AZD5363,Inhibited Phosphorylation Of AKT Downstream Molecules,Activated Phosphorylation Of MTOR And SMG-1 Dependent Of Liver Cancer Cells Type

1 Objectives(1) To observe the effect of AZD5363 on cells proliferation and migration in liver cancer cells after the cell lines (Hep-G2 and Huh-7) exposed to AZD5363.(2) To investgate the effect of AZD5363 on AKT signaling pathway in liver cancer cell lines.(3) To investgate the effect of AZD5363 on mTOR signaling pathway and SMG-1 in liver cancer cell lines.2 Methods2.1 Cell culture reagentsHuman liver cancer cell lines Hep-G2 cells and Huh-7 cells line were obtained from Shandong Provincial Hospital Afflicted to Shandong University, the center of liver disease. All cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) medium containing 10% fetal bovine serum(FBS),1% Penicillin-Streptomycin Solutionandl% none essential amino acids(NEAA). All cells were maintained in a humidified incubator with 5% CO2 at 37℃. The structure and synthesis of AKT inhibitor AZD5363 has been described previously.2.2 Cell counting kit-8 (CCK-8) assayCell growth rate was measured by CCK-8(cell counting kit-8) (Dojindo Corp). Briefly, cells seeded at 1000-2000/well density in 96-well plates were cultured overnight with 90μl medium, and then treated with AZD5363 at different concentrations for 24h,48h and 72 h. CCK-8 One Solution Reagent was added to each well according to the manufacturer’s instructions. After 1.5 h in culture the cell viability was determined by measuring the absorbance at 450 nm.2.3 Transwell migration assayMonolayers of serum-starved adherent cells are trypsynized and 50000 cell suspensions are placed in 200μl serum-free DMEM medium into the upper well of a transwell filter apparatus. The filter is suspended in a well of a 24-well plate and the lower reservoir is filled with 800μl of DMEM containing 10% FBS. The cells are incubated under normal conditions for 24h. Migration assays are terminated by retrieving the filter, rubbing off non-migrated cells from top surface, then treated with formaldehyde, methanol, and Giemsa staining, and counting cells that are found on the underside of the filter.2.4 Western blot analysisExtraction of protein in a 6-well plate, add the mixture of RIPA and PMSF (RIPA:PMSF=100:1)(Beyotime Biotechnology), proteins concentration were quantified by the Pierce BCA protein assay kit (Thermo Scientific Pierce. Rockford, IL, USA).Then soluble proteins (50μg) subjected to SDS-PAGE followed by immunoblotting. Proteins were separated electrophoretically in SDS-polyacrylamide gels and transferred to NC membranes, then incubated at 4℃ overnight with a primary antibody, incubated with HRP-conjugated secondary antibody at room temperature for 2 h, Immunoreactivity was detected using the FluorChem E Chemiluminescent Western Blot Imaging System (Cell Biosciences, Santa Clara, CA, USA) according to the manufacturer’s instructions. All antibodies were obtained from Cell Signaling Technology, including phosphor-mTOR (ser2448) (D9C2) (#5536S), phosphor-Akt (Thr450) D5G4 (#12178), phosphor-GSK-3β (ser9) (5B3) Rabbit mAb (#9323), mTOR (7C10) (#2983S), AKT (#9272), GSK-3β (27C10) Rabbit mAb (#9315), SMG-1 (Q25) Rabbit mAb (#4993s), except GAPDH (ZSGB-BIO), besides, the secondary antibody is Peroxidase-Conjugated Affini Pure Goat Anti-Mouse IgG (H+L) (ZSGB-BIO) or Peroxidase-Conjugated Affini Pure Goat Anti-Rabbit IgG (H+L) (ZSGB-BIO).2.5 Data Analysis and StatisticsStatistical analyses were performed using SPSS statistical software (version 18.0) and GraphPad Prism 5.0 software.The data are presented as mean±S.E.M. The differences between groups were performed with one-way analysis of variance followed by Student-Newman-Keuls post hoc test for pairwise comparison, in which P<0.05 is considered to be statistically significant.3 Results3.1 AZD5363 inhibited proliferation of liver cancer cells in dose-and time-dependent manner.To determine the effect of AZD5363on cell proliferation, a panel of two liver cancer cell lines was tested for anti-proliferative sensitivity using an in vitro cell growth assay. Hep-G2 and Huh-7 cells were exposed to AZD5363 at different concentrations ranging from 5 to 30μM, cells seeded in 96-well plates were cultured overnight, and then treated with AZD5363 at different concentrations for 24h,48h and 72h. Cell growth rate was measured by a CCK-8 assay, a dose-dependent inhibition of cell viability was observed in both cell lines (Fig.lA and B). The drug concentration to inhibit the two cell lines is similar, it inhibited proliferation of liver cancer cells in dose and time-dependent manner, the drug concentration of half maximal inhibitory concentration (IC50) is lower with prolonging the time of exposing to the cells, when the time is up to 72 h, the IC50 of hep-G2 is 18.476μM and the IC50 of Huh-7 is 17.80μM, besides, in order to obtain better inhibition curve, a higher density of huh-7 than hep-G2 was needed given. Thus we can indicate that AZD5363 activity was obviously selective for liver cancer cells.3.2 AZD5363 suppressed liver cancer cells migration To explore the effect of AZD5363 on liver cancer cells migration extensively, we do the transwell migration assay. Cells and drugs were inoculated in the upper chamber with serum-free medium, in the lower chamber was medium containing serum, cells were cultured for 24h, then treated with formaldehyde, methanol, and Giemsa staining, we get the result shown in figure2, AZD5363 can significantly reduce the activity of both in Hep-G2 and Huh-7 cells lines (fig.2A).3.3 AZD5363 inhibited phosphorylation of AKT substrates AKT functions in cell survival signaling by phosphorylating downstream targets, and dephosphorylation of these substrates indicates the inhibition of AKT activity. AKT plays a key role in glucose metabolism; its substrateGSK3βcan modulate glycogen synthesis and glucose transporter function respectively, We thereby investigated whether AZD5363 could inhibit the phosphorylation of AKT substrates; as expected, AZD5363 inhibited the phosphorylation of GSK3(3 but increased the phosphorylation of AKT with the increasing exposed time to AZD5363 and the increasing concentration of AZD5363 in Hep-G2 cells line and Huh-7 cells line (Fig.3A and B).3.4 AZD5363 activated phosphorylation of mTOR dependent of liver cancer cells typeTo explore the effect of AZD5363 on the mTOR pathway, we detected the phosphorylation levels of mTOR, In contrast to inhibited phosphorylation of AKT substrates, AZD5363 has much less broad activity with mTOR pathway in panels of tumor cell lines in vitro. As is shown, AZD5363 attenuated the phosphorylation of mTOR only in the cells line of huh-7, and observed that AZD5363 attenuated the phosphorylation of mTOR which indicated that AZD5363 significantly stimulated mTOR signaling dependent of liver cancer types (Fig.4A and B,5 A and B).3.5 AZD5363 activated the SMG-1 dependent of liver cancer cells line SMG-1 and mTOR both belong to the PIKK family. To explore the effect of AZD5363 on the SMG-1, we detected the levels of SMG-1. We observed that AZD5363 could promote the expression of SMG-1 in Huh-7 cells line, while in the Hep-G2 cells line, we did not find the situation (Fig.6A and B). However, the mechanism by which AZD5363 induced SMG-1was different from that of the AKT signaling, moreover, we observed that the effect becomes more visible with prolonging the time or increasing the dose, which indicated that AZD5363 activated SMG-1 in dose-and time-dependent manners (Fig.6A and B,7A and B).4 Conclusion(1) AZD5363 inhibited proliferation of liver cancer cells and suppressed liver cancer cells migration in dose- and time-dependent manner.(2) AZD5363 inhibited phosphorylation of AKT substrates(3) AZD5363 activated phosphorylation of mTOR dependent of liver cancer cells type(4)AZD5363 activated the SMG-1 dependent of liver cancer cells line1 Objectives(1) To investgate the effect of high fat diet in liver regeneration after partial hepatectomy in mice.(2) To investgate the effect of high-fat diet on the expression of Wip1 and NF-KB signaling pathway inliver regeneration tissue.2 Methods2.1 Animal model48 male mice on a C57BL/6 background (8 weeks old) were randomly divided into two groups:normal diet group (n=15) fed a standard mice chow containing 6.5% fat (17.3% of calories)and the high fat diet group (HFD) was fed a high-fat diet containing 21% fat (40.8% of calories). All the mice were performed 2/3 partial hepatectomy, At 48h,72h and 120h after PHx, mice of each group were sacrificed and all the regenerated liver samples were collected. Liver tissues were either fixed with 10% buffered formalin for histological examination, or immediately frozen in liquid nitrogen and stored at-80℃ for RNA and protein isolation.2.2 Western blot analysisTotal protein was extracted from the mouse liver tissue in liquid nitrogento detect the expression of proliferating cell nuclear antigen in liver tissue (PCNA), Wip1, cyclin D and NF-KB signaling pathway.2.3 Quantitative real-timePCRTotal RNA was extracted from liver tissue in liquid nitrogen, the expression levels of PCNA and Wip1 mRNA were analysis by RT-PCR.3 Results3.1 Animal liver weight/body weight ratio and liver regeneration rateThe ratios of liver/body weight and liver regeneration at each time points after 2/3 PHx were showing in the table. Compared with the control group, the ratios of liver/body weight (%)were increased significantly at 48h (3.05±0.20vs2.31±0.16) and 72h (3.72±0.23vs3.02±0.19) in the HFD group (p<0.001). Liver regeneration (%) were also increased at 48h (67.25±6.73vs51.50±4.78) and 72h (78.98±4.77vs69.00±5.17) in the HFD group in comparison with the control group (p<0.01).3.2 The expression of Proliferating cell nuclear antigen (PCNA) in liver tissueAt 48 h and the 120 h after PHx the PCNA protein were upregulated in HFD mice compared with that in the control group(p<0.05). while at 72h after PHx, the expression were similar between the groups. The study showed that the peak of PCNA was found at 48h in the HFD group, while that was at 72h in control group. The mRNA level of PCNA was also observed higher in HFD mice than that of control mice at 48h (p<0.002). These results indicated that the remnant liver of HFD mice showed increased hepatocyte DNA replication and cell cycle progression.3.3The expression of p-NF-κB in liver tissues of p65 and cyclin DThe results showed that compared with the control group, the level of NF-κB and phosphorylation of NF-κB significantly increased at 48h after PHx (p< 0.001).while at the others assessed time points after PH, saw no changes in the expression of NF-κB and phosphorylation of NF-κB. Cyclin D1was regulated by NF-κB, we then detected the level of cyclin D1 using immunoblotting. Levels of cyclin D1 in HFD mice was higher than the control group at 48 hours and 72 hours after PHx(p< 0.001).3.4 The expression of Wipl in liver tissue:To explore the effect of HFD on Wipl, we detected the expression level of Wipl. Immunoblotting showed that Wipl was significantly inhibited in HFD group at 48h and 72h after 2/3 PHx (p<0.001).The mRNA level of Wipl was downregulated at 48h in HFD mice (p<0.05), at the other time points,analysis of RT-PCR indicated that HFD has a tiny effect on Wipl mRNA. Given all the results, the result showed that HFD can decrease the levels of Wipl protein and mRNA in hepatocytes after 2/3 PHx.4 Conclusions(1) High fat diet can promote the expression of PCNA and enhance the liver regeneration after 2/3PH in mice.(2) High fat diet can promote the expression of Wipl and activate NF-KB signaling pathway as well as increase the expression of cyclin D.