OBJECTIVE: To investigate the effect of SPARC gene overexpression on the chemotherapeutic sensitivity of AML-MDS cell line SKM-1 to Ara-C and to further explore its mechanism. METHODS: Subjects were divided into 6 groups: SKM-1 cells (Control), Negative control (LV-NC), SPARC overexpression (LV-SPARC), SKM-1 cells+30 ng/ml Ara-C (30 ng/ml Ara-C), LV-NC+30 ng/ml Ara-C and LV-SPARC+30 ng/ml Ara-C. Cell activity was detected by CCK-8 assay, cell cycle distribution and apoptosis were detected by flow cytometry, mRNA expression levels of SPARC, CPBP and MLKL were detected by RT-qPCR, and the expression levels of related protein were detected by Western blot. RESULTS: After co-treatment with SPARC overexpression and Ara-C, the cell viability decreased and apoptosis increased significantly, with obvious up-regulation of Bax and down-regulation of BCL-2 (P<0.05). Compared with the control group, the cell cycle of LV-SPARC+30 ng/ml Ara-C group was significantly arrested in S phase with obvious down-regulation of CDK2 and up-regulation of p27^KIP1 (P<0.05). Compared with LV-SPARC group and 30 ng/ml Ara-C group, the mRNA and protein expression levels of CPBP and MLKL (p-MLKL) were significantly elevated in LV-SPARC+30 ng/ml Ara-C group (P<0.05). In addition, after co-treatment with SPARC overexpression and Ara-C, the protein expression level of p-AKT decreased and the protein expression level of p53 increased (P<0.05). CONCLUSION SPARC overexpression enhanced the sensitivity of SKM-1 cells to Ara-C and promoted cell cycle arrest and apoptosis, the mechanism of which may be related to the regulation of CPBP/MLKL pathway.
Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Cytarabine ; Humans ; Kruppel-Like Factor 6/metabolism* ; Osteonectin/pharmacology* ; Protein Kinases/pharmacology* ; Proto-Oncogene Proteins c-akt/metabolism* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; RNA, Messenger ; Sincalide/pharmacology* ; Tumor Suppressor Protein p53 ; bcl-2-Associated X Protein/pharmacology*

OBJECTIVETo investigate the regulatory mechanism of the transcription of tumor metastasis suppressor gene TMSG-1.

METHODSLuciferase reporter assay and site-directed mutagenesis were used to analyze the regulatory region of TMSG-1. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) were carried out to verify the interaction of KLF6 and Sp1 with the regulatory region of TMSG-1. Co-immunoprecipitation (CoIP) was performed to analyze the interaction between KLF6 and Sp1. TMSG-1 and wt-KLF6 mRNA expressions in cells with different metastatic capacities were quantitated by real-time PCR. Cell invasive capability was determined by Matrigel invasion assay.

RESULTSA 63 bp inducible regulatory region (+59 bp - +123 bp) in exon 1 was identified by luciferase assay using reporter plasmids with a series of TMSG-1 regulatory region deletions. Mutations in KLF6/Sp1 binding sites of this region resulted in a decrease of luciferase activity, while cotransfection with KLF6 or Sp1 expressing plasmids led to a remarkable increase of luciferase activity. EMSA and ChIP demonstrated that KLF6 as well as Sp1 interacted with this region. CoIP also indicated a possible interaction between KLF6 and Sp1 proteins. In the highly metastatic cell sublines, a low level of wild type KLF6 was associated synchronously with a low TMSG-1 level. Prostate carcinoma cells overexpressing KLF6 exhibited a higher TMSG-1 level and a lower invasive capability.

CONCLUSIONSTranscription factor complex of KLF6 and Sp1 may participate in the inducible transcriptional regulation of TMSG-1, and a decreased wild type KLF6 expression is likely associated with a low TMSG-1 level in the highly metastatic cell sublines.

Binding Sites ; genetics ; Cell Line, Tumor ; Electrophoretic Mobility Shift Assay ; Humans ; Immunoprecipitation ; Kruppel-Like Factor 6 ; Kruppel-Like Transcription Factors ; genetics ; metabolism ; Lung Neoplasms ; metabolism ; pathology ; Male ; Membrane Proteins ; genetics ; metabolism ; Mutagenesis, Site-Directed ; Mutation ; Neoplasm Invasiveness ; Prostatic Neoplasms ; metabolism ; pathology ; Proto-Oncogene Proteins ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Sp1 Transcription Factor ; genetics ; metabolism ; Sphingosine N-Acyltransferase ; genetics ; metabolism ; Transcriptional Activation ; Transfection ; Tumor Suppressor Proteins ; genetics ; metabolism

OBJECTIVETo study the effects of rosiglitazone on Kruppule-like factor 6 (KLF6) and its target gene TGFâ1 during the development of nonalcoholic fatty liver fibrosis.

METHODThirty-six Wistar rats were divided into three groups: a control group, a high fat model group and an intervention group. Their blood serum TG, FFA, AST, ALT, HA, LN and CIV were measured. Hepatic expressions of KLF6, TGFbeta1 and alpha-SMA were detected by RT-PCR and immunohistochemistry. The pathological features and the degree of liver fibrosis before and after the rosiglitazone intervention were also studied.

RESULTSThe contents of TG, FFA, AST, ALT, HA, LN and CIV, the expression of KLF6, TGFbeta1 and alpha-SMA mRNA, and the degree (score) of liver fibrosis at the 24th week in the model group were significantly higher than those in the control group (P<0.01) but they were lower in the rosiglitazone intervention group (P<0.05). The protein expression of a-SMA was also lower in the intervention group compared with that of the model group.

CONCLUSIONRosiglitazone, to a certain extent, can inhibit KLF6-TGFbeta1 signal transduction by inducing expression of PPAR-gamma, and then inhibit the activation of hepatic stellate cells and minimize hepatic fibrosis.

Animals ; Fatty Liver ; metabolism ; pathology ; Kruppel-Like Factor 6 ; Kruppel-Like Transcription Factors ; metabolism ; Liver ; metabolism ; Male ; PPAR gamma ; metabolism ; Proto-Oncogene Proteins ; metabolism ; Rats ; Rats, Wistar ; Signal Transduction ; drug effects ; Thiazolidinediones ; pharmacology ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo observe the effect of KLF6 on prostate cancer cell line PC-3 by transgenic method.

METHODSWe obtained KLF6 cDNA by RT-PCR method from the liver cell, transfected plasmid pEGFP-C, recombinated with KLF6 into PC-3 cells, and used them as a transfection group and a control group. MTT, flow cytometer and immunocytochemical methods were used to observe the effect of anti-oncogene wild type KLF6 on prostate cancer cell line PC-3 by transgenic method for 48 hours.

RESULTSAfter transfected into PC-3 cells, KLF6 enhanced growth suppression, (30.0 +/- 5.4)% in the transfection group and 0% in the control, P < 0.01, apoptosis, (24.3 +/- 2.3)% in the transfection group and (5.2 +/- 0.7)% in the control, P < 0.01, the down-regulation of the expression of cyclin D1, (25.3 +/- 3.7)% in the transfection group and (38.5 +/- 4.6)% in the control, P < 0.05 and Bcl-2, (18.7 +/- 3.2)% in the transfection group, and (41.8 +/- 5.9)% in the control, P < 0.01 in PC-3 cells. It also decreased the ratio of the cell phase G2/M, increased the ratio of G0/G1 from (58.6 +/- 7.3)% in the control to (80.0 +/- 9.8)% in the transfection group, P < 0.05.

CONCLUSIONPC-3 cells transfected with wild type KLF6 can enhance its growth suppression and apoptosis. It shows great potential for the gene therapy of androgen-independent carcinoma of the prostate.

Apoptosis ; physiology ; Cell Cycle ; physiology ; Cell Line, Tumor ; Cyclin D1 ; biosynthesis ; Down-Regulation ; Flow Cytometry ; Humans ; Immunohistochemistry ; Kruppel-Like Factor 6 ; Kruppel-Like Transcription Factors ; genetics ; physiology ; Male ; Prostatic Neoplasms ; metabolism ; pathology ; Proto-Oncogene Proteins ; genetics ; physiology ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

BACKGROUND AND AIMThe Krüppel-like transcription factor KLF6 is a novel tumor-suppressor gene. It was inactivated in human prostate cancer and other tumors tissue, as the result of frequent mutation and loss of heterozygosity (LOH). However, there is no data reporting the levels of KLF6 both mRNA and protein in hepatocellular carcinomas (HCCs). We therefore detected mutations and expression of KLF6 in HCC tissues and further observed the effect of it on cell growth in HCC cell lines.

METHODSWe analyzed the exon-2 of KLF6 gene by direct DNA sequencing, and detected the expression of KLF6 by RT-PCR and Western blot in 23 HCC tissues and corresponding nontumorous tissues. Loss of growth suppressive effect of the HCC-derived KLF6 mutant was characterized by in vitro growth curves plotted, flow cytometry and Western blotting.

RESULTSKLF6 mutations were found in 2 of 23 HCC tissues and one of mutations was missense. Expression of KLF6 mRNA or protein was down-regulated in 8 (34.7%) or 9 (39.1%) of 23 HCC tissues. Wild-type KLF6 (wtKLF6) inhibited cellular proliferation and prolonged G1-S transition by inducing the expression of p21WAF1 following stable transfection into cultured HepG2 cells, but tumor-derived KLF6 mutant (mKLF6) had no effects.

CONCLUSIONOur findings suggest that KLF6 may be involved in pathogenesis of HCC.

Blotting, Western ; Carcinoma, Hepatocellular ; genetics ; pathology ; Cell Cycle ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Gene Expression Regulation, Neoplastic ; Humans ; Kruppel-Like Factor 6 ; Kruppel-Like Transcription Factors ; genetics ; Liver Neoplasms ; genetics ; pathology ; Mutation ; Proto-Oncogene Proteins ; genetics ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; Transfection

OBJECTIVETo investigate the expressions and clinical implications of kruppel-like factor 6(KLF-6) and APC in human colorectal carcinoma.

METHODSThe expressions of KLF-6 and APC in tumor and normal tissues from 32 patients with colorectal carcinoma were investigated by RT-PCR and immunohistochemical technique.

RESULTSThe expression rates of KLF-6 and APC mRNA were 37.5% and 34.3% in tumor tissue, 96.9% and 93.8% respectively in normal tissues (both P< 0.05). The expression rates of KLF-6 and APC protein were 28.1% and 25.0% in colorectal carcinomas, 81.3% and 84.43% respectively in normal tissues (both P< 0.05). There was a significant correlation between the expressions of KLF-6 and APC in colorectal carcinomas (P < 0.05). The expressions of KLF-6 and APC were significantly correlated with tumor differentiation, depth of infiltration, lymph node metastasis and clinical stage (P< 0.05).

CONCLUSIONDown-regulations of KLF-6 and APC might play an important role in the carcinogenesis, development, metastasis of human colorectal carcinoma.

Adenomatous Polyposis Coli Protein ; genetics ; Colorectal Neoplasms ; genetics ; metabolism ; pathology ; Female ; Gene Expression ; Genes, APC ; Humans ; Intestinal Mucosa ; metabolism ; pathology ; Kruppel-Like Factor 6 ; Kruppel-Like Transcription Factors ; genetics ; Male ; Middle Aged ; Neoplasm Staging ; Proto-Oncogene Proteins ; genetics ; RNA, Messenger ; genetics

To investigate the expression of KLF6mRNA in primary hepatocellular carcinoma (HCC), nomal liver tissues and the tissues adjacent to the cancers, reverse-transcription polymerase chain reaction (RT-PCR) was employed to investigate the expression of the KLF6 gene in HCC, the corresponding adjacent non-cancerous tissues and normal liver tissue. Our results showed that an amplified fragment of 427 bp DNA was detected in 18 of 19 (94.7%) adjacent non-cancerous tissues and normal liver tissue, and in 12 (85.7%) of 14 HCC. There were no significant differences in the levels of KLF6 mRNA between normal liver and liver tumors (P>0.05). It is concluded that KLF6 mRNA is generally expressed in HCC.
Carcinoma, Hepatocellular ; metabolism ; Humans ; Kruppel-Like Factor 6 ; Kruppel-Like Transcription Factors ; biosynthesis ; genetics ; Liver ; metabolism ; Liver Neoplasms ; metabolism ; Proto-Oncogene Proteins ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

Animals ; Cells, Cultured ; Cyclic AMP Response Element-Binding Protein ; metabolism ; Hepatocytes ; drug effects ; metabolism ; Humans ; Kruppel-Like Factor 6 ; Kruppel-Like Transcription Factors ; NF-kappa B ; metabolism ; Proto-Oncogene Proteins ; RNA, Messenger ; metabolism ; Trans-Activators ; genetics ; metabolism ; Transcription Factor AP-1 ; metabolism ; Transcription Factors ; pharmacology