Purpose The PI3K/AKT/mTOR pathway is one of the most highly activated cellular signaling pathways in advanced ovarian cancer. Although several PI3K/AKT/mTOR inhibitors have been developed to treat various types of cancer, the antitumor efficacy of many of these compounds against ovarian cancer has remained unclear. Methods Here, we tested and compared a panel of 16 PI3K/AKT/mTOR inhibitors (XL765, Miltefosine, Rapamycin, CCI-779, RAD001, FK506, XL147, GSK2110183, IPI-145, GSK2141795, BYL719, GSK458, CAL-101, XL765 analogue SAR245409, Triciribine, and GDC0941) that have entered clinical trials for antitumor activity against ovarian cancer, as well as the front line drug, paclitaxel. Antitumor efficacy was measured in both ovarian cancer cell lines and patient-derived ovarian primary tumor cell lines in vitro and in vivo. Results We identified the PI3K/mTOR dual inhibitor GSK458 as a potent inhibitor of proliferation in all cell lines tested at half maximal inhibitory concentrations ( IC_(50)\mathrm{IC}_{50} ) of approximately 0.01-1muM0.01-1 \mu \mathrm{M}, a range tens to hundreds fold lower than that of the other PI3K/AKT/mTOR inhibitors tested. Additionally, GSK458 showed the highest inhibitory efficacy against ovarian cancer cell migration. GSK458 also inhibited tumor growth and metastasis in nude mice intraperitoneally engrafted with SKOV3 cells or a patient-derived tumor cell xenograft (PDCX). Importantly, the inhibitory efficiency of GSK458 on cell proliferation and migration both in vitro and in vivo was comparable to that of paclitaxel. Mechanistically, the anti-tumor activity of GSK458 was found to be associated with inactivation of AKT and mTOR, and induction of cell cycle arrest at the G0/G1 phase. Conclusions Based on our results, we conclude that GSK458 may serve as an attractive candidate to treat ovarian cancer. 目的 PI3K/AKT/mTOR 通路是晚期卵巢癌中激活程度最高的细胞信号通路之一。尽管已经开发了几种 PI3K/AKT/mTOR 抑制剂来治疗各种类型的癌症，但其中许多化合物对卵巢癌的抗肿瘤疗效仍不清楚。方法 在这里，我们测试和比较了一组 16 种 PI3K/AKT/mTOR 抑制剂 （XL765、Miltefosine、Rapamycin、CCI-779、RAD001、FK506、XL147、GSK2110183、IPI-145、GSK2141795、BYL719、GSK458、CAL-101、XL765 类似物 SAR245409、Triciribine 和 GDC0941），以及一线药物紫杉醇。在体外和体内测量卵巢癌细胞系和患者来源的卵巢原发性肿瘤细胞系的抗肿瘤疗效。结果我们确定了 PI3K/mTOR 双重抑制剂 GSK458 在所有细胞系中是一种有效的增殖抑制剂，以大约 0.01-1muM0.01-1 \mu \mathrm{M} 一半最大抑制浓度 （ IC_(50)\mathrm{IC}_{50} ） 测试，范围比其他测试的 PI3K/AKT/mTOR 抑制剂低数十至数百倍。此外，GSK458 对卵巢癌细胞迁移的抑制效果最高。GSK458 还抑制腹膜内植入 SKOV3 细胞或患者来源的肿瘤细胞异种移植物 （PDCX） 的裸鼠的肿瘤生长和转移。重要的是，GSK458 在体外和体内对细胞增殖和迁移的抑制效率与紫杉醇相当。从机制上讲，发现 GSK458 的抗肿瘤活性与 AKT 和 mTOR 的失活以及 G0/G1 期细胞周期停滞的诱导有关。 结论 根据我们的结果，我们得出结论，GSK458 可能是治疗卵巢癌的有吸引力的候选药物。
Over the past two decades, the five-year overall survival (OS) rate for ovarian cancer patients has remained low ( 30%-40%30 \%-40 \% ). 在过去的二十年里，卵巢癌患者的五年总生存率 （OS） 一直很低 （ 30%-40%30 \%-40 \% ）。
There were an estimated 295,414 cases and 184,799 deaths of ovarian cancer around the world in 2018 [1]. Although the ovarian cancer death rate has plateaued over the last two decades, it remains high worldwide, making ovarian cancer the 据估计，2018 年全球卵巢癌病例为 295,414 例，死亡人数为 184,799 例 [1]。尽管卵巢癌死亡率在过去二十年中趋于稳定，但在全球范围内仍然很高，这使得卵巢癌成为
deadliest tumor among all gynecological malignancies [2-4]. As symptoms of ovarian cancer are inapparent at early stages, about two-thirds of patients are at an advanced disease stage at diagnosis. Several treatments have entered the clinic. Carboplatin and paclitaxel exhibit excellent antitumor effects as first-line treatments, however, around two-thirds of the patients become resistant to these drugs and ultimately relapse [5]. Several targeted (poly ADP ribose polymerase (PARP) inhibitors) and immuno-therapy (anti-PD-1 and PD-L1 antibodies) strategies have been evaluated, with only modestly increased OS rates (2%-4%)(2 \%-4 \%) [6]. Although PARP inhibitors (Olaparib, Rucaparib or Niraparib) can increase progression-free survival (PFS) durations of ∼5\sim 5 to 16 months in patients with or without BRCA1/2 mutations, they only marginally improve the fiveyear OS. In phase 2 and phase 3 clinical trials, for example, the reported OS was only prolonged for about 2 to 5 months by Olaparib therapy ( 58%58 \% and 24%24 \% data maturity, respectively) [7, 8]. Anti-PD-1 and/or PDL-1 antibodies are two of the most successful immuno-therapeutics presently used in the clinic [9]. However, only 6-15% of ovarian cancer patients respond to PD-1/PDL-1 blockade, with a median PFS of anti-PD-1 or PDL-1 antibody treatment being only ∼6\sim 6 months [10,11][10,11]. Therefore, novel effective treatment strategies are urgently needed to improve the OS of ovarian cancer patients [5]. 所有妇科恶性肿瘤中最致命的肿瘤 [2-4]。由于卵巢癌的早期症状不明显，因此约三分之二的患者在诊断时已处于晚期。几种治疗方法已进入临床。卡铂和紫杉醇作为一线治疗表现出优异的抗肿瘤作用，然而，约三分之二的患者对这些药物产生耐药并最终复发 [5]。已经评估了几种靶向策略（poly ADP ribose polymerase， PARP）抑制剂）和免疫治疗（抗 PD-1 和 PD-L1 抗体）策略，但 OS 率 (2%-4%)(2 \%-4 \%) 仅略有增加 [6]。尽管 PARP 抑制剂（奥拉帕尼、鲁卡帕尼或尼拉帕尼）可以将有或没有 BRCA1/2 突变的患者的无进展生存期 （PFS） 持续时间延长 ∼5\sim 5 至 16 个月，但它们仅略微改善了五年 OS。例如，在 2 期和 3 期临床试验中，奥拉帕尼治疗仅将报道的 OS 延长了约 2 至 5 个月（分别为 58%58 \% 和数据 24%24 \% 成熟度）[7， 8]。抗 PD-1 和/或 PDL-1 抗体是目前临床上使用的两种最成功的免疫疗法 [9]。然而，只有 6-15% 的卵巢癌患者对 PD-1/PDL-1 阻断有反应，抗 PD-1 或 PDL-1 抗体治疗的中位 PFS 仅为 ∼6\sim 6 数月 [10,11][10,11] 。因此，迫切需要新的有效治疗策略来改善卵巢癌患者的 OS [5]。
In this study, we found that the phosphatidylinositol 3-kinase (PI3K)/AKT pathway is highly activated in advanced (stage III and IV) compared to early stage (stage II) ovarian tumors by KEGG pathway enrichment analysis of mRNA sequencing data from The Cancer Genome Atlas (TCGA) database. This observation suggests that the PI3K/AKT signaling pathway may play a critical role in ovarian cancer metastasis and recurrence. Consistent with our observation, previous studies have shown that the PI3K/AKT pathway may be an attractive therapeutic target for treating ovarian cancer patients [12, 13]. Also, the TCGA consortium reported that the PI3K/AKT/mTOR (mammalian target of rapamycin, mTOR) pathway was activated in 34%34 \% of the tumors from 316 high grade serous ovarian cancer patients [14]. PI3K/AKT/mTOR is one of the most studied signaling pathways in cancer biology because of its wide involvement in carcinogenesis, metastasis, recurrence and chemotherapeutic drug resistance [15]. It has been reported that the PI3K/AKT/mTOR pathway is highly activated in approximately 70%70 \% of ovarian cancers [16, 17]. Several PI3K/AKT/mTOR inhibitors have shown some therapeutic effects against ovarian cancers, alone or in combination with other drugs such as paclitaxel or carboplatin, in clinical trials [15, 16]. Among the most widely studied PI3K/AKT/mTOR inhibitors are the PI3K inhibitors (BKM120 [18], XL147, GDC0941, PX866, ETP46321 [19], and CH5132799 [20]), AKT inhibitors (Perifosine, MK2206, GSK2141795 [16] and GSK690693), mTOR inhibitors (Rapamycin, RAD001 [21], CCI-779 [16, 22-24], Ridaforolimus, AZC8055, OSI-027, AZD2014 [25] and PP242 [26]), PI3K/mTOR dual inhibitors (SF1126 [16], 在这项研究中，我们发现磷脂酰肌醇 3-激酶 （PI3K）/AKT 通路在晚期 （III 期和 IV） 与早期 （II 期） 卵巢肿瘤相比，通过癌症基因组图谱 （TCGA） 数据库的 mRNA 测序数据的 KEGG 通路富集分析。这一观察表明 PI3K/AKT 信号通路可能在卵巢癌转移和复发中起关键作用。与我们的观察一致，先前的研究表明，PI3K/AKT 通路可能是治疗卵巢癌患者的有吸引力的治疗靶点 [12， 13]。此外，TCGA 联盟报道，PI3K/AKT/mTOR （雷帕霉素的哺乳动物靶标，mTOR） 通路在 316 例高级别浆液性卵巢癌患者的肿瘤中 34%34 \% 被激活 [14]。PI3K/AKT/mTOR 是癌症生物学中研究最多的信号通路之一，因为它广泛参与致癌、转移、复发和化疗耐药 [15]。据报道，PI3K/AKT/mTOR 通路在大约 70%70 \% 卵巢癌中高度激活 [16， 17]。在临床试验中，几种 PI3K/AKT/mTOR 抑制剂单独使用或与其他药物（如紫杉醇或卡铂）联合使用已显示出对卵巢癌的一些治疗效果 [15， 16]。研究最广泛的 PI3K/AKT/mTOR 抑制剂包括 PI3K 抑制剂（BKM120 [18]、XL147、GDC0941、PX866、ETP46321 [19] 和 CH5132799 [20]）、AKT 抑制剂（Perifosine、MK2206、GSK2141795 [16] 和 GSK690693）、mTOR 抑制剂（雷帕霉素、RAD001 [21]、CCI-779 [16、22-24]、利达福莫司、AZC8055、OSI-027、AZD2014 [25] 和 PP242 [26]）、PI3K/mTOR 双重抑制剂（SF1126 [16]、
GSK458 [16], XL765 [16], PF-04691502 [27], PF-05212384 [27], BEZ235 [28] and DS-7423 [29]) and the mTOR/AKT dual inhibitor (MKC1 [16]). However, in most studies these inhibitors only showed modest antitumor activity. In addition, many of them were reported in independent publications making it difficult to compare them side by side. Most importantly, the effects of these PI3K/AKT/mTOR inhibitors on ovarian cancer have not been elucidated yet, which may be due to study limitations such as limited numbers of cases included or high toxicities of the inhibitors in clinical trials. GSK458 [16]、XL765 [16]、PF-04691502 [27]、PF-05212384 [27]、BEZ235 [28] 和 DS-7423 [29]） 以及 mTOR/AKT 双重抑制剂 （MKC1 [16]）。然而，在大多数研究中，这些抑制剂仅显示出适度的抗肿瘤活性。此外，其中许多研究成果在独立出版物中进行了报道，因此很难并列比较。最重要的是，这些 PI3K/AKT/mTOR 抑制剂对卵巢癌的影响尚未阐明，这可能是由于研究局限性，例如纳入的病例数有限或临床试验中抑制剂的高毒性。
Patient-derived tumor xenograft (PDX) and patient-derived tumor cell xenograft (PDCX) mouse models have been shown to replicate the tumor biology of patients [30-32]. Accordingly, our previous work showed similar morphologies (i.e., glands, papillae, stromal cores, and desmoplastic stroma) between xenograft models and patient tumor tissues [33]. Here, we compared, in parallel, the antitumor effects of 16 PI3K/AKT/mTOR inhibitors and paclitaxel, not only in ovarian cancer cell lines but also in primary tumor cells in vitro. Also, we tested the effects of high potent candidates in PDX and PDCX models in vivo. Surprisingly, we found that many of the tested inhibitors had little or even no inhibitory effect on ovarian cancer cell proliferation or migration. GSK458, also known as GSK2126458, exhibited the highest antiproliferative and -migrative activities in both ovarian cancer cell lines and patient-derived primary tumor cell lines among all PI3K/AKT/mTOR inhibitors tested. GSK458 has previously been reported to be an efficient PI3K/mTOR dual inhibitor with an inhibitory constant (Ki) ranging from 0.01 to 0.3 nM by Steven and colleagues in 2010 [34]. A previous phase I clinical trial study showed that GSK458 has few adverse effects following oral administration and can produce durable objective responses (OR) in multiple tumor types (sarcoma, kidney, breast, endometrial, oropharyngeal, and bladder cancer) with two patients having ongoing responses lasting more than 4 years [35]. However, to date there are no thorough reports on antitumor activity of GSK458 in ovarian cancer. Here, we show that GSK458 may serve as a potent candidate for the development of targeted therapies against ovarian cancer. We also provide comparative data on the efficacy of various PI3K/AKT/mTOR inhibitors for further preclinical or clinical studies on ovarian cancer. Since the safety and antitumor activity of GSK458 has already been reported in a phase I clinical trial study [35], we believe it is a strong candidate for future preclinical and/or clinical studies. 研究显示，患者来源的肿瘤异种移植物（PDX）和患者来源的肿瘤细胞异种移植物（PDCX）小鼠模型可以复制患者的肿瘤生物学特性[30-32]。因此，我们之前的工作显示异种移植模型和患者肿瘤组织之间存在相似的形态（即腺体、、基质核心和结缔组织增生性基质）[33]。在这里，我们同时比较了 16 种 PI3K/AKT/mTOR 抑制剂和紫杉醇的抗肿瘤作用，不仅在卵巢癌细胞系中，而且在体外原发性肿瘤细胞中。此外，我们还在体内测试了高效候选药物在 PDX 和 PDCX 模型中的效果。令人惊讶的是，我们发现许多测试的抑制剂对卵巢癌细胞增殖或迁移几乎没有抑制作用。GSK458，也称为 GSK2126458，在所有测试的 PI3K/AKT/mTOR 抑制剂中，在卵巢癌细胞系和患者来源的原发性肿瘤细胞系中均表现出最高的抗增殖和迁移活性。GSK458 之前曾被 Steven 及其同事在 2010 年报道为一种有效的 PI3K/mTOR 双重抑制剂，抑制常数 （Ki） 范围为 0.01 至 0.3 nM [34]。先前的 I 期临床试验研究表明，GSK458 口服给药后几乎没有不良反应，并且可以在多种肿瘤类型（肉瘤、肾癌、乳腺癌、子宫内膜癌、口咽癌和膀胱癌）中产生持久的客观缓解 （OR），其中 2 例患者的持续缓解持续超过 4 年[35]。然而，迄今为止，还没有关于 GSK458 在卵巢癌中的抗肿瘤活性的全面报道。在这里，我们表明 GSK458 可能是开发针对卵巢癌的靶向疗法的有效候选者。 我们还提供了有关各种 PI3K/AKT/mTOR 抑制剂疗效的比较数据，用于卵巢癌的进一步临床前或临床研究。由于 GSK458 的安全性和抗肿瘤活性已在 I 期临床试验研究中报道 [35]，我们相信它是未来临床前和/或临床研究的有力候选者。
Ovarian tumor messenger RNA sequencing data from The Cancer Genome Atlas (TCGA) were downloaded (https:// 下载来自癌症基因组图谱 （TCGA） 的卵巢肿瘤信使 RNA 测序数据 （https:// tcga.xenahubs.net/download/TCGA.OV.sampleMap/ HiSeqV2_exon.gz) and analyzed. The data were divided into two groups (“early” and “advanced”) according to clinical stages determined by the International Federation of Gynecology and Obstetrics (FIGO) stage criteria. The early group includes patients at stage II ( 21 patients), and the advanced group includes patients at stage III ( 233 patients) and IV (38 patients). KEGG pathway enrichment analysis was performed using the DESeq (2012) R package with default parameters according to a previous study [36] to detect the most activated pathways in advanced ovarian tumors. tcga.xenahubs.net/download/TCGA.OV.sampleMap/HiSeqV2_exon.gz） 并进行了分析。根据国际妇产科联合会 （FIGO） 分期标准确定的临床分期，将数据分为两组 （“早期” 和 “晚期”）。早期组包括 II 期患者 （ 21 例患者），晚期组包括 III 期 （233 例患者） 和 IV 期 （38 例患者）。根据之前的研究 [36]，使用具有默认参数的 DESeq （2012） R 包进行 KEGG 通路富集分析，以检测晚期卵巢肿瘤中最活跃的通路。
2.2 Inhibitors 2.2 抑制剂
In total 16PI3K//AKT//mTOR16 \mathrm{PI} 3 \mathrm{~K} / \mathrm{AKT} / \mathrm{mTOR} inhibitors were acquired from Selleck (Houston, TX, USA). This inhibitory panel includes 5 PI3K inhibitors [XL-147 (#S7645), IPI-145 (#S7028), BYL719 (#S2814), CAL-101 (#S2226), and GDC-0491 (#S1065)], 3 AKT inhibitors [GSK2141795 (#S7492), GSK2110183 (#S7521) and Triciribine (#S1117)], 4 mTOR inhibitors [Rapamycin (#S1039), CCI-779 (#S1044), RAD001 (#S1120) and FK506 (#S5003)], 1 PI3K/AKT inhibitor [Miltefosine (#S3056)] and 3 PI3K/mTOR dual inhibitors [GSK458 (#S2658), XL765 (#S7646) and SAR245409 (#S1523)]. Paclitaxel (#T7191) was purchased from SigmaAldrich (St. Louis, MO, USA). Cisplatin (#HY-17394) was purchased from MedChemExpress (MCE, NJ, USA). All compounds were dissolved in DMSO at a concentration of 10 mM and stored at -80 before use. 总共 16PI3K//AKT//mTOR16 \mathrm{PI} 3 \mathrm{~K} / \mathrm{AKT} / \mathrm{mTOR} 从 Selleck （美国德克萨斯州休斯顿） 获得抑制剂。该抑制性组合包括 5 种 PI3K 抑制剂 [XL-147 （#S7645）、IPI-145 （#S7028）、BYL719 （#S2814）、CAL-101 （#S2226） 和 GDC-0491 （#S1065）]、3 种 AKT 抑制剂 [GSK2141795 （#S7492）、GSK2110183 （#S7521） 和曲瑞立滨 （#S1117）]、4 种 mTOR 抑制剂 [雷帕霉素 （#S1039）、CCI-779 （#S1044）、RAD001 （#S1120） 和 FK506 （#S5003）]、1 种 PI3K/AKT 抑制剂 [Miltefosine （#S3056）] 和 3 种 PI3K/mTOR 双重抑制剂 [GSK458 （#S2658）、XL765 （#S7646） 和 SAR245409 （#S1523）]。紫杉醇 （#T7191） 购自 SigmaAldrich（美国密苏里州圣路易斯）。顺铂 （#HY-17394） 购自 MedChemExpress （MCE， NJ， USA）。所有化合物均以 10 mM 的浓度溶于 DMSO 中，并在使用前以 -80 储存。
2.3 Ovarian cancer cell lines and primary ovarian tumor cell lines 2.3 卵巢癌细胞系和原代卵巢肿瘤细胞系
The cell lines used in this study cover the 4 main subtypes of epithelial ovarian cancers: serous carcinoma (OVCAR3, OVCAR8), endometrioid carcinoma (TOV112D, COV362), mucinous carcinoma (MCAS) and clear cell carcinoma (ES2, OVISE, OVTOKO). Additionally, several widely used epithelial ovarian cancer cells of unclear subtype (A2780, A2780CP, SKOV3, SKOV3TR and SKOV3IP) or with a mixed subtypes (IGROV-1 and HO8910PM), were included [37, 38]. A normal ovarian epithelial cell line HOSEPIC was used as control. All epithelial cell lines were cultured in complete RPMI-1640 culture medium (GIBCO, NE, USA) containing 10%10 \% fetal bovine serum (FBS; GIBCO), 100U//ml100 \mathrm{U} / \mathrm{ml} penicillin (GIBCO) and 100 mug//ml100 \mu \mathrm{~g} / \mathrm{ml} streptomycin (GIBCO). 本研究中使用的细胞系涵盖上皮性卵巢癌的 4 种主要亚型：浆液性癌 （OVCAR3， OVCAR8）、子宫内膜样癌 （TOV112D， COV362）、粘液癌 （MCAS） 和透明细胞癌 （ES2， OVISE， OVTOKO）。此外，还包括几种广泛使用的亚型不明（A2780、A2780CP、SKOV3、SKOV3TR 和 SKOV3IP）或混合亚型 （IGROV-1 和 HO8910PM） 的上皮性卵巢癌细胞 [37， 38]。使用正常的卵巢上皮细胞系 HOSEPIC 作为对照。所有上皮细胞系均在含有 10%10 \% 胎牛血清 （FBS;GIBCO）、 100U//ml100 \mathrm{U} / \mathrm{ml} 青霉素 （GIBCO） 和 100 mug//ml100 \mu \mathrm{~g} / \mathrm{ml} 链霉素 （GIBCO）。
Primary ovarian tumor cells were cultured as previously described [33]. Briefly, freshly isolated tumor tissues from 4 patients (GFY005, CZ001, CZ006 and CZ008) undergoing surgery or from a third generation patient (GFY004)-derived xenograft (PDX) inoculated in the front flank of BALB/c nude mice, were cut into 1-2mm1-2 \mathrm{~mm} diameter pieces. Then tissues were digested using a Tumor Dissociation Kit (130-095-929, 如前所述培养原代卵巢肿瘤细胞 [33]。简而言之，将 4 名接受手术的患者 （GFY005、CZ001、CZ006 和 CZ008） 或来自接种在 BALB/c 裸鼠前侧的第三代患者 （GFY004） 衍生的异种移植物 （PDX） 的新鲜分离的肿瘤组织切成 1-2mm1-2 \mathrm{~mm} 直径块。然后使用肿瘤解离试剂盒（130-095-929，
Miltenyl, Teterow, Germany) in a water bath at 37 for 45 min . Single cell suspensions were centrifugated and washed twice in RPMI-1640 medium at 150 xxg150 \times \mathrm{g} for 5 min . Primary ovarian tumor cells were cultured in RPMI-1640 medium containing 100U//ml100 \mathrm{U} / \mathrm{ml} penicillin, 100 mug//ml100 \mu \mathrm{~g} / \mathrm{ml} streptomycin and 20%20 \% FBS for 10 to 20 generations before experiments. The characteristics of the patients included are listed in supplemental table S1. Specifically, GFY004, GFY005 and CZ006 are high-grade serous carcinomas, CZ001 a is clear cell carcinoma and CZ008 is a poorly differentiated carcinoma. Miltenyl， Teterow， Germany） 在 37 度的水浴中浸泡 45 分钟。将单细胞悬液离心并在 RPMI-1640 培养基中洗涤两次，每次 150 xxg150 \times \mathrm{g} 洗涤 5 分钟。实验前，将原代卵巢肿瘤细胞在含有 100U//ml100 \mathrm{U} / \mathrm{ml} 青霉素、 100 mug//ml100 \mu \mathrm{~g} / \mathrm{ml} 链霉素和 20%20 \% FBS 的 RPMI-1640 培养基中培养 10 至 20 代。纳入患者的特征列于补充表 S1 中。具体而言，GFY004、GFY005 和 CZ006 是高级别浆液性癌，CZ001 a 是透明细胞癌，CZ008 是低分化癌。
2.4 Cell proliferation assay 2.4 细胞增殖测定
Cells (2xx10^(4)//ml)\left(2 \times 10^{4} / \mathrm{ml}\right) in a total volume of 100 mul100 \mu \mathrm{l} were seeded into 96 well plates and cultured overnight before treatment with various inhibitors. Inhibitors were added at 3-fold serial dilutions from 33.33 to 0.015 muM0.015 \mu \mathrm{M}. After culturing for an additional 72 h , cell viability was assayed by a 3 - (4,5-dimethylthiazol-2-yl)- 5- (3-carboxymethoxyphenyl)- 2- (4- sulfophenyl)- 2Htetrazolium (MTS) assay using a Cell Proliferation Assay kit (#G5430, Promega, Wisconsin, USA) according to the manual. IC_(50)\mathrm{IC}_{50} was calculated using untreated cells as controls. All inhibitors were added at the same time to the same cell lines. 100 mul100 \mu \mathrm{l} 将总体积的细胞 (2xx10^(4)//ml)\left(2 \times 10^{4} / \mathrm{ml}\right) 接种到 96 孔板中，并在用各种抑制剂处理前培养过夜。以 3 倍连续稀释的剂量加入抑制剂，从 33.33 至 0.015 muM0.015 \mu \mathrm{M} 。再培养 72 小时后，使用细胞增殖测定试剂盒（#G5430，Promega，Wisconsin，USA）通过 3-（4,5-二甲基噻唑-2-基）-5-（3-羧基甲氧基苯基）-2-（4-磺基苯基）-2Htetrazolium （MTS） 测定细胞活力。 IC_(50)\mathrm{IC}_{50} 使用未处理的细胞作为对照计算。所有抑制剂同时添加到相同的细胞系中。
2.5 Cell migration assay 2.5 细胞迁移测定
Cell migration assays were performed as described before [33]. Briefly, to compare the inhibitory effects on migration of PI3K/AKT/mTOR inhibitors, a total of 1xx10^(5)1 \times 10^{5} cells in 200 mul\mu \mathrm{l} RPMI-1640 medium containing different inhibitors, without FBS, were seeded into 8mum8 \mu \mathrm{~m} pore-transwell polycarbonate membrane filters (Costar Group, DC, USA) followed by insertion into 24 well culture plates. All inhibitors were used at a concentration of 1muM1 \mu \mathrm{M}, except for GSK458 and paclitaxel which were used at a concentration of 0.1 muM0.1 \mu \mathrm{M}. To determine whether GSK458 inhibited cell migration in a dose dependent manner, 5xx10^(4)5 \times 10^{4} cells per transwell were treated with a 10-fold serial dilution of GSK458 from 1 to 0.001 muM\mu \mathrm{M}. A volume of 700 mul700 \mu \mathrm{l} RPMI-1640 medium containing 10% FBS was added into each well of the 24 well plates. After culturing for another 18 h , cells were fixed with 4%4 \% paraformaldehyde and stained with 2%2 \% crystal violet. Nonmigrating cells on the upper surface of the chamber were scraped off with cotton swabs. Migrated cells at the bottom of the membrane were photographed at five random fields under a microscope with a magnification of 200 x and were counted. Migration inhibition (percentage of control) was defined as the difference between migrating cells in the untreated control compared to inhibitor treatment divided by the control value. All inhibitors were added at the same time to the same cell lines. 如前所述进行细胞迁移测定 [33]。简而言之，为了比较对 PI3K/AKT/mTOR 抑制剂迁移的抑制作用，将含有不同抑制剂的 200 mul\mu \mathrm{l} RPMI-1640 培养基中的总共 1xx10^(5)1 \times 10^{5} 细胞接种到 8mum8 \mu \mathrm{~m} 孔转孔聚碳酸酯膜过滤器（Costar Group，DC，USA）中，然后插入 24 孔培养板中。除 GSK458 和紫杉醇外，所有抑制剂的浓度均为 1muM1 \mu \mathrm{M}0.1 muM0.1 \mu \mathrm{M} ，其余抑制剂的浓度为 。为了确定 GSK458 是否以剂量依赖性方式抑制细胞迁移， 5xx10^(4)5 \times 10^{4} 用 10 倍连续稀释的 GSK458 从 1 到 0.001 处理每个 transwell 的细胞 muM\mu \mathrm{M} 。将含有 10% FBS 的体积 700 mul700 \mu \mathrm{l} RPMI-1640 培养基添加到 24 孔板的每个孔中。再培养 18 h 后，用 4%4 \% 多聚甲醛固定细胞，并用 2%2 \% 结晶紫染色。用棉签刮掉腔室上表面的非迁移细胞。在放大倍数为 200 倍的显微镜下，以 5 个随机视野拍摄膜底部迁移的细胞，并进行计数。迁移抑制（对照百分比）定义为未处理对照与抑制剂处理中迁移细胞之间的差异除以对照值。所有抑制剂同时添加到相同的细胞系中。
1 Department of Obstetrics and Gynecology, Shanghai Fengxian District Central Hospital, Southern Medical University, 201499 Shanghai, China 1 南方医科大学上海市奉贤区中心医院妇产科，201499 上海市
2 State Key Laboratory of Respiratory Disease, Guangzhou Medical University, 510182 Guangzhou, China 2 广州医科大学呼吸疾病国家重点实验室，510182广州
3 Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA 3 宾夕法尼亚大学佩雷尔曼医学院，美国宾夕法尼亚州费城 19104
4 Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences, School of Life Sciences, East China Normal University, Shanghai 200241, China 4 华东师范大学生命科学学院，生物医学研究所，上海市调控生物学重点实验室，上海200241
5 Department of Gynecology, Changzhou Second People’s Hospital Affiliated to Nanjing Medical University, Changzhou 213004, China 5 南京医科大学附属常州市第二人民医院妇科，常州213004
6 Department of Gynecology, The International Peace Maternity & Child Health Hospital, The China Welfare Institute, Shanghai Jiaotong University, Shanghai 200030, China 6 上海交通大学中国福利研究所国际和平妇幼保健院妇科，上海 200030，中国