Anti-Bcl-2 Antikörper [Bcl-2/100] (A86278)

Background: Recently double-hit lymphoma or double protein expressor lymphoma has been identified as a distinct group of diffuse large B cell lymphoma with poor prognosis. However, the expression status, clinical and prognostic effect of combined overexpression of MYC and BCL2 in extranodal NK/T-cell lymphoma, nasal type (ENKTL) are not known. Materials and methods: Paraffin-embedded lymphoma samples from 53 patients with newly diagnosed ENKTL were studied using immunohistochemistry for MYC and BCL2, and fluorescent in situ hybridization (FISH) for MYC and BCL2 were done on 5 tissue sections with highest percentages of both MYC and BCL2 positive lymphoma cells. Results: The median percentage of MYC-positive lymphoma cells and BCL2-positive lymphoma cells were 20% (range, 5%-45%) and 70% (10%-95%), respectively. Using median scores as cutoffs, we assigned each patient an IHC double-hit score (DHS) that ranged from 0 to 2. Using this DHS, 15 patients (28.3%) had a DHS of 0, 24 patients (45.3%) had a DHS of 1, and the remaining 14 patients (26.4%) had a DHS of 2. FISH analysis was performed on 5 tissue sections with DHS of 2, and none of them had MYC or BCL2 rearrangement. The DHS was not associated with patients' age, gender, disease stage, LDH level, B symptoms, performance status, or local tumor invasiveness. However, patients with tumor localized in extranasal sites seemed to have higher expression of BCL2 and higher DHS than nasal lesions (p=0.014 and 0.042, respectively). In univariate survival analysis, either high expression of MYC or BCL2 was significantly correlated with inferior PFS and OS (p<0.05). According to the DHS, patients with ENKTL could be divided into three significantly different risk groups for PFS and OS (3-year PFS rate for DHS of 0, 1, and 2 was 60%, 41%, and 21%, respectively, p=0.008; 3-year OS rate for DHS of 0, 1, and 2 was 79%, 49%, and 33%, respectively, p=0.015). In multivariate survival analysis, it was found that DHS was an independent prognostic factor for both PFS and OS (p=0.006 and 0.011, respectively). Conclusions: Our study demonstrated that DHS can help identify patients with newly diagnosed ENKTL who are at a high risk for a poor clinical outcome, which needs to be validated in prospective clinical trials with patients treated uniformly.

Cisplatin (DDP) has been one of the most widely used chemotherapy drugs for advanced non-small cell lung cancer. However, the increase in the number of DDP-resistant cancer cells has become a major impediment in the clinical management of cancer. In the present study, for the first time, the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay was used to demonstrate that nedaplatin (NDP) could have a stronger inhibitory effect than DDP alone in DDP-resistant A549 (A549DDP) cells and that it could attenuate the resistance of these cells. Additionally, flow cytometry analysis showed that the apoptosis rate of these resistant cells when exposed to NDP was markedly increased and the number of cells in the G2 stage of the cell cycle was significantly increased. Furthermore, western blot analysis indicated that NDP decreased the protein expression of P-glycoprotein, tumor protein p53 and B-cell lymphoma 2, and increased the expression of Bcl-2-associated X protein, all of which could possibly improve the NDP intracellular drug concentration and promote cell apoptosis. These observations suggested that NDP could have higher efficacy in DDP-resistant lung cancer cells, and further studies applying more detailed analyses are warranted to elucidate the mechanism(s) behind this effect.

The inhibition of apoptosis in cancer cells is the major pathological feature of hepatic carcinoma. Rosiglitazone (RGZ), a ligand for peroxisome proliferator-activated receptor ? (PPAR-?), has been shown to induce apoptosis in hepatic carcinoma cells. However, the mechanism underlying this effect remains to be elucidated. The present study aimed to investigate the effect of RGZ on cell viability and apoptosis, and its mechanisms in cultured HepG2 cells using MTT assay, flow cytometry and western blotting. The results revealed that treatment with RGZ may attenuate HepG2 cell viability and induce the apoptosis of the cells. The mechanism of RGZ-induced apoptosis involves an increase in the level of activated PPAR-? (p-PPAR-?) and a decrease in p85 and Akt expression. In addition, the PPAR-? antagonist GW9662 suppressed the effect of RGZ in the HepG2 cells. Taken together, the results suggest that RGZ induces the apoptosis of HepG2 cells through the activation of PPAR-?, suppressing the activation of the PI3K/Akt signaling pathway. Such mechanisms may contribute to the favorable effects of treatment using RGZ in HepG2 cells.

Hypoxia is associated with various pathophysiological events, including cancer, lung and cardiovascular diseases. A number of studies have indicated that alterations in microRNA (miRNA) expression may be involved in the regulation of the cellular response to hypoxia. In the present study, miR-18a expression was revealed to be markedly downregulated under hypoxic conditions in MGC-803 and HGC-27 gastric carcinoma cell lines. Furthermore, miR-18a was demonstrated to affect the rate of cell apoptosis and the cell invasion ability in MGC-803 and HGC-27 cells under hypoxic conditions. Cell apoptosis was were analyzed using flow cytometry and cell invasiveness was evaluated using a Transwell-matrigel assay. The results showed that miR-18a overexpression was able to promote cell apoptosis and inhibit cell invasion. Using bioinformatic analysis, hypoxia-inducible factor (HIF)-1a was identified as one of the target genes of miR-18a, and based on the function of HIF-1a in hypoxia, miR-18a was predicted to regulate HIF-1a expression. This hypothesis was confirmed by a further luciferase assay and the detection of the mRNA and protein expression levels of HIF-1a following the induction of miR-18a overexpression. In addition, the expression levels of mitochondrial apoptosis-associated genes were detected following the induction of miR-18a overexpression. In the cells overexpressing miR-18a, the Bcl-2 protein expression level was downregulated, while the protein expression levels of Bax, caspase 3 and caspase 9 were upregulated in the MGC-803 and HGC-27 cell lines. Therefore, miR-18a was hypothesized to induce apoptosis through the HIF-1a/mitochondrial apoptosis pathway.

Kv1.5 (also known as KCNA5) is a protein encoded by the KCNA5 gene, which belongs to the voltage-gated potassium channel, shaker-related subfamily. Recently, a number of studies have suggested that Kv1.5 is overexpressed in numerous cancers and plays crucial roles in cancer development. However, until now, the expression and functions of Kv1.5 in osteosarcoma are still unclear. To characterize the potential biological functions of Kv1.5 in osteosarcoma, herein, we examined the expression levels of Kv1.5 in osteosarcoma cells and tissues using quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and immunohistochemistry assays. Four short hairpin RNAs (shRNAs) targeting Kv1.5 were designed and homologous recombination technology was used to construct pGeneSil-Kv1.5 vectors. In addition, the vectors were transfected into osteosarcoma MG63 cells and Kv1.5 mRNA level was measured by qRT-PCR and the Kv1.5 protein level was examined by western blot. We also examined the effects of Kv1.5 silencing on proliferation, cell cycle and apoptosis of the osteosarcoma cells using CCK-8, colony formation, flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. Our results showed that Kv1.5 was aberrantly expressed in osteosarcoma and that the synthesized shRNA targeting Kv1.5 reduced Kv1.5 mRNA and protein expression effectively. Silencing Kv1.5 expression in the osteosarcoma cells significantly inhibited the proliferation of osteosarcoma cells, induced cell cycle arrest at G0/G1 phase, and induced cell apoptosis through up-regulation of p21, p27, Bax, Bcl-XL and caspase-3 and down-regulation of cyclins A, cyclins D1, cyclins E, Bcl-2 and Bik. In summary, our results indicate that Kv1.5 silencing could suppress osteosarcoma progression through multiple signaling pathways and suggest that Kv1.5 may be a novel target for osteosarcoma therapeutics.

Breast cancer is the most common cause of female cancer-associated mortality. Although treatment options, including chemotherapy, radiotherapy and surgery have led to a decline in the mortality rates associated with breast cancer, drug resistance remains one of the predominant causes for poor prognosis and high recurrence rates. The present study investigated the potential effects of the natural product, isorhamnetin on breast cancer, and examined the effects of isorhamnetin on the Akt/mammalian target of rapamycin (mTOR) and the mitogen-activated protein kinase (MAPK)/MAPK kinase (MEK) signaling cascades, which are two important signaling pathways for endocrine therapy resistance in breast cancer. The results of the present study indicate that isorhamnetin inhibits cell proliferation and induces cell apoptosis. In addition, isorhamnetin was observed to inhibit the Akt/mTOR and the MEK/extracellular signal-regulated kinase phosphorylation cascades. The inhibition of these two signaling pathways was attenuated by the two Akt and MEK1 inhibitors, but not by the nuclear factor-?B inhibitor. Furthermore, epidermal growth factor inhibited the effects of isorhamnetin via activation of the Akt and MEK signaling pathways. These results indicate that isorhamnetin exhibits antitumor effects in breast cancer, which are mediated by the Akt and MEK signaling pathways.

Prunella vulgaris L. belongs to the Prunella genus and has been proven effective in the treatment of gastric cancer, however, the therapeutic activity of the endophytic fungi is not yet well understood. The results of the present study suggest that the ethyl acetate extract (S03-EA) of the endophytic fungus XKC-S03, isolated from Prunella vulgaris L. spica, is a potent anticancer agent with the potential to treat gastric cancer. In the present study, the effects of S03-EA on gastric cancer in vitro and in vivo were determined using the 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan assay and the human gastric cancer SGC 7901 cell xenograft model. The tumor tissue was fixed with 10% formaldehyde solution and the levels of the apoptotic proteins, B-cell lymphoma protein-2 (Bcl-2), Bcl-2-associated X protein (Bax) and pro-angiogenic vascular endothelial growth factor (VEGF), were measured by immunohistochemistry. The results indicated that treating SGC 7901 cells with petroleum ether (S03-PE), ethyl acetate (S03-EA) or dichloromethane (S03-DM) extracts from the XKC-S03 fermentation broth inhibited cell proliferation. S03-EA demonstrated the best activity, with a half maximal inhibitory concentration of 25.89 µg/ml and dose-dependent suppression of the SGC 7901 tumor cells in vivo, without any evident adverse effects. In addition, the 100-mg/kg/day S03-EA-treated tumor tissue revealed a downregulation of Bcl-2 and VEGF expression and an upregulation of Bax expression. In conclusion, the S03-EA extract of XKC-S03, isolated from Prunella vulgaris L. spica, exhibits a growth-suppressive activity on gastric cancer in vitro and in vivo.

OBJECTIVE:

Combination therapy for cancer is more effective than using only standard chemo- or radiotherapy. Our previous results showed that dendritic cell-activated a-fetoprotein (AFP)-specific T-cells inhibit tumor in vitro and in vivo. In this study, we focused on antitumor function of CD8(+) T-cells combined with or without JAK2 inhibitor.

METHODS:

Proliferation and cell cycle were analyzed by CCK-8 and flow cytometry. Western blot was used to analyze the expression level of related protein and signaling pathway.

RESULTS:

We demonstrated reduced viability and induction of apoptosis of tumor cells with combination treatment. Intriguingly, cell cycle was blocked at the G1 phase by using AFP-specific CD8(+) T-cells combined with JAK2 inhibitor (AG490). Furthermore, an enhanced expression of BAX but no influence on Fas/FasL was detected from the tumor cells.

CONCLUSION:

These results indicate a Fas/FasL-independent pathway for cellular apoptosis in cancer therapies with the treatment of AFP-specific CD8(+) T-cells combined with JAK2 inhibitor.

BACKGROUND:

Long noncoding RNAs (lncRNAs) have recently emerged as important regulators in governing fundamental biological processes, and many of which are likely to have functional roles in tumorigenesis. Maternally expressed gene 3 (MEG3) gene encodes a lncRNA whose expression is lost in an expanding list of primary human tumors and tumor cell lines, however its biological role and regulatory mechanism in gastric cancer (GC) development and progression are poorly defined.

METHODS:

Quantitative RT-PCR analysis was used to determine whether aberrant MEG3 expression was associated with GC patients pTNM stage and pM state. Furthermore, the effect of ectopic expression of MEG3 on cell proliferation, migration, invasion and cell apoptosis was assessed by using CCK-8, wound healing, transwell invasion assays and flow cytometric analysis, respectively, in GC cell lines HGC-27 and MGC-803. Moreover, the competing endogenous RNA (ceRNA) activity of MEG3 on miR-181a was investigated via luciferase reporter assay and immunoblot analysis.

RESULTS:

MEG3 is decreased in GC patients and cell lines, and its expression was associated with metastatic GC. Furthermore, ectopic expression of MEG3 in HGC-27 and MGC-803 cells inhibited cell proliferation, migration, invasion, and promoted cell apoptosis, which might be due to MEG3 sequestering oncogenic miR-181 s in GC cells. Furthermore, MEG3 could up-regulated Bcl-2 via its competing endogenous RNA (ceRNA) activity on miR-181a.

CONCLUSIONS:

These findings suggest that lncRNA MEG3, a ceRNA of miR-181 s, could regulate gastric carcinogenesis and may serve as a potential target for antineoplastic therapies.

The clinical prognosis of malignant gliomas is poor and PCDH9 down-regulation is strongly associated with its poor prognosis. But the mechanism of PCDH9 down-regulation is unknown. Abnormal miRNAs profiles regulate tumor phenotypes through inhibiting their target genes and miRNAs could inhibit target genes more efficiently by binding to both the promoter and 3'UTR of target genes. In this study, to search the dual inhibitory miRNAs which suppress PCDH9 expression in gliomas, we performed an integrative analysis of databases including miRDB, TargetScan, microPIR and miRCancer. We identified three candidate miRNAs which were predicted to bind both the promoter and 3'UTR of PCDH9 and up-regulated in gliomas. Then, we validated miR-215-5p up-regulation and PCDH9 down-regulation in glioma samples and demonstrated that miR-215-5p could inhibit the mRNA and protein levels of PCDH9 in glioma cell lines by targeting its promoter and 3' UTR at the same time. Moreover, miR-215-5p could increase glioma cell proliferation, clone formation, in-vitro migration and reduce apoptosis via inhibiting PCDH9 expression. Our study provides evidence for a novel dual inhibition of PCDH9 by miR-215-5p in gliomas and suggests that miR-215-5p might be a therapeutic target for the treatment of gliomas.

Recently, the human ether à go-go (eag) related gene 1 (hERG1) channel, a member of the voltage-dependent potassium channel (Kv) family, was determined to have a critical role in cancer cell proliferation, invasion, tumorigenesis and apoptosis. However, the expression levels and functions of hERG1 in osteosarcoma cells remain poorly characterized. In this study, hERG1 transcript and protein levels in osteosarcoma cells and tissues were measured using semi-quantitative real time PCR (RT-PCR), Western blot, and immunohistochemistry. The effects of hERG1 knockdown on osteosarcoma cell proliferation, apoptosis and invasion were examined using CCK-8, colony formation, flow cytometry, caspase-3 activity, wound healing and transwell based assays. Furthermore, semi-quantitative RT-PCR, Western blot and a luciferase reporter assay were used to assess the effects of hERG1 inhibition on the nuclear factor-?B (NF-?B) pathway. In addition, the effect of NF-?B p65-siRNA and NF-?B p65 expression on the survival of osteosarcoma cells was investigated. Through this work, a relationship for hERG1 with the NF-?B pathway was identified. Osteosarcoma cells and tissues were found to express high levels of hERG1. Knockdown of hERG1 significantly suppressed cellular proliferation and invasion, and induced apoptosis, while inhibition of hERG1 significantly decreased activation of NF-?B. Overall, hERG1 may stimulate nuclear translocation of p65, thus regulating the NF-?B pathway through the activation of the hERG1/beta1 integrin complex and PI3K/AKT signaling. Taken together, these results demonstrate that hERG1 is necessary for regulation of osteosarcoma cellular proliferation, apoptosis and migration. Furthermore, this regulation by hERG1 is, at least in part, through mediation of the NF-?B pathway.

Cigarette smoking has been shown to be the most significant risk factor for lung cancer. Recent studies have also indicated that RNA-binding motif protein 5 (RBM5) can modulate apoptosis and suppress tumor growth. The present study focused on the role of RBM5 in the regulation of cigarette smoke extract (CSE)-induced transformation of bronchial epithelial cells into the cancerous phenotype and its mechanism of action. Herein, we exposed normal BEAS-2B cells for 8 days to varying concentrations of CSE or dimethylsulfoxide (DMSO), followed by a recovery period of 2 weeks. Next, the RBM5 protein was overexpressed in these transformed BEAS-2B cells though lentiviral infection. Later, the morphological changes, cell proliferation, cell cycle, apoptosis, invasion and migration were assessed. In addition, we analyzed the role of RBM5 in xenograft growth. The expression of RBM5 along with the genes related to cell cycle regulation, apoptosis and invasion were also examined. Finally, our results revealed that BEAS-2B cells exposed to 100 µg/ml CSE acquired phenotypic changes and formed tumors in nude mice, indicative of their cancerous transformation and had reduced RBM5 expression. Subsequent overexpression of RBM5 in these cells significantly inhibited their proliferation, induced G1/S arrest, triggered apoptosis and inhibited their invasion and migration, including xenograft growth. Thus, we established an in vitro model of CSE-induced cancerous transformation and concluded that RBM5 overexpression inhibited the growth of these transformed cells through cell cycle arrest and induction of apoptosis. Therefore, our study suggests the importance of RBM5 in the pathogenesis of smoking-related cancer.

Ether á go-go 1 (Eag1) is frequently highly expressed in various malignant cancers and its excessive expression is correlated with poor prognosis in various cancers. However, the relationship of Eag1 expression with the clinical outcome of patients having ovarian cancer treated with cisplatin-based adjuvant chemotherapy is still unknown. In this study, we measured the expression of Eag1 in ovarian cancer and investigated the association between cisplatin chemosensitivity of ovarian cancer cells and Eag1 expression level. We demonstrate that decreased expression of Eag1 correlates with favorable prognosis in patients treated with cisplatin-based adjuvant chemotherapy and predicts higher cisplatin sensitivity in ovarian cancer cells. In vitro, knockdown of Eag1 by small interfering RNA facilitated the sensitivity of ovarian cancer cells (SKOV3 and TYK) to cisplatin-induced apoptosis via nuclear factor ?-light chain-enhancer of activated B cells (NF-?B) pathway. Furthermore, knockdown of Eag1 expression was associated with decreased expression of the P-glycoprotein without affecting multidrug resistance-associated protein 1 expression. Taken together, Eag1 may serve as a potential indicator to predict Eag1 chemosensitivity, and silencing Eag1 may represent a potential therapeutic strategy for ovarian cancer.

© The Author(s) 2015.

The aim of the present study was to determine the expression and function of B cell translocation gene 1 (BTG1) in kidney carcinoma. Kidney samples were obtained from cancer lesions (n=85) and the adjacent normal tissue (n=40) in kidney cancer patients immediately following endoscopic biopsy. The effect of BTG1 overexpression was examined in vitro utilizing a human kidney cancer cell line, ACHN, stably transfected with a recombinant lentivirus (LeBTG1 cells) and compared to empty vector-transfected controls (LeEmpty). BTG1 protein expression was significantly lower in kidney cancer tissue biopsies compared to normal tissue, as measured by immunohistochemistry (34.1 vs. 77.8% of tissues; P<0.05) and western blotting (0.481±0.051 vs. 0.857±0.081; P<0.05). In vitro analyses revealed that LeBTG1 cells had a reduced survival fraction compared to control LeEmpty cells, with higher rates of apoptosis (16.6±2.5 vs. 6.1±0.7%; P<0.05). The proportion of LeBTG1 cells in G(0)/G(1) stage and S phase was also significantly different from LeEmpty cells (66.8±5.3 and 22.2±1.5% vs. 44.4±3.1 and 34.5±2.3%, respectively; P<0.05), and the migration and invasion of LeBTG1 cells was significantly impaired with respect to LeEmpty cells (74.0±9.0 and 53.0±7.0 vs. 118.0±15.0 and 103.0±13.0, respectively; P<0.05). These effects were accompanied by decreased protein expression of cyclin D1, B-cell lymphoma 2 and matrix metalloproteinase 9 in LeBTG1 cells (0.118±0.018, 0.169±0.015 and 0.207±0.027, respectively) compared to control LeEmpty cells (0.632±0.061, 0.651±0.063 and 0.443±0.042, respectively; P<0.05). Reduced BTG1 expression is associated with increased disease severity, suggesting it is a negative regulator of kidney cancer and can serve as a prognostic indicator. The results of the present study show that BTG1 protein levels were significantly reduced in kidney cancer biopsy specimens and were associated with disease progression and prognosis.