Overcoming cisplatin resistance by mTOR inhibitor in lung cancer

Abstract

Background: Cisplatin resistance is complex and involves several different mechanisms. Employing cDNA microarray analysis, we have found that cisplatin resistant cells share the common characteristic of increase in ribosomal proteins and elongation factors. We hypothesize that in order to survive cisplatin treatment, cells have to synthesize DNA repair proteins, antiapoptotic proteins and growth-stimulating proteins. Thus, by blocking the translation of these proteins, one should be able to restore cisplatin sensitivity. We have studied the role of CCI-779, an ester analog of rapamycin which is known to inhibit translation by disabling mTOR, in restoring cisplatin sensitivity in a panel of cisplatin resistant cell lines. We have also determined the role of CCI-779 in P-gp1 and MRP1 mediated resistance.

Results: Our data show that CCI-779 possess antiproliferative effects in both cisplatin sensitive and resistant cell lines, but shows no effect in P-gp1 and MRP1 overexpressing cell lines. Importantly, CCI-779 at 10 ng/ml (less that 10% of the growth inhibitory effect) can increase the growth inhibition of cisplatin by 2.5-6 fold. Moreover, CCI-779 also enhances the apoptotic effect of cisplatin in cisplatin resistant cell lines. In these resistant cells, adding CCI-779 decreases the amount of 4E-BP phosphorylation and p-70S6 kinase phosphorylation as well as lower the amount of elongation factor while cisplatin alone has no effect. However, CCI-779 can only reverse P-gp mediated drug resistance at a higher dose(1 ug/ml).

Conclusion: We conclude that CCI-779 is able to restore cisplatin sensitivity in small cell lung cancer cell lines selected for cisplatin resistance as well as cell lines derived from patients who failed cisplatin. These findings can be further explored for future clinical use. On the other hand, CCI-779 at achievable clinical concentration, has no growth inhibitory effect in P-gp1 or MRP1 overexpressing cells. Furthermore, CCI-779 also appears to be a weak MDR1 reversal agent. Thus, it is not a candidate to use in MDR1 or MRP1 overexpressing cells.

Figure 1

Apoptosis assay using annexin-V by FACS analysis in cisplatin resistant cell line (SR-2). Cells were treated with cisplatin alone or in combination with 0.15 ug of CCI-779. Left panels are dual staining for propidium iodide uptake and annexin-V^FITC, Right panels are corresponding distribution of annexin V^FITC staining in populations of cells. 1A: control. 1B: SR-2 treated with 6 ug of cisplatin (No annexin V^FITC was detected) 1C: SR-2 treated with 6 ug of cisplatin+CCI-779 (small peak of annexin V^FITC was detected) 1D: SR-2 treated with 10 ug of cisplatin (a peak of annexin V^FITC was detected) 1E: SR-2 treated with 10 ug of cisplatin+CCI-.779 (a large peak of annexin V^FITC was detected) Note: CCI-779 alone at 0.15 and 0.3 ug/ml did not induce apoptosis (data not shown).

Figure 2

Westernblot analysis of mTOR in Lane1: SCLCB, Lane 2: SCLCBC. Lane 3: SR-2, Lane 4: SCLC1. Note: there are no differences in mTOR levels in these 4 cell lines.

Figure 3

Westernblot analysis of 4E-BP phosphorylation in parental and cisplatin resistant small cell lung cancer cell lines. A: SCLC1 (parental line) was treated with 0.1 ug/ml of cisplatin alone, CCI-779 alone or in combination. Lane1: control (untreated cells) which showed high amount of phosphorylated 4E-BP. Lane 2: SCLC1 treated with 0.1 ug/ml of cisplatin which showed a decrease in the amount of phosphorylated 4E-BP, lane 3&4: SCLC1 treated with 150 ng/ml of CCI-779 alone and CCI-779 with cisplatin 0.1 ug/ml respectively. Both showed comparable amount of phosphorylated 4E-BP and are less than control cells and or cells treated with cisplatin alone. B: 4E-BP phosphorylation in SR-2 (cisplatin resistant cell line derived from SCLC1) after treatment with cisplatin with and without CCI-779. Lane 1: Control untreated cells which showed an abundant amount of phospho-4E-BP, lane 2, 3, and 4: SR-2 treated with 5,10 and 0.1 ug/ml of cisplatin. The amount of phospho-4E-BP are not affected by cisplatin at low dose, but slightly decreased at high dose. Lane 5,6 SR-2 treated with CCI-779 at 150 ng and 300 ng/ml respectively. Predominantly unphosphorylated 4E-BP is seen, Lane 7&8: SR-2 treated with 150 ng/ml of CCI-779 combined with cisplatin 5 ug and 10 ug/ml respectively. Small amounts of phosphorylated 4E-BP are seen and much less than those with cisplatin alone (lane 2,3&4). C:4E-BP phosphorylation in SCLCB(parental line). Lane 1: Control untreated cells which showed an abundant amount of phospho-4E-BP, lane 2: SCLCB treated with 0.1 ug/ml of cisplatin showed a decrease amount of phospho-4E-BP. lane 3 & 4 SCLCB treated with 150 ng/ml of CCI-779 alone and CCI-779 150 ng/ml with 0.1 ug/ml of cisplatin respectively. Both showed comparable amount of phospho-4E-BP and is slightly less than cisplatin alone. D: 4E-BP phosphorylation in SCLCBC (cisplatin resistant cells). Lane 1,2,3: SCLCBC treated with CCI-779 150 ng/ml combined with cisplatin at 1,5 and 10 ug/ml of cisplatin respectively. The amount of phospho-4E-BP is minimum and predominantly unphosphorylated form, lane 4&5 SCLCBC treated with CCI-779 at 150 ng and 300 ng/ml, only unphosphorylated 4E-BP predominate. Lane 6,7,8 SCLCBC treated with 10,5 and 1 ug/ml of cisplatin respectively. The amount of phospho4E-BP are similar to the control cells. Lane 9. Control untreated cells. Actin is used as control for protein loading for all cell lines

Figure 4

Westernblot analysis of phospho-P70S6kinase in cisplatin resistant cells. A: SR-2 cells. Lane 1. SR-2 treated with cisplatin 10 ug/ml and CCI-779 150 ng/ml, lane 2: SR-2 treated with CCI-779 at 150 ng/ml. Both 1&2 showed only minimal amount of phospho-p70S6kinase. Lane 3&4 SR-2 treated with cisplatin at 0.1 and 10 ug/ml., cisplatin does not affect the amount of phospho-p70S6kinase and showed similar intensity to control cells. Lane 5: control untreated cells. B. SCLCBC cells. Lane 1: Control untreated cells, lane 2&3 SCLCBC treated with cisplatin at 0.1 and 10 ug/ml respectively. Similar amounts of phospho-p70S6kinase were seen. Lane 4 SCLCBC treated with cisplatin 10 ug/ml and CCI-779 150 ng/ml. The amount of phosphor-p70S6kinase is less. Lane 5 SCLCBC treated with CCI-779 at 150 ng/ml. No signal intensity for p-70S6kinase was seen. Actin was used as control for protein loading.

Figure 5

Westernblot analysis of eEF-1α. A eEF-1α in sensitive and resistant cells. Lane 1. SCLCB, lane 2: SCLCBC, lane 3: SR-2, lane 4: SCLC1. The signal intensity of the resistant cell line SCLCBC and SR-2 are much greater than their sensitive cells SCLCB and SCLC1 counterpart. B eEF-1α in SR-2 cell line. Lane 1: control, lane 2&3: SR-2 treated with 0.5 and 10 ug/ml of cisplatin respectively. Similar signal intensity are seen compared to control, lane 4&5: SR-2 treated with 10 ng and 50 ng/ml of CCI-779 respectively. There is no change in signal intensity of eEF-1α at 10 ng of CCI-779, but decreased after exposure to 50 ng/ml of CCI-779, lane 6: SR-2 treated with 10 ug/.ml of cisplatin with CCI-779 50 ng/ml, the signal intensity also less and similar to those treated with CCI-779 alone. C eEF-1α in SCLCBC. Lane 1: control, lane 2&3 SCLCBC treated with 0.5 ug and 10 ug/ml of cisplatin respectively. Similar signal intensity of eEF-1α is seen compared to control, lane 4&5: SR-2 treated with 10 ng and 50 ng/ml of CCI-779 respectively, there is no changes in signal intensity at 10 ng/ml of CCI-779, but decreased at 50 ng/ml of CCI-779, lane 6 SCLCBC treated with 10 ug/ml of cisplatin with CCI-779 50 ng/ml. The signal intensity is less and similar to those treated with CCI-779 alone.