Zewdu A, Lopez G, Braggio D, Kenny C, Constantino D, Bid H K, Batte K, Iwenofu O H, Oberlies N H, Pearce C J, Strohecker A M, Lev D, Pollock R E
Department of Surgical Oncology, Ohio State University Wexner Medical Center, Columbus, Ohio, USA; The James Cancer Center, Ohio State University Wexner Medical Center, Columbus, Ohio, USA.
Department of Surgical Oncology, Ohio State University Wexner Medical Center, Columbus, Ohio, USA; The James Cancer Center, Ohio State University Wexner Medical Center, Columbus, Ohio, USA; Resonant Therapeutics, Inc., Ann Arbor, Michigan, USA.
Clin Exp Pharmacol. 2016 Nov;6(6). doi: 10.4172/2161-1459.1000221. Epub 2016 Oct 24.
The heterogeneity of soft tissue sarcoma (STS) represents a major challenge for the development of effective therapeutics. Comprised of over 50 different histology subtypes of various etiologies, STS subsets are further characterized as either karyotypically simple or complex. Due to the number of genetic anomalies associated with genetically complex STS, development of therapies demonstrating potency against this STS cluster is especially challenging and yet greatly needed. Verticillin A is a small molecule natural product with demonstrated anticancer activity; however, the efficacy of this agent has never been evaluated in STS. Therefore, the goal of this study was to explore verticillin A as a potential STS therapeutic.
We performed survival (MTS) and clonogenic analyses to measure the impact of this agent on the viability and colony formation capability of karyotypically complex STS cell lines: malignant peripheral nerve sheath tumor (MPNST) and leiomyosarcoma (LMS). The in vitro effects of verticillin A on apoptosis were investigated through annexin V/PI flow cytometry analysis and by measuring fluorescently-labeled cleaved caspase 3/7 activity. The impact on cell cycle progression was assessed via cytometric measurement of propidium iodide intercalation. studies were performed using MPNST xenograft models. Tumors were processed and analyzed using immunohistochemistry (IHC) for verticillin A effects on growth (Ki67) and apoptosis (cleaved caspase 3).
Treatment with verticillin A resulted in decreased STS growth and an increase in apoptotic levels after 24 h. 100 nM verticillin A induced significant cellular growth abrogation after 24 h (96.7, 88.7, 72.7, 57, and 39.7% reduction in LMS1, S462, ST88, SKLMS1, and MPNST724, respectively). We observed no arrest in cell cycle, elevated annexin, and a nearly two-fold increase in cleaved caspase 3/7 activity in all MPNST and LMS cell lines. Control normal human Schwann (HSC) and aortic smooth muscle (HASMC) cells displayed higher tolerance to verticillin A treatment compared to sarcoma cell lines, although toxicity was seen in HSC at the highest treatment dose. studies mirrored the in vitro results: by day 11, tumor size was significantly reduced in MPNST724 xenograft models with treatment of 0.25 and 0.5 mg/kg verticillin A. Additionally, IHC assessment of tumors demonstrated increased cleaved caspase 3 and decreased proliferation (Ki67) following treatment with verticillin A.
Advancement in the treatment of karyotypically complex STS is confounded by the high level of genetic abnormalities found in these diseases. Consequently, the identification and investigation of novel therapies is greatly needed. Our data suggest that verticillin A selectively inhibits MPNST and LMS growth via induction of apoptosis while exhibiting minimal to moderate effects on normal cells, pointing to verticillin A as a potential treatment for MPNST and LMS, after additional preclinical validation.
软组织肉瘤（STS）的异质性是有效治疗方法开发面临的主要挑战。STS由50多种不同组织学亚型组成，病因各异，其亚组进一步分为核型简单或复杂两类。由于与基因复杂的STS相关的基因异常数量众多，开发对该STS集群有效的疗法极具挑战性，但又非常必要。绿胶霉素A是一种具有抗癌活性的小分子天然产物；然而，该药物在STS中的疗效从未得到评估。因此，本研究的目的是探索绿胶霉素A作为一种潜在的STS治疗药物。
我们进行了生存（MTS）和克隆形成分析，以测量该药物对核型复杂的STS细胞系：恶性外周神经鞘瘤（MPNST）和平滑肌肉瘤（LMS）的活力和集落形成能力的影响。通过膜联蛋白V/PI流式细胞术分析并测量荧光标记的裂解型半胱天冬酶3/7活性，研究绿胶霉素A对细胞凋亡的体外影响。通过碘化丙啶嵌入的细胞计数测量评估对细胞周期进程的影响。使用MPNST异种移植模型进行体内研究。对肿瘤进行处理，并使用免疫组织化学（IHC）分析绿胶霉素A对生长（Ki67）和凋亡（裂解型半胱天冬酶3）的影响。
用绿胶霉素A处理24小时后，STS生长减少，凋亡水平增加。100 nM绿胶霉素A在24小时后诱导显著的细胞生长抑制（LMS1、S462、ST88、SKLMS1和MPNST724分别减少96.7%、88.7%、72.7%、57%和39.7%）。我们在所有MPNST和LMS细胞系中均未观察到细胞周期停滞、膜联蛋白升高以及裂解型半胱天冬酶3/7活性几乎增加两倍。对照正常人雪旺氏（HSC）细胞和主动脉平滑肌（HASMC）细胞与肉瘤细胞系相比，对绿胶霉素A处理表现出更高的耐受性，尽管在最高处理剂量下HSC中可见毒性。体内研究结果与体外结果相似：到第11天，用0.25和0.5 mg/kg绿胶霉素A处理的MPNST724异种移植模型中的肿瘤大小显著减小。此外，对肿瘤的IHC评估显示用绿胶霉素A处理后裂解型半胱天冬酶3增加，增殖（Ki67）减少。
核型复杂的STS治疗进展因这些疾病中发现的高水平基因异常而受阻。因此，迫切需要鉴定和研究新的治疗方法。我们的数据表明，绿胶霉素A通过诱导凋亡选择性抑制MPNST和LMS生长，同时对正常细胞表现出最小至中等的影响，这表明在进行额外的临床前验证后，绿胶霉素A可能是MPNST和LMS的一种治疗方法。
