Inhibition of Autophagy Enhances Cell Death Induction by 5-Fluorouracil in Human Colon Cancer Cells

Abstract

G A A b st ra ct s resistance in MKN-28 cells (25 μM gefitinib, treated overnight, P<0.01). We also found that DARPP-32 expressing MKN-28 cells had activated the PI3K-AKT pathway as compared to control cells. We next examined the binding of DARPP-32 to EGFR and ERBB3. The threeway co-immunoprecipitation experiments demonstrated the existence of DARPP-32, EGFR, and ERBB3 in the same protein complex. The enhanced EGFR-ERBB3 heterodimerization promotes phosphorylation of ERBB3, then activated the PI3K-AKT pathway, as indicated by p-p85 (Tyr458) subunit of PI3K and p-AKT (Ser473) levels. Following treatment with gefitinib (25 μM) overnight, MKN-28 cells expressing DARPP-32 displayed stable protein levels of EGFR. In contrast, control cells exhibited a significant reduction in EGFR protein levels, suggesting that DARPP-32 promotes stability and suppresses degradation of EGFR protein. After the gefitinib treatment of DARPP-32-expressing MKN-28 cells, p-ERBB3 and activated PI3K-AKT pathway were maintained at higher levels than in control cells. The knockdown of endogenous DARPP-32 by lentiviral DARPP-32 shRNA in SNU-16 cell lines reversed these signaling effects and increased sensitivity to gefitinib (p<0.01). Conclusion: Our results suggest that DARPP-32 overexpression may participate in the transition to intestinal metaplasia and progression to neoplasia. The In Vitro studies indicate that DARPP32 plays a role in increasing the stability of EGFR protein by delaying gefitinib-induced EGFR degradation; maintaining EGFR-ERBB3 heterodimerization promotes phosphorylation of ERBB3, which regulates the PI3K/Akt pathway. DARPP-32 may have potential as a predictive biomarker in gastric cancer prognosis and clinical response to treatment.