Combined treatment with TNF-α/gefitinib alleviates the resistance to gefitinib in PC-9 cells

Gefitinib has been approved for the treatment of patients with non-small cell lung cancer. However, its efficiency is limited by the development of drug resistance. Additional treatments for cases of non-small cell lung cancer relapsing with treatment with gefitinib are urgently required. To investigate the mechanisms of acquired resistance to gefitinib, we established PC-9-ZD, a human lung cancer cell line resistant to gefitinib after long-term exposure to the drug. PC-9-ZD cells showed more resistance to gefitinib than their parental PC-9 cells. We show that gefitinib reduces p-Akt levels, concomitant with elevation of p21 levels and suppression of cdk2/4 and cyclinE/D1 activities, which result in impaired cell cycle progression through G1 arrest only in parental PC-9 cells, in which it inhibits growth. Our present data suggested that after long-term exposure to gefitinib, the survival of PC-9-ZD cells with heightened levels of p-Akt and reduced levels of p21 resisted further gefitinib-induced inhibition of cell growth. To explore a new strategy to improve the efficacy of gefitinib, we treated the cells with tumor necrosis factor-alpha (TNF-alpha) and found that the cells with acquired resistance to gefitinib showed increasing sensitivity to TNF-alpha, which correlated with the low activation level of nuclear factor (NF)kappaB/p65 in PC-9-ZD cells. TNF-alpha treatment induced an elevated activated NFkappaB/p65, concomitant with induced p21 levels, which resulted in increased sensitivity to gefitinib in PC-9-ZD cells. Consistent with our earlier observation that p21 is induced in an NFkappaB/p65-dependent manner, we conclude that p21 plays an important role in mediating cell growth inhibition by gefitinib. Thus, we proposed that combined treatment with TNF-alpha/gefitinib is an efficient therapeutic strategy for tumors that develop resistance to gefitinib.