Glutamine provides carbon and nitrogen to support the proliferation of cancer cells. However, the precise reason why cancer cells are particularly dependent on glutamine remains unclear. In this study, we report that glutamine modulates the tumor suppressor F-box and WD repeat domain-containing 7 (FBW7) to promote cancer cell proliferation and survival. Specifically, lysine 604 (K604) in the sixth of the 7 substrate-recruiting WD repeats of FBW7 undergoes glutaminylation (Gln-K604) by glutaminyl tRNA synthetase. Gln-K604 inhibits SCFFBW7-mediated degradation of c-Myc and Mcl-1, enhances glutamine utilization, and stimulates nucleotide and DNA biosynthesis through the activation of c-Myc. Additionally, Gln-K604 promotes resistance to apoptosis by activating Mcl-1. In contrast, SIRT1 deglutaminylates Gln-K604, thereby reversing its effects. Cancer cells lacking Gln-K604 exhibit overexpression of c-Myc and Mcl-1 and display resistance to chemotherapy-induced apoptosis. Silencing both c-MYC and MCL-1 in these cells sensitizes them to chemotherapy. These findings indicate that the glutamine-mediated signal via Gln-K604 is a key driver of cancer progression and suggest potential strategies for targeted cancer therapies based on varying Gln-K604 status.
Glutamine/metabolism* ; Myeloid Cell Leukemia Sequence 1 Protein/genetics* ; Humans ; Proto-Oncogene Proteins c-myc/genetics* ; Cell Proliferation ; Signal Transduction ; Neoplasms/pathology* ; F-Box-WD Repeat-Containing Protein 7/genetics* ; Cell Survival ; Cell Line, Tumor ; Apoptosis

Hepatocellular carcinoma (HCC) is a major cause of cancer-related mortality in part due to its high resistance to chemotherapeutic drugs. The anti-apoptotic Mcl-1 expression has been reported as a resistance factor in various types of tumors. Here, we investigated the expression of Mcl-1 in hepatoma cells and HCC tissues and its relationship with p53, and analyzed the possibility of the gene as a molecular target for HCC therapy. HCC specimens of 30 patients were examined by immunohistochemistry for Mcl-1 and p53 expression. Mcl-1 expression in hepatoma cell lines was measured by RT-PCR and Western blotting. The suppression of Mcl-1 by RNA interference or specific phosphatidylinositol-3 kinase (PI3K) inhibitor, LY294002, was evaluated as monotherapy, and it was combined with mitomycin C (MMC) in treating hepatoma cell line HepG2. Cell viability and apoptosis were assessed by MTT and FACS analysis. Finally, changes of Mcl-1 or p53 expression in various hepatoma cell lines were examined after transfection with Mcl-1 siRNA, the Mcl-1 expression plasmid, or the wide-type p53 expression plasmid, respectively. Mcl-1 protein was remarkably enhanced in HCC tissues as compared with adjacent non-tumor liver tissues. In addition, Mcl-1 was prominently expressed in HepG2 and Hep3B cells, weakly in SMMC7721 cells, and not in L02 cells. P53 protein was also overexpressed in HCC tissues and there was a significant correlation between the expression of p53 and Mcl-1. Silencing Mcl-1 by RNAi or LY294002 downregulated Mcl-1 expression and led to decreased cell viability and increased apoptosis. Combination of MMC and Mcl-1 RNAi or LY294002 exhibited a significant chemosensitizing effect. The expression of p53 was not influenced by Mcl-1 siRNA in HepG2 cells or transfection with the Mcl-1 expression plasmid in L02 cells. Furthermore, the expression of Mcl-1 in Hep3B cells was also not significantly changed after transfection with the wild-type p53 expression plasmid. It is concluded that Mcl-1 is overexpressed in HCC tissues. The mechanisms by which silencing Mcl-1 sensitizes hepatoma cells towards chemotherapy may be not attributed to the upregulated expression of p53 but the dysfunction of p53 through Mcl-1/p53 interaction. Mcl-1 may be a potential target of gene therapy for HCC.
Adenoma, Liver Cell ; drug therapy ; genetics ; pathology ; Apoptosis ; drug effects ; Biomarkers, Tumor ; biosynthesis ; genetics ; Chromones ; administration & dosage ; Gene Expression Regulation, Neoplastic ; drug effects ; Hep G2 Cells ; Humans ; Liver Neoplasms ; drug therapy ; genetics ; pathology ; Morpholines ; administration & dosage ; Myeloid Cell Leukemia Sequence 1 Protein ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Transfection ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics

OBJECTIVETo explore the effect of downregulation of Tiam1 by siRNA on the esophageal squamous cell carcinoma (ESCC) EC9706 cells, and provide theoretical basis for gene therapy of ESCC using Tiam1 as a molecular target.

METHODSTiam1 siRNA was transfected into EC9706 cells, and expression changes of Tiam1 mRNA and protein after transfection were detected by quantitative real-time PCR and Western blotting. Cell proliferation was analyzed using CCK-8 kit. Cell cycle and apoptosis of the EC9706 cells were assessed by flow cytometry. Cell cycle-related proteins and cell apoptosis-associated proteins were analyzed by Western blotting.

RESULTSCompared with the untreated group and control siRNA group, the relative expression levels of Tiam1 mRNA (1.00 and 0.11 ± 0.02) were not significantly different (P > 0.05). The relative expression levels of Tiam1 mRNA in the Tiam1 siRNA group at 24, 48 and 72 h after transfection were 0.30 ± 0.04, 0.09 ± 0.01 and 0.09 ± 0.006, respectively, significantly lower than that of the untreated group (P < 0.05 for all). The expression level of Tiam1 protein at 24 h after Tiam1 siRNA transfection in the EC9706 cells was 0.11 ± 0.02, significantly lower than that in the un-treated group (0.44 ± 0.05) and control siRNA group (0.44 ± 0.04, P < 0.05 for all). The percentages of G0/G1 cells in the Tiam1 siRNA group, untreated group and control siRNA group were (54.48 ± 2.14)%, (40.69 ± 1.85)% and (41.78 ± 1.31)%, respectively (P < 0.01). The percentages of S phase cells in the Tiam1 siRNA group, untreated group and control siRNA group were (27.18 ± 1.65)%, (32.32 ± 1.15)% and (30.35 ± 1.09)%, respectively (P < 0.01). The expression levels of cyclin D1 protein in the untreated group, control siRNA group and Tiam1 siRNA group were 0.43 ± 0.02, 0.41 ± 0.01 and 0.11 ± 0.02, respectively (P < 0.05). The expression levels of p27 protein in the untreated group, control siRNA group and Tiam1 siRNA group were 0.10 ± 0.01, 0.09 ± 0.02 and 0.20 ± 0.02, respectively (P < 0.05). The ratios of early apoptotic cells in the untreated group, control siRNA group and Tiam1 siRNA group were (10 ± 0.9)%, (10 ± 0.5)% and (27 ± 0.7)%, respectively (P < 0.01). The expression levels of Mcl-1 protein in EC9706 cells of untreated group, control siRNA group and Tiam1 siRNA group were 0.47 ± 0.12, 0.48 ± 0.13 and 0.16 ± 0.02, respectively (P < 0.05). The expression levels of Bcl-2 protein in EC9706 cells of the untreated group, control siRNA group and Tiam1 siRNA group were 0.49 ± 0.08, 0.50 ± 0.05 and 0.04 ± 0.03, respectively (P < 0.05). The caspase-3 activities in the untreated group, control siRNA group and Tiam1 siRNA group were 2.3 ± 0.09, 2.3 ± 0.10 and 16.0 ± 1.50, respectively; and that of caspase-9 were 2.3 ± 0.08, 2.3 ± 0.11 and 14.5 ± 0.9, respectively (P < 0.05 for all).

CONCLUSIONSTiam1 siRNA can significantly inhibit the proliferation of esophageal cancer EC9706 cells, induce cell cycle arrest and cell apoptosis. These effects are related to the regulation of the expressions of cell cycle-related genes (cyclin D1 and p27) and cell apoptosis-related genes (Mcl-1, Bcl-1, caspase-3 and caspase-9) by Tiam1 siRNA.

Apoptosis ; Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cell Cycle Checkpoints ; Cell Line, Tumor ; Cell Proliferation ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; Down-Regulation ; Esophageal Neoplasms ; genetics ; metabolism ; pathology ; Guanine Nucleotide Exchange Factors ; genetics ; metabolism ; Humans ; Myeloid Cell Leukemia Sequence 1 Protein ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; T-Lymphoma Invasion and Metastasis-inducing Protein 1 ; Transfection

Mcl-1 and Bcl-xL, key anti-apoptotic proteins of the Bcl-2 family, have attracted attention as important molecules in the cell survival and drug resistance. In this study, we investigated whether inhibition of Bcl-xL influences cell growth and apoptosis against simultaneous treatment of resveratrol and clofarabine in the human malignant mesothelioma H-2452 cells. Resveratrol and clofarabine decreased Mcl-1 protein levels but had little effect on Bcl-xL levels. In the presence of two compounds, any detectable change in the Mcl-1 mRNA levels was not observed in RT-PCR analysis, whereas pretreatment with the proteasome inhibitor MG132 led to its accumulation to levels far above basal levels. The knockdown of Bcl-xL inhibited cell proliferation with cell accumulation at G2/M phase and the appearance of sub-G0/G1 peak in DNA flow cytometric assay. The suppression of cell growth was accompanied by an increase in the caspase-3/7 activity with the resultant cleavages of procaspase-3 and its substrate poly (ADP-ribose) polymerase, and increased percentage of apoptotic propensities in annexin V binding assay. Collectively, our data represent that the efficacy of resveratrol and clofarabine for apoptosis induction was substantially enhanced by Bcl-xL-lowering strategy in which the simultaneous targeting of Mcl-1 and Bcl-xL could be a more effective strategy for treating malignant mesothelioma.
Adenine Nucleotides/*pharmacology ; Antimetabolites, Antineoplastic/*pharmacology ; Apoptosis/*drug effects ; Arabinonucleosides/*pharmacology ; Caspase 3/metabolism ; Caspase 7/metabolism ; Cell Line, Tumor ; Cell Proliferation/drug effects ; G2 Phase Cell Cycle Checkpoints/drug effects ; Gene Knockdown Techniques ; Humans ; Leupeptins/pharmacology ; Lung Neoplasms/metabolism/pathology ; M Phase Cell Cycle Checkpoints/drug effects ; Mesothelioma/metabolism/pathology ; Myeloid Cell Leukemia Sequence 1 Protein/antagonists & inhibitors/genetics/metabolism ; RNA Interference ; RNA, Messenger/metabolism ; RNA, Small Interfering/metabolism ; Stilbenes/*pharmacology ; bcl-X Protein/antagonists & inhibitors/*genetics/*metabolism

Resistance to chemotherapy is a major obstacle for the effective treatment of advanced ovarian cancer. The mechanism of chemoresistance is still poorly understood. Recently, more and more evidence showed microRNAs (miRNAs) modulated many key molecules and pathways involved in chemotherapy. microRNA-106a (miR-106a) has been implicated in many cancers, but its role in ovarian cancer and drug resistance still remains unexplored. This study was to investigate whether miR-106a mediated resistance of the ovarian cancer cell line A2780 to the chemotherapeutic agent cisplatin (DDP). The different levels of miR-106a in A2780 cells and their resistant variant A2780/DDP cells were identified by using real-time PCR. MTT assay and flow cytometry were used to analyze the effect of miR-106a on cisplatin resistance of these paired cells. Real-time PCR, Western blotting and luciferase reporter assay were applied to explore whether Mcl-1 was a target of miR-106a. As compared to A2780 cells, the expression of miR-106a was down-regulated in the cisplatin resistant cell line A2780/DDP. Moreover, knockdown of miR-106a dramatically decreased antiproliferative effects and apoptosis induced by cisplatin in A2780 cells, while overexpression of miR-106a significantly increased antiproliferative effects and apoptosis induced by cisplatin in A2780/DDP cells. Furthermore, miR-106a inhibited cell survival and cisplatin resistance through downregulating the expression of Mcl-1. Mcl-1 was a direct target of miR-106a. These results suggest that miR-106a may provide a novel mechanism for understanding cisplatin resistance in ovarian cancer by modulating Mcl-1.
Antineoplastic Agents ; pharmacology ; Cell Line, Tumor ; Cisplatin ; pharmacology ; Drug Resistance, Neoplasm ; genetics ; Female ; Humans ; MicroRNAs ; genetics ; Myeloid Cell Leukemia Sequence 1 Protein ; genetics ; Ovarian Neoplasms ; drug therapy ; genetics

The purpose of this study was to investigate the effects of Celecoxib on the proliferation of the FLT3-ITD positive and negative acute myeloid leukemia cells and its mechanism. The proliferation inhibition effect of Celecoxib with different doses on the FLT3-ITD positive cells MV4-11 and the FLT3-ITD negative K562 cells was detected by CCK-8 method, the cell apoptosis was determined by flow cytometry, and the MEK, Mcl-1, pAKT expression was tested by Western blot. The results showed that Celecoxib inhibited the proliferation of both MV4-11 and K562 cells, but the IC50 for MV4-11 was (29.14 ± 2.4) µmol/L, which was significantly lower than that of K562 cells (39.84 ± 1.0) µmol/L (P < 0.05); The induced apoptosis rate of Celecoxib at 20-80 µmol/L on MV4-11 was not observed, but there was apparent influence on K562 at the same concentration. Western blot showed that Celecoxib down-regulated the expression of MEK and Mcl-1 but did not change the expression of pAKT obviously on MV4-11 cells, while the expression of Mcl-1 was reduced a little, but no obvious change were found in the expression of MEK and pAKT on K562 cells. It is concluded that the Celecoxib can inhibit the proliferation of FLT3-ITD positive AML cells distinctly, and the potential mechanism may be related to the inhibition of the MEK/Mcl-1 signaling pathway.
Apoptosis ; drug effects ; Celecoxib ; Cell Proliferation ; drug effects ; Cyclooxygenase 2 Inhibitors ; pharmacology ; Gene Expression Regulation, Leukemic ; Humans ; K562 Cells ; Leukemia, Myeloid, Acute ; drug therapy ; metabolism ; pathology ; MAP Kinase Kinase 1 ; genetics ; Myeloid Cell Leukemia Sequence 1 Protein ; genetics ; Proto-Oncogene Proteins c-akt ; genetics ; Pyrazoles ; pharmacology ; Signal Transduction ; Sulfonamides ; pharmacology ; fms-Like Tyrosine Kinase 3 ; genetics

OBJECTIVETo study the function and clinicopathological significance of RNA-29c (miR-29c) in the carcinogenesis and development of gastric cancer.

METHODSMicroRNA microarray was applied to assess the miRNAs expression profile of gastric cancer. Quantitative real-time PCR was used to detect the expression of miR-29c in 64 cases of gastric cancer tissues and corresponding normal gastric epithelium, as well as cell lines GES-1, BGC-823 and SGC-7901 cells. MTT assay and flow cytometry were applied to detect the effects of forced expression of miR-29c in gastric cancer BGC-823 cells including cell proliferation, apoptosis, cell cycle and drug sensitivity. Quantitative real-time PCR, Western blot and luciferase reporter assay were used to explore the targeted relationship between miR-29c and myeloid cell leukemia-1 (Mcl-1).

RESULTSCompared with normal gastric epithelium, seven microRNAs (miR-374b*, miRPlus-E1212, miR-338-5p, miR-297, miR-21, miR-135b, miR-18a) were significantly up-regulated more than 2-folds, and nine microRNAs (miR-29b-2*, miR-1260, miRPlus-E1241, miR-S1-5p, miR-148a, miR-29c, miR-647, miR-196b*, ebv-miR-BART5) were significantly down-reguated in gastric cancer tissues. The average expression level of miR-29c in gastric cancer tissues was 0.70 ± 0.34 and in corresponding normal epithelium was 1.00 ± 0.06 (P < 0.05). miR-29c expression was related to tumor size, lymph node metastasis, clinical stage, Laurén classification, Borrmann classification and Ming classification (P < 0.05). The poorer differentiation degree of gastric cell lines, the lower was miR-29c expression level (P < 0.05). Overexpression of miR-29c in gastric cancer BGC-823 cells suppressed cell proliferation, stimulated cell apoptosis, induced cell cycle arrest in S phase and increased the chemotherapy sensitivity to drug docetaxel (all were P < 0.05). The average expression level of Mcl-1 mRNA in gastric cancer tissues was 3.47 ± 1.34 and corresponding epithelialium was 1.00 ± 0.20 (P < 0.05). The expression level of miR-29c was negatively related with that of Mcl-1 mRNA in gastric cancer tissues. miR-29c directly targeted to regulation of Mcl-1 expression.

CONCLUSIONSThere are special miRNA expression profile in gastric cancer. The expression of miR-29c is closely related to biological behavior of human gastric cancer. miR-29c is involved in targeted regulation of Mcl-1, and may be one of mechanisms of the carcinogenesis of gastric cancer.

Antineoplastic Agents ; therapeutic use ; Apoptosis ; Cell Cycle Checkpoints ; Cell Differentiation ; Cell Line, Tumor ; Cell Proliferation ; Drug Resistance, Neoplasm ; Gene Expression Regulation, Neoplastic ; Humans ; Lymphatic Metastasis ; MicroRNAs ; genetics ; metabolism ; Microarray Analysis ; Myeloid Cell Leukemia Sequence 1 Protein ; genetics ; metabolism ; Neoplasm Staging ; RNA, Messenger ; metabolism ; Stomach Neoplasms ; drug therapy ; genetics ; metabolism ; pathology ; Taxoids ; therapeutic use ; Transcriptome ; Tumor Burden

Animals ; Carcinogenesis ; Cell Cycle Proteins ; genetics ; metabolism ; Cyclin E ; metabolism ; F-Box Proteins ; genetics ; metabolism ; F-Box-WD Repeat-Containing Protein 7 ; Gene Silencing ; Genes, Tumor Suppressor ; Humans ; Mutation ; Myeloid Cell Leukemia Sequence 1 Protein ; metabolism ; Neoplasms ; genetics ; metabolism ; pathology ; Proto-Oncogene Proteins c-myc ; metabolism ; Receptors, Notch ; metabolism ; Signal Transduction ; TOR Serine-Threonine Kinases ; metabolism ; Ubiquitin-Protein Ligases ; genetics ; metabolism

OBJECTIVETo detect the expression of phosphorylated-signal transducer and activator of transcription 3 (p-Stat3) and myeloid leukemia-1 (Mcl-1) as well as their correlation, and to investigate the functional role of Stat3 and Mcl-1 in the pathogenesis of esophageal squamous cell carcinoma (ESCC).

METHODSStat3 activity in ESCC cells was inhibited with JAK/Stat3 inhibitors (AG490 or JSI-124). Specific siRNA was used to inhibit the Stat3 expression. Cell apoptosis was detected by flow cytometry. Expression of Mcl-1 protein was determined by Western blotting. Expression of phospho-Stat3 (Tyr705) and myeloid leukemia-1 (Mcl-1) proteins in ESCC tissues was detected by tissue microarray and immunohistochemistry. The relationship between p-Stat3 or Mcl-1 aberrant expression and clinicopatholohical features of ESCC was analyzed. The correlation of their expression was also analyzed.

RESULTSSuppression of the Stat3 signaling activation in ESCC cells led to marked apoptosis, and dramatic reduction of Mcl-1 protein. The positive rate of phospho-Stat3 (Tyr705) expression was 45.0% in 50/111 of the ESCC tissue samples. The lower the degree of tumor differentiation, the higher the positive rate of phospho-Stat3 (Tyr705), showing a significant difference (P = 0.018). The positive rate of Mcl-1 protein expression was 72.1% (80/111), and the lower the degree of tumor differentiation was, the higher there was the positive rate of Mcl-1, with a significant difference (P = 0.026). There was a positive correlation between the expressions of p-Stat3 and Mcl-1 proteins (P = 0.012).

CONCLUSIONSIn a subset of ESCC tissues, p-Stat3 (Tyr705) and Mcl-1 are overexpressed and positively correlated with each other, and both are correlated with tumor differentiation. Persistent activation of Stat3 contributes to apoptotic resistance in ESCC cells, and may be at least partly mediated through upregulation of Mcl-1.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Differentiation ; Cell Line, Tumor ; Cell Survival ; drug effects ; Esophageal Neoplasms ; metabolism ; pathology ; Humans ; Myeloid Cell Leukemia Sequence 1 Protein ; metabolism ; Neoplasm Grading ; Neoplasm Staging ; Phosphorylation ; RNA, Small Interfering ; genetics ; STAT3 Transcription Factor ; antagonists & inhibitors ; genetics ; metabolism ; Tyrphostins ; pharmacology

This study was aimed to explore the expression level and correlation of MCL-1 and miR-29a in extranodal NK/T-cell lymphoma (ENKTCL) tissue. Maxvision immunohistochemistry technique and real time fluorescent quantitative PCR were used to detect the expression level of MCL-1 and miR-29a in tissue of 20 patients with ENKTCL and 10 patients with proliferative lymphadenitis, respectively. The results showed that the expression of MCL-1 protein were higher in patients with ENKTCL than that in patients with proliferative lymphadenitis, but there were no significant correlation between MCL-1 overexpression and age, sex, Ann Arbor stage and International Prognostic Index (IPI), respectively. Correlation analysis indicated that there was significant negative correlation between miR-29a expression and MCL-1 expression (r = -0.59, P = 0.016). It is concluded that miR-29a may target MCL-1 gene, regulate its expression, then participate in tumorigenesis and development of ENKTCL.
Adolescent ; Adult ; Aged ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Lymphoma, Extranodal NK-T-Cell ; genetics ; pathology ; Male ; MicroRNAs ; genetics ; Middle Aged ; Myeloid Cell Leukemia Sequence 1 Protein ; genetics ; Young Adult