RESUMEN
Objective: To explore new multidrug resistant genes of pancreatic cancer by establishment and characterization of chemo-resistant cell lines. Methods: The cisplatin-resistant cell line JF305/CDDP and the gemcitabine-resistant cell line PANC-1/GEM were induced by high-dose intermittent treatment. CCK-8 assay was used to detect the 50% inhibiting concentration (IC(50)), drug resistance index (R), cross-resistance, and growth difference of different cells. The changes of cell cycle and migration ability of drug-resistant cells were determined by flow cytometry and transwell assay, respectively. And then real-time fluorescence quantitative PCR was used to detect the expression of multidrug resistance-related genes. Results: The drug resistance indexes of JF305/CDDP and PANC-1/GEM were 15.3 and 27.31, respectively, and there was cross-resistance. Compared with the parental cells, the proliferation rate of JF305/CDDP was decreased by 40% on the fourth day (P<0.05); the proportion of S phase was decreased from (45±2)% to (30±2)% (P<0.05), and the migration ability was enhanced from (32 ±1) cells per field to (158±5) cells per field (P<0.01). The expression of multidrug resistance-related genes MRP2, MDR1, LRP and MSX2 was increased in JF305/CDDP cells (P<0.05). Knockdown of MSX2 in JF305 cells reduced the expression of MRP2, whereas overexpression of MSX2 in PANC-1 cells upregulated MRP2 level (P<0.05). Conclusions: Two stable multidrug resistant cell lines of pancreatic cancer, JF305/CDDP and PANC-1/GEM, were successfully established. MSX2 might be a new drug resistance related gene in pancreatic cancer cells by up-regulation of MRP2 expression.
Asunto(s)
Resistencia a Antineoplásicos/genética , Proteínas de Homeodominio/genética , Neoplasias Pancreáticas/genética , Antineoplásicos/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/farmacología , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Resistencia a Múltiples Medicamentos/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , GemcitabinaRESUMEN
This study aimed to examine anti-prostate cancer immune response induced by dendritic cells (DCs) transduced with PSMA/4-1BBL recombinant adenoviruses in vitro. Ad-PSMA, Ad-4-1BBL, and Ad-GFP were transfected into DCs derived from peripheral blood of healthy volunteers. Ad-PSMA/4-1BBL-DC, Ad-PSMA-DC, Ad-4-1BBL-DC, Ad-GFP-DC, and normal-DC, PSMA and 4-1BBL protein levels in DCs were detected by western blot. IL-12, IFN-γ and IL-10 were measured by ELISA. Mixed lymphocyte reaction and the cytotoxicity of each group targeted to LNCap, Du145, and 22RV prostate cancer cells were determined by CCK-8 assay. PSMA and 4-1BBL protein could express on DC successfully, the IL-12 supernatant content (134.29 ± 2.22 pg) was higher than others (P < 0.05). The ability to stimulate autologous T lymphocyte proliferation in the co-transfection group was higher than others (P < 0.05). When the DCs were co-cultured with CTLs, the PSMA/4-1BBL-DC-CTL group showed the highest content of IFN-γ (1176.10 ± 14.37pg/5 x 10(6) cells), but the lowest IL-10 content (75.14 ± 2.01 pg/5 x 10(6) cells) (P < 0.05), and the strongest anti-tumor effect when the effector to target ratio was 40:1, along with a higher killing ratio of LNCap cells than others (P < 0.05). Overall, Mature DCs transfected with Ad-PSMA/4- 1BBL not only showed high secretion of IL-12, but also induced CTLs to stimulate and enhance the killing effect of PSMA specific effector cells to PSMA positively expressing prostate cancer cells. Furthermore, the DCs infected with two kinds of tumor-associated antigens would induce more effective tumor-specific CTL induction.