Department of Biochemistry & Biophysics, University of Kalyani, Kalyani, 741235, India.
Radiation Biology Laboratory, Inter-University Accelerator Centre, New Delhi, 110067, India.
BMC Cancer. 2019 Aug 22;19(1):829. doi: 10.1186/s12885-019-6015-4.
Carbon ion (C) 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 C 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 C 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 C 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 C 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 C 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 C 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 C ion exposure and in combination with PARP-1 inhibitor olaparib.
Our experiments show that C 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 C ion and olaparib/siRNA. Hence, C ion radiotherapy could potentially be combined with olaparib as chemotherapeutic agent for better control of cancer metastasis.
碳离子（C）放疗在杀死高转移性癌细胞的同时，对相邻的正常细胞的影响最小，因此越来越有前途。我们之前的研究表明，联合 PARP-1 抑制与 C 离子放疗协同降低 HeLa 细胞中的 MMP-2 和 MMP-9，但详细机制尚不清楚。为了理解这种机制以及使用 PARP-1 抑制剂与 C 离子放疗联合治疗以更好地控制转移的原理，我们使用 siRNA 或奥拉帕利研究了在存在和不存在 PARP-1 抑制剂的情况下，C 离子对两种非小细胞肺癌（NSCLC）A549 和 H1299（p53 缺失）细胞的转移潜能的影响。
我们使用 siRNA 或奥拉帕利监测了 C 离子暴露后 A549 和 p53 缺失的 H1299 细胞系中细胞增殖、体外细胞迁移、划痕愈合、MMP-2、MMP-9 的表达和活性。还观察了 C 离子暴露后 A549 和 H1299 细胞中 NF-kB、EGFR、Akt、p38、ERK 的表达和磷酸化。我们还检查了 A549 和 H1299 细胞中几个参与上皮-间充质转化（EMT）途径的标记基因的表达，如 N-钙粘蛋白、波形蛋白、肌球蛋白、闭合蛋白-1、-2。为了确定 C 离子和奥拉帕利在抑制细胞转移潜能方面的普遍作用，我们还在 C 离子暴露后以及与 PARP-1 抑制剂奥拉帕利联合使用时，研究了 HeLa 和 MCF7 细胞系中的划痕愈合和 MMP-2、MMP-9 的活性。
我们的实验表明，C 离子和 PARP-1 抑制分别减少细胞增殖、细胞迁移、划痕愈合、EGFR、Akt、p38、ERK 的磷酸化，导致 NF-kB 失活。联合治疗消除了 NF-kB 的表达，从而协同降低了 MMP-2 和 MMP-9 的表达。每种单一治疗均减少了 N-钙粘蛋白、波形蛋白、肌球蛋白，但增加了闭合蛋白-1、-2，从而抑制 EMT 过程。然而，联合治疗协同地显著改变了这些蛋白以抑制 EMT 途径。
C 离子和奥拉帕利/siRNA 通过 NF-kB 靶向 MMP-2、MMP-9 的转录激活途径和 EMT 途径中的关键标记蛋白。因此，C 离子放疗可能与奥拉帕利作为化疗药物联合使用，以更好地控制癌症转移。
