Activation of odorant receptor in colorectal cancer cells leads to inhibition of cell proliferation and apoptosis

The analysis and functional characterization of ectopically expressed human olfactory receptors (ORs) is becoming increasingly important, as many ORs have been identified in several healthy and cancerous tissues. OR activation has been demonstrated to have influence on cancer cell growth and progression. Here, ORs were identified using RNA-Seq analyses and RT-PCR. We demonstrated the OR protein localization in HCT116 cells using immunocytochemistry (IHC). In order to analyze the physiological role of OR51B4, we deorphanized the receptor by the use of CRE-Luciferase assays, conducted calcium imaging experiments as well as scratch-and proliferation assays. Furthermore, western blot analyses revealed the involvement of different protein kinases in the ligand-dependent signaling pathway. Receptor knockdown via shRNA was used to analyze the involvement of OR51B4. We identified OR51B4, which is highly expressed in the colon cancer cell line HCT116 and in native human colon cancer tissues. We deorphanized the receptor and identified Troenan as an effective ligand. Troenan stimulation of HCT116 cells has anti-proliferative, anti-migratory and pro-apoptotic effects, mediated by changes in the intracellular calcium level upon PLC activation. These effects cause changes in the phosphorylation levels of p38, mTor and Akt kinases. Knockdown of the receptor via shRNA confirmed the involvement of OR51B4. This study emphasizes the importance of ectopically expressed ORs in the therapy for several diseases. The findings provide the basis for alternative treatments of colorectal cancer

Activation of odorant receptor in colorectal cancer cells leads to inhibition of cell proliferation and apoptosis

The analysis and functional characterization of ectopically expressed human olfactory receptors (ORs) is becoming increasingly important, as many ORs have been identified in several healthy and cancerous tissues. OR activation has been demonstrated to have influence on cancer cell growth and progression. Here, ORs were identified using RNA Seq analyses and RT-PCR. We demonstrated the OR protein localization in HCT116 cells using immunocytochemistry (IHC). In order to analyze the physiological role of OR51B4, we deorphanized the receptor by the use of CRE-Luciferase assays, conducted calcium imaging experiments as well as scratch- and proliferation assays. Furthermore, western blot analyses revealed the involvement of different protein kinases in the ligand-dependent signaling pathway. Receptor knockdown via shRNA was used to analyze the involvement of OR51B4. We identified OR51B4, which is highly expressed in the colon cancer cell line HCT116 and in native human colon cancer tissues. We deorphanized the receptor and identified Troenan as an effective ligand. Troenan stimulation of HCT116 cells has anti-proliferative, anti-migratory and pro-apoptotic effects, mediated by changes in the intracellular calcium level upon PLC activation. These effects cause changes in the phosphorylation levels of p38, mTor and Akt kinases. Knockdown of the receptor via shRNA confirmed the involvement of OR51B4. This study emphasizes the importance of ectopically expressed ORs in the therapy for several diseases. The findings provide the basis for alternative treatments of colorectal cancer

Diagram of the proposed signaling cascade that is induced by Troenan stimulation.

<p>PLC: phospholipase C, DAG: diacylglycerol, PKC: protein kinase C, CREB: cAMP response element-binding protein, SOCE: store-operated calcium entry.</p

Impaired actin filament formation and induction of apoptosis upon stimulation of HCT116 cells with Troenan.

<p><b>(A)</b> HCT116 cells treated with Troenan (500 μM) and control cells. <b>(B) and (C)</b> Phalloidin staining of control cells (B) and cells treated with Troenan (300 μM) (C). Scale bar: 10 μm. <b>(D) and (E)</b> Immunocytochemical staining of HCT116 cells with an antibody against caspase-3 after treatment with control (D) or Troenan (300 μM) (E). Cells treated with Troenan (300 μM) for 48 hours. Scale bar: 10 μm. <b>(F)</b> HCT116 cells show decreased serotonin release after application of Troenan (700 μM) for 60 minutes.</p

Transcript abundance of potential effector channels in HCT116 cells determined by RNA-Seq.

<p><b>(A)</b> Bar chart showing the FPKM values of different possible effector channels. Voltage-dependent L- and T-Type channels: CACNA1S, CACNA1C, CACNA1D, CACNA1F, CACNA2D1, CACNA2D2; CACNA1G, CACNA1H, CACNA1I; CACNB1, CACNB3. Cyclic nucleotide-gated ion channels: CNGA1, CNGA2, CNGA3, CNGA4, CNGB1, CNGB3. Voltage-dependent Ca²⁺ channel: CATSPER1. Transient receptor potential channels: TRPCI, TRPC6, TRPV1, TRPM8, TRPC6, TRPV2, TRPM7, TRPM8. Calcium release-activated calcium channels: ORAI1, ORAI2, ORAI3. <b>(B)</b> Transcript expression of PLC isoforms in the colon cancer cell line HCT116. Bar chart showing FPKM values of different PLCs in the colon cancer cell line HCT116.</p

Pharmacological analysis of the signaling pathway involved in the activation of OR51B4.

<p>Representative calcium imaging traces of HCT116 cells stimulated with Troenan and different specific inhibitors. Grey area represents the duration of the inhibitor applicated. Bar chart showing mean amplitudes of Troenan-induced Ca²⁺ signals in HCT116 cells. <b>(A)</b> Localization of Ca²⁺ by use of EGTA-Ringer. Investigation of different specific inhibitors of calcium signaling upon Troenan stimulation. (<b>B</b>) Gallein (10 μM), <b>(C)</b> U-73522 (10 μM), <b>(D)</b> SQ22.536 (50 μM), <b>(E)</b> H89 (10 μM), <b>(F)</b> RR; Ruthenium red (5 μM), <b>(G)</b> L-cis-diltiazem (150 μM), <b>(H)</b> Mibefradil (10 μM), <b>(I)</b> BTP-2 (25 μM), <b>(J)</b> Thapsigargin (1 μM). N > 3 with n = 18 measurements in 9 cell culture dishes with approximately 200 cells. The data are shown as the mean SEM.</p

Expression of OR51B4 in HCT116 cells and colon cancer tissues.

<p><b>(A)</b> Bar chart displays the FPKM values of the most highly expressed ORs in HCT116 cells. <b>(B)</b> Immunocytochemical staining of OR51B4 in HCT116 cells with a specific OR51B4-antibody. Left: HCT116 cells. Right: Negative control: HCT116 cells stained with second antibody alone. Bottom left: Hana3A cells transfected with OR51B4 plasmid. Bottom right: untransfected Hana3A cells. Scale bar: 10 μm. <b>(C)</b> The most highly expressed ORs in NGS analyses, validated by RT-PCR. + = +RT, cDNA;— = -RT, RNA; g = genomic DNA as a control; M = marker. <b>(D)</b> OR51B4 expressed in human colon cancer tissues: RT-PCR analysis shows OR51B4 expression in human colon cancer tissues. Expression in A: Colon tissue B: Colorectal cancer tissue C, D, E: Colon carcinoma tissues. M = marker.</p

Western blot analysis of HCT116 cells stimulated with Troenan (300 μM; T) or control (C) for 5 and 25 minutes.

<p><b>(A)</b> Phosphorylation of different isoforms of PKC. <b>(B)</b> Reduced phosphorylation of Stat2, Stat3 and Stat5 upon Troenan stimulation (300 μM). <b>(C)</b> Stimulation of Troenan (300 μM) leads to the time-dependent phosphorylation of p38 MAPK. Reduced phosphorylation was observed for Akt, mTor and Fyn. Troenan stimulation did not affect ERK and SAPK. The total amounts of p38, Akt, ERK, Stat3 and Stat5 and β-actin served as controls. n = 3. <b>(D)</b> Quantification of the mean pixel intensities of the phosphorylated protein kinases Akt, ERK1/2, p38, Stat5 and Stat3. The pixel intensities of duplicates were averaged. Total amounts of the protein kinases were determined and served as controls.</p

Analysis of the Troenan-induced effect in HCT116 cells containing a doxycycline-sensitive OR51B4-knockdown-sequence.

<p><b>(A)</b> Confirmation of knockdown functionality by qRT-PCR and calcium imaging experiments. M = Marker. Stimulation of HCT116/EV (left) and HCT116/10F1 cells (right) with Troenan (100 μM/ 300 μM). <b>(B)</b> Representative calcium signal of HCT116/EV (above) and HCT116/10F1 (below) cells stimulated with Troenan (300 μM) in calcium imaging analysis. <b>(C)</b> Migration analysis via scratch assay with HCT116/EV and HCT116/10F1 cells with and without doxycycline induction. Stimulation of the cells with Troenan (300 μM) for 48 hours. N = 3 assays with 3 dishes. <b>(D)-(G)</b> Proliferation analysis of HCT116/EV (D, E) and HCT116/10F1 (F, G) cells after treatment with Troenan (300 μM) with and without doxycycline induction. Troenan (300 μM) was applied for 72 hours. N = 20.</p

Characterization of Troenan-induced calcium signals in HCT116 cells.

<p><b>(A)</b> Representative image of HCT116 cells stimulated with Troenan (300 μM) in calcium imaging analysis. <b>(B)</b> Number of cells responding to Troenan in different concentrations. <b>(C)</b> Repetitive activation of HCT116 cells upon repetitive Troenan application. <b>(D)</b> Dose-dependent activation of HCT116 cells by Troenan. Troenan was applied at concentrations of 50 μM, 100 μM and 300 μM. Peak amplitudes show the increases in intracellular calcium concentration. To ensure viability of the cells, ATP was applied last, which serves as a positive control. N > 3 with n = 18 measurements in 9 cell culture dishes with approximately 200 cells.</p