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Objective To study the effects and mechanisms of molecular targeted drug combination on multi-driven proliferation hepatocellular carcinoma SK-Hep-1 cells. Methods Four molecular targeted drugs (HG6-64-1, Dasatinib, Crizotinib, and Sunitinib) were used to treat SK-Hep-1 cells, and the monophasic kinetic analysis curve and two-phase analysis curve were drawn. Western blot analysis was used to detect the effects of the above drugs on key signaling pathways in SK-Hep-1 cells. MTT assay was used to detect the effects of the above drugs and their combination on the proliferation of SK-Hep-1 cells. Results Compared with the monophasic kinetic analysis curve, the biphase analysis curve could better fit the effects of molecular targeted drugs on SK-Hep-1 cells, which predicted that the combination of HG6-64-1, Dasatinib, and MK-2206 could effectively inhibit the proliferation of SK-Hep-1 cells. Conclusion Two-phase kinetic analysis can quantitatively describe the response of multi-driven proliferation hepatocellular carcinoma SK-Hep-1 cells to molecular targeted therapy. The combination of HG6-64-1, Dasatinib, and MK-2206 is a potential drug combination for the treatment of hepatocellular carcinoma.
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BACKGROUND:A group of nuclease-like proteins were previously purified from Eisenia foetida tissues, exploring primary structures of these proteins wil help to uncover basic structure characteristics of them and provide foundations for the study addressing the relationship of their structures and functions. OBJECTIVE:To explore primary structures of nuclease-like proteins EWD1 and EWD2. METHODS:Edman degradation method was used to sequence the N-terminal amino acids of EWD1 and EWD2, acid hydrolisis method was used to analyze amino acid compositions of EWD1 and EWD2, LC-MS/MS was used to analyze some peptide sequences within the proteins, and MALDI-TOF-MS was used to calculate the number of the disulfide bonds and the contents of polysaccharides. RESULTS AND CONCLUSION:Among the amino acid compositions in EWD1 and EWD2, the sum contents of aspartate and asparagines were the highest (al nearly 10%), the contents of hydrophobic amino acids were also high, and the contents of cysteine was low. The EWD1 and EWD2 had similar amino acid compositions with other nucleases. Edman degradation results showed that, the N-terminal sequences of the large subunit of EWD1 were in turn as fol ows:D, E, W, V, Y, P;the N-terminal sequences of EWD2 were as fol ows:L, L, G, P, Y, K, P, K, C. The results of LC-MS/MS indicated the two proteins were novel proteins;MALDI-TOF-MS results showed that 8 cysteine residues formed 4 disulfide bonds in EWD1, 6 cysteine residues formed 3 disulfide bonds in EWD2. EWD1 and EWD2 were al glycoproteins, the content of polysaccharides was 17.3%in EWD1 and 15.6%in EWD2.
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<p><b>BACKGROUND</b>Herpes simplex virus thymidine kinase phosphorylates ganciclovir to ganciclovir monophosphate, which is then converted to ganciclovir triphosphate by endogenous cellular nucleoside kinases. The ganciclovir triphosphate acts as a DNA chain terminator due to the lack of a functional 3'-OH group and terminates the process of DNA replication, hence leading to cell apoptosis. At present, HSVtk gene usually acts as suicide gene to kill tumor cells. The aim of this study was to investigate the selective cytotoxicity of the herpes simplex virus thymidine kinase/ganciclovir (HSVtK/GCV) suicide gene system controlled by the a-fetoprotein (AFP) promoter on hepatocellular carcinoma (HCC) cells in vitro.</p><p><b>METHODS</b>pAFP-HSVtk-IRES2-EGFP recombinant plasmid vectors driven by the AFP promoter were constructed. HL-7702 liver cells, HUH-7 HCC, and HepG2 HCC were transfected with the recombinant plasmids. HSVtK gene expression was detected using Western blotting analysis. HepG2 cells line stably expressing HSVtk gene was selected by G418 reagent. The cytotoxicity of HSVtK/GCV suicide gene system on hepatoma cells was measured by CCK-8 reagents when different doses of ganciclovir were added.</p><p><b>RESULTS</b>Plasmid pAFP-TK-IRES2-EGFP-expressed HSVtk gene was constructed successfully. HSVtk gene expression level was significantly higher in AFP-positive hepatoma cells than in AFP-negative liver cells. After G418 selection, a HepG2 cells line stably expressing HSVtk gene was acquired. With the increase of the dose of ganciclovir the optical density at 450 nm of HepG2 cells stably expressing HSVtk gene gradually decreased (P < 0.05).</p><p><b>CONCLUSION</b>The HSVtK gene-specific expression in hepatoma cells as well as the cytotoxicity of the suicide gene system in HepG2 cells provided the basis for the targeted gene therapy of HCC.</p>
Subject(s)
Humans , Apoptosis , Genetics , Carcinoma, Hepatocellular , Genetics , Cell Line, Tumor , Fetal Proteins , Genetics , Ganciclovir , Pharmacology , Hep G2 Cells , Liver Neoplasms , Genetics , Promoter Regions, Genetic , Genetics , TransfectionABSTRACT
Humanin (HN, its analogue [Gly14]-Humanin, HNG) was originally identified as an endogenous peptide that protects neuronal cells from apoptosis induced by various types of Alzheimer's disease-related insults. But the relative low content of this peptide in its natural sources limits its further characterization. An expression vector pET32a/HNG was corstructed and transformed it into E. coli BL21 trxB (DE3). HNG was expressed as a fusion protein in the soluble fraction and was purified by nickel affinity chromatography. Subsequently, the purified fusion protein was cleaved by enterokinase and was further purified by reverse-phase HPLC. A 23 mg recombinant HNG (rHNG) from 1 L bacterial culture was purified. The molecular weight of rHNG determined by ESI-MS was 2876.5 Da which was the expected size for correctly processed peptide. The N-terminal amino acid sequence of rHNG determined by Edman degradation method is identical to the theoretical sequence. Neuroprotective bioassay studies of rHNG exhibited its potential neuroprotective effect comparable to that of the natural HNG peptide.