RESUMO
Simultaneous use of cisplatin (CIS) and gemcitabine (GEN) for treating bladder cancer has increased because of their complementary effects. However, the molecular mechanisms underlying the activities of these two antineoplastic drugs are not fully known. Here, molecular biology techniques and microscopy were used to investigate transcriptomic and morphological changes in low and high-grade urinary bladder transitional carcinoma cell lines [RT4 - wild type TP53; 5637 - two TP53 mutations, one in codon 72 (Arg-Pro) and other in codon 280 (Arg-Thr) and T24 - in-frame deletion of tyrosine 126 in the TP53 allele] simultaneously treated with CIS/GEN. Gene expression profile was evaluated by PCR arrays; cell morphology by scanning and transmission electron microscopy, and apoptosis was analyzed using fluorescent dye. Results showed concomitantly upregulation of CDKN2B (G1/S transition), GADD45A (DNA repair and apoptosis) and SERTAD1 (regulation of transcription) gene, increased number of nuclear chamfers and apoptotic cells, and reduced number of microfilaments, organelles and in the size of the nucleus in 5637 and T24 cells after simultaneous treatment with CIS/GEN. In conclusion, independently of the TP53 mutation status and tumor grade, CIS/GEN induced gene modulation accompanied by changes in cell morphologies, which confirm the antiproliferative activity of the treatment protocol. These findings help to understand the pathways modulated by these antineoplastic agents and may provide insights for anti-cancer chemotherapy.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma de Células de Transição/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias da Bexiga Urinária/patologia , Apoptose/efeitos dos fármacos , Carcinoma de Células de Transição/metabolismo , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cisplatino/farmacologia , Inibidor de Quinase Dependente de Ciclina p15/biossíntese , Inibidor de Quinase Dependente de Ciclina p15/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Humanos , Mutação , Proteínas Nucleares/biossíntese , Proteínas Nucleares/efeitos dos fármacos , Transativadores/biossíntese , Transativadores/efeitos dos fármacos , Fatores de Transcrição , Proteína Supressora de Tumor p53/genética , Regulação para Cima , Neoplasias da Bexiga Urinária/metabolismo , GencitabinaRESUMO
We evaluated the influence of heme oxygenase-1 (HO-1) gene inhibition in myelodysplastic syndrome (MDS) cell line SKM-1 on enhancement of the demethylating effects of decitabine on p15, and explored the possible mechanism. DNMT1 gene expression in SKM-1 cells was silenced by being transfected by a constructed siRNA with liposomes. The proliferation inhibition rates after drug treatment were detected by cell counting kit-8 assay. The apoptotic rates were detected by Annexin V/PI assay with flow cytometry. The expressions of p16, p15, TP73, CDH1, ESR1, and PDLIM4 mRNAs were detected by real-time PCR, and those of HO-1, DNMT1, DNMT3A, DNMT3B, HDAC, and p15 proteins were measured by western blot. The degree of methylation of the p15 gene was analyzed by using methylation-specific PCR (MSP). CCK-8 assay showed that after HO-1 gene expression was inhibited; the proliferation rate of SKM-1 cells treated by decitabine (70.91 ± 0.05%) was significantly higher than that of the control group (53.67 ± 0.05%). Flow cytometry showed that the apoptotic rate of SKM- 1 cells treated by decitabine in combination with HO-1 expression inhibition (44.25 ± 0.05%) exceeded that of the cells treated by this drug alone (37.70 ± 0.05%). MSP showed that inhibiting HO-1 expression significantly increased the degree of methylation of the p15 gene. As suggested by western blot, the degree of methylation of the p15 protein was changed after decitabine treatment when the expression of the HO-1 protein was changed, being associated with the affected DNMT1 expression. Inhibited HO-1 expression attenuated the hypermethylation of CDKN2B by suppressing DNMT1, which was conducive to treatment by cooperating with decitabine. In conclusion, the findings of this study provide valuable experimental evidence for targeted MDS therapy, and a theoretical basis for further studies.
Assuntos
Inibidor de Quinase Dependente de Ciclina p15/genética , DNA (Citosina-5-)-Metiltransferases/biossíntese , Heme Oxigenase-1/biossíntese , Síndromes Mielodisplásicas/tratamento farmacológico , Apoptose/efeitos dos fármacos , Azacitidina/administração & dosagem , Azacitidina/análogos & derivados , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p15/biossíntese , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , Decitabina , Regulação Neoplásica da Expressão Gênica , Heme Oxigenase-1/genética , Humanos , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Proteínas de Neoplasias/genética , RNA Interferente Pequeno/genéticaRESUMO
The aim of this study was to investigate the effect of the p15 gene combined with Bcr-abl-specific siRNA and STI571 on the proliferation, cell cycle and apoptosis of K562 chronic myeloid leukemia cells. Using the gene sequence results, we amplified the p15 gene from normal peripheral blood by RT-PCR, and constructed a p15-pcDNA3.1 vector. The K562 cell line with G418 resistance was screened, synthesized and transfected for bcr-abl gene fusion point for 21-nt siRNA. In p15-pcDNA3.1-K562 cells, the growth rate was slower than that of the control K562 cells, G0/G1-phase was increased and S-phase was decreased significantly. In the siRNA group, bcr-abl fusion gene expression was significantly decreased in K562 cells accompanied by the downregulation of BCL-xL protein expression and G1-phase arrest. Cell survival rate was significantly decreased compared with the sole p15-K562 cell group and the sole RNA interference-K562 cell group. In the combination of p15-pcDNA3.1-K562 cells with STI571, the proportion of apoptosis was significantly increased and the cell survival rate was significantly decreased compared with the p15-K562 cell group and STI571-K562 cell group. siRNA at 30 pM combined with 0.5 µM STI571 promoted apoptosis compared with sole application. The p15 gene combined with siRNA had a synergistic effect on the inhibition of proliferation and the induction of apoptosis in K562 cells. Exogenous p15 protein expression combined with STI571 appeared to have a synergistic effect on proliferation inhibition and apoptosis induction in K562 cells. The combination of low-dose RNA interference with STI571 showed a synergistic effect in inducing apoptosis.