Upregulated Expression of Transient Receptor Potential Cation Channel Subfamily V Receptors in Mucosae of Patients with Oral Squamous Cell Carcinoma and Patients with a History of Alcohol Consumption or Smoking

<div><p>Objectives</p><p>Transient receptor potential cation channel (subfamily V, members 1–4) (TRPV1–4) are expressed in skin and neurons and activated by external stimuli in normal mucosae of all oral cavity sites. The oral cavity is exposed to various stimuli, including temperature, mechanical stimuli, chemical substances, and changes in pH, and, notably, the risk factors for oncogenic transformation in oral squamous epithelium are the same as the external stimuli received by TRPV1–4 receptors. Hence, we examined the relationship between oral squamous cell carcinoma (SCC) and TRPV1–4 expression.</p><p>Materials and Methods</p><p>Oral SCC patients (n = 37) who underwent surgical resection were included in this study. We investigated the expression of TRPV1–4 by immunohistochemical staining and quantification of <i>TRPV1–4</i> mRNA in human oral mucosa. In addition, we compared the TRPV1–4 levels in mucosa from patients with SCC to those in normal oral mucosa.</p><p>Results</p><p>The receptors were expressed in oral mucosa at all sites (tongue, buccal mucosa, gingiva, and oral floor) and the expression was stronger in epithelia from patients with SCC than in normal epithelia. Furthermore, alcohol consumption and tobacco use were strongly associated with the occurrence of oral cancer and were found to have a remarkable influence on TRPV1–4 receptor expression in normal oral mucosa. In particular, patients with a history of alcohol consumption demonstrated significantly higher expression levels.</p><p>Conclusion</p><p>Various external stimuli may influence the behavior of cancer cells. Overexpression of TRPV1-4 is likely to be a factor in enhanced sensitivity to external stimuli. These findings could contribute to the establishment of novel strategies for cancer therapy or prevention.</p></div

Clinical, pathological, and epidemiologic characteristics of 37 patients with oral squamous cell carcinoma.

<p>Clinical, pathological, and epidemiologic characteristics of 37 patients with oral squamous cell carcinoma.</p

Box plot illustrating relative levels of <i>TRPV1</i>, <i>2</i>, <i>3</i>, and <i>4</i> expression compared by alcohol consumption and smoker status.

<p>Expression levels of each target gene were normalized to those of <i>ACTB</i> in the same samples and are expressed relative to control group expression levels (DDCT method; Applied Biosystems). Box plots illustrate the median, and lower, and upper quartiles of <i>TRPV1–4</i> expression. Whiskers were calculated using the Tukey method; filled circles represent outliers. **p < 0.01, *p < 0.05. (a) Relative expression levels of <i>TRPV1</i>, <i>2</i>, <i>3</i>, and <i>4</i> were all significantly higher in alcohol users than non-users. (b) Relative expression levels of <i>TRPV1</i>, <i>2</i>, <i>3</i>, and <i>4</i> were higher in the smoking than the non-smoking group. For <i>TRPV2</i>, <i>3</i> and <i>4</i>, the difference was significant, whereas for <i>TRPV1</i>, the difference was not significant, although expression levels of this receptor were higher among smokers.</p

Immunostaining images of TRPV1–4 in human squamous cell carcinoma of the tongue (by grade).

<p>Merged image of anti-TRPV1–4 (green), counterstained with Evance blue (red) and DAPI (blue).</p

Comparisons of relative levels of expression of <i>TRPV1–4</i> in all oral cavity sites between normal oral mucosae and oral mucosae from patients with SCC.

<p>The levels of each target gene were normalized to those of <i>ACTB</i> in the same samples and are expressed relative to expression in the control group (DDCT method; Applied Biosystems). Box plots illustrate the median, lower, and upper quartiles of <i>TRPV1–4</i> expression. Whiskers were calculated using the Tukey method; filled circles represent outliers. **p < 0.01, *p < 0.05. Immunostaining images of squamous cell cancer in each respective site are presented below the graphs. Merged image of anti-TRPV1-4 (green), counterstained with Evance blue (red) and DAPI (blue). Comparisons of relative expression levels of <b>a.</b> <i>TRPV1</i>; <b>b.</b> <i>TRPV2</i>; <b>c.</b> <i>TRPV3</i>; and <b>d.</b> <i>TRPV4</i>.</p

Immunostaining images of TRPV1–4 in normal human oral mucosae.

<p>Merged image of anti-TRPV1–4 (green), counterstained with Evance blue (red) and DAPI (blue).</p

Box plot of relative levels of expression of <i>TRPV1</i>, <i>2</i>, <i>3</i>, and <i>4</i> compared by tumor grade.

<p>Expression levels of each target gene were normalized to those of <i>ACTB</i> in the same sample and are expressed relative to control group expression (DDCT method; Applied Biosystems). Box plots illustrate the median, lower, and upper quartiles of <i>TRPV1–4</i> expression. Whiskers were calculated using the Tukey method; filled circles represent outliers. No consistent trends or significant differences in relative levels of expression of <i>TRPV1</i>, <i>2</i>, <i>3</i>, or <i>4</i> were observed according to tumor grade of human oral SCC.</p

Box plot demonstrating <i>TRPV1–4</i> expression in normal human oral mucosae.

<p>The relative expression level of each target gene was normalized to <i>ACTB</i> levels in the same sample and expressed relative to control group expression (DDCT method; Applied Biosystems). Box plots illustrate the median, lower and upper quartile values of <i>TRPV1–4</i> expression. Whiskers were calculated using the Tukey method; filled circles represent outliers. (a) Relative levels of expression of <i>TRPV1</i>, <i>2</i>, <i>3</i>, and <i>4</i> in the oral cavity (all regions, tongue, buccal mucosa, gingiva, and oral floor). A comprehensive examination by qPCR of all regions in the oral cavity showed that expression of <i>TRPV1</i> and <i>TRPV3</i> was significantly higher than that of <i>TRPV2</i> and <i>TRPV4</i> (p < 0.01). In the tongue, buccal mucosa, and oral floor, <i>TRPV1</i> expression was significantly greater than that of <i>TRPV2</i> and <i>TRPV4</i> (p < 0.05). In the gingiva, relative expression levels of <i>TRPV1</i> and <i>TRPV3</i> were significantly higher than those of <i>TRPV2</i> and <i>TRPV4</i> (p < 0.01). (b) Relative levels of <i>TRPV1</i>, <i>2</i>, <i>3</i>, and <i>4</i> expression by site in normal human oral mucosae. No significant differences were observed in relative expression levels; however, expression levels were relatively high in the tongue and oral floor in comparison with the gingiva and buccal mucosa.</p

Relationship between c-D-index and oral complications.

<p>Relationship between c-D-index and oral complications.</p

Box plot of the D-index in each grade of chemotherapy.

<p>Box plot of the D-index in each grade of chemotherapy.</p