Oral Cancer Pain Includes Thermal Allodynia That May Be Attenuated by Chronic Alcohol Consumption.

BACKGROUND: Oral cancer is one of the most painful cancer types, and is often refractory to existing analgesics. Oral cancer patients frequently develop a tolerance to opioids, the mainstay of current cancer pain therapy, leaving them with limited therapeutic options. Thus, there is a great need to identify molecular mechanisms driving oral cancer pain in an effort to develop new analgesics. Previous reports demonstrate that oral cancer patients experience intense mechanical pain and pain in function. To date, no studies have examined thermal pain in oral cancer patients or the role that alcohol consumption plays in oral cancer pain. This study aims to evaluate patient-reported pain levels and thermal allodynia, potential molecular mechanisms mediating thermal allodynia, and the effects of alcohol consumption on patient-perceived pain. METHODS: This study evaluated human oral squamous cell carcinoma (OSCC) cell lines for their ability to activate thermosensitive channels in vitro and validated these findings in a rat model of orofacial pain. Patient-reported pain in a south Texas OSCC cohort (n = 27) was examined using a visual analog scale (VAS). Covariant analysis examined variables such as tobacco and alcohol consumption, ethnicity, gender, and cancer stage. RESULTS: We determined that OSCC secretes factors that stimulate both the Transient Receptor Potential Ankyrin type 1 channel (TRPA1; noxious cold sensor) and the Transient Receptor Potential Vanilloid type 1 channel (TRPV1; noxious heat sensor) in vitro and that OSCC-secreted factors sensitize TRPV1 nociceptors in vivo. These findings were validated in this cohort, in which allodynia to cold and heat were reported. Notably, subjects that reported regular alcohol consumption also reported lower pain scores for every type of pain tested, with significantly reduced cold-induced pain, aching pain, and burning pain. CONCLUSION: Oral cancer patients experience multiple types of cancer pain, including thermal allodynia. Alcohol consumption correlates with reduced OSCC pain and reduced thermal allodynia, which may be mediated by TRPA1 and TRPV1. Hence, reduced pain in these patients may contribute to a delay in seeking care, and thus a delay in early detection and treatment.

Inhibition of Ca2+ entry by capsazepine analog CIDD-99 prevents oral squamous carcinoma cell proliferation.

Oral cancer patients have a poor prognosis, with approximately 66% of patients surviving 5-years after diagnosis. Treatments for oral cancer are limited and have many adverse side effects; thus, further studies are needed to develop drugs that are more efficacious. To achieve this objective, we developed CIDD-99, which produces cytotoxic effects in multiple oral squamous cell carcinoma (OSCC) cell lines. While we demonstrated that CIDD-99 induces ER stress and apoptosis in OSCC, the mechanism was unclear. Investigation of the Bcl-family of proteins showed that OSCC cells treated with CIDD-99 undergo downregulation of Bcl-XL and Bcl-2 anti-apoptotic proteins and upregulation of Bax (pro-apoptotic). Importantly, OSCC cells treated with CIDD-99 displayed decreased calcium signaling in a dose and time-dependent manner, suggesting that blockage of calcium signaling is the key mechanism that induces cell death in OSCC. Indeed, CIDD-99 anti-proliferative effects were reversed by the addition of exogenous calcium. Moreover, electrophysiological properties further established that calcium entry was via the non-selective TRPC1 channel and prolonged CIDD-99 incubation inhibited STIM1 expression. CIDD-99 inhibition of calcium signaling also led to ER stress and inhibited mitochondrial complexes II and V in vitro. Taken together, these findings suggest that inhibition of TRPC mediates induction of ER stress and mitochondrial dysfunction as a part of the cellular response to CIDD-99 in OSCC.

Basics for Improved Use of Phages for Therapy.

Blood-borne therapeutic phages and phage capsids increasingly reach therapeutic targets as they acquire more persistence, i.e., become more resistant to non-targeted removal from blood. Pathogenic bacteria are targets during classical phage therapy. Metastatic tumors are potential future targets, during use of drug delivery vehicles (DDVs) that are phage derived. Phage therapy has, to date, only sometimes been successful. One cause of failure is low phage persistence. A three-step strategy for increasing persistence is to increase (1) the speed of lytic phage isolation, (2) the diversity of phages isolated, and (3) the effectiveness and speed of screening phages for high persistence. The importance of high persistence-screening is illustrated by our finding here of persistence dramatically higher for coliphage T3 than for its relative, coliphage T7, in murine blood. Coliphage T4 is more persistent, long-term than T3. Pseudomonas chlororaphis phage 201phi2-1 has relatively low persistence. These data are obtained with phages co-inoculated and separately assayed. In addition, highly persistent phage T3 undergoes dispersal to several murine organs and displays tumor tropism in epithelial tissue (xenografted human oral squamous cell carcinoma). Dispersal is an asset for phage therapy, but a liability for phage-based DDVs. We propose increased focus on phage persistence-and dispersal-screening.

[Corrigendum] Novel polygodial analogs P3 and P27: Efficacious therapeutic agents disrupting mitochondrial function in oral squamous cell carcinoma.

After the publication of the above article, the authors have realized that they failed to properly acknowledge the work performed by their Center for Innovative Drug Discovery High Throughput Screening Core Facility in the paper. The Declarations section of their paper should therefore have also included the following statement: "The UT Health San Antonio Center for Innovative Drug Discovery HTS Facility was funded in part by the Cancer Prevention Research Institute of Texas (CPRIT; grant no. RP160844)". The authors regret their oversight in failing to include this information in the Declarations section of their paper. [the original article was published in International Journal of Oncology 53: 2627-2636, 2018; DOI: 10.3892/ijo.2018.4585].

Loss of Ca2+ entry via Orai-TRPC1 induces ER stress, initiating immune activation in macrophages.

Activation of cellular stresses is associated with inflammation; however, the mechanisms are not well identified. Here, we provide evidence that loss of Ca2+ influx induces endoplasmic reticulum (ER) stress in primary macrophages and in murine macrophage cell line Raw 264.7, in which the unfolded protein response is initiated to modulate cytokine production, thereby activating the immune response. Stressors that initiate the ER stress response block store-dependent Ca2+ entry in macrophages prior to the activation of the unfolded protein response. The endogenous Ca2+ entry channel is dependent on the Orai1-TRPC1-STIM1 complex, and the presence of ER stressors decreased expression of TRPC1, Orai1 and STIM1. Additionally, blocking Ca2+ entry with SKF96365 also induced ER stress, promoted cytokine production, activation of autophagy, increased caspase activation and induced apoptosis. Furthermore, ER stress inducers inhibited cell cycle progression, promoted the inflammatory M1 phenotype, and increased phagocytosis. Mechanistically, restoration of Orai1-STIM1 expression inhibited the ER stress-mediated loss of Ca2+ entry that prevents ER stress and inhibits cytokine production, and thus induced cell survival. These results suggest an unequivocal role of Ca2+ entry in modulating ER stress and in the induction of inflammation.

The Highly Pure Neem Leaf Extract, SCNE, Inhibits Tumorigenesis in Oral Squamous Cell Carcinoma via Disruption of Pro-tumor Inflammatory Cytokines and Cell Signaling.

Oral squamous cell carcinoma (OSCC) is a deadly disease that comprises 60% of all head and neck squamous cell cancers. The leaves of the Neem tree (Azadirachta indica) have been used in traditional Ayurvedic medicine for centuries to treat numerous oral maladies and are known to have significant anti-inflammatory properties. We hypothesize that a highly pure super critical CO2 Neem leaf extract (SCNE) prevents initiation and progression of OSCC via downregulation of intra-tumor pro-inflammatory pathways, which promote tumorigenesis. Hence, we investigated the anticancer effects of SCNE using in vitro and in vivo platforms. OSCC cell lines (SCC4, Cal27, and HSC3) were treated with SCNE while inflammation, proliferation, and migration were analyzed over time. SCNE treatment significantly inhibited OSCC cell proliferation and migration and reduced MMP activity in vitro, suggesting its potential to inhibit tumor growth and metastasis. The preventive effects of SCNE in ectopic xenograft and 4NQO-1 (4-Nitroquinoline-1-oxide) carcinogen-induced mouse models of OSCC were also evaluated. Indeed, xenografted nude mice showed significant reduction of OSCC tumor volumes. Likewise, SCNE significantly reduced the incidence of tongue dysplasia in the 4NQO-1 OSCC initiation model. In both OSCC animal models, SCNE significantly depressed circulating pro-cancer inflammatory cytokines (host and tumor-secreted) including NFkB, COX2, IL-1, IL-6, TNFα, and IFNγ. In addition, we demonstrate that SCNE downregulates STAT3 and AKT expression and activity in vitro. We also demonstrate that the primary active component, nimbolide (NIM), has significant anticancer activity in established OSCC xenografts. Lastly, we show that SCNE induces an M1 phenotype in tumor associated macrophages (TAMS) in vivo. Taken together, these data strongly support SCNE as means of preventing OSCC via downregulation of pro-cancer inflammatory cascades and NIM as a potential new therapy for existing OSCC.

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.

Synthesis and SAR of novel capsazepine analogs with significant anti-cancer effects in multiple cancer types.

We previously demonstrated that capsazepine (CPZ), a synthetic transient receptor potential Vanilloid subtype 1 (TRPV1) antagonist, has significant anti-cancer effects in vivo. The purpose of this study was to develop more potent analogs based upon CPZ pharmacophore and structure-activity relationships (SAR) across analogs. We generated 30 novel compounds and screened for their anti-proliferative effects in cultured HeLa cervical cancer cells. Cell viability assays identified multiple compounds with IC50s < 15 µM and one compound, 29 with an IC50 < 5 µM; six fold more potent than CPZ. We validated the anti-proliferative efficacy of two lead compounds, 17 and 29, in vivo using HeLa-derived xenografts in athymic nude mice. Both analogs significantly reduced tumor volumes by day 8 compared to control treated animals (p < 0.001) with no observable adverse effects. Calcium imaging determined that compound 17 activates TRPV1 whereas 29 neither activates nor inhibits TRPV1; indicating a unique mechanism-of-action that does not involve TRPV1 signaling. Cell viability assays using a panel of additional tumor types including oral squamous cell carcinoma, non-small cell lung cancer (NSCLC), breast cancer, and prostate cancer cell lines (HSC-3, H460, MDA-231, and PC-3 respectively) demonstrated that both lead compounds were efficacious against every cancer type tested. Compounds 29 displayed IC50s of 1-2.5 µM in HSC-3and PC-3cells. Thus, we propose that these novel CPZ analogs may serve as efficacious therapeutic agents against multiple tumor types that warrant further development for clinical application.

Novel polygodial analogs P3 and P27: Efficacious therapeutic agents disrupting mitochondrial function in oral squamous cell carcinoma.

Polygodial, a drimane sesquiterpenoid dialdehyde isolated as a pungent component of the water pepper Persicaria hydropiper, exhibits antifeedant, antimicrobial, anti-inflammatory and anticancer effects. Polygodial also activates transient receptor potential vanilloid subtype 1 (TRPV1) channels. Previously, we described the synthesis of a C12-Wittig derivative of polygodial, termed P3, with significant antiproliferative effects against multiple cancer types including oral squamous cell carcinoma (OSCC). In the present study, a more potent derivative, P27, with superior anti-proliferative effects in vitro and antitumor effects in Cal-27 derived xenografts is described. Polygodial, P3, and P27 all significantly decreased OSCC tumor growth, with P27 being equipotent with polygodial and P3 being the least efficacious. However, neither analog elicited the adverse effect observed with polygodial: Profound transient inflammation. Although P3 and P27 pharmacophores are based on polygodial, novel effects on OSCC cell cycle distribution were identified and shared anticancer effects that are independent of TRPV1 activity were observed. Polygodial elicits an S-phase block, whereas P3 and P27 lead to G2/M phase arrest. Pretreatment of OSCC cells with the TRPV1 antagonist capsazepine does not affect the antiproliferative activity of P3 or P27, indicating that TRPV1 interactions do not regulate OSCC cell proliferation. Indeed, calcium imaging studies identified that the analogs neither activate nor antagonize TRPV1. Behavioral studies using a rat model for orofacial pain confirmed that these analogs fail to induce nocifensive responses, indicating that they are non-noxious in vivo. All compounds induced a significant concentration-dependent decrease in the mitochondrial transmembrane potential and corresponding apoptosis. Considering that P27 is equipotent to polygodial with no TRPV1-associated adverse effects, P27 may serve as an efficacious novel therapy for OSCC.

Thymol inhibits oral squamous cell carcinoma growth via mitochondria-mediated apoptosis.

BACKGROUND: Thymol is a transient receptor potential ankyrin subtype 1 channel, (TRPA1) agonist found in thyme and oregano. Thymol has antioxidant, anti-inflammatory, and antimicrobial properties; thus, thymol is added to many commercially available products including Listerine mouthwash. Thymol is also cytotoxic to HL-60 (acute promyelocytic leukemia) cells in vitro. Therefore, we evaluated the effects of thymol against oral squamous cell carcinoma (OSCC) and its anticancer mechanism-of-action. METHODS: The antiproliferative effects of thymol in OSCC Cal27 cells were determined by MTS assays. Antitumor effects were evaluated in Cal27- and HeLa-derived mouse xenografts. Calcium imaging, mitochondrial transmembrane potential (ΔΨm) studies, and Western blot analysis of cleaved PARP (c-PARP) evaluated thymol's mechanism-of-action. RESULTS: Thymol had significant, long-lasting antiproliferative effects in vitro. In vivo, thymol displayed significant antitumor effects in Cal27-derived tumors. Thymol's anticancer effects were confirmed in HeLa-derived xenografts demonstrating that thymol effects are not tumor-type specific. Calcium imaging verified calcium influx in Cal27 cells that were reversed with the TRPA1 antagonist, HC030031. However, no calcium influx was seen in HeLa cells indicating that TRP channels do not regulate thymol cytotoxicity. This was confirmed using cell viability assays in which pre-treatment with HC030031 had no effect on thymol cytotoxicity. Instead, ΔΨm studies revealed that thymol induces significant ΔΨm depolarization and apoptosis. CONCLUSION: Our findings provide the first evidence of thymol's novel antitumor effects against OSCC in vivo, which do not rely on TRPA1 activity. Instead, we show that thymol induces mitochondrial dysfunction and apoptosis and may be efficacious against multiple cancers.

Co-targeting ALK and EGFR parallel signaling in oral squamous cell carcinoma.

Squamous cell carcinoma (SCC) comprises 90% of all head and neck cancers and has a poor survival rate due to late-stage disease that is refractive to traditional therapies. Epidermal growth factor receptor (EGFR) is over-expressed in greater than 80% of head and neck SCC (HNSCC). However, EGFR targeted therapies yielded little to no efficacy in clinical trials. This study investigated the efficacy of co-targeting EGFR and the anaplastic lymphoma kinase (ALK) whose promoter is hypomethylated in late-stage oral SCC (OSCC). We observed increased ALK activity in late-stage human OSCC tumors and invasive OSCC cell lines. We also found that while ALK inhibition alone had little effect on proliferation, co-targeting ALK and EGFR significantly reduced OSCC cell proliferation in vitro. Further analysis showed significant efficacy of combined treatment in HSC3-derived xenografts resulting in a 30% decrease in tumor volumes by 14days (p<0.001). Western blot analysis showed that co-targeting ALK and EGFR significantly reduced EGFR phosphorylation (Y1148) in HSC3 cells but not Cal27 cells. ALK and EGFR downstream signaling interactions are also demonstrated by Western blot analysis in which lone EGFR and ALK inhibitors attenuated AKT activity whereas co-targeting ALK and EGFR completely abolished AKT activation. No effects were observed on ERK1/2 activation. STAT3 activity was significantly induced by lone ALK inhibition in HSC3 cells and to a lower extent in Cal27 cells. Together, these data illustrate that ALK inhibitors enhance anti-tumor activity of EGFR inhibitors in susceptible tumors that display increased ALK expression, most likely through abolition of AKT activation.

Synthetic and Biological Studies of Sesquiterpene Polygodial: Activity of 9-Epipolygodial against Drug-Resistant Cancer Cells.

Polygodial, a terpenoid dialdehyde isolated from Polygonum hydropiper L., is a known agonist of the transient receptor potential vanilloid 1 (TRPV1). In this investigation a series of polygodial analogues were prepared and investigated for TRPV1-agonist and anticancer activities. These experiments led to the identification of 9-epipolygodial, which has antiproliferative potency significantly exceeding that of polygodial. 9-Epipolygodial was found to maintain potency against apoptosis-resistant cancer cells as well as those displaying the multidrug-resistant (MDR) phenotype. In addition, the chemical feasibility for the previously proposed mechanism of action of polygodial, involving the formation of a Paal-Knorr pyrrole with a lysine residue on the target protein, was demonstrated by the synthesis of a stable polygodial pyrrole derivative. These studies reveal rich chemical and biological properties associated with polygodial and its direct derivatives. These compounds should inspire further work in this area aimed at the development of new pharmacological agents, or the exploration of novel mechanisms of covalent modification of biological molecules with natural products.

Wittig derivatization of sesquiterpenoid polygodial leads to cytostatic agents with activity against drug resistant cancer cells and capable of pyrrolylation of primary amines.

Many types of cancer, including glioma, melanoma, non-small cell lung cancer (NSCLC), among others, are resistant to proapoptotic stimuli and thus poorly responsive to current therapies based on the induction of apoptosis in cancer cells. The current investigation describes the synthesis and anticancer evaluation of unique C12-Wittig derivatives of polygodial, a sesquiterpenoid dialdehyde isolated from Persicaria hydropiper (L.) Delabre. These compounds were found to undergo an unprecedented pyrrole formation with primary amines in a chemical model system, a reaction that could be relevant in the biological environment and lead to the pyrrolation of lysine residues in the target proteins. The anticancer evaluation of these compounds revealed their promising activity against cancer cells displaying various forms of drug resistance, including resistance to proapoptotic agents. Mechanistic studies indicated that compared to the parent polygodial, which displays fixative general cytotoxic action against human cells, the C12-Wittig derivatives exerted their antiproliferative action mainly through cytostatic effects explaining their activity against apoptosis-resistant cancer cells. The possibility for an intriguing covalent modification of proteins through a novel pyrrole formation reaction, as well as useful activities against drug resistant cancer cells, make the described polygodial-derived chemical scaffold an interesting new chemotype warranting thorough investigation.

Vanilloids induce oral cancer apoptosis independent of TRPV1.

OBJECTIVE: To investigate the mechanisms of vanilloid cytotoxicity and anti-tumor effects in oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: Immunohistochemistry and qPCR analyses demonstrated expression of the TRP vanilloid type 1 (TRPV1) receptor in OSCC. Using cell proliferation assays, calcium imaging, and three mouse xenograft models, prototypical vanilloid agonist (capsaicin) and antagonist (capsazepine) were evaluated for cytotoxic and anti-tumor effects in OSCC. RESULTS: OSCC cell lines treated with capsaicin displayed significantly reduced cell viability. Pre-treatment with capsazepine failed to reverse these effects. Moreover, capsazepine alone was significantly cytotoxic to tumor cells, suggesting the mechanism-of-action is independent of TRPV1 activation. This was further confirmed by calcium imaging indicating that TRPV1 channels are not functional in the cell lines tested. We then examined whether the observed vanilloid cytotoxicity was due to the generation of reactive oxygen species (ROS) and subsequent apoptosis. Induction of ROS was confirmed by flow cytometry and reversed by co-treatment with the antioxidant N-acetyl-cysteine (NAC). NAC also significantly reversed vanilloid cytotoxicity in cell proliferation assays. Dose-dependent induction of apoptosis with capsazepine treatment was demonstrated by FACS analyses and c-PARP expression in treated cells. Our in vivo xenograft studies showed that intra-tumoral injections of capsazepine exhibited high effectiveness in suppressing tumor growth with no identifiable toxicities. CONCLUSIONS: These findings confirm TRPV1 channel expression in OSCC. However anti-tumor effects of vanilloids are independent of TRPV1 activation and are most likely due to ROS induction and subsequent apoptosis. Importantly, these studies demonstrate capsazepine is a potential therapeutic candidate for OSCC.