MKP1 mediates chemosensitizer effects of E1a in response to cisplatin in non-small cell lung carcinoma cells

Abstract

The adenoviral gene E1a is known to enhance the antitumor effect of cisplatin, one of the cornerstones of the current cancer chemotherapy. Here we study the molecular basis of E1a mediated sensitivity to cisplatin in an experimental model of Non-small cell lung cancer. Our data show how E1a blocks the induction of autophagy triggered by cisplatin and promotes the apoptotic response in resistant cells. Interestingly, at the molecular level, we present evidences showing how the phosphatase MKP1 is a major determinant of cisplatin sensitivity and its upregulation is strictly required for the induction of chemosensitivity mediated by E1a. Indeed, E1a is almost unable to promote sensitivity in H460, in which the high expression of MKP1 remains unaffected by E1a. However, in resistant cell as H1299, H23 or H661, which display low levels of MKP1, E1a expression promotes a dramatic increase in the amount of MKP1 correlating with cisplatin sensitivity. Furthermore, effective knock down of MKP1 in H1299 E1a expressing cells restores resistance to a similar extent than parental cells. In summary, the present work reinforce the critical role of MKP1 in the cellular response to cisplatin highlighting the importance of this phosphatase in future gene therapy approach based on E1a gene.

Keywords: E1a; MKP1; chemotherapy; cisplatin; lung cancer.

Figure 1. E1a promotes sensitivity to cDDP in NSCLC

(A) H23, H1299, H460 and H661 were infected with empty vector or E1a 13s. 50 μg of total cell lysates (TCL) were blotted against E1a. Membranes were reproved against Tubulin as loading control. (B) NSCLC cell lines with/without E1a 13s were treated with the indicated doses of cDDP during 48 hours and viability was evaluated by crystal violet method. Bars indicate standard deviation (SD). (C) NSCLC cells lines carrying empty vector or E1a 13s were treated with the IC₇₅ obtained from Figure 1B during 5 days and viability was evaluated by crystal violet method. Bars indicate standard deviation (SD).

Figure 2. E1a enhances the antitumor effect of cDDP in NSCLC trough promotion of apoptosis

(A) H1299 cells infected with E.V or E1a 13s were treated with 6 μg/ml of cDDP for 24 h and caspase 3/7 activity was evaluated. (B) H1299 cells infected with E.V or E1a 13s were treated with 6 μg/ml of cDDP for 48 h. Then, 50 μg of TCL were blotted against LC3 and p62. Membranes were reproved against tubulin as loading control. (C) Cells were treated as in (A) in the presence or absence of 10 μM Q-VD and then survival ratio was evaluated 48 hours later by using MTT assay. (D) H1299 cells infected with E.V. or E1a 13s were treated with chloroquine, in the absence of serum, at the indicated time points (time 0 means no chloroquine). Then 50 μg of TCL were blotted against LC3. Membranes were reproved against Tubulin as loading control.

Figure 3. Sensitivity to cDDP correlates with low expression of MKP1 in NSCLC

(A) NSCLC derived cell lines were treated with the indicated doses of cDDP during 48 hours and viability was evaluated by crystal violet method. Bars indicate standard deviation (SD). (B) H1299, H23, H661 and H460 were subjected to qRT-PCR assay in order to quantify DUSP5 mRNA expression levels. (C) Same approach was performed to measure MKP1 mRNA expression levels. (D) 50 μg of TCL from our panel of NSCLC cells were blotted against endogenous MKP1, p38MAPK and the respective active form. Tubulin was used as loading control.

Figure 4. Abrogation of MKP1 mediates resistance to cDDP in NSCLC

(A) Expression of MKP1 was evaluated in H460 infected with sh-RNA scrambled or MKP1 by qRT-PCR. (B) 50 μg of TCL from H460 cells infected with MKP1 sh-RNA or empty vector were blotted against MKP1. Tubulin was used as loading control. (C) H460 cell line infected with sh-RNA scrambled or MKP1 was treated with the indicated doses of cDDP during 48 hours and viability was evaluated by the crystal violet method. Bars indicate standard deviation (SD). (D) 50 μg of TCL from cDDP resistant H460 generated by co-culturing (cDDP-R) or parental H460 cell line were blotted against MKP1. Tubulin was used as loading control. (E) H460 cDDP-R or parental H460 were treated with the indicated doses of cDDP during 48 hours and viability was evaluated by crystal violet method. Bars indicate standard deviation (SD).

Figure 5. Upregulation of MKP1 mediates E1a associated sensitivity to cDDP in a p38MAPK dependent fashion

(A) 50 μg of the TCL from NSCLC expressing E1a 13s or empty vector were used to evaluate the MKP1, P-p38MAPK and p38MAPK. Tubulin was used as loading control. (B) H1299 stably transfected with pcDNA E1a 13s (H1299 E1a) or empty vector (E.V.) were infected with lentivirus carrying sh-RNA against MKP1 (H1299 E1a shMKP1) and then mRNA MKP1 levels were evaluated by qRT-PCR. (C) 50 μg of TCL from cells used in B) were blotted against MKP1 and E1a. Tubulin was used as loading control. (D) 50 μg of TCL from cell used in C) were blotted against p38, ERK1/2 and the respective active forms. Tubulin was used as loading control. (E) H1299 E.V, H1299 E1a and H1299 E1a and sh-RNA MKP1 were treated with the indicated doses of cDDP during 48 hours and viability was evaluated by crystal violet method. Bars indicate standard deviation (SD). (F) H1299 E.V and H1299 E1a were treated with the indicated doses of cDDP during 48 hours in the presence/absences of PD98059 (10 μM). Viability was evaluated by crystal violet method. Bars indicate standard deviation (SD). (G) H1299 E.V and H1299 E1a were treated with the indicated doses of cDDP during 48 hours in the presence/absences of SB203580 (10 μM). Viability was evaluated by crystal violet method. Bars indicate standard deviation (SD).