The novel capsazepine analog, CIDD-99, significantly inhibits oral squamous cell carcinoma in vivo through a TRPV1-independent induction of ER stress, mitochondrial dysfunction, and apoptosis.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a deadly disease with a mere 40% five-year survival rate for patients with advanced disease. Previously, we discovered that capsazepine (CPZ), a transient receptor potential channel, Vanilloid subtype 1 (TRPV1) antagonist, has significant anti-tumor effects against OSCC via a unique mechanism-of-action that is independent of TRPV1. Thus, we developed novel CPZ analogs with more potent anti-proliferative effects (CIDD-24, CIDD-99, and CIDD-111). METHODS: Using OSCC xenograft models, we determined the efficacy of these analogs in vivo. TRPV1 interactions were evaluated using calcium imaging and a rat model of orofacial pain. Anti-cancer mechanism(s)-of-action were assessed by cell cycle analysis and mitochondrial depolarization assays. RESULTS: CIDD-99 was the most potent analog demonstrating significant anti-tumor effects in vivo (P < 0.001). CIDD-24 was equipotent to the parent compound CPZ, but less potent than CIDD-99. CIDD-111 was the least efficacious analog. Calcium imaging studies confirmed that CIDD-99 neither activates nor inhibits TRPV1 confirming that TRPV1 activity is not involved in its anti-cancer effects. All analogs induced an S-phase block, dose-dependent mitochondrial depolarization, and apoptosis. Histological analyses revealed increased apoptosis and reduced cell proliferation in tumors treated with these analogs. Importantly, CIDD-99 had the most dramatic anti-tumor effects with 85% of tumors resolving leaving only minute traces of viable tissue. Additionally, CIDD-99 was non-noxious and demonstrated no observable adverse reactions CONCLUSION: This study describes a novel, highly efficacious, CPZ analog, CIDD-99, with dramatic anti-tumor effects against OSCC that may be efficacious as a lone therapy or in combination with standard therapies.

Clinical significance of kallikrein-related peptidase-4 in oral cancer.

Kallikrein-related-peptidase-4 (KLK4), a serine protease originally discovered in developing tooth with broad target sequence specificity, serves vital functions in dental enamel formation. KLK4 is involved in degradation of extracellular matrix proteins and it is thought that this proteolytic activity could also promote tumor invasion and metastasis. Recent studies have associated KLK4 expression with tumor progression and clinical outcome, particularly in prostate and ovarian cancer. Very little is known in regard KLK4 involvement in oral squamous cell carcinomas (OSCCs). Our objective was to investigate KLK4 expression in OSCC pathogenesis and disease progression. KLK4 expression was evaluated by immunohistochemistry, western blots and zymograms in OSCC lines. Invasion assays using high versus low/undetectable KLK4-expressing OSCC cell lines were performed jointly with KLK4 siRNA inhibition. A large collection of OSCC specimens was evaluated for KLK4 expression and correlation with patients' characteristics and outcomes were determined. Our data indicate that KLK4 is differentially expressed in oral carcinomas. OSCC cell lines with high invasive and metastatic potential have the highest levels of KLK4 expression. KLK4 mRNA and protein expression correlated with enzyme activity detected by zymograms. Inhibition of KLK4 expression results in diminished invasive potential in OSCC cell lines. Consistently, KLK4 expression is stronger in primary tumors that later either recurred or developed metastases, suggesting that its preferential expression in OSCC might contribute to individual tumor biology. Therefore, this study provides supportive evidence in favor of a prognostic value for KLK4 in OSCC and suggests that KLK4 could serve as a potential therapeutic target in patients with oral cancer.