Using folic acid-modified polyethylenimine-SPIO nanoparticles for PD-L1 siRNA delivery to target gastric cancer / 中国病理生理杂志

AIM: To synthesis and characterize a multi-functional siRNA delivery agent with effective thera-peutic effects and MR-tracing ability for programmed death ligand-1 ( PD-L1 ) positive gastric cancer SGC-7901 cell line . METHODS:The characterization , binding ability , cytotoxicity , transfection efficiency and cellular internalization of the polyplex were determined .The PD-L1 knockdown effect was analyzed , and cytokines secreted by cocultured T cells were measured.RESULTS:We developed folic acid (FA)-PEG-SS-PEI-SPION as siRNA delivery agent for PD-L1 knockdown. At N/P ratio of 10, the FA-PEG-SS-PEI-SPION bound PD-L1 siRNA to form polyplex in a diameter of (116.7 ±2.5) nm with zeta potential of (9.14 ±0.80) mV.Transfection efficiency of the targeted polyplex was (95.06 ±0.44)%, com-pared with ( 93.87 ±1.05 )% of the untargeted polyplex .Mean fluorescence intensity of the targeted polyplex was 1892.67 ±81.51, significantly higher than 1324.33 ±186.58 of the untargeted.The cellular magnetic resonance (MR) imaging showed the polyplex also acted as T 2 weighted contrast agent for cancer MR imaging .The relative mRNA level of PD-L1 in polymer/siRNA-2 treatment group was (9.07 ±0.79)%.Decreased protein expression of PD-L1 was showed by Western blot .The secretion levels of IFN-γand TNF-αin cocultured T cells increased , while that of IL-10 decreased . CONCLUSION:Our findings highlighted the potential of the multifunctional theranostic nanoparticles for effective targe -ting PD-L1 knockdown therapy and MR imaging diagnosis in gastric cancers .

Inhibitory effect of a novel histone deacetylases inhibitor FK228 on human colon cancer HCT-116 cells in vitro and in vivo / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the inhibitory effects of a novel histone deacetylases inhibitor FK228 on human colon cancer HCT-116 cells in vitro and in vivo, and evaluate its toxicity and side effects.</p><p><b>METHODS</b>The in vitro growth inhibitions of HCT-116 cells by different concentrations of FK228 and 5-Fu for 24, 48 and 72 h were assessed by CCK-8 assay. BALB/c nude mouse models of tumor xenografts were prepared by subcutaneous implantation of tumor tissue, and 4 mg/kg FK228 and 50 mg/kg 5-Fu were i.p. injected, respectively. The inhibitory effects on tumor growth, hematology, and liver and kidney function were evaluated.</p><p><b>RESULTS</b>CCK-8 assay indicated that FK228 had an obvious growth inhibitory effect on HCT-116 cells in a dose- and time-dependent manner. The IC50 of FK228 in HCT-116 cells was 12.05 ng/ml for 48 h, while the IC50 of 5-Fu was 18.92 µg/ml. At 20 days after FK228 and 5-Fu treatment, the tumor volume of the FK228 group was (139.71 ± 44.54)mm(3), significantly lower than that of the 5-Fu group [(282.28 ± 58.81)mm(3)] and that of the model group [(520.65 ± 39.73)mm(3), P < 0.01 for both]. The average tumor weight was (0.07 ± 0.02)g in the FK228 group, significantly lower than that of the 5-Fu group [(0.20 ± 0.08)g, P < 0.01]. The tumor growth inhibition rate of the FK228 group was 73.2%, significantly higher than that of the 5-Fu group (45.8%, P < 0.01). The ALT levels of the FK228 and 5-Fu groups were significantly higher than that of the model group (P < 0.01). The BUN of 5-Fu group was significantly higher than that of the model group (P < 0.01), but the BUN of FK228 group was not significantly different from that of the blank and control groups (P > 0.05 for both). Routine blood test showed that WBC, RBC, Hb and PLT of the 5-Fu group were significantly lower than those of the model group (P < 0.05 for all), but only WBC of the FK228 group was significantly lower than that of the model group (P < 0.05). The pathological examination using HE staining revealed that in the FK228 group, there were fibrosis and inflammatory cell infiltration in the liver tissue, and mild edema of the renal tubules in the kidney. However, in the 5-Fu group there were extensive hepatocyte edema and necrosis in the liver, and evident deformation and necrosis of glomeruli and tubules, and tubular wall thinning in the kidney.</p><p><b>CONCLUSIONS</b>The results of this study indicate that FK228 can more effectively than 5-Fu inhibit the growth of HCT-116 cells in vitro and vivo, and without obvious toxic effect on the kidney and hematology. Its clinical value in colon cancer treatment deserves further investigation.</p>

Cloning of human migfilin N-terminal domain and preparation of anti-migfilin polyclonal antibody / 南方医科大学学报

<p><b>OBJECTIVE</b>To clone migfilin-N terminal sequence into E.coli and obtain a fusion protein for preparing rabbit polyclonal antibody against migfilin, thereby facilitating the study of the role of migfilin in the biological behavior of colon cancer.</p><p><b>METHODS</b>Based on human migfilin cDNA sequence, a pair of primers was designed to amplify migfilin-N terminal sequence by PCR. The PCR product was subcloned into the bacterial expression vector pGEX-4T-1 with EcoRI/XhoI sites, and the target recombinant plasmids were identified with enzymatic cleavage followed by DNA sequence analysis. By transforming the expression vector into component E.coli BL(21) cells, the GST-migfilin-N fusion protein was expressed with IPTG induction. Glutathione-sepharose beads were used to purify the fusion protein, and anti-migfilin polyclonal antibody was produced by immunization of rabbits with the purified GST-migfilin N-terminal fusion protein. The resultant anti-migfilin polyclonal antibody was purified by protein A beads and used for Western blotting for detecting migfilin expression in different cell lines.</p><p><b>RESULTS AND CONCLUSION</b>The migfilin-N terminal gene fragment was cloned successfully, and purified GST-migfilin N-terminal fusion protein and anti-rabbit migfilin polyclonal antibodies were obtained. Western blot analysis demonstrates that the antibodies specifically detected migfilin expression in the cell lines, which may facilitate further investigation of the role of migfilin in the biology of colon cancer.</p>