Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Colorado School of Medicine, 12631 East 17th Avenue B198-6, Aurora, CO, 80045, USA.
Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Colorado School of Medicine, Aurora, CO, USA.
Mol Cell Biochem. 2019 Nov;461(1-2):37-46. doi: 10.1007/s11010-019-03586-9. Epub 2019 Jul 10.
We tested the hypothesis that stimulation of adiponectin receptors with the synthetic agonist AdipoRon suppresses proliferation and induces apoptotic death in human high grade serous ovarian tumor cell lines and in ex vivo primary tumors, mediated by activation of 5' AMP-activated protein kinase (AMPK) and inhibition of mechanistic target of rapamycin (mTOR). We determined the effect of AdipoRon on high grade serous ovarian tumor cells lines (OVCAR3, OVCAR4, A2780) and ex vivo primary tumor tissue. Western blotting analysis was performed to examine changes in activation of AMPK and mTOR signaling and flow cytometry was utilized to examine changes in cell cycle progression. Immunofluorescence of cleaved caspase-3 positive cells and flow cytometry of annexin V positive cells were used to determine changes in apoptotic response. The CyQUANT proliferation assay was used to assess cell proliferation. AdipoRon treatment increased AMPK phosphorylation (OVCAR3 P = 0.01; A2780 P = 0.02) but did not significantly alter mTOR activity. AdipoRon induced G1 cell cycle arrest in OVCAR3 (+ 12.1%, P = 0.03) and A2780 (+ 12.0%, P = 0.002) cells. OVCAR3 and OVCAR4 cells treated with AdipoRon underwent apoptosis based on cleaved caspase-3 and annexin V staining. AdipoRon treatment resulted in a dose dependent decrease in cell number versus vehicle treatment in OVCAR3 (-61.2%, P < 0.001), OVCAR4 (-79%, P < 0.001), and A2780 (-56.9%, P < 0.001). Ex vivo culture of primary tumors treated with AdipoRon resulted in an increase in apoptosis measured with cleaved caspase-3 immunohistochemistry. AdipoRon induces activation of AMPK and exhibits an anti-tumor effect in ovarian cancer cell lines and primary tumor via a mTOR-independent pathway.
我们验证了这样一个假设，即合成激动剂 AdipoRon 刺激脂联素受体可通过激活 5' AMP 激活的蛋白激酶 (AMPK) 和抑制雷帕霉素的靶蛋白 (mTOR)，抑制人高级别浆液性卵巢肿瘤细胞系和离体原发性肿瘤的增殖并诱导其凋亡。我们确定了 AdipoRon 对高级别浆液性卵巢肿瘤细胞系（OVCAR3、OVCAR4、A2780）和离体原发性肿瘤组织的影响。通过 Western blot 分析检测 AMPK 和 mTOR 信号转导激活的变化，通过流式细胞术检测细胞周期进程的变化。用免疫荧光法检测 cleaved caspase-3 阳性细胞的变化，用流式细胞术检测 annexin V 阳性细胞的变化，以确定细胞凋亡反应的变化。用 CyQUANT 增殖测定法评估细胞增殖。AdipoRon 处理增加了 AMPK 磷酸化（OVCAR3 P=0.01；A2780 P=0.02），但并未显著改变 mTOR 活性。AdipoRon 诱导 OVCAR3（+12.1%，P=0.03）和 A2780（+12.0%，P=0.002）细胞的 G1 细胞周期停滞。用 AdipoRon 处理的 OVCAR3 和 OVCAR4 细胞通过 cleaved caspase-3 和 annexin V 染色发生凋亡。与对照相比，AdipoRon 处理导致 OVCAR3 细胞（-61.2%，P<0.001）、OVCAR4 细胞（-79%，P<0.001）和 A2780 细胞（-56.9%，P<0.001）的细胞数呈剂量依赖性减少。AdipoRon 处理离体原发性肿瘤导致用 cleaved caspase-3 免疫组化检测到的凋亡增加。AdipoRon 通过非 mTOR 依赖性途径在卵巢癌细胞系和原发性肿瘤中诱导 AMPK 激活并发挥抗肿瘤作用。
