Reply: A morpho-molecular prognostic model for hepatocellular carcinoma

10.1038/bjc.2012.515British Journal of Cancer1083741-BJCA

The Pharmacochaperone Activity of Quinine on Bitter Taste Receptors.

Bitter taste is one of the five basic taste sensations which is mediated by 25 bitter taste receptors (T2Rs) in humans. The mechanism of bitter taste signal transduction is not yet elucidated. The cellular processes underlying T2R desensitization including receptor internalization, trafficking and degradation are yet to be studied. Here, using a combination of molecular and pharmacological techniques we show that T2R4 is not internalized upon agonist treatment. Pretreatment with bitter agonist quinine led to a reduction in subsequent quinine-mediated calcium responses to 35 ± 5% compared to the control untreated cells. Interestingly, treatment with different bitter agonists did not cause internalization of T2R4. Instead, quinine treatment led to a 2-fold increase in T2R4 cell surface expression which was sensitive to Brefeldin A, suggesting a novel pharmacochaperone activity of quinine. This phenomenon of chaperone activity of quinine was also observed for T2R7, T2R10, T2R39 and T2R46. Our results suggest that the observed action of quinine for these T2Rs is independent of its agonist activity. This study provides novel insights into the pharmacochaperone activity of quinine and possible mechanism of T2R desensitization, which is of fundamental importance in understanding the mechanism of bitter taste signal transduction

Agonist promoted desensitization of T2R4 expressed in HEK293T cells.

<p><b>[A]</b>. Representative calcium traces of T2R4-expressing HEK293T cells pretreated with assay buffer, quinine or yohimbine. <b>[B]</b>. Representative calcium traces of T2R4-expressing HEK293T cells pretreated with assay buffer or yohimbine. Fluo-4 NW loaded cells were pretreated with buffer alone or 1 mM each of quinine or yohimbine for 15 min, washed with assay buffer, and exposed to same concentrations of the agonist by the Flexstation 3 microplate reader. Changes in intracellular calcium were measured in terms of relative fluorescence units (RFU) at 525 nm following excitation at 494 nm. The arrow at 20 sec indicates the addition of agonist by Flexstation 3 microplate reader. <b>[C]</b>. Bar plot representation of the measured intracellular calcium. Buffer pretreated T2R4-expressing cells stimulated with quinine were represented as 100%, and calcium responses of other treatments were normalized to it. Data is representative of four independent experiments and expressed as mean ± SEM. Significance was checked by one-way ANOVA using <i>Dunnette’s</i> post hoc test, *p<0.05, ***p<0.001.</p

Time course studies of quinine incubation times on T2R4 surface expression.

<p>[<b>A</b>]. Time-dependent effect of quinine incubation on cell surface expression of T2R4 in HEK293T cells. T2R4 expressing cells were treated with quinine for 15 min, 30 min, 60 min or 120 min at 37°C. T2R4 cell surface expression was analyzed by flow cytometry using mouse monoclonal anti-FLAG antibody. [<b>B</b>]. Time-dependent effect of quinine incubation on surface expression of endogenous T2R4 in hASMCs. T2R4 surface expression was also determined after denatonium benzoate (DB) incubation for 15 min, which produced no change in surface expression of T2R4 in hASMCs. Data represents values from three independent experiments performed in duplicates. Significance was checked by one-way ANOVA using <i>Tukey’s</i> post hoc test, *p<0.05, **p<0.01, ***p<0.001.</p

T2R trafficking and chaperone activity of quinine.

<p>[<b>A</b>]. Effect of agonist treatment on T2R4 cell surface expression. T2R4-HEK293T cells were treated with buffer (control) or 1 mM each of quinine, yohimbine, dapsone and parthenolide, or 2 mM denatonium benzoate (DB) for 1 h at 37°C. TPα expressing HEK293T cells treated with its agonist, 1 μM U46619, were used as a positive control. [<b>B</b>]. Effect of BFA treatment on T2R4 trafficking. T2R4 expressing HEK293T cells were treated with vehicle or 5 μg/ml BFA for 4 h at 37°C. The cells were incubated with 1 mM quinine, in the presence of BFA, for 15 min or 60 min at 37°C. Cell surface expression of T2R4 was determined by flow cytometry and expressed as percentage increase over FLAG-T2R4 without quinine stimulation (basal, represented as quinine-0), which was considered as 100%. Values represent the mean ± SEM of 3–7 independent experiments. [<b>C</b>]. Effect of quinine treatment on cell surface expression of FLAG-tagged T2R1, T2R3, T2R4 and TPα transiently expressed in HEK293T cells. [<b>D</b>]. Effect of DXM treatment on surface expression of transiently expressed T2R1 and T2R4 in HEK293T cells. Receptor-expressing cells were incubated with 500 μM DXM for 15 min before determining cell surface expression by flow cytometry. Receptor expression was analyzed using mouse monoclonal anti-FLAG antibody, which was detected by goat anti-mouse Alexa 488 antibody. Significance was checked by one-way ANOVA using <i>Tukey’s</i> post test or by Student’s <i>t-test</i>. <b>[E].</b> Effect of FLAG sequence on T2R4 internalization or expression. HEK293T cells expressing either FLAG-T2R4 or untagged T2R4 were treated with buffer (control) or 1 mM quinine for 1 hr at 37°C. Cell surface expression of T2R4 was determined by flow cytometry using polyclonal anti-T2R4 antibody targeting the extracellular surface of T2R4. Data was represented as percentage change over FLAG-T2R4 without quinine stimulation (represented as untreated), which was considered as 100%. Values represent the mean ± SEM of 3–4 independent experiments performed in duplicates (Mean ± SEM). Statistical significance was checked by Two-way ANOVA using <i>Bonferroni’s</i> multiple comparison test. * <i>p</i> <0.05. [F]. Effect of quinine on TAS2R4 mRNA levels. Serum-starved TAS2R4 expressing HEK293T cells were treated either with assay buffer or 1 mM quinine for 15 min or 60 min. Total RNA was extracted and reverse transcribed into cDNA. TAS2R4 expression was examined by performing real-time PCR using TAS2R4-specific primers. The mRNA expression is shown as fold change over TAS2R4 without any quinine stimulation. Data are mean ± SEM from three independent experiments.</p

Agonist and chaperone activity of quinine on T2Rs.

<p><b>[A].</b> Quinine agonistic activity on 11 T2Rs (left panel). HEK293T cells expressing the 11 T2Rs and mock-transfected control (pcDNA3.1 vector alone) were treated with 1 mM quinine and calcium responses were recorded as relative fluorescence unit (RFU). Representative calcium traces of quinine response for different T2Rs expressed in HEK293T cells and mock-transfected cells (control) were presented in the right panel. Data represents values from at least three independent experiments performed in triplicates. Significance was checked by one-way ANOVA using <i>Dunnet’s</i> post hoc test, *p<0.05, **p<0.01, ***p<0.001. <b>[B].</b> Trafficking and chaperone activity of quinine on 11 T2Rs. HEK293T cells expressing FLAG-T2Rs were incubated with buffer or 1 mM quinine for 1 hr at 37°C. The cells were stained with FLAG-antibody (1:500 dilution) for 1 hr on ice in the dark and washed with FACS buffer for 2–3 times. Flow cytometry analysis was performed to study the effect of quinine on cell surface expression of T2Rs. The MFI values of the mock-transfected cells were subtracted from both treated and untreated cells expressing the respective T2Rs. The data was then normalized to untreated cells taken as 100% for each T2R. Data represents values from at least three independent experiments performed in duplicates. Significance was checked by Student t-test, *p<0.05, **p<0.01, ***p<0.001.</p

Quinine induced desensitization of T2Rs.

<p>Representative calcium traces showing desensitization of the quinine response to quinine pretreatment in HEK293T cells and hASMCs. <b>[A]</b>. Fluo-4 NW loaded T2R4 expressing HEK293T cells and mock-transfected HEK293T cells were pretreated with 1 mM quinine for 15 min, washed with assay buffer and re-exposed to 1 mM quinine. The intracellular calcium mobilized was measured in terms of relative fluorescence units (RFU) at 525 nm following excitation at 494 nm, using a FlexStation 3 fluorescence microplate reader. <b>[B]</b>. Human ASM cells pretreated with quinine for 15 min, washed with assay buffer and re-exposed to quinine were used as control. Cells pretreated with assay buffer and then exposed to quinine represent first treatment. Cells pretreated with quinine and re-exposed to same concentration of quinine represent second treatment. The arrow at 20 sec indicates the addition of agonist by Flexstation 3 microplate reader. <b>[C]</b>. Bar plot representation of the calcium responses in T2R4-HEK293T cells and hASMCs in response to quinine treatments. The results are represented with the calcium mobilized in untreated cells taken as 100%. Results are from a minimum of three independent experiments. Statistical significance was determined by student <i>t-</i>test.</p