Claudin-3 gene silencing with siRNA suppresses ovarian tumor growth and metastasis

Abstract

Claudin-3 (CLDN3) is a tight junction protein that is overexpressed in 90% of ovarian tumors. Previous in vitro studies have indicated that CLDN3 overexpression promotes the migration, invasion, and survival of ovarian cancer cells. Here, we investigated the efficacy of lipidoid-formulated CLDN3 siRNA in 3 different ovarian cancer models. Intratumoral injection of lipidoid/CLDN3 siRNA into OVCAR-3 xenografts resulted in dramatic silencing of CLDN3, significant reduction in cell proliferation, reduction in tumor growth, and a significant increase in the number of apoptotic cells. Intraperitoneal injection of lipidoid-formulated CLDN3 siRNA resulted in a substantial reduction in tumor burden in MISIIR/TAg transgenic mice and mice bearing tumors derived from mouse ovarian surface epithelial cells. Ascites development was reduced in CLDN3 siRNA-treated mice, suggesting the treatment effectively suppressed metastasis. Toxicity was not observed after multiple i.p. injections. Importantly, treatment of mice with nonimmunostimulatory 2 -OMe modified CLDN3 siRNA was as effective in suppressing tumor growth as unmodifed siRNA. These results suggest that lipidoid-formulated CLDN3 siRNA has potential as a therapeutic for ovarian cancer.lipidoid ͉ ovarian cancer ͉ cancer therapy O varian cancer has the highest mortality rate among gynecologic malignancies, and ranks 4th as the most common cancer in women in the United States (1). Treatment of early stage ovarian carcinoma improves the survival rate up to 90%. However, most women have advanced stage metastatic cancer at the time of diagnosis due to the asymptomatic nature of early stages of the disease and the lack of effective screening modalities. The standard treatment for patients with advanced stage epithelial ovarian cancer is surgical debulking followed by chemotherapy with paclitaxel plus a platinum-based therapy (cisplatin or carboplatin). Although Ϸ80% of patients receiving this therapeutic regimen have an initial favorable response, recurrent disease will occur in a majority of cases. New effective therapies are urgently needed for those patients with advanced-stage ovarian cancer who either do not respond to initial therapy or develop recurrent disease.Claudins are integral membrane proteins associated with tight junctions. Two members of the claudin protein family, claudin-3 (CLDN3) and CLDN4, are overexpressed in epithelial ovarian tumors relative to normal ovarian tissue (2-5). In fact, they are among the most highly expressed proteins in ovarian tumors. High amounts of CLDN3 and CLDN4 are associated with increased cellular motility and survival of ovarian tumor cells, and an increase in matrix metalloproteinase type 2 (MMP-2) (4). These observations implicate a role for CLDN3 and CLDN4 in ovarian tumorigenesis and metastasis, and suggest their importance as target proteins for development of new diagnostic and therapeutic reagents. CLDN3 and CLDN4 have also been identified as receptors for cytotoxic Clostridium perfringens enterotoxin (CPE). Binding of CPE to ...

Delivery of Therapeutics Targeting the mRNA-Binding Protein HuR Using 3DNA Nanocarriers Suppresses Ovarian Tumor Growth

“…Cell culture, transfection, and viability assay A2780 cells (T. (22), were authenticated and maintained as described previously (23). Frozen aliquots of cells were prepared upon receipt.…”

Efficient inhibition of ovarian cancer by short hairpin RNA targeting claudin-3

“…Its overexpression has been identified relating to migration, invasion and proliferation of cancer cells (16,19,35). Interestingly, CLDN3 is identified as one of the only two claudin family members capable of mediating cpe binding and cytolysis (36).…”

“…According to shrnA design principles and published sirnA sequences for clDn3, shrnA primers targeting clDn3 (sense 5'-tcccgcAAcAtcAtcAcgtcgcAttcAAgAcgtg cgAcgt gAt gAt gt t g c t t t t t t g -3' a nt is ens e, 5'-AgctcAAAAAAgcAAcAtcAtcAcgtcgcAcgt cttgAAtgcgAcgtgAtgAtgttgc-3') were designed and chemically synthesized for plasmid construction (19). the HK sequence, which has no homology with any known mammalian gene sequences, was used as the negative control.…”

“…The abnormal high expression of CLDN3 plays a crucial role in the proliferation, survival, invasion, cellular motility and metastasis of ovarian cancer, which makes clDn3 a promising therapeutic target for the treatment of ovarian cancer (16). recently, various new antitumor strategies involving clDn3 have emerged, including therapeutic antibody, cpe meditated cytolysis and rnA interference (17)(18)(19). Among these, shrnA meditated knocking down of clDn3 seemed of great prospect.…”

Novel siRNA Delivery System Using a Ternary Polymer Complex with Strong Silencing Effect and No Cytotoxicity

“…Both cationic and neutral lipoplexes have exhibited good efficacy with different toxicity and protein adsorption. [11][12][13][14][15] Previously, we also reported that lipoplexes of siRNA constructed with trimethyl[2,3-(dioleoxy)-prolyl] ammonium chloride and cholesteryl 3β-N-(dimethylaminoethyl) carbamate hydrochloride showed a significant silencing effect in a mouse colon carcinoma cell line expressing luciferase regularly. 16) On the other hand, cationic polymers are one of the most popular non-viral gene vectors due to their excellent ability to compact anionic genes, synthetic controllability, better flexibility achieved simply by varying the chemical composition, molecular weight (MW), and architecture (linear, randomly branched, dendrimer, block, and graft copolymer).…”