Expression of c-FLIPL in Leukemia and Its Clinical Significance / 中国医科大学学报

Objective To investigate the expression of c-FLIPL in leukemia and explore its clinical significance. Methods The expression level of c-FLIPL mRNA in bone marrow mononuclear cells was determined by real-time polymerase chain reaction(PCR)in 103 leukemia patients with different types of leukemia,including 54 cases of acute lymphocytic leukemia(ALL)with 37 newly diagnosed,5 relapsed,and 12 complete remis-sion,38 cases of acute myeloid leukemia(AML)with 24 newly diagnosed,6 relapsed,and 8 complete remission,newly diagnosed 2 cases of acute undifferentiated leukemia(AUL),6 cases of chronic myelocytic leukemia(CML),and 3 cases of chronic myelomonocytic leukemia(CM-ML). The immunophenotype of patients were detected by flow cytometry. Results Expression level of c-FLIPL mRNA was higher in newly diag-nosed and relapsed leukemia patients. There was no significant difference between newly diagnosed and relapsed leukemia patients(P>0.05). Ex-pression level of c-FLIPL mRNA of AUL and CML was higher than that in other patients ,but there was no significant difference(P>0.05). Ex-pression level of c-FLIPL mRNA of all newly diagnosed and relapsed leukemia patients was significantly higher than that in control group and com-plete remission group(P<0.05). The expression level of c-FLIPL mRNA was correlated with risk stratification ,white blood cell(WBC),lactate dehydrogenase(LDH),hydroxybutyrate dehydrogenase(HBDH),CD45 and TEL-AML1,but was not associated with age,sex,fibrinogen and chromosome abnormality. Conclusion c-FLIPL mRNA is highly expressed in leukemia patients ,and is closely related with risk stratification , WBC,LDH,HBDH and prognosis.

Effects of c-FLIPL knockdown in cervical uterine carcinoma cell lines / Efectos de la inhibición de C-Flipl en líneas celulares de cáncer de cuello uterino

Overexpression of Short and Raji variants of Cellular FLICE-like inhibitory protein (c-FLIP) is capable of inhibiting apoptosis, while the function of the Long isoform depends of c-FLIPL concentration in cells. The aim of this study was to determine the effects of c-FLIPL knockdown in cervical cell lines. SiHa, C-4I and C-33A cervical cancer cell lines were analyzed. c-FLIPL level expression was determined by quantitative real-time PCR and western blotting. c-FLIPL was transiently downregulated by siRNA. The effects of knockdown of c-FLIPL on cell viability, proliferation and apoptosis were assessed by comparing with scrambled siRNA-transfected cells. SiHa and C-4I c-FLIPL knockdown cells showed increased viability compared with scrambled siRNA-transfected cells (P<0.05), while C-33A cells did not show significant differences. Ki-67 and PCNA immunocytochemistry was performed to evaluate proliferation on these cervical cancer cell lines. SiHa cells with c-FLIPL knockdown showed elevated expression of Ki-67 protein compared with their scrambled counterparts (P<0.0001), while C-33A c-FLIPL knockdown cells showed a significantly lower in PCNA expression (P<0.01) compared with control. All three c-FLIP-transfected cell lines showed a higher level of apoptosis compared with their scrambled controls. Our results suggest that c-FLIPL could have effects in proliferation and apoptosis in cervical cancer cell lines.

Cuando las variantes Short y Raji de la proteína Cellular FLICE-like inhibitory protein (c-FLIP) se encuentran sobrexpresadas son capaces de inhibir la apoptosis, mientras la función de la isoforma Long (c-FLIPL), depende de la concentración de esta molécula en las células. El objetivo de este estudio fue determinar los efectos de la inhibición de c-FLIPL en líneas celulares de cáncer de cuello uterino. Para realizar el estudio fueron utilizadas SiHa, C-4I y C-33A, líneas celulares de cáncer cervical. La expresión de c-FLIPL en estas líneas fue establecida mediante PCR en tiempo real y western blot. Posteriormente la expresión de c-FLIPL fue inhibida, mediante transfeción transiente con siRNA complementario al mRNA mensajero de c.-FLIPL. Los efectos de esta inhibición en la viabilidad celular, proliferación y apoptosis fue comparada con células transfectadas con un siRNA control (scrambled). Una vez reprimido c-FLIPL, las líneas celulares SiHa y C-4I presentaron un aumento de la viabilidad celular (P<0,05). Para evaluar la proliferación celular se utilizó inmunocitoquímica de los marcadores Ki-67 y PCNA. Las células SiHa transfectadas con siRNA c-FLIPL, mostraron una elevada expresión de Ki-67 (P<0,0001), mientras que las células C-33A con c-FLIPL inhibido mostraron una menor expresión de PCNA (P<0,01). Las tres líneas celulares con c-FLIPL reprimido mostraron un mayor nivel de apoptosis que las células control. Estos resultados sugieren que c-FLIPL puede tener efectos en la proliferación y apoptosis de líneas celulares de cáncer de cuello uterino.

The Histone Deacetylase Inhibitor Trichostatin A Sensitizes Human Renal Carcinoma Cells to TRAIL-Induced Apoptosis through Down-Regulation of c-FLIP(L)

Histone acetylation plays a critical role in the regulation of transcription by altering the structure of chromatin, and it may influence the resistance of some tumor cells to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) by regulating the gene expression of components of the TRAIL signaling pathway. In this study, we investigated the effects and molecular mechanisms of trichostatin A (TSA), a histone deacetylase inhibitor, in sensitizing TRAIL-induced apoptosis in Caki human renal carcinoma cells. Our results indicate that nontoxic concentrations of TSA substantially enhance TRAIL-induced apoptosis compared with treatment with either agent alone. Cotreatment with TSA and TRAIL effectively induced cleavage of Bid and loss of mitochondrial membrane potential (MMP), which was associated with the activation of caspases (-3, -8, and -9) and degradation of poly (ADP-ribose) polymerase (PARP), contributing toward the sensitization to TRAIL. Combined treatment with TSA and TRAIL significantly reduced the levels of the cellular Fas-associated death domain (FADD)-like interleukin-1beta-converting enzyme (FLICE) inhibitory protein (c-FLIP), whereas those of death receptor (DR) 4, DR5, and FADD remained unchanged. The synergistic effect of TAS and TRAIL was perfectly attenuated in c-FLIP(L)-overexpressing Caki cells. Taken together, the present study demonstrates that down-regulation of c-FLIP contributes to TSA-facilitated TRAIL-induced apoptosis, amplifying the death receptor, as well as mitochondria-mediated apoptotic signaling pathways.

Expression of cellular FLICE-like inhibitory protein long form mRNA in colon cancer and its clinical significance / 第二军医大学学报

Objective: To observe the expression of cellular FLICE-like inhibitory protein long form (cFLIPL) mRNA in colon cancer and to assess its relationship with the biological behavior and prognosis of colon cancer. Methods: Expression of cFLIPL mRNA was examined in 86 colon cancer samples and their corresponding normal tissues by semi-quantitative RT-PCR technique. Expression of CEA and P53 in the colon cancer samples was assayed by SABC immunohistochemistry staining. The plasma levels of CEA and CA19-9 were determined by microparticle enzyme-linked immunoassay (MEIA) before operation. Colon cancer tissues were divided into cFLIPL mRNA high expression group and low expression group based on the average value of all the 86 patients. The relationship between the high expression of cFLIPL mRNA and the different clinical features and pathological characters was determined. We also compared the 5-year accumulative survival rates of the 2 groups. Results: The expression of cFLIPL mRNA in the colon cancer tissues was significantly higher than that in the matched normal tissues (0.59±0.10 vs 0.36±0.10, P

Roles of cFLIPL in Fas-mediated Apoptosis of Colon Cancer

PURPOSE: Substantial numbers of the colon cancer cells have been observed to express Fas/Fas ligands, but are resistant to Fas-mediated apoptosis, suggesting that colon tumors might develop the specific mechanisms to overcome Fas-mediated apoptosis. Recently, cellular FLICE-like inhibitory protein (cFLIP) has been identified as an endogenous inhibitor of Fas- or other receptor-mediated apoptosis and its altered high expression has been suspected to be associated with tumor development or progression. This study investigated the prevalence of cFLIPL alterations in colon carcinomas and possible implications in the progression of colon cancers. METHODS: In order to investigate the function of cFLIPL for the development and progression of the colon cancer, we analyzed the expression of the cFLIPL in 58 colon cancer and 6 colon cancer cell lines. RESULTS: The study results demonstrated that cFLIPL is one of the major determinants for sensitivity to Fas-mediated apoptosis in colon cancer cell lines. In addition, we analyzed cFLIPL expression in 58 cases of adenocarcinoma of the colon and showed that cFLIPL, expression was increased in adenocarcinoma as compared to matched normal mucosa. CONCLUSION: Taken together, our data strongly suggested that abnormal overexpression of cFLIPL is a frequent event in colon carcinomas and might contribute to in vivo tumor transformation.