November 05, 2025
Abstract
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, −99, and −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.
Keywords: oral cancer, capsazepine analog, mitochondria
Introduction
Head and neck squamous cell carcinoma (HNSCC) is an insidious disease resulting in over 50,000 new cases and 10,000 deaths in the United States annually (1, 2). Greater than 50% of all HNSCC patients will die within five years of their diagnosis (1). This is due to late-detection and consequent advanced disease, which is resistant to conventional treatments. Notably, 75% of new patients have advanced disease at the time of diagnosis (3–5). In addition, the majority of tumors (60%) are found in the oral cavity (OSCC); the site with the highest recurrence rates and a median survival of a mere 6 months (3–7). Hence there is a great need to develop new methods of treatment for OSCC.
Previously, we identified a novel anti-cancer function of Vanilloids in which both capsaicin (TRPV1 agonist) and its synthetic analog CPZ (TRPV1 antagonist) have anti-proliferative effects against multiple OSCC cell lines (8). We also demonstrated significant anti-tumor effects of CPZ in vivo using three OSCC xenograft models, which yielded no adverse effects on non-malignant tissues and no liver or kidney toxicities. Interestingly, we showed that the anti-cancer effects of CPZ are through a second mechanism-of-action that does not involve TRPV1; rather, induction of reactive oxygen species (ROS) and apoptosis (8). Based upon these findings, we generated potent CPZ analogs (30 total) with significantly greater anti-proliferative effects in vitro (9). Lead compounds CIDD-24 (compound 17) and CIDD-111 (compound 29) displayed significant anti-tumor effects in vivo, which were more potent than the parent compound CPZ. We also confirmed that these lead compounds are efficacious against multiple cancer types including OSCC. Here we describe the efficacy of CIDD-24, CIDD-111, and the novel, more potent analog, CIDD-99, against a panel of OSCC cell lines and validate their anti-tumor effects in vivo. Mechanisms-of-action are investigated using calcium imaging, behavior studies, and mitochondrial function testing.
Materials and Methods
Reagents
Working stocks of CPZ and analogs were made fresh by diluting each compound in 100% EtOH to a final stock concentration of 100mM.
