Nanoengineered Gallium Ion Incorporated Formulation for Safe and Efficient Reversal of PARP Inhibition and Platinum Resistance in Ovarian Cancer

Platinum-based chemotherapy remains the main systemic treatment of ovarian cancer(OC).However,the inevitable development of platinum and poly(adenosine diphosphate-ribose)polymerase inhibitor(PARPi)resistance is associated with poor outcomes,which becomes a major obstacle in the management of this disease.The present study developed“all-in-one”nanoparticles that contained the PARPi olaparib and gallium(Ga)(III)(olaparib-Ga)to effectively reverse PARPi resistance in platinum-resistant A2780-cis and SKOV3-cis OC cells and in SKOV3-cis tumor models.Notably,the olaparib-Ga suppressed SKOV3-cis tumor growth with negligible toxicity.Moreover,the suppression effect was more evident when combining olaparib-Ga with cisplatin or carboplatin,as evaluated in A2780-cis and SKOV3-cis cells.Mechanistically,the combined treatment induced DNA damage,which elicited the activation of ataxia telangiectasia mutated(ATM)/AMT-and Rad3-related(ATR)checkpoint kinase 1(Chk1)/Chk2 signal transduction pathways.This led to the arrest of cell cycle progression at S and G2/M phases,which eventually resulted in apoptosis and cell death due to unrepairable DNA damage.In addition,effective therapeutic responses to olaparib-Ga and cisplatin combination or olaparib-Ga and carboplatin combination were observed in SKOV3-cis tumor-bearing animal models.Altogether,the present findings demonstrate that olaparib-Ga has therapeutic implications in platinum-resistant OC cells,and the combination of olaparib-Ga with cisplatin or carboplatin may be promising for treating patients with OC who exhibit resistance to both PARPi and platinum.

奥拉帕利联合IL-1β抑制剂对HRD阴性卵巢上皮性癌细胞生长的抑制作用

目的:探讨聚二磷酸腺苷核糖聚合酶(PARP)抑制剂联合白细胞介素1β(IL-1β)抑制剂对同源重组修复缺陷(HRD)阴性卵巢上皮性癌(卵巢癌)细胞的抑制作用。方法:(1)使用HRD阴性的卵巢癌细胞系OVCAR3、CAOV3细胞,先分别给予PARP抑制剂奥拉帕利(122 μmol/L)和二甲基亚砜(作为对照)处理OVCAR3细胞,RNA测序及筛选差异表达基因;随后采用酶联免疫吸附试验(ELISA)和蛋白印迹(western blot)法验证奥拉帕利处理后OVCAR3、CAOV3细胞中IL-1β蛋白的表达。(2)根据不同浓度(0、1.25、2.5、5、10、20、40、80、160、320 μmol/L)IL-1β抑制剂diacerein(为蒽醌类化合物)在OVCAR3和CAOV3细胞中的药物剂量反应曲线,确定两种细胞IL-1β抑制剂的50%抑制浓度(IC 50)。细胞实验分为4组,对照组、奥拉帕利组、IL-1β抑制剂组、奥拉帕利+IL-1β抑制剂组,活细胞计数(CCK-8)法检测4组OVCAR3、CAOV3细胞的存活率。(3)将稳定表达荧光素酶基因luciferase的OVCAR3细胞(OVCAR3-Luc细胞)按照1×10 7个/只接种于裸鼠腹腔中,建立裸鼠腹腔移植瘤模型。动物实验将16只成瘤裸鼠采用随机表法随机分为4组,对照组、奥拉帕利组、IL-1β抑制剂组、奥拉帕利+IL-1β抑制剂组,每组4只。采用荧光素酶小动物活体成像测定4组裸鼠移植瘤的荧光信号强度,免疫组化法检测移植瘤组织中增殖相关核抗原(Ki-67)蛋白的表达。(4)药物毒副反应:腹腔注射后第29天,裸鼠称重后取其外周血进行血常规和肝肾功能检测;随后处死裸鼠,取裸鼠重要器官进行HE染色评估药物对器官的损害情况。 结果:(1)RNA测序及差异表达基因筛选,共获得25个显著差异表达基因,尤以IL-1β mRNA的表达差异最显著。ELISA法检测显示,奥拉帕利处理后OVCAR3、CAOV3细胞分泌的IL-1β蛋白含量分别为(36.2±3.5)和(49.5±3.5)pg/ml,均显著高于对照OVCAR3、CAOV3细胞[分别为(5.3±0.7)和(14.7±0.7)pg/ml;P均<0.001];western blot检测显示,奥拉帕利处理后OVCAR3和CAOV3细胞中IL-1β蛋白的相对表达水平分别为2.87±0.37、2.05±0.08,均显著高于对照OVCAR3、CAOV3细胞(均设为1.00;P均<0.001)。(2)剂量反应曲线确定IL-1β抑制剂在OVCAR3细胞中的IC 50为75 μmol/L,CAOV3细胞中为100 μmol/L。CCK-8法检测显示,对照组、奥拉帕利组、IL-1β抑制剂组、奥拉帕利+IL-1β抑制剂组OVCAR3细胞的存活率分别为(100.0±0.4)%、(63.1±6.2)%、(61.6±4.7)%、(32.9±5.2)%,CAOV3细胞分别为(100.0±3.5)%、(63.3±3.8)%、(63.8±3.5)%、(30.0±1.3)%,奥拉帕利+IL-1β抑制剂组OVCAR3、CAOV3细胞的存活率均低于对照组、奥拉帕利组、IL-1β抑制剂组( P均<0.01)。(3)腹腔注射后第29天,荧光素酶小动物活体成像显示,奥拉帕利+IL-1β抑制剂组裸鼠移植瘤的荧光信号强度为(0.5±0.4)×10 10 p/s,显著低于对照组[(4.2±1.0)×10 10 p/s]、奥拉帕利组[(3.1±0.9)×10 10 p/s]、IL-1β抑制剂组[(2.2±0.9)×10 10 p/s;P均<0.05];奥拉帕利+IL-1β抑制剂组裸鼠瘤重为(0.09±0.03)g,显著低于对照组[(0.25±0.05)g]、奥拉帕利组[(0.17±0.03)g]、IL-1β抑制剂组[(0.19±0.04)g;P均<0.05]。免疫组化法检测显示,奥拉帕利+IL-1β抑制剂组裸鼠移植瘤组织中Ki-67蛋白的表达水平为0.33±0.10,显著低于对照组(1.00±0.20)、奥拉帕利组(0.76±0.07)、IL-1β抑制剂组(0.77±0.12;P均<0.05)。(4)药物毒副反应:腹腔注射后第29天,奥拉帕利+IL-1β抑制剂组裸鼠的体重、血常规和肝肾功能,分别与对照组、奥拉帕利组、IL-1β抑制剂组比较,差异均无明显统计学意义( P均>0.05)。重要器官包括心、肝、脾、肺和肾,镜下观察均未见明显致死性损害。 结论:PARP抑制剂奥拉帕利联合IL-1β抑制剂在HRD阴性的卵巢癌细胞中显示出显著的肿瘤抑制作用,且无明显的药物毒副反应。