Inhibition of mTOR restores cisplatin sensitivity through down-regulation of growth and anti-apoptotic proteins

Abstract

We show that cisplatin resistance in certain lung cancer cell lines can be reversed through inhibition of mTOR (mammalian Target of Rapamycin). These cell lines appear to possess high levels of phospho-mTOR, phospho-AKT and other growth-related proteins, such as hTERT (human telomerase reverse transcriptase), and Cyclin D3 which decrease upon inhibition of mTOR. Interestingly in one cisplatin resistant cell line which expresses BCL2/BCLxL, treatment with mTOR inhibitor (CCI-779) results in decreased levels of these anti-apoptotic proteins and may contribute to increasing apoptosis. Moreover, continuous exposure to CCI-779 was found to increase the expression of the multi-drug resistant P-gp1(P-gycoprotein1) efflux pump and therefore should be taken into consideration when designing clinical trials with this compound.

Figure 1

Increased AKT and mTOR activity in lung cancer cell lines correlates with increased sensitivity to mTOR inhibition. (A) ID₅₀ of lung cancer cell lines treated with cisplatin alone, CCI-779 alone and incombination for 72h. Each data point is the average of triplicate samples with S.D. (B) Immunoblots of pmTOR, p-P70S6K, and p4EBP in different lung cancer cell lines. Overall, SCLCs are shown to express higher level of pmTOR, p-P70S6K, and p4EBP when compared with NSCLCs.

Figure 1

Increased AKT and mTOR activity in lung cancer cell lines correlates with increased sensitivity to mTOR inhibition. (A) ID₅₀ of lung cancer cell lines treated with cisplatin alone, CCI-779 alone and incombination for 72h. Each data point is the average of triplicate samples with S.D. (B) Immunoblots of pmTOR, p-P70S6K, and p4EBP in different lung cancer cell lines. Overall, SCLCs are shown to express higher level of pmTOR, p-P70S6K, and p4EBP when compared with NSCLCs.

Figure 2

mTOR inhibition affects survival and anti-apoptotic proteins. (A) Western blot analysis of SR2 treated for 48 hr and 72 hr (Note the band intensity of p-mTOR as well as p4EBP is markedly decreased at 72 hr) confirming the effectiveness of siRNA in down-regulating mTOR. (B) Immunoblots of growth/proliferation and apoptotic protein in SCLC1 and SCLCSR2 treated with 0.5 μg/mL and 5 μg/mL of cisplatin, respectively. The combination of cisplatin and CCI-779 showed a decrease intensity of cyclin D3, hTERT and pAKT expressions in SCLCSR2 but not in SCLC1. Rictor expressions are much less in SCLCSR2 with minor changes upon drug treatment. Note the CCI-779 induced cleaved caspase-3 when combined with cisplatin is mediated by an increase in the ratio of BAX/BAK : BCL2 (6 folds), as well as BAX/BAK : BCLxL (16 folds). The number above each lane depicted the changes from its control which was arbitrarily set at 1. All the band intensity was normalized with actin. (Experiments were all done at 24hr time point)

Figure 2

mTOR inhibition affects survival and anti-apoptotic proteins. (A) Western blot analysis of SR2 treated for 48 hr and 72 hr (Note the band intensity of p-mTOR as well as p4EBP is markedly decreased at 72 hr) confirming the effectiveness of siRNA in down-regulating mTOR. (B) Immunoblots of growth/proliferation and apoptotic protein in SCLC1 and SCLCSR2 treated with 0.5 μg/mL and 5 μg/mL of cisplatin, respectively. The combination of cisplatin and CCI-779 showed a decrease intensity of cyclin D3, hTERT and pAKT expressions in SCLCSR2 but not in SCLC1. Rictor expressions are much less in SCLCSR2 with minor changes upon drug treatment. Note the CCI-779 induced cleaved caspase-3 when combined with cisplatin is mediated by an increase in the ratio of BAX/BAK : BCL2 (6 folds), as well as BAX/BAK : BCLxL (16 folds). The number above each lane depicted the changes from its control which was arbitrarily set at 1. All the band intensity was normalized with actin. (Experiments were all done at 24hr time point)