Oxidative stress-mediated colon cancer cell death by small molecule inhibitors of STAT3

Abstract

Extracellular signal에 의해 receptor-associated kinase들에 의해 signal transducer and activator of transcription (STAT) protein이 phosphorylation 되고 homo- 또는 heterodimer를 이룬다. Dimerized STAT protein들은 핵 내로 들어가게 되고 target gene의 발현을 조절한다. STAT3는 STAT family member 중 하나로 cell proliferation, survival 그리고 tumor growth을 promotion한다고 알려져 있다. 우리는 최근 개발된, STAT3 phosphorylation을 억제하는 small molecule inhibitor인 LLL12와 Stattic을 사용하여 colon cancer cell proliferation에서 STAT3의 효과를 분석하였다. WST-1 assay를 통하여 이 두 inhibitor들이 cytotoxic하며 24 시간 이내 급격한 cell death를 일으키는 것을 확인할 수 있었다. 또한 apoptotic cell death의 marker 중 하나인 PARP-1의 cleavage를 관찰할 수 없었다. 흥미롭게도 우리는 이 두 inhibitor들이 ROS 생성을 증가시키는 것을 확인 하였으며, antioxidant인 N-acetylcysteine을 전처리 함으로써 STAT3 inhibitor들에 의한 cell death를 억제하고 STAT3의 phosphorylation의 감소 역시 억제하는 것을 확인 했다. 이러한 결과들은 LLL12와 Stattic에 의한 cell death에 ROS 생성에 의한 cytotoxicity가 우선적으로 관여한다는 것을 알 수 있게 해준다.;In response to extracellular signals, signal transducer and activator of transcription (STAT) proteins are phosphorylated by receptor-associated kinases and form homo- or heterodimers. The dimerized STAT proteins then translocate to the nucleus and activate the expression of target genes. STAT3 belongs to a member of the STAT family whose activation is known to promote cell proliferation, survival, and tumor growth in vivo. In the present study, we analyzed the effect of STAT3 on colon cancer cell proliferation using LLL12 and Stattic, recently developed small molecule inhibitors of STAT3 phosphorylation. WST-1 assay revealed that both inhibitors were cytotoxic, inducing rapid cell death within 24 h. Considering the morphological change and only a small portion of Parp1 cleavage, marker of apoptotic cell death, the main cell death was likely due to the necrosis. Interestingly, we have found that both inhibitors resulted in generation of reactive oxygen species (ROS) and pretreatment of cells with N-acetylcysteine, an antioxidant, abolished not only the cell death but also the reduction of STAT3 phosphorylation induced by STAT3 inhibitors. Taken together these results suggest that ROS-induced cytotoxicity is the primary reason for cell death by LLL12 and Stattic.