RAF Kinase Inhibitory Protein Expression and Phosphorylation Profiles in Oral Cancers.

Raf Kinase Inhibitory Protein (RKIP) expression has been profiled for a number of unique tissue cancers. However, certain tissues have not been explored, and oral and oropharyngeal cancers stand out as high priority targets, given their relatively high incidence, high morbidity rate, and in many cases, preventable nature. The purpose of this study was to examine changes in RKIP expression and phosphorylation in tissues resected from oral cancer patients, and compare to results generated from immortalized cell lines raised from primary oral cancer tissues, including oral squamous cell carcinoma line 4 (SCC4) and human squamous cell carcinoma line 3 (HSC3). Out of 4 human samples collected from male and female patients across various ages with variable risk factors, we observed an across the board reduction in RKIP expression. Two human samples demonstrated a significant increase in phosphorylated RKIP when normalized to total RKIP, however all 4 were increased when normalized to total cellular protein. The immortalized oral cancer cell culture HSC3 revealed significant increases in phosphorylated RKIP with no change in total RKIP expression, while line SCC4 demonstrated an increase in both total and phosphorylated RKIP. Results presented here indicate that oral cancers behave similarly to other cancers in terms of changes in RKIP expression and phosphorylation, although immortalized cell line expression profiles significantly differ from human tissue biopsies.

Transient receptor potential V1 regulates activation and modulation of transient receptor potential A1 by Ca2+.

The transient receptor potential A1 (TRPA1) channel contributes to nociceptive signaling in certain pain models. It has been suggested that Ca(2+), which activates and modulates TRPA1, could play a critical regulatory role in this process. Since TRPA1 and transient receptor potential V1 (TRPV1) channels are co-expressed and interact in neurons, we investigated whether activation and modulation of TRPA1 by Ca(2+) is regulated by TRPV1. Cell-attached recordings showed that TRPA1 is activated by extracellular Ca(2+) ([Ca(2+)](e)) in concentration-response fashion. This activation, especially by 2 mM [Ca(2+)](e) was substantially suppressed by co-expression with TRPV1. Inside-out recordings demonstrated that intracellular Ca(2+) ([Ca(2+)](i))-triggered activation of TRPA1 was attenuated by the presence of TRPV1 only at 2 mM [Ca(2+)](e), but not in Ca(2+)-free conditions. Further, depletion of internal Ca(2+) stores by thapsigargin generated TRPA1-mediated currents, which is affected by TRPV1 in both Chinese hamster ovary cells and sensory neurons. Since mustard oil current (I(MO)) is modulated by [Ca(2+)](e), we next examined whether alterations in the Ca(2+)-permeability of TRPV1 by mutating Y671 effect I(MO) properties. First it was demonstrated that the mutations in TRPV1 did not affect association of the TRPA1 and TRPV1 channels. However, these TRPV1 mutations, particularly Y671K, altered the following characteristics of TRPA1: magnitude of I(MO) in presence and absence of [Ca(2+)](e); the influence of [Ca(2+)](e) on the voltage-dependency of I(MO), and open probability of single-channel I(MO). In summary, activation of TRPA1 by [Ca(2+)](e) and [Ca(2+)](i) is controlled by the TRPV1 channel, and characteristics of I(MO) depend on Ca(2+) permeability of the TRPV1 channel.

Tumor necrosis factor alpha enhances the sensitivity of rat trigeminal neurons to capsaicin.

Tumor necrosis factor alpha (TNFalpha), a pro-inflammatory cytokine, enhances the development of pain and hyperalgesia, although the molecular mechanisms are not well understood. This study evaluated the hypothesis that TNFalpha increases the sensitivity of rat trigeminal neurons to capsaicin via two different mechanisms triggered by either brief or sustained exposure to the cytokine. A brief (5 min) application of TNFalpha significantly sensitized capsaicin-evoked accumulation of intracellular calcium ([Ca2+]i) (226.4+/-37.7 nM vs. 167.5+/-31.3 nM) and increased capsaicin-evoked nocifensive behavior (78.3+/-9.7 vs. 30.9+/-3.6 s) as compared with vehicle pretreatment (P<0.01 for both). Sustained (30 min to 4 h) exposure of cultured neurons to TNFalpha evoked a twofold increase in mRNA transcript (P<0.05) and protein levels (P<0.01) of transient potential receptor vanilloid type 1 (TRPV1). This long-term up-regulation of TRPV1 expression by TNFalpha correlated with enhancement in capsaicin-induced calcitonin gene-related peptide release (P<0.05). Demonstration of colocalization of TNFalpha receptor subtypes I and II with TRPV1 in almost all (>90%) TRPV1 expressing neurons provides evidence consistent with a direct interaction on the same subpopulation of sensory neurons. In summary, our data demonstrate that TNFalpha directly enhances the sensitivity of rat trigeminal neurons to capsaicin via both rapid, non-genomic mechanisms as well as sustained genomic regulation in TRPV1 expression. Thus, increased sensitization and up-regulation of TRPV1 constitutes a potential mechanism by which TNFalpha mediates inflammatory hyperalgesia and pain.