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. 2019 Jul 3;20(13):3271.
doi: 10.3390/ijms20133271.

Carboplatin Enhances the Activity of Human Transient Receptor Potential Ankyrin 1 through the Cyclic AMP-Protein Kinase A-A-Kinase Anchoring Protein (AKAP) Pathways

Kanako Miyano  1 , Seiji Shiraishi  1 , Koichiro Minami  1   2   3 , Yuka Sudo  1   4 , Masami Suzuki  1 , Toru Yokoyama  1   2   3 , Kiyoshi Terawaki  1 , Miki Nonaka  1 , Hiroaki Murata  5 , Yoshikazu Higami  4   6 , Yasuhito Uezono  7   8   9   10
Affiliations

Carboplatin Enhances the Activity of Human Transient Receptor Potential Ankyrin 1 through the Cyclic AMP-Protein Kinase A-A-Kinase Anchoring Protein (AKAP) Pathways

Kanako Miyano et al. Int J Mol Sci. .

Abstract

Carboplatin, an anticancer drug, often causes chemotherapy-induced peripheral neuropathy (PN). Transient receptor potential ankyrin 1 (TRPA1), a non-selective cation channel, is a polymodal nociceptor expressed in sensory neurons. TRPA1 is not only involved in pain transmission, but also in allodynia or hyperalgesia development. However, the effects of TRPA1 on carboplatin-induced PN is unclear. We revealed that carboplatin induced mechanical allodynia and cold hyperalgesia, and the pains observed in carboplatin-induced PN models were significantly suppressed by the TRPA1 antagonist HC-030031 without a change in the level of TRPA1 protein. In cells expressing human TRPA, carboplatin had no effects on changes in intracellular Ca2+ concentration ([Ca2+]i); however, carboplatin pretreatment enhanced the increase in [Ca2+]i induced by the TRPA1 agonist, allyl isothiocyanate (AITC). These effects were suppressed by an inhibitor of protein kinase A (PKA). The PKA activator forskolin enhanced AITC-induced increase in [Ca2+]i and carboplatin itself increased intracellular cyclic adenosine monophosphate (cAMP) levels. Moreover, inhibition of A-kinase anchoring protein (AKAP) significantly decreased the carboplatin-induced enhancement of [Ca2+]i induced by AITC and improved carboplatin-induced mechanical allodynia and cold hyperalgesia. These results suggested that carboplatin induced mechanical allodynia and cold hyperalgesia by increasing sensitivity to TRPA1 via the cAMP-PKA-AKAP pathway.

Keywords: AKAP; CIPN; PKA; TRPA1; cAMP; carboplatin.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effects of carboplatin on mechanical allodynia, as well as cold and heat hyperalgesia. Carboplatin (10 mg/kg) or its vehicle (control) was administered intraperitoneally to mice twice a week for 2 weeks. von Frey test (A), acetone test (B), and hot plate test (C) were performed and body weight (D) was measured. The data are expressed as the mean ± standard error of the mean (n = 5–17). * and **** indicate p < 0.05 and p < 0.0001, respectively, compared with each control group; Bonferroni’s multiple comparison test following two-way analysis of variance.
Figure 2
Figure 2
Effects of a TRPA1 inhibitor on carboplatin-induced mechanical allodynia and cold hyperalgesia. HC-030031, a selective TRPA1 antagonist, was administered intraperitoneally to mice at Day 7 after treatment with carboplatin, which were then subjected to von Frey test (A,B) and acetone test (C,D). Injection of HC-030031 ameliorated mechanical allodynia in carboplatin-induced peripheral neuropathy model mice in a time (A)- and dose (B)-dependent manner. At 30 min after injection of HC-030031, carboplatin-induced peripheral neuropathy model mice showed significant amelioration of cold hyperalgesia (C,D). The data are expressed as the mean ± standard error of the mean (n = 4–8). * and **** indicate p < 0.05 and 0.0001, respectively, compared with the control group; ##, #### indicate p < 0.01 and 0.0001, respectively, compared with the carboplatin group; Bonferroni’s multiple comparison test following one (B,D)- or two (A,C)-way analysis of variance.
Figure 3
Figure 3
Effects of carboplatin on the expression of TRPA1 protein in mouse DRG. The protein expression of TRPA1 in the DRG of carboplatin-induced peripheral neuropathy model mice was measured by western blotting. The data are expressed as the mean ± standard error of the mean (n = 5–7): compared with the control group; Bonferroni’s multiple comparison test following one-way analysis of variance. n.s.; not significant.
Figure 4
Figure 4
Carboplatin enhances TRPA1 activation in a time- and dose-dependent manner in TRPA1-expressing HEK293 cells. Fura 2-loaded, hTRPA1-expressing cells were pretreated with vehicle (gray line) or carboplatin (10 µM, black line) for 10, 30, or 60 min and then treated with allyl isothiocyanate (AITC) (100 µM), respectively (A,B). Concentration-response relationship of carboplatin and increases in [Ca2+]i induced by AITC, respectively (C,D). Representative tracing of the mean [Ca2+]i in randomly selected cells expressing hTRPA1 (A,C). The extent of increases in [Ca2+]i induced by AITC was quantified by determining the differences between the ratio (340/380) of the basal and peak levels after treatment with each agonist (B,D). The data are expressed as the means ± standard error of the mean of at least triplicate experiments (n = 107–313). *, ***, and **** indicate p < 0.05, 0.01, and 0.0001, respectively, compared with the AITC alone group; Bonferroni’s comparison test following one-way analysis of variance.
Figure 5
Figure 5
Effects of inhibitors of phospholipase C (PLC) or protein kinase A (PKA) on carboplatin-induced enhancement of TRPA1 activation. (A) Fura 2-loaded cells expressing hTRPA1 were pretreated with both carboplatin (10 µM) and each inhibitor [the PLC inhibitor U73122 (1 µM) or the PKA inhibitor KT5720 (0.1 µM)] for 30 min and then treated with allyl isothiocyanate (AITC) (100 µM), respectively. The cells were pretreated with forskolin (FSK; 10 µM), a PKA activator, for 30 min, and treated with AITC (A). The extent of increases in [Ca2+]i induced by AITC was quantified by determining the differences between the ratio (340/380) of the basal and peak levels after treatment with each agonist (A). (B) hTRPA1-expressing cells were treated with vehicle or carboplatin (10 µM) for 0, 10, or 30 min, and the amount of intracellular cAMP was measured using cAMP-Screen Direct® systems. The data are expressed as the means ± standard error of the mean of at least triplicate experiments (A, n = 68–474; B, n = 3–4). *, **, and *** indicate p < 0.05, 0.01, and 0.001, respectively, compared with the vehicle group; Bonferroni’s multiple comparison test following one-way analysis of variance (ANOVA). # indicates p < 0.05, compared with the carboplatin alone group; Bonferroni’s multiple comparison test following one-way ANOVA.
Figure 6
Figure 6
Effects of an inhibitor of A-kinase anchoring protein (AKAP) on TRPA1 activation or mechanical allodynia induced by carboplatin. (A) Fura 2-loaded cells expressing hTRPA1 were pretreated with both carboplatin (10 µM) and AKAP St-Ht31 inhibitor peptide (AKAP I; 50 µM) for 30 min and then treated with AITC (100 µM). The extent of increases in [Ca2+]i induced by AITC was quantified by determining the differences between the ratio (340/380) of the basal and peak levels after treatment with each agonist (A). (B) The mice were intrathecally administered AKAP I (20 µg), and then subjected to von Frey test (B) and acetone test (C). At 60 min after injection of AKAP I (Day 8 and 9), mechanical hyperalgesia in model rats of carboplatin-induced peripheral neuropathy was ameliorated (B). At 30 min after injection of AKAP I, there was a significant amelioration of cold hyperalgesia in the model rats (C). The data are expressed as the mean ± standard error of the mean of at least triplicate experiments (A, n = 74–173; B, n = 4–11; C, n = 4–14). **** indicates p < 0.0001, compared with the vehicle or control group; #### indicates p < 0.001, compared with the carboplatin alone group; Bonferroni’s multiple comparison test following one -way analysis of variance.
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References

    1. Ruckdeschel J.C. The future role of carboplatin. Semin. Oncol. 1994;21(Suppl. 12):114–118. - PubMed
    1. Cavaletti G., Fabbrica D., Minoia C., Frattola L., Tredici G. Carboplatin toxic effects on the peripheral nervous system of the rat. Ann. Oncol. 1998;9:443–447. doi: 10.1023/A:1008231925889. - DOI - PubMed
    1. Go R.S., Adjei A.A. Review of the comparative pharmacology and clinical activity of cisplatin and carboplatin. J. Clin. Oncol. 1999;17:409–422. doi: 10.1200/JCO.1999.17.1.409. - DOI - PubMed
    1. Bolis G. Pilot study with fixed-dose carboplatin and escalating paclitaxel in advanced ovarian cancer. Semin. Oncol. 1995;22(Suppl. 14):32–34. - PubMed
    1. Broun E.R., Nichols C.R., Mandanas R., Salzman D., Turns M., Hromas R., Cornetta K., Einhorn L.H. Dose escalation study of high-dose carboplatin and etoposide with autologous bone marrow support in patients with recurrent and refractory germ cell tumors. Bone Marrow Transplant. 1995;16:353–358. - PubMed

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