Cepharanthine, a regulator of keap1-Nrf2, inhibits gastric cancer growth through oxidative stress and energy metabolism pathway.

Cepharanthine (CEP), a bioactive compound derived from Stephania Cephalantha Hayata, is cytotoxic to various malignancies. However, the underlying mechanism of gastric cancer is unknown. CEP inhibited the cellular activity of gastric cancer AGS, HGC27 and MFC cell lines in this study. CEP-induced apoptosis reduced Bcl-2 expression and increased cleaved caspase 3, cleaved caspase 9, Bax, and Bad expression. CEP caused a G2 cell cycle arrest and reduced cyclin D1 and cyclin-dependent kinases 2 (CDK2) expression. Meanwhile, it increased oxidative stress, decreased mitochondrial membrane potential, and enhanced reactive oxygen species (ROS) accumulation in gastric cancer cell lines. Mechanistically, CEP inhibited Kelch-like ECH-associated protein (Keap1) expression while activating NF-E2 related factor 2 (Nrf2) nuclear translocations, increasing transcription of Nrf2 target genes quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HMOX1), and glutamate-cysteine ligase modifier subunit (GCLM). Furthermore, a combined analysis of targeted energy metabolism and RNA sequencing revealed that CEP could alter the levels of metabolic substances such as D (+) - Glucose, D-Fructose 6-phosphate, citric acid, succinic acid, and pyruvic acid, thereby altering energy metabolism in AGS cells. In addition, CEP significantly inhibited tumor growth in MFC BALB/c nude mice in vivo, consistent with the in vitro findings. Overall, CEP can induce oxidative stress by regulating Nrf2/Keap1 and alter energy metabolism, resulting in anti-gastric cancer effects. Our findings suggest a potential application of CEP in gastric cancer treatment.

Acidic stress induces protective autophagy in SGC7901 cells.

Objective To investigate the effect of acidity on gastric cancer SGC7901 cells in terms of autophagy and provide a new strategy for therapeutically targeting gastric cancer autophagy in an acidic environment. Methods Transmission electron microscopy (TEM) and confocal laser scanning microscopy were used to examine the effect of an acidic environment on autophagosome formation. Light chain 3 (LC3) and p62 levels in SGC7901 cells exposed to acidic conditions were measured using Western blot analysis. To explore changes in autophagy flux, the cells were treated with an inhibitor of autophagy bafilomycin A1. The CCK-8 assay was performed to determine if inhibiting acid-induced autophagy affected cell proliferation. Results Increased autophagosome formation was observed by TEM. Punctate LC3 structures were observed in cells cultured under acidic conditions, whereas untreated cells exhibited diffuse and weak staining for punctate LC3 structures. Cytoplasmic LC3-I translocated to the autophagic membrane (LC3-II) levels increased under acidic conditions, whereas p62 levels decreased. The bafilomycin A1-induced inhibition of autophagy caused by the acidic environment inhibited cell proliferation. Conclusion The acidic environment upregulates autophagy in SGC7901 cells. In long-term culture, a stable and high level of autophagy is maintained in an acidic environment, which has a protective effect on cells.

Association of Mixed Lineage Kinase Domain-Like Protein Expression With Prognosis in Patients With Colon Cancer.

BACKGROUND: The mixed lineage kinase domain-like protein has recently been identified as a key downstream component of tumor necrosis factor-induced necroptosis, which is an important pathway of cancer cell death. The goal of the current study is to explore the expression of mixed lineage kinase domain-like protein in colon cancer tissues and evaluate the prognostic value in patients with colon cancer. METHODS: We collected normal and cancer colon tissues from 135 patients diagnosed with colon cancer after radical operation during July 2007 to April 2009 at The Affiliated Hospital of Qingdao University. Immunohistochemistry analysis was scored using an established scoring system. Kaplan-Meier survival curves were generated for recurrence-free survival and overall survival for all patients and 2 subsets of patients. The relationship between mixed lineage kinase domain-like protein expression and prognosis parameter (recurrence-free survival, overall survival) was analyzed by univariate and multivariate Cox regression analyses. RESULTS: The median age of all patients was 67 years and 56.3% were male. Low expression of mixed lineage kinase domain-like protein was associated with decreased overall survival (78.6 vs 81.2 months; P = .011) in all patients. In the subset of 79 patients who received adjuvant chemotherapy, low expression of mixed lineage kinase domain-like protein was associated with decreased recurrence-free survival (60.4 vs 72.8 months; P = .032) and decreased overall survival (66.3 vs 72.9 months; P = .005). Low expression of mixed lineage kinase domain-like protein was associated with decreased overall survival (74.9 vs 79.8 months; P = .006) and recurrence-free survival (69.6 vs 78.8 months; P = .005) among patients with Tumor Node Metastasis (TNM) stage II colon cancer. CONCLUSIONS: Low expression of mixed lineage kinase domain-like protein was associated with decreased overall survival in all patient-group with resected colon cancer. It is associated with decreased recurrence-free survival and overall survival in the subset of patients who receive adjuvant chemotherapy and patients who were TNM stage II. Mixed lineage kinase domain-like protein may provide important prognostic information in patients with colon cancer.

Tspan9 inhibits the proliferation, migration and invasion of human gastric cancer SGC7901 cells via the ERK1/2 pathway.

Tetraspanins are a heterogeneous group of 4-transmembrane proteins that recruit other cell surface receptors and signaling proteins into tetraspanin-enriched microdomains (TEMs). TEMs of various types are involved in the regulation of cell growth, migration and invasion of several tumor cell types, both as suppressors or promotors. Tetraspanin 9 (Tspan9, NET-5, PP1057), a member of the transmembrane 4 superfamily (TM4SF) of tetraspanins, reportedly regulates platelet function in concert with other platelet tetraspanins and their associated proteins. Our previous study demonstrated that Tspan9 is also expressed in gastric cancer (GC), but the role of Tspan9 in GC has not been well characterized. In this study, we investigated the influence of Tspan9 on proliferation, migration and invasion of human gastric cancer SGC7901 cells using CCK-8 assay, cell cycle analysis, wound-healing assay and Transwell assay. Western blot analysis and ELISA assay were also performed to identify the potential mechanisms involved. The proliferation, migration and invasion of human gastric cancer SGC7901 cells were significantly inhibited by overexpression of Tspan9. In addition, Tspan9 downregulated the phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and the secretion levels of proteins related to tumor metastasis, such as matrix metalloproteinase-9 (MMP-9) and urokinase plasminogen activator (uPA). Our study indicated that Tspan9 inhibited SGC7901 cell proliferation, migration and invasion through the ERK1/2 pathway.

DNA hypermethylation of the vimentin gene inversely correlates with vimentin expression in intestinal- and diffuse-type gastric cancer.

The vimentin gene is a hallmark of epithelial-to-mesenchymal transition and has been observed to be overexpressed in various types of tumor cell line and tissue. Previous studies have reported correlations between vimentin DNA methylation levels and subsequent vimentin expression levels in solid tumors, including breast and colorectal cancer; however, to the best of our knowledge, such a correlation has not been reported for gastric cancer (GC) using Lauren classification. Therefore, the present study aimed to quantify DNA methylation levels of the vimentin gene using quantitative (q) methylation-specific polymerase chain reaction (PCR) in intestinal-type GC cell lines (MKN-28, AGS and MKN-1), diffuse-type GC cell lines (SGC-7901, SNU-5 and KATO III), the GES-1 immortalized human non-neoplastic gastric epithelial cell line, as well as in tumor and paratumor normal tissue samples. Furthermore, the present study analyzed the messenger RNA expression of the vimentin gene in these cell lines and tissues by reverse transcription-qPCR. A comparison of the clinicopathological features was conducted between patients, grouped according to the Lauren classification. The present study identified that the vimentin promoter region was hypermethylated in all GC cell lines and tumor tissue samples when compared with immortalized normal gastric epithelial cells and paratumor normal tissues. In addition, vimentin promoter methylation levels were observed to be higher in intestinal-type cell lines when compared with those of diffuse-type lines and tissues. Correspondingly, vimentin expression levels were lower in intestinal-type gastric cell lines compared with those of diffuse-type cell lines and tissues, and were lowest in the non-neoplastic gastric cell line and paratumor normal tissues. Patients with diffuse-type GC were on average younger (P=0.023), and exhibited higher tumor (P=0.020), node (P=0.032) and TNM classification of malignant tumor stage (P=0.039) than those with intestinal-type GC. Following treatment of AGS cells (which demonstrated the highest methylation level of the vimentin gene) with 5-aza-2'-deoxycytidine, vimentin expression was restored significantly. Thus, the present study revealed that vimentin promoter methylation levels are inversely correlated with vimentin expression levels in GC (according to Lauren classification). High levels of methylation in the vimentin gene promoter region may be involved in carcinogenesis and the development of GC, and may provide a novel molecular classification for GC.

[Effect of Selenious Yeast Tablets on the Thyroglobulin Antibody Level in ThyroglobulinAntibody-positive Patients with Differentiated Thyroid Cancer].

OBJECTIVE: To investigate the change of thyroglobulin antibodies (TgAb) after the application of selenious yeast tablet (SYT) in differentiated thyroid cancer (DTC) patients with positive TgAb (>115 U/ml). METHODS: We enrolled 41 DTC patients with positive TgAb who had undergone total thyroidectomy and subsequent ¹³¹I therapy as well as applied SYT in group 1 (G1). Patients with an interval of more than 6 months between SYT use and ¹³¹I therapy or with repeated TgAb measurements before the use of SYTs were divided into group 2 (G2) and group 3 (G3), respectively. Changes in TgAb after application of SYT in both G1 and G2 were observed and analyzed by rank sum test. Comparison of TgAb gradient over certain time before and after the application was analyzed by t-test. RESULTS: The proportions of patients with decreased or elevated TgAb were 85.4% and 14.6% in G1 and 90.9% and 9.1% in G2, respectively. Compared with the previous TgAb levels, TgAb decreased significantly after the application of SYT in either G1 (P=0.000) or G2(P=0.003). In G3, the TgAb level rose by 5.6% every month before applying SYT and fell 8.3% every month after the application (P=0.086). CONCLUSION: Application of SYT in DTC patients with positive TgAb can effectively decrease the TgAb level.

miR-141 confers docetaxel chemoresistance of breast cancer cells via regulation of EIF4E expression.

Resistance to docetaxel, a chemotherapy drug for breast cancer (BC) treatment, occurs in ~50% of patients, and the underlying molecular mechanisms of drug resistance are not fully understood. Gene regulation through miR-141 has been proven to play an important role in cancer drug resistance. The present study investigated the role of miR-141 expression in BC cells of acquired docetaxel resistance. Inhibition of miR-141 enhanced the response to docetaxel in docetaxel-resistant cells (MCF-7/DTX and MDA-MB-231/DTX, respectively), whereas overexpression of miR-141 confered resistance in docetaxel-sensitive cells (MCF-7 and MDA-MB-231, respectively). By directly targeting the eukaryotic translation initiation factor 4E (EIF4E) mRNA, miR-141 acts on genes that are necessary for drug induced apoptosis rendering the cells drug resistant. Modulation of miR-141 expression was correlated with EIF4E expression changes and a direct interaction of miR-141 with EIF4E was shown by a luciferase assay. Thus, the present study is the first to show an increased expression of miR-141 in an acquired model of docetaxel resistance in BC. This serves as a mechanism of acquired docetaxel resistance in BC cells, possibly through direct interactions with EIF4E, therefore presenting a potential therapeutic target for the treatment of docetaxel resistant BC.

Changes in thyroglobulin antibodies after treatment of differentiated thyroid cancer and its influencing factors.

OBJECTIVE: To investigate the changes in thyroglobulin antibodies (TgAb) and its influencing factors in differentiated thyroid cancer (DTC) patients with positive TgAb (>115 U/ml) after total thyroidectomy and radioiodine (¹³¹I) therapy. METHODS: We collected the clinical data of 118 DTC patients with positive TgAb and analyzed their TgAb levels before surgery, before ¹³¹I therapy, and after ¹³¹I therapy with a median follow-up of 2.3 months and 5.2 months. Multiple linear regression (MLR) was applied to analyze the time of TgAb concentration decreased by more than 50% (T50) and its influencing factors. RESULTS: Compared with the previous TgAb levels, TgAb decreased significantly 2.3 months and 5.2 months after surgery or after ¹³¹I therapy, respectively (both P=0.000). The proportions of patients with TgAb decreased by more than 50% in each stage were 28.6%,33.3%, and 37.2%,respectively. The negative conversion ratios were 23.4%,48.9%, and 62.8%,respectively. MLR showed that only the interval between surgery and ¹³¹I therapy was correlated with T50 (B=1.125, P=0.000). CONCLUSIONS: The TgAb levels in DTC patients remarkably decrease after surgery and after ¹³¹I therapy. The interval between surgery and ¹³¹I therapy remarkably influences the lowering speed of TgAb levels. Prompt application of ¹³¹I therapy after surgery helps to lower TgAb levels.

MOR1 expression in gastric cancer: a biomarker associated with poor outcome.

BACKGROUND: At present, the expression of MOR1 and its function in gastric cancer remains unclear with evidence suggesting that it is to be involved in tumor progression and metastasis. The study was to assess the clinicopathologic relevance and prognostic value of MOR1 expression in gastric cancer. METHODS: Real-time quantitative RT-PCR and immunohistochemical staining were used to detect MOR1 expression in primary gastric cancerous surgical specimens and adjacent nontumorous tissues. RESULTS: High MOR1 expression was detected in cancerous tumor compared with their adjacent nontumorous tissues. In addition, the chi-square test revealed that high MOR1 expression was significantly correlated with depth of invasion (p = 0.006), lymph node metastasis (p = 0.001), distant metastasis (p = 0.017), and TNM staging (p = 0.027). Moreover, Kaplan-Meier analysis revealed a significant association between MOR1 expression and overall survival. High expression of MOR1 was identified as an independent and significant predictor gene of reduced postoperative survival. CONCLUSION: We conclude that MOR1 expression may be a useful biomarker for better prediction of the clinical outcome and management of gastric cancer patients.

Prognosis significance of HER2 status and TACC1 expression in patients with gastric carcinoma.

HER2 amplification and/or expression occurs in gastric carcinoma (GC), but the role of HER2 in the prognosis of GC remains unclear. The dysregulation of transforming acidic coiled coil 1 (TACC1), a downstream gene of HER2, is thought to be involved in the development of GC. The aim of this study was to investigate the role and relationship of HER2 and TACC1 in GC. The expression of HER2 and TACC1 was analyzed using immunohistochemistry on 129 primary resected GC patients, and HER2 amplification was additionally determined by FISH. The data on clinicopathological features and relevant prognostic factors in these patients were analyzed. The expression (3+, 2+ and 1+) and the amplification of HER2 was observed in 57 cases (44.2 %) and 25 cases (19.4 %), respectively, and the correlation between HER2 expression and amplification was strong (p < 0.001). According to the FDA criteria, 24 cases (18.6 %) would have been considered as HER2 positive. A total 62 (48.1 %) GC tissues showed positive cytoplasmic staining of TACC1. There was a significant and positive association between TACC1 and HER2. HER2 positive was significantly associated with TNM stage (p = 0.019), and TACC1 expression was significantly associated with lymph node metastasis (p = 0.004) and TNM stage (p = 0.004). TNM stage, TACC1 expression and co-positive of both HER2 and TACC1 were independent prognostic factors. TACC1 expression is an independent prognostic indicator of GC. The correlation between TACC1 expression and HER2-positive status indicated a possible synergistic regulation of the two molecules and co-positive of both HER2 and TACC1 maybe a more valuable prognostic marker.

Significance of TFF3 protein and Her-2/neu status in patients with gastric adenocarcinoma.

Increased knowledge of molecular alterations involved in gastric carcinogenesis has provided useful information for diagnosis and prediction. In this study, we investigated the clinicopathological significance of TFF3 expression and Her-2/neu status in gastric adenocarcinoma and explored the correlation between TFF3 expression and Her-2/neu status. A hundred and twenty-six (126) patients having undergone curative gastrectomy were enrolled. Immunohistochemistry for TFF3 was performed on tumor tissues and non-neoplastic resection margins. Her-2/neu status was assessed by immunohistochemical staining and fluorescence in situ hybridization (FISH) on tumor tissues. As a result, TFF3 expression and Her-2/neu positivity were detected in 46.8% and 11.9% of the cases, respectively. Patients with negative TFF3 exhibited longer survival than the positive group (P=0.0142), while patients with positive Her-2/neu only showed a tendency toward longer overall survival (P>0.05). However, Her-2/neu was significantly associated with improved disease-free survival (P=0.0234). Multivariate analysis demonstrated Her-2/neu and TFF3 to be independent prognostic indicators of recurrence, and a significantly poor prognosis for expression of TFF3 in patients with Her-2/neu negative tumors. TFF3 is an independent indicator for overall survival in gastric cancer, while Her-2/neu, as a marker of long time survival prediction, seems to be limited.

Construction and expression of an eukaryotic expression vector containing the IER3 gene.

BACKGROUND: More and more research indicate that the immediately early response gene 3 (IER3) is involved in many biological processes, such as apoptosis and immunoreaction, as well as viral infection, tumorigenesis and tumour progression. METHODS: Here we describe the construction of an eukaryotic expression vector containing IER3 gene and its expression in A549 cells as assessed through fluorescence microscopyand Western- blotting. RESULTS: Fluorescence detection displayed that GFP in cytoplasm was high during 48 and 72 hours post-transfection. In addition, Western blotting showed significant increase in IER3 gene expression in the transfected cells compared with controls. CONCLUSION: The recombinate plasmid expression vector was constructed successfully, which may provide a basis for further exploration of function of IER3 in lung cancer.

A Randomised Controlled Phase II Trial of the Combination of XELOX with Thalidomide for the First-line Treatment of Metastatic Colorectal Cancer.

OBJECTIVE: To evaluate the efficacy and safety of the combination of XELOX regimen (oxaliplatin plus capecitabine) with thalidomide for the first-line treatment of metastatic colorectal cancer (MCRC). METHODS: All of the 89 patients with MCRC who fulfilled eligibility criteria were randomly assigned to treatment group (n=44) and control group (n=45). The treatment group received a combination of XELOX with thalidomide and the control group received XELOX alone. Each patient received at least 2 cycles of treatment (1 cycle=21 d). The primary endpoint was progression-free survival (PFS) and the secondary endpoints were objective response rate (ORR) as well as disease control rate (DCR). Drug safety and quality of life were also assessed. RESULTS: The median PFS of the treatment and control groups were 5.6 and 5.2 months, respectively. The difference did not have a statistical significance (P=0.307). The ORRs of the two groups also had no statistical difference (34.1% vs. 26.7%, P=0.446). The addition of thalidomide to XELOX significantly improved the DCR (63.6% vs. 42.2%, P=0.043). Among 24 patients with hepatic metastasis in the treatment group, 2 patients satisfied the surgical criteria after treatment but none of 23 patients in the control group did. Grade 3 or 4 constipation in patients treated with thalidomide was significantly increased (20.5% vs. 4.4%, P=0.022) but didn't result in treatment interruption. The rate of lethargy was increased but the difference between the two groups had no statistical significance (13.6% vs. 4.4%, P=0.130). The quality of life had no statistical difference between the two groups. CONCLUSIONS: The combination of XELOX with thalidomide for the first-line treatment of MCRC was well tolerated. Statistically significant improvement was achieved for the DCR but not for PFS.

Immunogenicity of C-terminus of Plasmodium falciparum merozoite surface protein 1 expressed as a non-glycosylated polypeptide in yeast.

The C-terminal region of the merozoite surface protein 1 (MSP119) is one of the most promising vaccine candidates against the erythrocytic forms of malaria. In the present study, a gene encoding Plasmodium falciparum MSP119 was expressed in yeast Pichia pastoris. A non-glycosylated form of the recombinant protein MSP119 was purified from culture medium. This recombinant protein maintains its antigenicity. Significant immune responses were seen in C57BL/6 mice after the second immunization. Moreover, the specific antibodies recognized the native antigens of P. falciparum. The prevailing isotypes of immunoglobulin (Ig) G associated with immunization were IgG1, IgG2a and IgG2b. The antibodies isolated from mouse sera immunized with MSP119 can inhibit parasite growth in vitro. Based on these immunological studies, we concluded that MSP119 deserves further evaluation in pre-clinical immunizations against P. falciparum.

Effects of low-dose radiation on tumor growth, erythrocyte immune function and SOD activity in tumor-bearing mice.

BACKGROUND: Activating on mammalian and human body LDR is thought to induce adaptive response, enhance immune function and increase anti-tumor ability. This study was designed to assess the effect of low-dose radiation on tumor growth and on erythrocyte immune function and superoxide dismutase (SOD) activity in tumor-bearing mice. METHODS: Male Kunming mice were subcutaneously implanted with S180 sarcoma cells in the right inguen to create an experimental in situ animal model. Six hours before implantation, the mice were given 75 mGy X-ray radiation, over the body. Tumor size was observed 5 days later while tumor volume was calculated every other day, allowing for the creation of a graph depicting tumor growth. Fifteen days after implantation, the mice were killed to measure tumor weight and observe the necrotic areas and the location of tumor-infiltrating lymphocytes (TILs). Erythrocyte immune function and SOD activity were also determined. RESULTS: Mice pre-exposed to low-dose radiation had a lower tumor formation rate than did those receiving no radiation (P < 0.05). Tumor growth was significantly lower in the mice pre-exposed to low-dose radiation; after 15 days, the average tumor weight in the mice pre-exposed to low-dose radiation was also lower (P < 0.05). Areas of tumor necrosis and infiltration of TILs were larger in the low-dose radiation group than in the non-radiation group. Erythrocyte immune function and SOD activity were higher in the low-dose radiation group than in the non-radiation group (P < 0.05). CONCLUSION: Low-dose radiation can markedly increase the anti-tumor ability of an organism and improve erythrocyte immune function and red blood cell SOD activity as well, suggesting that low-dose radiation might be useful in the clinical treatment of cancer.