Kang-Ai Injection Inhibits Gastric Cancer Cells Proliferation through IL-6/STAT3 Pathway.

OBJECTIVE: To explore the mechanisms underlying the proliferative inhibition of Chinese herbal medicine Kang-Ai injection (KAI) in gastric cancer cells. METHODS: Gastric cancer cell lines MGC803 and BGC823 were treated by 0, 0.3%, 1%, 3% and 10% KAI for 24, 48 and 72 h, respectively. The cell proliferation was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The apoptosis and cell cycle were evaluated by flow cytometry. Interleukin (IL)-6 mRNA and protein expression levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immune sorbent assay (ELISA), respectively. The protein expression levels of cyclin A, cyclin E, cyclin B1, cyclin D1, p21, retinoblastoma (RB), protein kinase B (AKT), extracellular regulated protein kinases (ERK), signal transducer and activator of transcription (STAT) 1 and STAT3 were detected by Western blot. RESULTS: KAI inhibited the proliferation of MGC803 and BGC823 gastric cancer cells in dose- and time-dependent manner. After treated with KAI for 48 h, the proportion of G1 phase was increased, expression level of cyclin D1 and phosphorylation-RB were down-regulated, whereas the expression of p21 was up-regulated (all P<0.01). Furthermore, 48-h treatment with KAI decreased the phosphorylation level of STAT3, inhibited the mRNA and protein expressions of IL-6 (all P<0.01). IL-6 at dose of 10 ng/mL significantly attenuated the proliferative effect of both 3% and 10% KAI, and recovered KAI-inhibited STAT3 phosphorylation and cyclin D1 expression level (all P<0.01). CONCLUSION: KAI exerted an anti-proliferative function by inhibiting IL-6/STAT3 signaling pathway followed by the induction of G1 phase arrest in gastric cancer cells.

FEN1 is a prognostic biomarker for ER+ breast cancer and associated with tamoxifen resistance through the ERα/cyclin D1/Rb axis.

BACKGROUND: Tamoxifen is an important choice in endocrine therapy for patients with oestrogen receptor-positive (ER+) breast cancer, and disease progression-associated resistance to tamoxifen therapy is still challenging. Flap endonuclease-1 (FEN1) is used as a prognostic biomarker and is considered to participate in proliferation, migration, and drug resistance in multiple cancers, especially breast cancer, but the prognostic function of FEN1 in ER+ breast cancer, and whether FEN1 is related to tamoxifen resistance or not, remain to be explored. METHODS: On-line database Kaplan-Meier (KM) plotter, GEO datasets, and immunohistochemistry were used to analyse the prognostic value of FEN1 in ER+ breast cancer from mRNA and protein levels. Cell viability assay and colony formation assays showed the response of tamoxifen in MCF-7 and T47D cells. Microarray data with FEN1 siRNA versus control group in MCF-7 cells were analysed by Gene Set Enrichment Analysis (GSEA). The protein levels downstream of FEN1 were detected by western blot assay. RESULTS: ER+ breast cancer patients who received tamoxifen for adjuvant endocrine therapy with poor prognosis showed a high expression of FEN1. MCF-7 and T47D appeared resistant to tamoxifen after FEN1 over-expression and increased sensitivity to tamoxifen after FEN1 knockdown. Importantly, FEN1 over-expression could activate tamoxifen resistance through the ERα/cyclin D1/Rb axis. CONCLUSIONS: As a biomarker of tamoxifen effectiveness, FEN1 participates in tamoxifen resistance through ERα/cyclin D1/Rb axis. In the future, reversing tamoxifen resistance by knocking-down FEN1 or by way of action as a small molecular inhibitor of FEN1 warrants further investigation.

Biological and clinical significance of flap endonuclease­1 in triple­negative breast cancer: Support of metastasis and a poor prognosis.

Flap endonuclease­1 (FEN1), a structure­specific nuclease participating in DNA replication and repair processes, has been confirmed to promote the proliferation and drug resistance of tumor cells. However, the biological functions of FEN1 in cancer cell migration and invasion have not been defined. In the present study, using online database analysis and immunohistochemistry of the specimens, it was found that FEN1 expression was associated with a highly invasive triple­negative breast cancer (TNBC) subtype in both breast cancer samples from the Oncomine database and from patients recruited into the study. Furthermore, FEN1 was an important biomarker of lymph node metastasis and poor prognosis in patients with TNBC. FEN1 promoted migration of TNBC cell lines and FEN1 knockdown reduced the number of spontaneous lung metastasis in vivo. Ingenuity Pathway Analysis of FEN1­related transcripts in 198 patients with TNBC demonstrated that the polo­like kinase family may be the downstream target of FEN1. PLK4 was further identified as a critical target of FEN1 mediating TNBC cell migration, by regulating actin cytoskeleton rearrangement. The results of the present study validate FEN1 as a therapeutic target in patients with TNBC and revealed a new role for FEN1 in regulating TNBC invasion and metastasis.

Integrated bioinformatics analysis for the identification of potential key genes affecting the pathogenesis of clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (CCRCC) is a typical type of RCC with the worst prognosis among the common epithelial neoplasms of the kidney. However, its molecular pathogenesis remains unknown. Therefore, the aim of the present study was to screen for effective and potential pathogenic biomarkers of CCRCC. The gene expression profile of the GSE16441, GSE36895, GSE40435, GSE46699, GSE66270 and GSE71963 datasets were downloaded from the Gene Expression Omnibus database. First, the limma package in R language was used to identify differentially expressed genes (DEGs) in each dataset. The robust and strong DEGs were explored using the robust rank aggregation method. A total of 980 markedly robust DEGs were identified (429 upregulated and 551 downregulated). According to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, these DEGs exhibited an obvious enrichment in various cancer-related biological pathways and functions. The Search Tool for the Retrieval of Interacting Genes/Proteins database was used for the construction of a protein-protein interaction (PPI) network, the Cytoscape MCODE plug-in for module analysis and the cytoHubba plug-in to identify hub genes from the aforementioned DEGs. A total of four key modules were identified in the PPI network. A total of six hub genes, including C-X-C motif chemokine ligand 12, bradykinin receptor B2, adenylate cyclase 7, calcium sensing receptor (CASR), kininogen 1 and lysophosphatidic acid receptor 5, were identified. The DEG results of the hub genes were verified using The Cancer Genome Atlas database, and CASR was found to be significantly associated with the prognosis of patients with CCRCC. In conclusion, the present study provided new insight and potential biomarkers for the diagnosis and prognosis of CCRCC.

Inhibition of FEN1 Increases Arsenic Trioxide-Induced ROS Accumulation and Cell Death: Novel Therapeutic Potential for Triple Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer, which is very difficult to treat and commonly develops resistance to chemotherapy. The following study investigated whether the inhibition of Flap Endonuclease 1 (FEN1) expression, the key enzyme in the base excision repair (BER) pathway, could improve the anti-tumor effect of arsenic trioxide (ATO), which is a reactive oxygen species (ROS) inducer. Our data showed that ATO could increase the expression of FEN1, and the knockdown of FEN1 could significantly enhance the sensitivity of TNBC cells to ATO both in vitro and in vivo. Further mechanism studies revealed that silencing FEN1 in combination with low doses of ATO might increase intracellular ROS and reduce glutathione (GSH) levels, by reducing the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2); elevating ROS leaded to apoptosis and p38 and JNK pathway activating. In conclusion, our study suggested the combination of FEN1 knockdown and ATO could induce TNBC cell death by promoting ROS production. FEN1 knockdown can effectively decrease the application concentrations of ATO, thus providing a possibility for the treatment of TNBC with ATO.

DNMT3a promotes proliferation by activating the STAT3 signaling pathway and depressing apoptosis in pancreatic cancer.

BACKGROUND: Although aberrant DNA methyltransferase 3a (DNMT3a) expression is important to the tumorigenesis of pancreatic ductal adenocarcinoma (PDAC), the role of DNMT3a in PDAC prognosis is not clarified yet due to the limited studies and lacking of underlying molecular mechanism. METHODS: The expression of DNMT3a was examined by immunohistochemistry in PDAC tissues. Gene expression profiles assays were conducted to explore the impact of DNMT3a on biological processes and signal pathways. Cell cycle and apoptosis were measured by flow cytometry. Western blotting and real-time qPCR assays were used to explore the impact of DNMT3a on expression of protein and mRNA related to cell cycle, STAT3 signaling pathway and apoptosis. RESULTS: DNMT3a was overexpressed and closely associated with poor outcomes of PDAC. DNMT3a knockdown restrained PDAC cell proliferation, induced cell cycle arrest and promoted apoptosis in vitro. Affymetrix GeneChip Human Transcriptome Array identified that the cell cycle-related process was most significantly associated with DNMT3a. DNMT3a knockdown induced G1-S phase transition arrest by decreasing the expression of cyclin D1, which was mediated by the reduction of IL8 and the subsequent inactivation of STAT3 signaling pathway. Furthermore, exogenous apoptosis was also promoted after DNMT3a knockdown, probably via up-regulation of DNA transcription and expression in CASP8. CONCLUSION: These findings indicate that DNMT3a plays an important role in PDAC progression. DNMT3a may serve as a prognostic biomarker and a therapeutic strategy candidate in PDAC.

Bufalin induces protective autophagy by Cbl-b regulating mTOR and ERK signaling pathways in gastric cancer cells.

Bufalin, a natural small-molecule compound derived from the traditional Chinese medicine Chan su, has shown promising anti-cancer effects against a broad variety of cancer cells through different mechanisms. It has been reported to induce autophagy in gastric cancer cells. However, the molecular mechanism involved is not fully elucidated. In the present study, we aimed to investigate the molecular mechanism by which bufalin induce autophagy in human gastric cancer cells. We found that bufalin induced apoptosis and autophagy in gastric cancer cells, and autophagy prevented human gastric cancer cells from undergoing apoptosis. Bufalin treatment changed the expression of autophagy-related proteins. Moreover, phosphorylated Akt, mTOR, and p70S6K were all significantly decreased, while phosphorylated ERK1/2 was increased by bufalin. Pretreatment of MGC803 cells with the ERK1/2-specific inhibitor PD98059 led to the down-regulation of LC3 II. Further study showed that Cbl-b positively regulated autophagy by suppressing mTOR and enhancing ERK1/2 activation. Therefore, our data provide evidence that bufalin induces autophagy in MGC803 cells via both Akt/mTOR/p70S6K and ERK signaling pathways, and Cbl-b-mediated suppression of mTOR and activation of ERK1/2 might play an important role.

FEN1 knockdown improves trastuzumab sensitivity in human epidermal growth factor 2-positive breast cancer cells.

Trastuzumab has been widely applied as a treatment for human epidermal growth factor 2 (HER2)-overexpressing breast cancer. However, the therapeutic efficacy of trastuzumab is limited. Flap endonuclease 1 (FEN1) is a multifunctional endonuclease that has a crucial role in DNA recombination and repair. Inhibition of FEN1 is associated with the reversal of anticancer drug resistance. However, it is unclear whether FEN1 is involved in trastuzumab resistance. In the present study, it was demonstrated that trastuzumab increases the expression of FEN1, and FEN1 knockdown significantly enhanced the sensitivity of BT474 cells to trastuzumab (P<0.05). It was also revealed that trastuzumab induced HER receptor activation, increased binding with FEN1 and estrogen receptor α (ERα), and upregulated ERα-target gene transcription (P<0.05). Upon silencing of FEN1 expression with siRNA, activation of HER receptor and FEN1 binding to ERα were decreased, and trastuzumab-induced ERα target gene upregulation was partially ameliorated (P<0.05). These results suggest that FEN1 may mediate trastuzumab resistance via inducing HER receptor activation and enhancing ERα-target gene transcription. The findings of the present study indicate a novel role of FEN1 in trastuzumab resistance, suggesting that targeting FEN1 may enhance the efficiency of trastuzumab as a treatment for HER2-positive breast cancer.

SPARC expression in gastric cancer predicts poor prognosis: Results from a clinical cohort, pooled analysis and GSEA assay.

BACKGROUND: The prognostic role of Secreted Protein Acidic and Rich in Cysteine (SPARC) in gastric cancer (GC) remains controversial. We investigated the clinical significance, the survival relevance, and potential function of SPARC in GC with resected samples, online gene set GSE62254, and cell line SGC7901. RESULTS: High immunostaining of SPARC significantly correlated with tumor differentiation (P = 0.004), and independently predicted shorter overall survival (OS) (HR = 1.446, P = 0.022), based on the current IHC evaluation. The accuracy of the results was further validated with 1000 times bootstrapping and the time-dependent receiver-operating characteristics (ROC) curves. The meta-analysis (pooled HR = 1.60, 95% CI: 1.01-2.53) confirmed SPARC as the predictor for reduced OS in GC. Moreover, the association between enhanced SPARC expression and Adriamycin (Adr) sensitivity was revealed by GSEA, and then confirmed by comparative cellular experiments, such as the protein level analysis of SGC7901and SGC7901/Adr cell line. MATERIALS AND METHODS: Immunohistochemistry (IHC) method was used to detect SPARC expression in 137 GC cases. Meta-analysis was performed based on 5 studies published in English on PubMed up to March 2016. GSEA was performed using online data set GSE62254 and GC-related functional gene sets derived from molecular signatures database (MSigDB). Western Blot was carried out to compare protein-level differences between gastric carcinoma SGC7901 cell line and Adr resistant SGC7901/Adr cell line. MTT assay was done to confirm the induction of SPARC on Adr sensitivity. CONCLUSIONS: Increased SPARC expression in GC led to a worse clinical outcome of patients and might induce Adr sensitivity of GC cells.

The role of E3 ubiquitin ligase Cbl proteins in interleukin-2-induced Jurkat T-cell activation.

Interleukin- (IL-) 2 is the major growth factor for T-cell activation and proliferation. IL-2 has multiple functions in the regulation of immunological processes. Although most studies focus on T-cell immunomodulation, T-cell activation by IL-2 is the foundation of priming the feedback loop. Here, we investigated the effect of MAPK/ERK and PI3K/Akt signaling pathways on IL-2-induced cell activation and the regulatory mechanisms of upstream ubiquitin ligase Cbl-b and c-Cbl. Morphological analysis of Jurkat T cells was performed by cytospin preparations with Wright-Giemsa stain. CD25 expression on Jurkat T cells was determined by flow cytometry. Changes in cell activation proteins such as p-ERK, ERK, p-Akt, Akt, and ubiquitin ligase Casitas B-cell Lymphoma (Cbl) proteins were analyzed by western blot. Following IL-2-induced activation of Jurkat T cells, p-ERK expression was upregulated, while there was no change in p-Akt, ERK, or Akt expression. Thus, the MAPK/ERK signaling pathway, but not PI3K/Akt, was involved in IL-2-induced T-cell activation. Either using PD98059 (a specific inhibitor for p-ERK) or depletion of ERK with small interfering RNA (siRNA) reduced the expression of CD25. This study also showed that ubiquitin ligase proteins Cbl-b and c-Cbl might be involved in IL-2-induced Jurkat T-cell activation by negatively regulating the MAPK/ERK signaling pathway.

ß-Elemene induces apoptosis in human renal-cell carcinoma 786-0 cells through inhibition of MAPK/ERK and PI3K/Akt/ mTOR signalling pathways.

BACKGROUND: Renal-cell carcinoma (RCC) is resistant to almost all chemotherapeutics and radiation therapy. ß-Elemene, a promising anticancer drug extracted from a traditional Chinese medicine, has been shown to be effective against various tumors. In the present study, anti-tumor effects on RCC cells and the involved mechanisms were investigated. METHODS: Human RCC 786-0 cells were treated with different concentrations of ß-elemene, and cell viability and apoptosis were measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry, respectively. Protein expression was assayed by western blotting. Autophagy was evaluated by transmission electron microscopy. RESULTS: ß-Elemene inhibited the viability of 786-0 cells in a dose- and time-dependent manner. The anti-tumor effect was associated with induction of apoptosis. Further study showed that ß-elemene inhibited the MAPK/ERK as well as PI3K/Akt/mTOR signalling pathways. Moreover, robust autophagy was observed in cells treated with ß-elemene. Combined treatment of ß-elemene with autophagy inhibitors 3-methyladenine or chlorochine significantly enhanced the anti-tumor effects. CONCLUSIONS: Our data provide first evidence that ß-elemene can inhibit the proliferation of RCC 786- 0 cells by inducing apoptosis as well as protective autophagy. The anti-tumor effect was associated with the inhibition of MAPK/ERK and PI3K/Akt/mTOR signalling pathway. Inhibition of autophagy might be a useful way to enhance the anti-tumor effect of ß -elemene on 786-0 cells.

ß-Elemene induces apoptosis as well as protective autophagy in human non-small-cell lung cancer A549 cells.

OBJECTIVES: ß-Elemene, a novel traditional Chinese medicine, has been shown to be effective against a wide range of tumours. In this study, the antitumour effect of ß-elemene on human non-small-cell lung cancer (NSCLC) A549 cells and the mechanism involved have been investigated. METHODS: Cell viability and apoptosis were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively. Protein expression was assayed by Western blotting. Autophagy was evaluated under fluorescence microscopy and transmission electron microscopy. KEY FINDINGS: ß-Elemene inhibited the viability of A549 cells in a dose-dependent manner. This suppression of cell viability was due to the induction of apoptosis. Further study showed that ß-elemene inhibited the activity of the PI3K/Akt/mTOR/p70S6K1 signalling pathway, and at the same time it triggered a robust autophagy. The autophagy was characterized by the accumulation of punctate LC3 dots in the cytoplasm, morphological changes, and the increased levels of LC3-II as well as Atg5-Atg12 conjugated proteins. Inhibition of autophagy with chlorochine significantly enhanced the antitumour effect of ß-elemene. CONCLUSIONS: Our data indicated that ß-elemene inhibited the activity of the PI3K/Akt/mTOR/p70S6K1 signalling pathway in human NSCLC A549 cells, which resulted in apoptosis as well as protective autophagy. A combination of ß-elemene with autophagy inhibitor might be an effective therapeutic option for advanced NSCLC.

[Cisplatin enhances TRAIL-induced apoptosis in gastric cancer cells through clustering death receptor 4 into lipid rafts].

OBJECTIVE: Gastric cancer cells are insensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). To sensitize gastric cancer cells to TRAIL, we treated gastric cancer MGC803 cells with TRAIL and cisplatin. METHODS: Cell proliferation was measured using MTT assay. Cell apoptosis was determined by flow cytometry. Expression of proteins was analyzed by Western blot. The distribution of lipid rafts and death receptors was analyzed by immunofluorescence microscopy. MGC803 cells were pretreated with 50 mg/L nystatin for 1 h, and followed by the treatment of cisplatin and TRAIL. RESULTS: 100 µg/L TRAIL resulted in (8.51 ± 3.45)% inhibition of cell proliferation and caused (3.26 ± 0.89)% cell apoptosis in MGC803 cells. Compared with the treatment with cisplatin alone, treatment with TRAIL (100 µg/L) and cisplatin (8.49 mg/L, IC(50) dose of 24 h) led to a dramatic increase in both inhibition of cell proliferation [(52.58 ± 4.57)% vs. (76.43 ± 5.35)%, P < 0.05] and cell apoptosis [(23.10 ± 3.41)% vs. (42.56 ± 4.11)%, P < 0.05]. Moreover, cleavage of caspase-8 and caspase-3 was detected. TRAIL (100 µg/L) did not induce obvious lipid rafts aggregation and death receptor 4 (DR4) clustering, while cisplatin (8.49 mg/L) significantly promoted the localization of DR4 in aggregated lipid rafts. Pretreatment with 50 mg/L nystatin, a cholesterol-sequestering agent, triggered (3.66 ± 0.52)% cell apoptosis after 24 h. Pretreatment with nystatin for 1 h before the addition of 8.49 mg/L cisplatin for 24 h caused a decreased tendency to cell apoptosis [(25.74 ± 3.28)% vs. (22.76 ± 2.97)%]. While, pretreatment with nystatin before the addition of cisplatin and TRAIL, the proportion of apoptotic cells decreased from (43.16 ± 4.26)% to (31.52 ± 3.99)% (P < 0.05). CONCLUSION: Cisplatin enhances TRAIL-induced apoptosis in gastric cancer MGC803 cells through clustering death receptors into lipid rafts.

Protective autophagy antagonizes oxaliplatin-induced apoptosis in gastric cancer cells.

Oxaliplatin-based chemotherapy is used for treating gastric cancer. Autophagy has been extensively implicated in cancer cells; however, its function is not fully understood. Our study aimed to determine if oxaliplatin induce autophagy in gastric cancer MGC803 cells and to assess the effect of autophagy on apoptosis induced by oxaliplatin. MGC803 cells were cultured with oxaliplatin. Cell proliferation was measured using MTT assay, and apoptosis was determined by flow cytometry. Protein expression was detected by Western blot. Autophagy was observed using fluorescent microscopy. Our results showed that the rate of apoptosis was 9.73% and 16.36% when MGC803 cells were treated with 5 and 20 µg/mL oxaliplatin for 24 h, respectively. In addition, caspase activation and poly ADP-ribose polymerase (PARP) cleavage were detected. Furthermore, when MGC803 cells were treated with oxaliplatin for 24 h, an accumulation of punctate LC3 and an increase of LC3-II protein were also detected, indicating the activation of autophagy. Phosphorylation of Akt and mTOR were inhibited by oxaliplatin. Compared to oxaliplatin alone, the combination of autophagy inhibitor chlorochine and oxaliplatin significantly enhanced the inhibition of cell proliferation and the induction of cell apoptosis. In conclusion, oxaliplatin-induced protective autophagy partially prevents apoptosis in gastric cancer MGC803 cells. The combination of autophagy inhibitor and oxaliplatin may be a new therapeutic option for gastric cancer.

Ubiquitin ligase c-Cbl is involved in tamoxifen-induced apoptosis of MCF-7 cells by downregulating the survival signals.

BACKGROUND: Tamoxifen (TAM) is a nonsteroidal antiestrogen that has been widely used in the treatment of breast cancer through its anti-estrogen activity. Recent studies show that TAM is cytotoxic to both estrogen receptor (ER)-positive and ER-negative cells via the induction of apoptosis. However, the molecular mechanisms of this effect are not well understood. In the present study, we investigated the roles of c-Src, ERK, AKT and c-Cbl ubiquitin ligases during TAM-induced apoptosis of MCF-7 cells. MATERIAL AND METHODS: MCF-7 cell proliferation and apoptosis were measured by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and flow cytometry, respectively. c-Cbl expression, and the activity of c-Src, ERK, AKT were assayed by Western blotting. Overexpression of the wild and the dominant-negative type of c-Cbl (70Z/Cbl) were achieved by transient transfection of plasmids encoding c-Cbl and 70Z/Cbl, respectively, and were confirmed by Western blotting. Statistical analysis was performed using the t-test, and a p-value <0.05 was considered to be statistically significant. RESULTS: A high concentration of TAM (25 µM) induced a time-dependent apoptosis of MCF-7 cells. ERK1/2 and AKT were activated during TAM-induced apoptosis. The ERK1/2 inhibitor PD98059, the PI3K/Akt inhibitor LY294002, and the c-Src inhibitor PP2 all enhanced TAM action. Moreover, the ubiquitin ligase c-Cbl was up-regulated during this process. Over-expression of c-Cbl significantly enhanced the apoptosis-inducing effects of TAM, while 70Z/Cbl suppressed the apoptosis-inducing effects of TAM. Further investigation revealed that, overexpression of c-Cbl significantly downregulated the c-Src protein levels and TAM-induced AKT activity. But 70Z/Cbl significantly upregulated TAM-induced ERK and AKT activity. CONCLUSIONS: This study demonstrates that c-Src, ERK, and AKT played a protective role during TAM-induced apoptosis, and that c-Cbl sensitized MCF-7 cells to TAM by modulating the expression of c-Src, and TAM-induced ERK and AKT activity.

Bortezomib synergizes TRAIL-induced apoptosis in gastric cancer cells.

Background/Aims Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as a newly identified biological agent has shown promising antitumor effects in a wide range of cancers. However, gastric cancer cells are less sensitive than other cancer cells to TRAIL-induced apoptosis.Here, we combined TRAIL with bortezomib, a proteasomal inhibitor to induce apoptosis in three gastric cancer cell lines.Methods After the cells were treated with TRAIL and/or bortezomib, the cell viability, apoptosis and cell cycle distribution were examined. The levels of death receptors and the mitochondrial membrane potential were also detected. The expression of apoptosis-associated proteins was determined by Western blot.Results Bortezomib at low concentration significantly(P<0.05) enhanced the cytotoxic effect of TRAIL by enhancing apoptosis as well as cell cycle arrest at G2/M phase. The enhancement of efficiency of TRAIL by bortezomib involved up-regulation of death receptor 4 and 5, as well as reduction of the mitochondrial membrane potential. Further study showed that combined treatment with TRAIL and bortezomib down-regulated anti-apoptotic protein cIAP-1, and over expression of cIAP-1 significantly(P\0.05) reduced the synergistic effect between TRAIL and bortezomib.Conclusions Bortezomib synergizes TRAIL-induced apoptosis in human gastric cancer cells. The synergistic effect between these two drugs is associated with up-regulation of death receptors and down-regulation of cIAP-1.The combination of TRAIL and bortezomib might be an effective regimen for the treatment of advanced gastric cancer.

[Expression of c-Cbl, Cbl-b, and epidermal growth factor receptor in gastric carcinoma and their clinical significance].

BACKGROUND AND OBJECTIVE: c-Cbl and Cbl-b are two ubiquitous members of the Casitas B-lineage lymphoma (Cbl) family, which play important roles in the downregulation of epidermal growth factor receptor (EGFR) by acting as E3 ubiquitin ligases and multiadaptor proteins. This study investigated the expression of c-Cbl, Cbl-b, and EGFR in gastric carcinoma and its clinical significance. METHODS: The expressions of c-Cbl, Cbl-b, and EGFR were detected by immunohistochemistry using tissue microarrays consisting of 124 specimens of gastric carcinoma and 16 specimens of normal gastric mucosa. The relationship between the expressions of c-Cbl, Cbl-b, and EGFR and clinicopathologic factors of gastric carcinoma were analyzed statistically. RESULTS: The positive rates of c-Cbl, Cbl-b, and EGFR were higher in the gastric carcinoma group than in the normal group (71.0% vs. 18.0%, P<0.01; 82.3% vs. 25.0%, P<0.01; 56.5% vs. 12.5%, P<0.01, respectively). The expression of c-Cbl was positively correlated with depth of invasion (r=0.219, P=0.015), and TNM staging (r=0.266, P=0.003). The expression of Cbl-b was positively correlated with lymph node metastasis (r=0.190, P<0.034) and TNM staging (r=0.298, P<0.001). The expression of EGFR was positively correlated with depth of invasion (r=0.286, P<0.001) and TNM staging (r=0.362, P=0.000). The expression of both c-Cbl and Cbl-b was positively correlated with EGFR (r=0.241, P=0.007; r=0.183, P=0.042, respectively). Synchronous strong-positive expressions of c-Cbl, Cbl-b, and EGFR were observed in 27 specimens of gastric carcinoma, most of which were at advanced stage. CONCLUSIONS: Overexpressions of c-Cbl, Cbl-b, and EGFR are closely related to the invasion and progression of gastric carcinoma. c-Cbl and Cbl-b may serve as novel molecular markers for gastric carcinoma.

The role of cbl family of ubiquitin ligases in gastric cancer exosome-induced apoptosis of Jurkat T cells.

BACKGROUND. Exosomes are nanometer-sized vesicles with immunomodulatory functions, which are released by a diverse range of living cells. Although recent studies have shown that tumor-derived exosomes can suppress the function of T cells, the molecular mechanisms are not well understood. In the present study, we investigated the role of the Casitas B lineage lymphoma (cbl) family of ubiquitin ligases in gastric cancer exosome-induced apoptosis of Jurkat T cells. MATERIALS AND METHODS. By serial centrifugation and sucrose gradient ultracentrifugation, we isolated and purified the exosomes from gastric cancer SGC7901 cells, and identified them by electron microscopy and Western blotting. Cell apoptosis was detected using propidium iodide staining. Western blotting and RT-PCR was exploited to evaluate the expression of proteins and mRNA, respectively. RESULTS. Gastric cancer exosomes induced Jurkat T cell apoptosis in a time- and dose-dependent manner and activated caspases 3, 8 and 9. The expression of Cbl-b and c-Cbl was up-regulated during exosome-induced apoptosis of cells. Meanwhile, exosomes induced ubiquitination of the p85 subunit of phosphoinositide 3-kinase (PI3K) and reduced downstream Akt activity. Inhibition of proteasome led to partial restoration of Akt activity and cell apoptosis. DISCUSSION AND CONCLUSIONS. The Cbl family of ubiquitin ligases might be involved in regulation of exosome-induced apoptosis of Jurkat T cells by increasing PI3K proteasome degradation, inactivation of PI3K/Akt signaling, thus mediating some effects of caspase activation.

Reversal effect of PI3-K inhibitor LY294002 on P-glycoprotein-mediated multidrug resistance of human leukemia cell line K562/DNR and gastric cancer cell line SGC7901/ADR.

BACKGROUND AND OBJECTIVE: Phosphatidylinositol-3-kinase/protein kinase B (PI3-K/Akt) signaling pathway plays an important role in cell survival. This study was to explore the reversal effect of PI3-K inhibitor LY294002 on p-glycoprotein (P-gp)-mediated multidrug resistance in human leukemia cell line K562/DNR and gastric cancer cell line SGC7901/ADR. METHODS: The cells were divided into simple drug-treated groups and LY294002 pretreated groups: the former groups received treatment of daunorubicin (DNR), adriamycin (ADR), vincristine (VCR) and etoposide (VP-16), respectively; the latter groups received pretreatment of LY294002 before drug treatment. Trypan blue dye exclusion method and MTT assay were used to detect the drug sensitivity of K562/DNR and SGC7901/ADR cells, and the effect of LY294002 on the drug resistance. The expression of P-gp and phosphorylated Akt (p-Akt) in K562/DNR, SGC7901/ADR and their parental cell lines K562 and SGC7901 was detected by Western blot. Intracellular drug accumulation was measured by flow cytometry (FCM). RESULTS: LY294002 pretreatment significantly decreased the 50% inhibition concentration (IC(50)) of DNR, ADR, VCR and VP-16 for K562/DNR cells, with reverse efficiencies of 72.4%, 64.9%, 60.4% and 52.8%. In SGC7901/ADR cells, the similar result was obtained with a reverse efficiency of 31.0%. LY294002 pretreatment partially inhibited the expression of p-Akt and P-gp, and promoted the intracellular accumulation of DNR and ADR in K562/DNR and SGC7901/ADR cells, respectively. CONCLUSION: LY294002 could partially reverse multidrug resistance in K562/DNR and SGC7901/ADR cells in vitro via inhibiting PI3-K/Akt pathway.

[Effects of Bufalin on SYK and CBL family proteins in induction of HL-60 cell apoptosis].

The study was aimed to explore the mechanism of SYK and CBL family of ubiquitin ligases in Bufalin-induced HL-60 cells apoptosis. Cell viability was tested by trypan blue staining and apoptosis was detected by using flow cytometry. The expressions of CBL and CBL-b and the phosphorylation of SYK were detected by using immunoprecipitation and Western blot. The results showed that Bufalin inhibited HL-60 cell proliferation in time- and dose-dependent manners. IC(50) of suppressing cell viability at 24, 48 and 72 hours were about 26.3, 7.8 and 2.0 nmol/L respectively. The high dose of bufalin already induced apoptosis of HL-60 cells at 8 hours. SYK was quickly phosphorylated, and the expressions of CBL and CBL-b were down-regulated after treatment with Bufalin. It is concluded that SYK activation and CBL protein down-regulation may be involved in Bufalin-induced HL-60 cell apoptosis.