulfite sequencing were performed to assess the expression and methylation status of the ECRG4 gene in primary NPC samples, NPC-derived cell lines and patient-derived peripheral blood samples. The NPC-derived cell line CNE1 was selected for treatment with a methylation inhibitor to restore ECRG4 expression. In addition, cell proliferation, invasion and colony formation assays were performed to assess the inhibitory effects of exogenous ECRG4 expression in CNE1 cells. RESULTS: Down-regulated ECRG4 expression was found to occur in 82.5% (33/40) of the primary NPC biopsies tested. This down-regulation was significantly correlated with its tumor-specific promoter methylation status (72.5%, 29/40) and was also observed in the matching peripheral blood samples from the NPC patients (57.5%, 23/40). Pharmacologic demethylation through 5-aza-dC treatment led to gene reactivation in ECRG4 methylated and silenced NPC cell lines. Moreover, exogenous expression of ECRG4 in the CNE1 cell line strongly inhibited its growth and invasive capacities, as well as its enhanced chemosensitivity to cisplatin through autophagy induction. CONCLUSION: Our data suggest that methylation-mediated suppression of the ECRG4 gene occurs frequently in NPC and that restoration of its expression may have therapeutic benefits.

The enhancement of radiosensitivity in human esophageal squamous cell carcinoma cells by zoledronic acid and its potential mechanism.

Esophageal squamous cell carcinoma (ESCC) has a low 5-year patient survival rate. Radiotherapy, as a preoperative or postoperative treatment of surgery, has a crucial role in improving local control and survival of ESCC. Various chemotherapeutic and biologic agents have been used as radio-sensitizers in combination with radiotherapy. Here, we demonstrate that zoledronic acid (ZOL) has a radio-sensitizing effect on ESCC cells. Exposure of ESCC cancer cells to ZOL plus radiation resulted in increased cell death through arresting the cell cycle between S and G2/M phases. ZOL appeared to inhibit proliferation, tube formation and invasion of endothelial cells. These anti-angiogenetic effects were more marked concurrently with irradiation. In addition, synergistic suppressive effects on VEGF expression were observed after combined treatment. Our data suggest that the combination of ZOL and radiation is a promising therapeutic strategy to enhance radiation therapy for ESCC patients.

Mimotope vaccination for epitope-specific induction of anti-VEGF antibodies.

BACKGROUND: Tumor angiogenesis is critical for tumor growth, infiltration and metastasis. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor and targeting it is important in reducing angiogenesis. Bevacizumab (Avastin), a monoclonal antibody that reacts directly against VEGF, has been demonstrated to be an effective treatment for various cancers such as rectal cancer, colon carcinoma, and non-small cell lung cancer, etc. RESULTS: In the current study, we used the phage display technique to generate mimotopes that complemented the screening Avastin antibody (Ab). The candidate mimotopes of VEGF were isolated from a 12-mer peptide library. The phage displaying peptide DHTLYTPYHTHP (designated as 12P) exhibited high affinity to Avastin. The chemically synthesized 12P was conjugated to keyhole limpet hemocyanin (KLH) by glutaraldehyde (GA) to form vaccine KLH-12 peptide (KLH-12P). This epitope vaccine significantly induced humoral immunity in mice. The blood serum from KLH-12P-immunized mice associated with VEGF and blocked its binding to VEGFR, thus inhibiting vascular endothelial cell proliferation and migration. CONCLUSIONS: Our data indicate that the isolated mimotope 12P reported here could potentially elicit specific antibodies against VEGF and result in the induction of anti-angiogenesis responses.

Promoter hypermethylation contributes to the frequent suppression of the CDK10 gene in human nasopharyngeal carcinomas.

BACKGROUND: Previous studies have shown a down-regulation of the gene encoding cyclin-dependent kinase 10 (CDK10) in hepatocellular carcinomas. Here we provide evidence that down-regulation of the CDK10 gene is mediated by promoter hypermethylation in primary human nasopharyngeal carcinomas (NPC) and NPC-derived cell lines. METHODS: RT-PCR, Western blotting, methylation-specific PCR and bisulfite sequencing were performed to assess the expression and methylation status of the CDK10 gene in primary NPC samples, NPC-derived cell lines and patient-derived peripheral blood samples. The NPC-derived cell line CNE-2 was selected for treatment with a methylation inhibitor to restore CDK10 expression. In addition, cell proliferation, invasion and colony formation assays were performed to assess the inhibitory effects of ectopic CDK10 expression in CNE-2 cells. RESULTS: Down-regulation of CDK10 expression in primary NPC samples (23/40, 57.5%) was found to be significantly correlated with the methylation status of its promoter CpG island (21/40, 52.5%). Demethylation by 5-aza-dC treatment led to reactivation of the CDK10 gene in the CNE-2 cell line. Additionally, exogenous expression of CDK10 in CNE-2 cells strongly suppressed its growth, invasion and colony formation capacities. The high sensitivity (15/40, 37.5%) and specificity (0% false positives) of detecting CDK10 promoter hypermethylation in NPC patient-derived peripheral blood samples suggest that it could be employed as an epigenetic marker for noninvasive cancer diagnosis and recurrence screening. CONCLUSION: Our findings implicate that aberrant methylation of the CDK10 gene promoter occurs frequently in NPC, and that reactivation of CDK10 might be utilized as a novel epigenetic strategy for the treatment of NPC patients.

Demethylation of E-cadherin gene in nasopharyngeal carcinoma could serve as a potential therapeutic strategy.

E-cadherin has been proven to be widely down-regulated and tightly associated with tumour invasion and metastasis in multiple human cancer types. Recent research demonstrated that aberrant methylation around gene promoter region attributes to E-cadherin silencing. However, the detailed information about this epigenetic inactivation in nasopharyngeal carcinoma (NPC) is rare. The aim of this study was to probe more into the basic mechanism of E-cadherin methylation in NPC and elucidate the application of demethylating agents to restore E-cadherin expression. To address this question, we initially studied E-cadherin methylation status in NPC primary tumours and cell lines by methylation-specific PCR, and compared it with E-cadherin expression. Methylated E-cadherin was detected in 13 of 20 (65%) NPC clinical specimens and 2 of 2 (100%) NPC cell lines (HNE-1 and CNE-2), which was inversely correlated with E-cadherin expression. The detailed methylation profile at individual CpGs within CpG island of E-cadherin promoter region was confirmed by bisulphite sequencing. E-cadherin gene could be demethylated and reactivated in HNE-1 and CNE-2 cells upon treatment with 5-aza-dC, a DNA demethylating agent. Our findings indicate that frequent aberrant methylation of E-cadherin may play an important role in downregulation of E-cadherin, and demethylation therapy could serve as a promising strategy for NPC patients. Furthermore, a high frequency of E-cadherin methylation (9/20, 45%) in peripheral blood of NPC patients suggests its potential clinical application as an early diagnostic or predictive marker.

[Study on promoter methylation status of E-cadherin gene in nasopharyngeal carcinoma cell lines].

OBJECTIVE: To investigate the role of methylation on E-cadherin inactivation in nasopharyngeal carcinoma (NPC) cell line HNE1 and CNE2, as well as evaluate the inhibitory effect of 5-aza-2'-deoxycytidine (5-Aza-dC) on cell abilities of proliferation and invasion. METHODS: The expression level of E-cadherin was measured by RT-PCR, Western blot and immunohistochemistry (polymer method), the methyaltion status was analyzed by methylation-specific PCR (MSP), and cell proliferation and invasion were examined by MTT and invasion assay, separately before and after treatment with demethylating agent 5-Aza-dC. RESULTS: The expression level of E-cadherin was down-regulated compared with the normal tissue, simultaneously partially methylated in gene promoter. Treatment with 20 µmol/L 5-Aza-dC increased the expression of E-cadherin and reduced the methylation degree. Moreover, it also significantly suppressed cell growth (27.6% for HNE1 cells and 34.3% for CNE2 cells, P < 0.05) and invasiveness (37.2% for HNE1 cells and 29.7% for CNE2 cells, P < 0.05). CONCLUSIONS: Aberrant methylation around gene promoter region may play an important part in down regulation of E-cadherin in NPC, suggesting a potential therapeutic strategy for demethylating agents such as 5-Aza-dC.

Anti-cancer effects of celecoxib on nasopharyngeal carcinoma HNE-1 cells expressing COX-2 oncoprotein.

Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor with antitumor and antiangiogenic activities. To investigate the effects of celecoxib on nasopharyngeal carcinoma (NPC), HNE-1 cells were treated with celecoxib at various concentrations. MTT assay, migration assay and invasion assay were performed to observe the inhibitory activity of celecoxib on HNE-1 cells. Additionally, VEGF-A expression and radiation survival of NPC cell were also examined after treatment with celecoxib. Celecoxib treatment presented an anti-proliferation function in a time and dose-dependent manner on HNE-1 cells which highly express COX-2 protein. Celecoxib also displayed an obvious inhibitory activity on invasive capacity of NPC cells. Moreover, the celecoxib's effects to suppress VEGF-A expression and enhance radiosensitivity were detected in HNE-1 cells. These findings implicate that application of celecoxib may be an effective strategy for NPC therapy.

The preventive effect of adjuvant-free administration of TNF-PADRE autovaccine on collagen-II-induced rheumatoid arthritis in mice.

Adjuvants are necessary to elicit high titers of antibodies in vaccine-immunization procedures. We previously developed a mouse tumor necrosis factor-alpha (TNF-alpha) autovaccine (mTNF-PADRE) capable of inducing anti-TNF-alpha antibodies. In this study, we investigated the therapeutic effect of adjuvant-free administration of the autovaccine on collagen-type-II-induced rheumatoid arthritis (CIA) in mice. Our results showed that the vaccine could ameliorate the symptoms of CIA in mice. In addition, this study suggests that it is possible to control the antibody levels in mice immunized with mTNF-PADRE without adjuvant.

Inhibition effect of pcDNA-tum-5 on the growth of S180 tumor.

Tumor growth and metastasis depend on vessel formation, and inhibition of angiogenesis of tumor by production of anti-angiogenic drugs should be a promising approach for cancer therapy. Tumstatin is an angiogenesis inhibitor. The anti-angiogenic activity of tumstatin is localized to the 54-132 amino acids. The gene fragment encoding amino acids 45-132 of tumstatin (tum-5) was subcloned into pcDNA3.1 (pcDNA-tum5). Tum-5 protein could be expressed and secreted in CHO cells after transfection. The conditioned medium (containing tum-5 protein) from the transfectant has an anti-angiogenic effect on HUVEC cells in vitro. The anti-tumor effect of pcDNA-tum5 on mice bearing S180 tumors was evaluated. The results showed that pcDNA-tum-5 has a significant inhibition activity in the growth of the tumors. This study suggests that the gene delivery of tum-5 may be an effective strategy for cancer therapy.

Amelioration of experimental autoimmune encephalomyelitis by BLyS autovaccine.

The B-lymphocyte stimulator (BLyS) is implicated in various pathophysiological processes. The overexpression of BLyS has been observed in some human diseases, including systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome, and multiple sclerosis. This feature suggests that BLyS may be a therapeutic target for some human autoimmune diseases. We developed a therapeutic vaccine by coupling a tetanus toxoid T-helper cell epitope with the C-terminal of BLyS (TT-BLyS). This vaccine can induce high titers of neutralizing antibodies against BLyS in an animal model; the antibody has markedly protective effects on experimental autoimmune encephalomyelitis in rats, which is induced by inoculation of spinal cord homogenate. Our data suggest that the BLyS autovaccine may be a useful candidate for the treatment of some autoimmune diseases associated with the production of BLyS.

Prepared and screened a modified TNF-alpha molecule as TNF-alpha autovaccine to treat LPS induced endotoxic shock and TNF-alpha induced cachexia in mouse.

Overexpression of TNF-alpha in the body is critically involved in many diseases. A strategy to construct TNF-alpha autovaccine by introducing a T cell helper epitope to the protein has been developed and may be an alternative because it is cheaper and highly efficient. However, the induction of high level anti-TNF-alpha neutralizing autoantibodies by TNF-alpha autovaccine is depend on a proper T cell help epitope. In order to evaluate the effect of different T helper cell epitopes on the immunogenicity of mouse TNF-alpha (mTNF-alpha), three T helper cell epitopes, TT (QYIKANSKFIGITEL), HEL (NTDGSTDYGILQINSR), and PADRE (AKFVAAWTLKA), were chosen for this study. The sequence (amino acids 126-140) of mTNF-alpha was replaced with those of the T cell help epitopes, respectively. The three fusion proteins (mTNF-TT, mTNF-HEL, mTNF-PADRE) were expressed in Escherichia coli and purified with a simple strategy. The abilities of the proteins elicited TNF-alpha autoantibodies in BALB/c mice were investigated. The results showed that mTNF-PADRE is the most effective among the three modified TNF-alpha molecules. In the absence of adjuvant, the therapeutic effect of TNF-PADRE on LPS induced endotoxic shock mice and mTNF-alpha induced cachexia mice was observed. This study suggests that mTNF-PADRE may be a better candidate of mTNF-alpha autovaccine.

Identification of a novel peptide ligand of human vascular endothelia growth factor receptor 3 for targeted tumour diagnosis and therapy.

Human vascular endothelia growth factor receptor 3 (VEGFR-3) is up-regulated in a variety of human cancers. It is a potentially rational target for drug delivery. To identify novel ligands with specific binding capabilities to VEGFR-3, we screened a phage display peptide library and found a consensus motif of the peptides which is displayed by the positive phages CSDxxHxWC (x is any amino acid). The phage displaying peptide CSDSWHYWC (designated as P1) exhibited the highest affinity to VEGFR-3 in phage ELISA and the chemically synthesized P1 could bind to VEGFR-3 specifically in a dose-dependent manner. In addition, the flow cytometry assay and immunofluorescence showed that the FITC labelled P1 could bind to VEGFR-3 positive carcinoma cells with specificity. Our study suggests that P1 may be a homing peptide for treatment of tumours.