Reduction of metastatic potential by inhibiting EGFR/Akt/p38/ERK signaling pathway and epithelial-mesenchymal transition after carbon ion exposure is potentiated by PARP-1 inhibition in non-small-cell lung cancer

Abstract

Background: Carbon ion (¹²C) radiotherapy is becoming very promising to kill highly metastatic cancer cells keeping adjacent normal cells least affected. Our previous study shows that combined PARP-1 inhibition with ¹²C ion reduces MMP-2,-9 synergistically in HeLa cells but detailed mechanism are not clear. To understand this mechanism and the rationale of using PARP-1 inhibitor with ¹²C ion radiotherapy for better outcome in controlling metastasis, we investigated metastatic potential in two non-small cell lung cancer (NSCLC) A549 and H1299 (p53-deficient) cells exposed with ¹²C ion in presence and absence of PARP-1 inhibition using siRNA or olaparib.

Methods: We monitored cell proliferation, in-vitro cell migration, wound healing, expression and activity of MMP-2, - 9 in A549 and p53-deficient H1299 cell lines exposed with ¹²C ion with and without PARP-1 inhibitor olaparib/DPQ. Expression and phosphorylation of NF-kB, EGFR, Akt, p38, ERK was also observed in A549 and H1299 cells exposed with ¹²C ion with and without PARP-1 inhibition using siRNA or olaparib. We also checked expression of few marker genes involved in epithelial-mesenchymal transition (EMT) pathways like N-cadherin, vimentin, anillin, claudin-1, - 2 in both NSCLC. To determine the generalized effect of ¹²C ion and olaparib in inhibition of cell's metastatic potential, wound healing and activity of MMP-2, - 9 was also studied in HeLa and MCF7 cell lines after ¹²C ion exposure and in combination with PARP-1 inhibitor olaparib.

Results: Our experiments show that ¹²C ion and PARP-1 inhibition separately reduces cell proliferation, cell migration, wound healing, phosphorylation of EGFR, Akt, p38, ERK resulting inactivation of NF-kB. Combined treatment abolishes NF-kB expression and hence synergistically reduces MMP-2, - 9 expressions. Each single treatment reduces N-cadherin, vimentin, anillin but increases claudin-1, - 2 leading to suppression of EMT process. However, combined treatment synergistically alters these proteins to suppress EMT pathways significantly.

Conclusion: The activation pathways of transcription of MMP-2,-9 via NF-kB and key marker proteins in EMT pathways are targeted by both ¹²C ion and olaparib/siRNA. Hence, ¹²C ion radiotherapy could potentially be combined with olaparib as chemotherapeutic agent for better control of cancer metastasis.

Keywords: Carbon ion exposure; Epithelial-mesenchymal transition (EMT); Matrix metalloproteinases (MMPs); Metastatic potential; Non-small-cell lung cancer; PARP-1.

Fig. 1

a Survival fraction (SF) of A549 and H1299 cells exposed with ¹²C ion with and without olaparib (O). The survival fraction (SF) of olaparib treated cells at 0 Gy was corrected to 1 and all the olaparib treated cells were normalized accordingly. b represent the same data where survival fraction of untreated control was assumed as 1 and of all other treated cells (either ¹²C ion or olaparib or combined) normalized accordingly. c Time course cell proliferation in A549 cells after treatment with PARP-1 inhibitor DPQ (D) and olaparib (O) separately and in combination with ¹²C ion exposure (0–4 Gy). Each bar represents mean cell proliferation ± standard deviation obtained from three independent experiments. All the differences with respect to control are significant and p-values at each dose were p ≤ 0.001 in each cell-type

Fig. 2

Cell migration and MMP-2, − 9 activity in A549 and H1299 cells. Percent cell migration after PARP-1 inhibition with olaparib (O), DPQ (D) and combined with ¹²C ion in A549 (a) and H1299 (b) cells. Each bar with different pattern represents mean percent cell migration with standard deviation obtained from three independent experiments in triplicates. c-d-e Typical photograph of gelatin zymogram to determine MMP-2 (72 kDa) and MMP-9 (92 kDa) activity after exposure with ¹²C in presence and absence of DPQ (d) (c) or olaparib (O) (d) in A549 cells and H1299 (e) cells

Fig. 3

Expression of MMP-2 and MMP-9 in A549 and H1299 cells treated with ¹²C with and without olaparib (O). a Relative mRNA expression of MMP-2 and MMP-9 in A549 cells after 24 h treatment as determined by Real Time PCR. Each bar represents mean expression ± standard deviation obtained from three independent experiments. All the differences with respect to control are significant and p-values at each dose was p ≤ 0.001 in each cell-type. b-c & d-e Typical western blot to determine expression of MMP-2 and MMP-9 from whole cell lysate in b (A549) & d (H1299) and their respective secretion in culture medium in c (A549) & e (H1299). f,g, h & i Relative expression as measured in fold of MMP-2 and MMP-9 from whole cell lysate f (A549) & h (H1299) and their respective secretion in culture medium in g (A549) &  i (H1299)

Fig. 4

Expression and phosphorylation status of the proteins involved in the upstream signaling pathway in the transcriptional regulation of MMP-2,-9 in H1299 (left panel) and A549 (right panel) cells after ¹²C ion exposure with and without olaparib (O) or siRNA against PARP-1. The bar graphs in each panel represents the quantitative analysis of the proteins as obtained from densitometric analysis of the bands from three independent experiments using imageJ

Fig. 5

Expression of few EMT markers after ¹²C ion exposure with and without olaparib or siRNA. a Typical western blots of N-cadherin and vimentin in A549 (left) and H1299 (right) cells. b & c Relative expression of N-cadherin and vimentin in A549 and H1299 cells as measured in fold. d, e, f & g Expression of the EMT markers N-cadherin, anillin, claudin-1 and claudin-2 respectively by Real Time PCR after normalizing with GAPDH in A549 cell line. All the differences with respect to control are significant and p-values at each dose were p ≤ 0.001

Fig. 6

The cartoon picture represents the mechanisms of inhibition of in-vitro metastatic potential after single and combined treatment. The downward or upward arrows with three different colours beside the protein represent down-regulation or up-regulation respectively of that protein for only PARP-1 inhibition (red), only ¹²C ion exposure (green) and combined treatment (blue). Phosphorylation of each protein is denoted by P within oval shape attached with the respective protein. The down arrow of different colours beside P denotes reduction of phosphorylation of the respective proteins after different treatments