ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of docosahexaenoic acid (DHA) on invasiveness of aflatoxin B1 (AFB1)-induced hepatocellular carcinoma cells in vitro.</p><p><b>METHODS</b>HepG2.2.15 cells were exposed to different concentrations of AFB1 and DHA plus AFB1. The cell migration and invasion were assessed using wound-healing and Transwell assay, and flow cytometry was used to analyze the cell cycle changes. The ultrastructural changes of the cells were observed by transmission electron microscopy.</p><p><b>RESULTS</b>Compared with the control group, the cells exposed to2 µmol/L AFB1 showed obviously enhanced migration and invasion with decreased cell ratio in G1/G1 phase and increased cell ratio in G2/M phase but no changes in S phase cells; transmission electron microscopy revealed the presence of multiple nucleoli and significantly increased mitochondria and Golgi apparatus in the exposed cells. Compared with AFB1-exposed cells, the cells treated with DHA and AFB1 showed decreased migration and invasion abilities, and the G1/G1 phase cells increased and G2/M phase cells decreased significantly; ultrastructurally, the cells contained single nucleoli with decreased mitochondria and vacuolization occurred in the cytoplasm.</p><p><b>CONCLUSION</b>DHA can significantly inhibit AFB1-induced enhancement of cell migration and invasion in hepatocellular carcinoma cells in vitro.</p>
Subject(s)
Humans , Aflatoxin B1 , Pharmacology , Carcinoma, Hepatocellular , Pathology , Cell Cycle , Cell Movement , Docosahexaenoic Acids , Pharmacology , Golgi Apparatus , Hep G2 Cells , Liver Neoplasms , Pathology , Mitochondria , Neoplasm InvasivenessABSTRACT
Objective To construct a recombinant adenovirus encoding the Akt gene and to investigate its influence on the survival of hepatic cancer patients. Methods The Akt-wt and Akt-dn genes were subcloned into pacAd5 CMVK-NpA plasmid separately, and then the adenoviral recombinant adenovirus encoding the Akt gene was constructed with the RAPAd® CMV Adenoviral Expression System and was identified by PCR. The expression of Akt, GSK3β and p-GSK3β protein was detected by Western blotting analysis. SK-HEP-1 cells were infected with pacAd5 CMV-Akt-wt, pacAd5 CMV-Akt-dn and pacAd5 CMV-GFP, respectively. The cell survival was observed under fluorescence microscope after 24 h-starvation. Results pacAd5 CMV-Akt-wt and pacAd5 CMV-Akt-dn were enzymatically digested into two fragments(6 kb and 1. 44 kb). PCR result of the viral supernatant yielded a band of about 1. 44 kb. Western blotting analysis showed p-GSK3β expression in the Akt-wt group was stronger than that in the Akt-dn group. The survival rate of the SK-HEP-1 cells significantly decreased in the Akt-dn group than in the Akt-wt group. Conclusion We have successfully constructed the recombinant adenoviruses pacAd5-Akt-wt and pacAd5-Akt-dn, and they have pro-death effect in hepatic cancer cells, which paves a way for further studying Akt gene and the related signal pathway in hepatocellular carcinoma.
ABSTRACT
<p><b>OBJECTIVE</b>To construct recombinant lentiviral vectors carrying Rheb gene and its mutant Rheb'D60K gene, and examine their expression in human liver cancer cells.</p><p><b>METHODS</b>Rheb gene was amplified by PCR to construct the recombinant plasmid LV31-Rheb-WT and LV31-Rheb-D60K. HEK-293 FT cells were contransfected with the recombinant lentiviral vector together with a lentiviral package plasmid to produce the lentiviral particles. The expression of PS6 protein was detected in the lentivirus-infected MCF-7 cells. The apoptosis of SK-HEP-1 cells transfected with LV31-Rheb-WT or LV31-Rheb-D60K was observed.</p><p><b>RESULTS</b>The recombinant LV31-Rheb-WT and LV31-Rheb-D60K vectors were confirmed by PCR and DNA sequencing. Western blotting showed that PS6 protein expression was increased in LV31-Rheb-WT-transfected cells while decreased in LV31-Rheb-D60K-transfected cells. LV31-Rheb-D60K-transfected SK-HEP-1 cells showed more obvious apoptosis after starvation than LV31-Rheb-WT-transfected cells.</p><p><b>CONCLUSION</b>Lentiviral vectors carrying Rheb gene and its mutant has been successfully constructed, which can be useful in further investigation of the role of Rheb gene in cancer cells.</p>