Fluoroquinophenoxazine Derivative as an Antiproliferative Agent and Potential Dual Inhibitor of Human Epidermal Growth Factor Receptor 2 and Topoisomerase IIα in Gastric Cancer Cells

Abstract

Human Epidermal Growth Factor Receptor 2 (HER2, known as ErbB-2) has been known as an oncoprotein giving higher aggressiveness in breast cancers. HER2, a 185kDa trans-membrane tyrosine kinase receptor, is a member of the ErbB protein family and is encoded by HER2 gene. The HER2 gene locates at human chromosome band 17q21-q22 which is very close to topoisomeraseIIα (Topo IIα) gene location. This proximal location in chromosome between HER2 and Topo IIα may induce an amplification or reduction of Topo IIα gene when HER2 gene is amplified. By these reasons, National Comprehensive Cancer Network Guidelines recommend the co-treatment of tratuzumab, monoclonal antibody targeting HER2, with doxorubin/cyclophosphamide followed by paclitaxel for HER2-positive breast cancer from 2009. Doxorubicin is known to inhibit potently topoisomerases. In case of gastric cancers, HER2 was over-expressed in 6-35% patient who showed worse prognosis for chemotherapy than HER2-negative gastric cancer patients. Therefore we were interested to discover a compound which can inhibit both HER2 and Topo IIα in gastric cancer cells. We examined activity of compounds through diverse processes. We evaluated the cytotoxicity of 63 compounds on gastric cancer cells which were characterized as HER2 positive, Topo IIα positive, HER2 and Topo IIα both positive, or both negative cell in mRNA and protein level by our laboratory. We chose the most potent compound and examined the mode of action of the compound. The compound inhibited HER2 expression and HER2 tyrosine kinase activity and induced G0/G1 arrest through the increase of p27kip1 and regulation of cyclin D1. The compound finally enhanced the apoptosis of gastric cancer cell, NCI-N87 characterized as aHER2 positive cell, through stimulating caspase-3 and PARP cleavage. These results demonstrate that the compound which we selected has potency of treatment in the HER2 positive gastric cancers.;인체 상피세포 성장인자 수용체 2 (HER2 or ErbB-2)는 원발암 단백질로 알려져 있으며 유방암에서 높은 비율로 나타난다. HER2는 185 kDa에 달하는 transmembrane tyrosine kinase 수용체로 ErbB family에 포함되며 HER2 유전자에 의해 발현된다. HER2 유전자는 염색체 17q21-q22에 위치해 있으며 topoisomerase IIα (Topo IIα) 유전자와 가까이 있다. HER2와 topo IIα의 근접한 위치는 HER2 유전자가 증폭되었을 때, Topo IIα 유전자의 증폭 또는 억제를 유발하는 요인으로 작용한다. 이러한 이유로 National Comprehensive Cancer Network Guidelines는 2009년에, HER2를 타깃으로 한 단일클론항체인 trastuzumab을 독소루비신/사이클로포스파마이드와 함께 처리한 뒤 파클리탁셀을 뒤이어 처리하는 방법을 권장했다. 독소루비신은 Topoisomerase의 잠재적인 억제제로 잘 알려져 있다. 위암의 경우, HER2가 6-35% 과발현되어 있는데 HER2 과발현 암환자는 HER2 음성 암환자에 비해 치료에 대한 반응성이 더 좋지 않았다. 따라서, 본 연구에서는 위암에서 HER2와 Topo IIα 모두를 억제하는 화합물 발견에 주목하였다. 합성된 63가지 화합물의 독성을 HER2 과발현 암세포, Topo IIα 과발현 암세포, HER2와 Topo IIα 동시 과발현 암세포, 또는 두 가지 모두 음성인 암세포에서 확인하였고, 그 중 HER2 과발현 암세포에 효과적인 화합물을 암세포에 처리해 mRNA와 단백질 발현정도를 측정하여 가장 효과가 있는 화합물 YK6를 선정하였다. YK6는 HER2의 발현과 HER2 tyrosine kinase 활성을 억제하였으며 p27kip1을 증가시키고 cyclinD1을 억제시키는 작용을 통해 G0/G1 arrest를 유발했다. HER2 과발현 세포인 NCI-N87에 YK6를 처리한 결과, caspase-3와 poly (ADP-ribose) polymerase (PARP)의 분절을 통한 apoptosis를 확인했다. 이러한 결과들로 YK6가 HER2 과발현 위암에 치료제로서의 잠재적 가능성이 있음을 증명할 수 있었다.