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

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, 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.

Keywords: capsazepine analog; mitochondria; oral cancer.

Figure 1.. Effects of CPZ analogs on OSCC cell proliferation in vitro.

Panels A-D: MTS cell viability assay in OSCC cancer cell lines Cal-27, HSC-3, SCC-4, and SCC-9 respectively treated with CIDD-24, CIDD-9, and CIDD-111 for 24h (n=4 per group). Panels E-G: Chemical structures of CIDD-24, CIDD-99, and CIDD-111. Panel H: MTS cell viability assay using a panel of solid tumor types (HeLa, H460, MDA-231, and PC-3) treated with CIDD-99 for 24h (n=4 per group). Data is presented as mean ± standard deviation (SD).

Figure 2.. Effects of intra-tumor injection of CPZ analogs on growth of Cal-27 xenografts and normal epithelium.

Panel A: Tumor volumes (mean ± SD) of Cal-27-derived xenografts treated by intra-tumor injection of 120μg of CPZ, CIDD-24, CIDD-99, and CIDD-111 every other day for 28 days. Significant reduction in tumor volumes is seen by day 18 for CIDD-99, day 24 for CIDD-24, day 26 for CPZ, and day 28 for CIDD-111 (n=5 per group; *p<0.05, **p<0.01, ***p<0.001). Panel B: Scatter plot of Cal-27-derived tumor volumes on day 28. Mean volume of vehicle control treated tumors is 541 mm³ whereas mean tumor volumes of CPZ, CIDD-24, CIDD-99, and CIDD-111 treated tumors are 269 mm³, 211 mm³, 134 mm³, and 333 mm³ respectively (n=5 per group). Panel C: Representative photograph of Cal-27-derived xenograft treated by intra-tumor injection with CIDD-99 (120μg) every other day for 28 days. Arrow indicates healthy adjacent non-malignant epithelium with no erythema, ulceration, or swelling. Panel D: Cell viability assay of OKF6-TERT-2 keratinocytes treated with CIDD-99 for 24h (n=4 per group).

Figure 3.. Effects of systemic administration of CPZ analogs on growth of Cal-27 xenografts and histological changes.

Panel A: Tumor volumes (mean ± SD) of Cal-27-derived xenografts treated by IP injections of 12 mg/kg CIDD-99 every other day for 22 days. Significant reduction in tumor volumes is seen by day 6 and throughout the remainder of the study (n=10 per group; *p<0.05, **p<0.01, ***p<0.001). Panel B: Scatter plot of Cal-27-derived tumor volumes on day 22. Mean volume of vehicle control treated tumors is 480 mm³ and CIDD-99 treated tumors is 65.6 mm³ (n=10 per group). Panels C-E: Representative photomicrographs of H & E stained tumors treated by intra-tumor injection; top row, 4x magnification (scale bar = 500 μM) and bottom row, 10x magnification of area outlined in 4x above (scale bar = 250 μM). Panel C: Representative photomicrographs of H & E stained tumors treated with vehicle control (left panels) and CPZ (right panels). Wide bands of viable tumor cells line the periphery of the tumors with islands of tumor cells within the necrotic cores. Tumor core is indicated by asterisk (**). Panel D: Representative photomicrographs of H & E stained tumors treated with CIDD-24 (left panels) and CIDD-111 (right panels). Narrower bands of tumor cells line the periphery of the tumors with large necrotic cores (indicated by asterisk; **) and inflammatory infiltrates. Panel E: Representative photomicrographs of H & E stained tumors treated with CIDD-99. CIDD-99–1 (left panels) is representative of four of the five tumors treated with CIDD-99 and demonstrates the extremely small size of the remaining tissue, which is devoid of viable tumor cells, has a large inflammatory infiltrate, and is necrosed. CIDD-99–2 (right panels) demonstrates the largest of the tumors treated with CIDD-99 via intra-tumor injection, which does have some viable tumor cells along the periphery, a large necrotic core (**), and a large inflammatory infiltrate. Panel F: Representative photograph of tumors treated by IP injection of vehicle control (top row) or 12mg/kg CIDD-99 (bottom row), which demonstrates the dramatic reduction in tumor volume following 22 days of treatment with CIDD-99.

Figure 4.. Evaluation CPZ analogs’ TRPV1 interactions in vitro and in vivo.

Panel A: Calcium imaging of CHO-TRPV1 cells treated with CIDD-24, CIDD-99, and CIDD-111 (1 μM), or capsaicin positive control (100 nM). Calcium influx as measured by mean relative fluorescent units (RFU) ± SD is depicted. Panel B: Eye-wipe testing in response to 0.01% w/v of CIDD-24, CIDD-99, and CIDD-111 vs. capsaicin (n=6 per group). Nocifensive behaviors measured in seconds (mean ± SD) is depicted. Panel C: CHO-TRPV1 cells treated with CIDD-99 or CPZ (1 μM) alone or pre-treated with CIDD-99 or CPZ (1 μM), followed by capsaicin (100 nM). Calcium influx as measured by mean RFU ± SD is depicted; **p<0.01, ***p<0.001.

Figure 5.. Effects of CPZ analogs on cell cycle distribution and apoptosis.

Panel A: Cell cycle distribution of Cal-27 cells treated with 10 μM CIDD-24, CIDD-99, and CIDD-111 for 24h. Panel B: Quantification of cell cycle distribution (% cells) in G1, S, and G2 phase following treatment with vehicle control, CPZ (30 μM), or 10 μM CIDD-24, CIDD-99, and CIDD-111 for 24 h. Panel C: Western blot of c-PARP in Cal-27 cells treated with increasing concentrations of CIDD-24, CIDD-99, and CIDD-111 for 48h. Panel D: MTS assay of Cal-27 cells treated with CIDD-24, CIDD-99, and CIDD-111 (+/−) NAC (10mM) for 24 h; *p<0.05 and **p<0.01.

Figure 6.. Effects of CPZ analogs on mitochondrial membrane potential and ER stress.

Panels A-D: Mitochondrial membrane potential analysis of Cal-27 cells treated for 2h with CIDD-24, CIDD-99, CIDD-111, and CPZ at the indicated concentrations. Quantification of JC-1 aggregates to monomers ratio (mean ± SD) is shown for CPZ (Panel A), CIDD-24 (Panel B), CIDD-99 (Panel C), and CIDD-111 (Panel D); n=3 per group. Panel E: Western blot analysis of BiP and CHOP expression in Cal-27 cells treated for 24h with 10, 25, and 50 μM CIDD-24, CIDD-99, and CIDD-111 compared to CPZ. Panel F: Cell viability assay of Cal-27 cells treated for 24h with CIDD-99 (500 nM) and cisplatin (25 μM) or gefitinib (100 nM). Panel G: Cell viability assay of Cal-27 cells treated for 24h with increasing concentrations of CIDD-99 following exposure to 2, 5, and 10 Gray (Gy) radiation; *p<0.05, **p<0.01, ***p<0.001 vs control and ###p<0.001 between test groups.