Abstract
BackgroundDevelopment of innovative, effective therapies against recurrent/chemotherapy-resistant ovarian cancer remains a high priority. Using high-throughput technologies to analyze genetic fingerprints of ovarian cancer, we have discovered extremely high expression of the genes encoding the proteins claudin-3 and claudin-4.MethodsBecause claudin-3 and -4 are the epithelial receptors for Clostridium perfringens enterotoxin (CPE), and are sufficient to mediate CPE binding, in this study we evaluated the in vitro and in vivo bioactivity of the carboxy-terminal fragment of CPE (i.e., CPE290-319 binding peptide) as a carrier for tumor imaging agents and intracellular delivery of therapeutic drugs. Claudin-3 and -4 expression was examined with rt-PCR and flow cytometry in multiple primary ovarian carcinoma cell lines. Cell binding assays were used to assess the accuracy and specificity of the CPE peptide in vitro against primary chemotherapy-resistant ovarian carcinoma cell lines. Confocal microscopy and biodistribution assays were performed to evaluate the localization and uptake of the FITC-conjugated CPE peptide in established tumor tissue.ResultsUsing a FITC-conjugated CPE peptide we show specific in vitro and in vivo binding to multiple primary chemotherapy resistant ovarian cancer cell lines. Bio-distribution studies in SCID mice harboring clinically relevant animal models of chemotherapy resistant ovarian carcinoma showed higher uptake of the peptide in tumor cells than in normal organs. Imunofluorescence was detectable within discrete accumulations (i.e., tumor spheroids) or even single chemotherapy resistant ovarian cancer cells floating in the ascites of xenografted animals while a time-dependent internalization of the FITC-conjugated CPE peptide was consistently noted in chemotherapy-resistant ovarian tumor cells by confocal microscopy.ConclusionsBased on the high levels of claudin-3 and -4 expression in chemotherapy-resistant ovarian cancer and other highly aggressive human epithelial tumors including breast, prostate and pancreatic cancers, CPE peptide holds promise as a lead peptide for the development of new diagnostic tracers or alternative anticancer agents.
Highlights
Development of innovative, effective therapies against recurrent/chemotherapy-resistant ovarian cancer remains a high priority
Claudin-3 and Claudin-4 Transcript Levels in Chemotherapy-Resistant Ovarian Tumors We used q-RT-PCR assays to get highly sensitive measurements of claudin-3 and claudin-4 Clostridium perfringens enterotoxin (CPE) receptor expression in normal tissue cells and primary human tumors derived from patients harboring chemotherapyresistant ovarian carcinomas. Both claudin-3 and/or claudin-4 genes were found highly expressed in all primary ovarian cancer studied including five high grade serous papillary tumors and two high grade clear cell carcinomas when compared to normal ovarian epithelial cells (NOVA) as well as other normal cells or other gynecologic tumors
Flow Cytometry experiments On the basis of the high expression of claudin-3 and/or claudin-4 detected by RT-PCR in primary ovarian cancer cell lines we predicted that the fluorescein isothiocyanate (FITC)-conjugated CPE peptide may bind to ovarian tumors in vitro
Summary
Development of innovative, effective therapies against recurrent/chemotherapy-resistant ovarian cancer remains a high priority. Many patients with ovarian cancer fully respond to the standard combination of surgery and chemotherapy, nearly 90% later develop recurrent chemotherapy-resistant cancer and inevitably cell lines, we and others have consistently identified the genes encoding tight junction (TJ) proteins claudin-3 and -4 as two of the most highly upregulated genes in primary ovarian carcinoma [3,4,5,6,7,8,9,10,11,12,13]. Because peptides are promising molecules to deliver radionuclides or chemotherapeutic drugs into tumors, we hypothesized that using the C- CPE290-319 peptide to target ovarian cancer cells based on their high levels of claudin-3 and -4 may represents a novel, potentially highly effective diagnostic and therapeutic approach to identify and treat chemotherapy-resistant ovarian cancer
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