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

摘要

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. 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. 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. 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.