IL-6 signaling contributes to cisplatin resistance in non-small cell lung cancer via the up-regulation of anti-apoptotic and DNA repair associated molecules - PubMed (original) (raw)

IL-6 signaling contributes to cisplatin resistance in non-small cell lung cancer via the up-regulation of anti-apoptotic and DNA repair associated molecules

Shanzhou Duan et al. Oncotarget. 2015.

Abstract

Cisplatin-based chemotherapy is currently the most effective treatment regimen for non-small cell lung cancer (NSCLC), but eventually tumor resistance develops which limits its success. The potential implication of IL-6 signaling in the cisplatin resistance of NSCLC was explored by testing whether NSCLC cells with different levels of intracellular IL-6 show different responses to the cytotoxic treatment of cisplatin. When the cisplatin cytotoxicity of the IL-6 knocked down human NSCLC cells (A549IL-6si and H157IL-6si) were compared with their corresponding scramble control cells (A549sc and H157sc), higher cisplatin cytotoxicity was found in IL-6 si cells than sc cells. Subcutaneous xenograft mouse models were developed using a pair of A549sc and A549IL-6si cells. When the tumor grew to about 400 mm2, mice were treated with cisplatin and tumor regression was monitored. Higher tumor regression was detected in the A549IL-6si xenografts compared to A549sc xenografts following cisplatin treatment. Immunostaining study results from tumor tissues also supported this finding. Expression of anti-apoptotic proteins Bcl-2 and Mcl-1 and DNA repair associated molecules ATM, CHK1, TP73, p53, and ERCC1 were significantly up regulated in cisplatin-treated A549sc and H157sc cells, but no increase was detected in A549IL-6si and H157IL-6si cells. Further inhibitor studies revealed that up regulation of these molecules by IL-6 may be through activation of IL-6 downstream signaling pathways like Akt, MAPK, Stat3, and Erk. These results provide potential for combining cisplatin and inhibitors of IL-6 signaling or its downstream signaling pathway as a future therapeutic approach in preventing development of cisplatin resistant NSCLC tumors.

Keywords: DNA repair; IL-6; apoptosis; cisplatin resistance; non-small cell lung cancer.

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CONFLICTS OF INTEREST

There are no conflicts of interest.

Figures

Figure 1

Figure 1. IL-6 knockdown in NSCLC cells increased in vitro and in vivo sensitivity to cisplatin

A. Cisplatin cytotoxicity tests of A549IL-6si/sc cells upon various concentrations of cisplatin for 2 days. B. Cisplatin cytotoxicity tests of H157IL-6si/sc cells upon various concentrations of cisplatin for 2 days. C. Tumor regression analyses of A549IL-6si and A549sc cells-derived xenografts in nude mice on cisplatin treatments. Xenografts were developed by subcutaneously injecting 1 × 106 A549IL-6si or A549sc cells into flanks of 8 week old female nude mice. When tumor volumes reached 400 mm3, cisplatin (3 mg/kg, i.p. two times per week) treatment started. Tumor growth was monitored twice per week and at the end of three weeks of treatment, mice were sacrificed. D. H&E and IHC staining of tumor tissues. Tumor tissues of A549IL-6si/sc xenografts were processed and subjected to H&E and IHC staining. Upper panels show H&E staining, middle panels present IHC staining with IL-6 antibody, and lower panels are the IHC staining results using antibody against Ki67 (magnification, 100x). Quantitation of IL-6 and Ki67 IHC staining is shown on right. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2

Figure 2. IL-6 protected A549 and H157 cells from apoptotic death after cisplatin treatment via up regulation of Bcl-2 and Mcl-1

A. Analysis of apoptosis following cisplatin treatment. A549IL-6si/sc and H157IL-6si/sc cells were treated with cisplatin (5 μM) for 48 hours and subjected into Annexin V based-flow cytometric analysis according to the manufacturer's instruction. The % of apoptotic cell is indicated in each graph. B. qPCR results of analyzing mRNA expressions of anti-apoptotic molecules in A549IL-6si/sc and H157IL-6si/sc cells after cisplatin treatment (5 μM, 48 hours). C. Western blot results showing expression of anti-apoptotic proteins in A549IL-6si/sc and H157IL-6si/sc cells following cisplatin treatment (5 μM, 48 hours). D. IHC staining of tumor tissues of A549IL-6si/sc xenografts showing expression of Bcl-2 and Mcl-1(Magnification, 100x). Quantitation of Bcl-2 and Mcl-1 IHC staining is shown on right. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Figure 3. IL-6 expressing A549sc and H157sc cells showed higher expression of DNA repair molecules upon cisplatin treatment compared to A549IL-6si and H157IL-6si cells

A. qPCR results of mRNA expressions of DNA repair associated molecules in A549IL-6si/sc and H157IL-6si/sc cells after cisplatin treatment (5 μM, 48 hours). B. Western blot analyses results showing expression of the DNA repair-associated molecules in A549IL-6si/sc and H157IL-6si/sc cells on cisplatin treatment (5 μM, 48 hours). C. IHC staining of tumor tissues of A549IL-6si/sc xenografts investigating expression of ATM and CHK1 (Magnification, 100x). Quantitation of positively stained cells is shown on right. *p < 0.05 **p < 0.01, ***p < 0.001.

Figure 4

Figure 4. Activation of IL-6 downstream signaling pathways on cisplatin treatment

A. Western blot analysis analyzing activations of several signaling pathways in A549IL-6si/sc and H157IL-6si/sc cells 48 hours after cisplatin treatments (5 μM). B. Analyses of qPCR results showing expression of Bcl-2 and ERCC1 in A549sc and H157sc cells, with or without cisplatin treatment, in the absence and presence of inhibitors of each signaling pathway. *p < 0.05 **p < 0.01, ***p < 0.001.

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