Non-transfected Control Group Research Articles

Apollon silencing enhanced the sensitivity to cisplatin-induced cytotoxicity of chemoresistant breast cancer cell line MCF-1/cisplatin

Objective To explore the effect of Apollon on cisplatin-induced chemoresistance of breast cancer cell line MCF-1/cisplatin (DDP). Methods Apollon silencing was performed by transient transfection of small interfering RNA (siRNA). The inhibitory effect of different concentrations of cisplatin (10, 20, 40, 80, and 100 μmol/L) on cell viability was measured by methyl thiazol tetrazolium (MTT) assays. The mRNA and protein expression levels were detected by real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting, respectively. Results As compared with MCF-7 cells, Apollon mRNA and protein levels were 1.70±0.21 folds and 2.23±0.33 folds enhanced in cisplatin-resistant breast cancer cell line MCF-1/DDP, respectively (P=0.007, 0.003). Knockdown of Apollon increased the sensitivity to cisplatin-induced cytotoxicity of MCF-1/DDP. As compared with non-transfected control group, the half maximal inhibitory concentration (IC50) value of cisplatin was significantly decreased from (81.39±3.67) μmol/L to (33.24±2.01) μmol/L (P=0.002). Meanwhile, the mRNA and protein expression levels of Apollon and β-catenin were remarkably declined to 0.43±0.16 (P=0.005), 0.54±0.10 (P=0.006) and 0.23±0.08 (P=0.003), 0.44±0.20 (P=0.005) after Apollon silencing, respectively. In addition, the mRNA and protein expression levels of β-catenin were significantly 1.91±0.23 folds (P=0.0044) and 2.21±0.31 (P=0.0023) folds enhanced with little influence on Apollon expression (P=0.890, 0.730) upon the treatment of WAY-262611 in siRNA-transfected cells. Conclusion Apollon silencing enhanced the sensitivity to cisplatin-induced cytotoxicity of chemoresistant breast cancer cell line MCF-1/DDP, which might be partially dependent on the reduced β-catenin expression. Key words: Breast cancer; Apollon; Cisplatin-resistant

ZEB1 promotes epithelial–mesenchymal transition in cervical cancer metastasis

To investigate role of Zinc finger E-box binding homeobox 1 (ZEB1) in cervical cancer tissue (squamous cell carcinoma, SCC). Exploratory study. University hospital. Sixty patients with SCC, including stage CINIII (n = 10), IB1 (n = 10), IB2 (n = 10), IIA1 (n = 10), IIA2 (n = 10), and IIB (n = 10) were studied. Caski cells were transfected with recombinant shZEB1 lentivirus or shCtrl lentivirus to generate stable ZEB1-knockdown Caski cells. ZEB1 expression was analyzed by quantitative real-time polymerase chain reaction and immunohistochemistry in cervical cancer tissues. ZEB1 expression in Caski cells was down-regulated by short-hairpin RNA (shRNA) interference, and changes in ZEB1 expression corresponded with changes in the proliferation and migratory ability of Caski cells. Quantitative real-time polymerase chain reaction and immunohistochemistry results revealed that ZEB1 expression and the ratio of Vimentin to E-cadherin were high in 27 of 50 SCC patients and correlated with advanced International Federation of Gynecology and Obstetrics stage, tumor size >4 cm, and parametrial invasion. However, the expression of ZEB1 in cervical cancer tissue was independent of age and SCC antigen level. Transfection of ZEB1 shRNA in Caski cells significantly decreased the messenger RNA and protein expression of ZEB1, parallel with increased expression of the epithelial marker E-cadherin and decreased expression of the mesenchymal marker Vimentin. Furthermore, the proliferation and migratory ability of Caski cells were significantly lower in the transfected group than in the nontransfected control group. Down-regulation of ZEB1 expression may protect the invasive front of the tumors from converting to a mesenchymal phenotype by reducing the proliferation and motility of cervical cancer cells, suggesting that ZEB1 might be a potential therapeutic target for SCC.

Kv2 channels regulate firing rate in pyramidal neurons from rat sensorimotor cortex

The largest outward potassium current in the soma of neocortical pyramidal neurons is due to channels containing Kv2.1 α subunits. These channels have been implicated in cellular responses to seizures and ischaemia, mechanisms for intrinsic plasticity and cell death, and responsiveness to anaesthetic agents. Despite their abundance, knowledge of the function of these delayed rectifier channels has been limited by the lack of specific pharmacological agents. To test for functional roles of Kv2 channels in pyramidal cells from somatosensory or motor cortex of rats (layers 2/3 or 5), we transfected cortical neurons with DNA for a Kv2.1 pore mutant (Kv2.1W365C/Y380T: Kv2.1 DN) in an organotypic culture model to manipulate channel expression. Slices were obtained from rats at postnatal days (P7-P14) and maintained in organotypic culture. We used biolistic methods to transfect neurons with gold 'bullets' coated with DNA for the Kv2.1 DN and green fluorescent protein (GFP), GFP alone, or wild type (WT) Kv2.1 plus GFP. Cells that fluoresced green, contained a bullet and responded to positive or negative pressure from the recording pipette were considered to be transfected cells. In each slice, we recorded from a transfected cell and a control non-transfected cell from the same layer and area. Whole-cell voltage-clamp recordings obtained after 3-7 days in culture showed that cells transfected with the Kv2.1 DN had a significant reduction in outward current (∼45% decrease in the total current density measured 200 ms after onset of a voltage step from -78 to -2 mV). Transfection with GFP alone did not affect current amplitude and overexpression of the Kv2.1 WT resulted in greatly increased currents. Current-clamp experiments were used to assess the functional consequences of manipulation of Kv2.1 expression. The results suggest roles for Kv2 channels in controlling membrane potential during the interspike interval (ISI), firing rate, spike frequency adaptation (SFA) and the steady-state gain of firing. Specifically, firing rate and gain were reduced in the Kv2.1 DN cells. The most parsimonious explanation for the effects on firing is that in the absence of Kv2 channels, the membrane remains depolarized during the ISIs, preventing recovery of Na(+) channels from inactivation. Depolarization and the number of inactivated Na(+) channels would build with successive spikes, resulting in slower firing and enhanced spike frequency adaptation in the Kv2.1 DN cells.

TRAF6 regulates proliferation, apoptosis, and invasion of osteosarcoma cell

TRAF6, a unique tumor necrosis factor receptor-associated factor (TRAF) family member, possesses a unique receptor-binding specificity that results in its crucial role as the signaling mediator for TNF receptor superfamily and interleukin-1 receptor/Toll-like receptor superfamily. TRAF6 plays an important role in tumorigenesis, invasion and metastasis. This study aimed to explore the expression of TRAF6 in osteosarcoma tissues and its correlation to the clinical pathology of osteosarcoma and to discuss the relationship between TRAF6 expression and osteosarcoma invasion. These data will provide the experimental base for the biological treatment of osteosarcoma in the future. Using RT-PCR and Western blot, the results showed that the expression rate of TRAF6 mRNA in osteosarcoma tissues was significantly higher than that in normal bone tissue (p < 0.05), that the expression rate of TRAF6 mRNA in the carcinoma tissues from patients with lung metastasis was significantly higher than that from patients without lung metastasis (p < 0.05), and that the expression rate of TRAF6 mRNA also increased with increasing Enneking stage (p < 0.05). However, the mRNA expression of TRAF6 in osteosarcoma was independent of the patient's gender, age, and tumor size (p > 0.05). The TRAF6 protein displayed an up-regulation in osteosarcoma tissues compared to normal bone tissue (p < 0.05), displayed an up-regulation in osteosarcoma tissues from patients with lung metastasis compared to from patients without lung metastasis (p < 0.05), and displayed a gradual increase with increasing Enneking stage (p < 0.05). By the technique of RNA interference, the expression of TRAF6 in the human osteosarcoma MG-63 cell line was down-regulated, and the invasive ability of MG-63 cells was examined. The results showed that TRAF6 protein expression was significantly decreased in the MG-63 cells from TRAF6 siRNA-transfected group (p < 0.05), and the proliferation ability of MG-63 cells and the number of MG-63 cells that passed through the Transwell chamber were significantly lower than that in the non-transfected control group as well as the transfected control group (p < 0.05). In addition, the percentage of MG-63 cells undergoing apoptosis was significantly higher in the TRAF6 siRNA-transfected group compared with the non-transfected control group as well as the transfected control group (p < 0.05). The expression of p-p65, cyclin D1, MMP-9 was down-regulated in the MG-63 cells from TRAF6 siRNA-transfected group. The expression of caspase 3 was up-regulated in the MG-63 cells from TRAF6 siRNA-transfected group compared to the non-transfected control group as well as the transfected control group (p < 0.05). To make a long story short, the overexpression of TRAF6 in osteosarcoma might be related to the tumorigenesis, invasion of osteosarcoma.

Twist expression associated with the epithelial-mesenchymal transition in gastric cancer

This study aimed to investigate the expression of Twist in gastric cancer tissues and its correlation between Twist and the epithelial-mesenchymal transition (EMT). By means of RT-PCR and Western blot, the mRNA and protein expressions of Twist, E-cadherin, and Vimentin in 61 gastric cancer tissues and adjacent normal tissues were detected. The positive rates of Twist, E-cadherin, and Vimentin mRNA expression in gastric cancer tissues were 73.9. 40.6, and 60.9 %, respectively; compared to the expression of these genes in adjacent normal tissues (2.9, 75.4, and 27.5 %), the differences were significant (p < 0.05). The E-cadherin protein expression level in gastric cancer tissues was significantly lower than that in the adjacent normal tissues (p < 0.05). After the transfection of Twist siRNA into the MKN45 cells, the protein expression of Twist was significantly reduced (p < 0.05), the protein expression of E-cadherin was significantly increased, and the number of cells that passed through the Transwell chamber was significantly lower than that in the non-transfected control group as well as the transfected control group (p < 0.05). Twist may be associated with the epithelial-mesenchymal transition in gastric cancer and the tumorigenesis, invasion, and metastasis of gastric cancer.

Overexpression of ZEB1 associated with metastasis and invasion in patients with gastric carcinoma

The aim of this study was to investigate the expression of ZEB1 in gastric carcinoma, its correlation with the clinicopathology of gastric carcinoma, and the role of ZEB1 in invasion and metastasis in gastric carcinoma. ZEB1 expression was analyzed by immunohistochemistry and Western blot in 45 gastric carcinoma tissue samples that contained the adjacent gastric mucosa. The correlation between ZEB1 expression, the occurrence and development of gastric cancer, and clinical pathology was investigated. ZEB1 expression in the human gastric carcinoma cell line AGS was downregulated by RNA interference, and changes in ZEB1 expression corresponded with changes in the invasive and metastatic ability of AGS cells. Immunohistochemistry revealed that ZEB1 protein expression in gastric carcinoma tissues was significantly higher than in normal gastric mucosa tissues (p < 0.001). A lower degree of differentiation of gastric cancer (p = 0.009), a higher TNM (tumor, node, and metastasis) stage (p = 0.010), and a larger scope of invasion were correlated with higher expression of ZEB1 (p = 0.041, 0.002). However, the expression of ZEB1 in gastric carcinoma tissue was independent of gender, age, and tumor size (p > 0.05). Western blot results also showed that ZEB1 protein expression was significantly higher in gastric carcinoma tissue than in the adjacent normal gastric mucosa tissue (p = 0.008). A lower degree of differentiation of the gastric carcinoma correlated with a higher TNM stage, and a larger scope of invasion correlated with increased ZEB1 expression (p = 0.023). Transfection of ZEB1 siRNA in AGS cells significantly decreased the expression level of ZEB1 protein (p = 0.035). Furthermore, the number of cells that could pass through the Transwell chamber was significantly lower in the transfected group than in the non-transfected control group (p = 0.039), indicating that the suppression of ZEB1 expression could significantly reduce the invasive and metastatic ability of AGS cells (p = 0.005). Concluding, in gastric carcinoma tissue, overexpression of ZEB1 may be related to the occurrence and development as well as invasion and metastasis of gastric carcinoma.

Expressions of GRP78 and Bax associate with differentiation, metastasis, and apoptosis in non-small cell lung cancer

The purpose of this study was to detect the expressions of GRP78 and Bax in human non-small cell lung cancer (NSCLC) tissues, to analyze their correlations with carcinogenesis and the development of NSCLC, and to investigate the relationship of GRP78 expression to metastasis and apoptosis in the NSCLC cell line HCC827. The positive expression rates of GRP78 and Bax in NSCLC lung tissues were 59.7% and 34.7% by RT-PCR, respectively. The mRNA and protein expression levels of GRP78 in NSCLC tissues were significantly higher than that in the relatively normal surrounding lung tissues (p < 0.05); the lesser the degree of tumor differentiation was, the higher the mRNA and protein expression levels of GRP78 were (p < 0.05). The mRNA and protein expression levels of GRP78 from patients in advanced pathological stages (III-IV) were significantly higher than the corresponding levels in patients in early pathological stages (I-II) (p < 0.05); the mRNA and protein expression levels of GRP78 in patients with positive lymph node metastasis were significantly higher than those in patients with negative lymph node metastasis (p < 0.05). The mRNA and protein expression levels of Bax in the above cases showed the opposite trend of the mRNA and protein expression levels of GRP78. However, the mRNA and protein expression levels of both GRP78 and Bax were independent of the patient’s sex, the patient’s age, the tumor size and the histological type (adenocarcinoma or squamous cell carcinoma) of NSCLC (p > 0.05). The mRNA expression level of GRP78 and the mRNA expression level of Bax in human NSCLC tissues were negatively correlated (r = -0.353, p = 0.002). After transfection of GRP78 siRNA in HCC827 cells, the GRP78 protein expression level was significantly decreased (p < 0.01), while the Bax protein expression level was significantly increased (p < 0.01); the number of cells that passed through the Transwell chamber was significantly less in the non-transfected control group compared to the transfected control group (p < 0.01). The number of apoptotic cells was significantly greater in the non-transfected control group compared to the transfected control group (p < 0.01). The expression levels of GRP78 and Bax were related to the carcinogenesis, development and metastasis of NSCLC. GRP78 expression with siRNA interference in the human NSCLC cell line HCC827 can reduce metastasis and promote apoptosis in HCC827 cells.

Overexpression of ZEB1 relates to metastasis and invasion in osteosarcoma

This study aimed to investigate the expression of ZEB1 in osteosarcoma tissues and to discuss the relationship between ZEB1 expression and osteosarcoma metastasis. Using RT-PCR and Western blotting, the mRNA and protein expressions of ZEB1 in the osteosarcoma and normal bone tissues were detected. Using the RNA interference technique, the expression of ZEB1 in the human osteosarcoma MG-63 cell line was downregulated, and the changes in the invasion of MG-63 cells were examined. The positive mRNA expression rate of ZEB1 in the osteosarcoma tissues was significantly higher than that in normal bone tissue (P < 0.05). The protein expression level of ZEB1 in the sarcoma tissues from patients with positive lung metastasis was significantly higher than that from patients without lung metastasis (P < 0.05). After the transfection of ZEB1 siRNA into the MG-63 cells, the protein expression of ZEB1 was significantly reduced (P < 0.05), and the number of cells that passed through the Transwell chamber was significantly lower than that in the non-transfected control group as well as the transfected control group (P < 0.05). The overexpression of ZEB1 in osteosarcoma may be related to the carcinogenesis and development as well as metastasis and invasion of osteosarcoma.

Potential of nucleofected human MSCs for insulin secretion

The goal of this experiment was to generate insulin-producing human mesenchymal stem cells (hMSCs) as a therapeutic source for type I diabetes mellitus, which is caused by insulin deficiency due to the destruction of islet β cells. In various trials for the treatment of type I diabetes, cell-based therapy using adult stem cells is considered to be one of the most useful candidates for the treatment. In this experiment, a non-viral method called nucleofection was used to transfect hMSCs with pEGFP-C2 and furin-cleavable human preproinsulin gene (hPPI) to produce insulin-secreting cells as surrogate β cells. Transfection efficiency was determined using flow cytometry analysis. Expression and production of insulin were tested using RT-PCR and ELISA. The expression, production and maturation of insulin from the genetically engineered hMSCs showed an increase when compared with a non-transfected control group. Insulin expression from hMSCs using nucleofection in this study has shown the potential for type I diabetes therapy. For further study, an evaluation for in vivo experiments and clinical applications must be supplemented.

Expression and Characterization of TWIK‐2 in CHO cells

We have recently demonstrated that TWIK-2, a two-pore domain potassium channel, is highly expressed in rat middle cerebral artery (MCA) (AJP 291:, 2006). In this study, we cloned TWIK-2 from rat MCA and expressed it heterologously as a fusion protein to GFP for characterization. Direct fluorescence analysis by deconvolution microscopy in transiently transfected COS cells revealed that GFP-rTWIK-2 localized to the plasma membrane in a punctate pattern by 48 hours after transfection. Stable transfectants of GFP-rTWIK-2 were selected in CHO cells and analyzed by whole-cell electrophysiology. Whole cell currents were 16-fold greater than the nontransfected control group, were non-rectifying in physiological K+, but were inwardly rectifying in symmetrical K+. The observed reversal potentials in different concentrations of extracellular K+ were similar to the theoretical reversal potentials. In physiological K+, membrane potential was -81 ± 2 mv (n=13) for transfected and −64 ± 4 mv (n=5) for control cells. 1 mM BaCl2 inhibited currents by 89 ± 1% with a calculated IC50 of 88 μM. The TWIK-2 currents were minimally affected by 10 mM TEA. Arachidonic acid (10 μM) increased the currents 27 ± 7% (n=4). Since vascular smooth muscle of the rat MCA expresses a functional TWIK-2 channel, TWIK-2 has the potential of being an important regulator of vascular tone. (RO1 NS 46666, NS 038660, and AHA Bugher Award 027011N)