The Activation Of PI3K/AKT Pathway And Gefitinib Efficacy In Pancreatic Cancer Cell Lines

ObjectiveTo study the inhibitory function of gefitinib to human pancreatic cell and itsrelationship with PI3K/Akt pathway in vitro and to provide theoretical base forclinical screening of gefitinib sensitive pancreatic cancer patients. By culturing humanpancreatic cell lines with different sensitivity to gefitinib and using medications toinhabit the activation of p13k/Akt pathway, the diversification patterns of differentpancreatic cancer cell lines under the inhibition of gefitinib or p13k/Akt wereanalysed and the potential mechanisms of gefitinib resistance was discussed. If pAktbe a key factor to judge gefitinib’s efficacy and a therapeutic target was also evaluated.This study also provide theoretical base for the reversal of gifitinib resistance bygefitinib and p13k/Akt inhibitor combination and provide subsequent therapeuticoptions for gefitinib resistant patients.Methods3cell lines–BxPc-3, MiaPaCa-2and PANC-1with different gene mutation statuswere selected. EGFR and Kras gene mutation were detected by Sanger sequencingmethod. The growth inhibition curve of gefitinib and LY294002were tested by MTTmethod. The sensitivity of different cell lines to LY294002and gefitinib werecompared and the sensitivity change of each cell lines to gefitinib under differentLY294002concentrations. Each of the3cell lines were exposed to blank control,5μM gefitinib,10μM LY294002and5μM gefitinib combined with10μM LY294002respectively. Protein expression of EGFR, pEGFR, Akt and pAkt were detected byWestern Blot immune-blotting method. The influence of drugs in each groups tohuman pancreatic cell and to the EGFR and Akt protein activation were analyzed.Results1. DNA sequencing shows the EGFR and Kras gene of BxPc-3cell were all wildtype, MiaPaCa-2cell was with EGFR wild type and Kras G12C mutation.EGFR T790M and Kras G12D mutation were detected in PANC-1cell.2. MTT detection shows both gefitinib and LY294002could inhibit BxPc-3、 MiaPaCa-2and PANC-1cell growth and their inhibitory efficacy wereconcentration dependent. The dose of each groups was negatively correlated tocell survival rate（p<0.05）. While combined with LY294002, gefitinib’sinhibitory efficacy to human pancreatic cell was improved and the higher theconcentration of LY294002, the much more improvement of the gefitinib’sinhibitory efficacy was achieved. The IC₅₀concentration of LY294002to the3cell lines BxPc-3, MiaPaCa-2and PANC-1was （24.65±1.87）μM,（17.04±0.78）μM,（11.32±1.48）μM and was significantly different in the3celllines（F=191.79，P<0.01）; the IC₅₀concentration of gefitinib was （6.34±1.68）μM,（15.41±2.69）μM,（36.67±1.39）μM and was also significantly different in3celllines（F=545.74，P<0.01）; the IC₅₀concentration of gefitinib with LY2940022μM was （3.48±1.75）μM,（5.39±1.87）μM,（11.55±2.49）μM； the IC₅₀concentration of gefitinib with LY29400210μM was （0.66±0.19）μM,（0.69±0.07）μM,（2.33±0.49）μM respectively and was significantly different in3cell lines （F=87.82，P<0.01）. Significant difference wasn’t detected in differentgroups.3. Western Blot immune-blotting detection shows that in the blank control group,as a gefitinib sensitive cell line, the BxPc-3cell has the highest level of EGFRexpression （1.11±0.21）, protein expression （0.81±0.11）, and relative activity（0.73±0.05） in3cell lines; but has the lowest level of the Akt expression（0.53±0.07）, protein expression （0.09±0.11）, and relative activity （0.17±0.12）.However, as a highly resistant cell line to gefitinib, the PANC-1cell has thelowest level of EGFR expression （0.61±0.06）, protein expression （0.38±0.04）,and relative activity （0.62±0.07）, but has the highest level of the Akt expression（1.51±0.18）, protein expression （1.54±0.16）, and relative activity （1.02±0.21）.After the exposure to gifitinib, the expression of EGFR and AKT of the3celllines weren’t significantly changed, but the quantity of protein expression andrelative activity were decreased. The EGFR and Akt relative activity wasdecreased by51.22%and76.91%（0.61±0.09） respectively in BxPc-3cell line.The EGFR and Akt relative activity was only decreased by2.05%and44.96% （0.56±0.10） respectively in PANC-1cell which is called highly resistant cellline. LY294002has obvious inhibitory efficacy to all the3cell lines. Whencombined with gefitinib, it can improve gefitinib’s efficacy by inhibiting EGFRand Akt phosphorylation. As the sensitive cell line, the BxPc-3’s relativeactivity of EGFR was0.02±0.04which decreased by97.51%and its Aktrelative activity was0.02±0.02which decreased by87.98%; PANC-1was ahighly resistant cell line and its relative activity of EGFR was0.11±0.04whichdecreased by82.71%, and Akt relative activity was0.08±0.05which decreasedby93.42%.ConclusionGefitnib inhibit the growth of human pancreatic cancer cell lines BxPc-3、MiaPaCa-2and PANC-1by blocking the EGFR pathway activation. The PI3K/Aktpathway is related to human pancreatic cell’s response to gefitinib. Human pancreaticcell lines with high level expression of pAkt indicate drug resistance to gefitinib.Combination of p13k and LY294002could stop p13k/Akt pathway and could inhibitthe activity of pAkt and reverse pAkt positive cell’s resistance to gefitinib, thusimprove its sensitivity to gefitinib.