RESUMO
Hepatocellular carcinoma (HCC) is considered as a disease of dysfunction of the stem cells. Studies on stem cells have demonstrated that Oct4 plays a pivotal role in embryo regulation. In order to understand the role of Oct4 in HCC and the relationship among Oct4 and wnt/ß-catenin and TGF-ß signal pathways, we have detected the expression of Oct4, Nanog, Sox2, STAT3 as well as the genes in wnt/ß-catenin, and TGF-ß families in HCC cell lines and in tumor specimens from HCC patients. The authors found that Oct4 was expressed in all of the four HCC cell lines and the tumor specimens from HCC patients. Some other genes were also expressed in them with different level including Nanog, Sox2, STAT3 and TCF3, wnt10b, ß-catenin, ELF, Smad3 and Smad4. The ability of the clone formation and migration of the HepG2 decreased after Oct4 was knockdowned. Silencing of Oct4 and TCF3 in HCC cell line HepG2 revealed that there were complicated relationships among Oct4, wnt/ß-catenin family and TGF-ß family genes. Knockdowning Oct4 reduced the expression of TGF-ß family genes ELF, Smad3, Smad4 and wnt/ß-catenin family genes, wnt10b, and ß-catenin but increased TCF3. In reverse, knockdowning TCF3 led to the increased expression of Oct4 and TGF-ß family genes. In conclusion, the expression of Oct4 in HCC may play an important role as in stem cell. Because Oct4 improves not only the function of wnt/ß-catenin, but also the TGF-ß signal pathways, the significance of its expression in HCC might be more complicated than we evinced before.
Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Sequência de Bases , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Primers do DNA , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Hepáticas/patologia , Fator 3 de Transcrição de Octâmero/genética , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Cryotherapy has been shown to be an important therapeutic alternative to surgery in the treatment of hepatocellular carcinoma (HCC). Here, the influence of cryo-chemotherapy on HCC was examined in vitro using the human HCC cell line Bel-7402, a drug-resistant HCC cell line originating from Bel-7402 cells (Bel-7402/R), as well as two control cell lines, the HCC cell line SMMC-7721 and a colorectal tumor cell line HIC-251. Cells were treated with either exposure to different freezing temperatures (ranging from -15 to -80 degrees C for 20 min), exposure to sub-lethal concentrations of anticancer chemotherapy drugs or a combination of cryotherapy and chemotherapy. Cell viability and apoptosis under each condition were investigated. We found that the combined treatment resulted in increases in both cell death and apoptosis compared to either treatment alone. The increased level of apoptosis observed in Bel-7402 cells after cryo-chemotherapy was inhibited in the presence of caspase inhibitors. Furthermore, Bax expression was increased 2- to 3-fold in cells exposed to the combination treatment compared with cells treated by freezing or drugs alone. In contrast, Bcl-2 levels remained constant. Although Bel-7402/R cells originated from the Bel-7402 cell line, they were more sensitive to the freezing procedure than the parental cell line. The level of Bax expression in Bel-7402/R cells was also higher than that observed in the parental cell line. In addition, we found that Bel-7402/R cells had lower levels of survivin mRNA than the parental Bel-7402 cells, in both untreated and treated cells. In conclusion, our data show that in HCC cells, apoptosis induced by cryotherapy can be synergistically enhanced using anticancer drugs.
Assuntos
Antineoplásicos/uso terapêutico , Apoptose , Carcinoma Hepatocelular/terapia , Crioterapia , Neoplasias Hepáticas/terapia , Anexina A5/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Morte Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Terapia Combinada , Congelamento , Humanos , Proteínas Inibidoras de Apoptose , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , RNA Mensageiro/metabolismo , Survivina , Regulação para Cima , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
AIM: To investigate the cytotoxicity of the cytokine-induced killer (CIK) cells from the post-operation patients with primary hepatocellular carcinoma (HCC) to multidrug-resistant (MDR) cell of HCC both in vitro and in vivo. METHODS: A drug-resistant cell line was established by culturing human HCC cell line Bel-7402 in complete RPMI 1640 medium with increasing concentrations of adriamycin from 10 to 2,000 nmol/L. CIK cells were obtained by inducing the peripheral blood mononuclear cells with rhIFN-gamma, monoclonal anti-CD3 antibody, rhIL-1alpha as well as rhIL-2, which were added into the culture. To detect the cytotoxicity of the CIK cells from HCC patients, the Bel-7402/R was taken as target (T) cells and CIK cells as effect (E) cells. Cytotoxic test was performed and measured by MTT. As to in vivo test, CIK cells were transfused into patients with HCC. The tumor specimens of the patients were obtained and immunohistochemistry was carried out to detect CD3, CD45, CD45RO as well as CD68. RESULTS: A MDR 1 HCC cell line Bel-7402/R was established. Its MDR1 mRNA overexpressed which was shown by RT-PCR; the P-glycoprotein expression increased from 1.32% of parent cells to 54%. CIK cells expanded vigorously by more than 70-fold and the CD3+CD56+ increased by more than 600-fold after 3-wk incubation on average. The cytotoxicity of CIK from HCC patients to Bel-7402/R was about 50% and to L-02 below 10% (t = 8.87, P<0.01), the same as that of CIK from normal individuals. Each of the 17 patients received 1-5 x 10(10) of CIK cell transfusion. No side effects were observed. After CIK treatment, the tumor tissue nodules formed and a large amount of lymphocytes infiltrated in the liver cancer tissue and CD3, CD45, CD45RO, and CD68 increased greatly which was shown by immunohistochemistry. CONCLUSION: A stable MDR1 HCC cell line has been established which could recover from liquid nitrogen and CIK from HCC patients has strong cytotoxicity to MDR HCC cell. CIK adoptive immunotherapy is safe and has no side effects. Receivers improved their immunity to tumor evidently. CIK treatment may be a better choice for HCC patients after operation to prevent the recurrence, especially when tumors have developed drug resistance.