Division of Surgical Oncology, Department of Surgery, Drexel University College of Medicine, Philadelphia, PA.
Division of Obstetrics and Gynecology, Department of Gynecologic Oncology, Jefferson University Hospitals, Philadelphia, PA.
Ann Clin Lab Sci. 2020 Sep;50(5):611-624.
Patients with epithelial ovarian cancers experience the highest fatality rates among all gynecological malignancies which require development of novel treatment strategies. Tumor cell necrosis was previously reported in a number of cancer cell lines following treatment with a p53-derived anti-cancer peptide called PNC-27. This peptide induces necrosis by transmembrane pore formation with HDM-2 protein that is expressed in the cancer cell membrane. We aimed to extend these studies further by investigating expression of membrane HDM-2 protein in ovarian cancer as it relates to susceptibility to PNC-27.
Herein, we measured HDM-2 membrane expression in two ovarian cancer cell lines (SKOV-3 and OVCAR-3) and a non-transformed control cell line (HUVEC) by flow cytometric and western blot analysis. Immunofluorescence was used to visualize colocalization of PNC-27 with membrane HDM-2. Treatment effects with PNC-27 and control peptide were assessed using a MTT cell proliferation assay while direct cytotoxicity was measured by lactate dehydrogenase (LDH) release and induction of apoptotic markers; annexin V and caspase-3.
HDM-2 protein was highly expressed and frequently detected in the membranes of SKOV-3 and OVCAR-3 cells; a prominent 47.6 kDa HDM-2 plasma membrane isoform was present in both cell lines whereas 25, 29, and 30 kDa isoforms were preferentially expressed in OVCAR-3. Notably, PNC-27 colocalized with HDM-2 in the membranes of both cancer cell lines that resulted in rapid cellular necrosis. In contrast, no PNC-27 colocalization and cytotoxicity was observed with non-transformed HUVEC demonstrating minimal expression of membrane HDM-2.
Our results suggest that HDM-2 is highly expressed in the membranes of these ovarian cancer cell lines and colocalizes with PNC-27. We therefore conclude that the association of PNC-27 with preferentially expressed membrane HDM-2 isoforms results in the proposed model for the formation of transmembrane pores and epithelial ovarian cancer tumor cell necrosis, as previously described in a number of solid tissue and hematologic malignancies.
上皮性卵巢癌患者的病死率在所有妇科恶性肿瘤中最高，这需要开发新的治疗策略。先前有报道称，一种名为 PNC-27 的 p53 衍生抗癌肽可使多种癌细胞系发生肿瘤细胞坏死。该肽通过与表达在癌细胞膜上的 HDM-2 蛋白形成跨膜孔诱导坏死。我们旨在通过研究卵巢癌中膜 HDM-2 蛋白的表达与 PNC-27 的易感性之间的关系，进一步扩展这些研究。
在此，我们通过流式细胞术和 Western blot 分析测量了两种卵巢癌细胞系（SKOV-3 和 OVCAR-3）和非转化对照细胞系（HUVEC）中 HDM-2 膜表达。免疫荧光用于观察 PNC-27 与膜 HDM-2 的共定位。通过 MTT 细胞增殖测定评估 PNC-27 和对照肽的治疗作用，通过乳酸脱氢酶（LDH）释放和诱导凋亡标记物（膜联蛋白 V 和 caspase-3）测量直接细胞毒性。
HDM-2 蛋白在 SKOV-3 和 OVCAR-3 细胞的膜中高度表达且频繁检测到；两种细胞系中均存在显著的 47.6 kDa HDM-2 质膜同工型，而 25、29 和 30 kDa 同工型则在 OVCAR-3 中优先表达。值得注意的是，PNC-27 与两种癌细胞系的膜中的 HDM-2 共定位导致细胞迅速坏死。相比之下，在非转化的 HUVEC 中未观察到 PNC-27 共定位和细胞毒性，表明其膜 HDM-2 的表达水平较低。
我们的结果表明，HDM-2 在这些卵巢癌细胞系的膜中高度表达，并与 PNC-27 共定位。因此，我们得出结论，PNC-27 与优先表达的膜 HDM-2 同工型的结合导致了跨膜孔的形成和上皮性卵巢癌肿瘤细胞坏死，正如先前在许多实体组织和血液恶性肿瘤中所描述的那样。
