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. 2017 Apr 18:10:915-925.
doi: 10.2147/JPR.S125045. eCollection 2017.

Identification of capsazepine as a novel inhibitor of system xc- and cancer-induced bone pain

Jennifer Fazzari  1 , Matthew D Balenko  1 , Natalie Zacal  1 , Gurmit Singh  1
Affiliations

Identification of capsazepine as a novel inhibitor of system xc- and cancer-induced bone pain

Jennifer Fazzari et al. J Pain Res. .

Abstract

The cystine/glutamate antiporter has been implicated in a variety of cancers as a major mediator of redox homeostasis. The excess glutamate secreted by this transporter in aggressive cancer cells has been associated with cancer-induced bone pain (CIBP) from distal breast cancer metastases. High-throughput screening of small molecule inhibitors of glutamate release from breast cancer cells identified several potential compounds. One such compound, capsazepine (CPZ), was confirmed to inhibit the functional unit of system xc- (xCT) through its ability to block uptake of its radiolabeled substrate, cystine. Blockade of this antiporter induced production of reactive oxygen species (ROS) within 4 hours and induced cell death within 48 hours at concentrations exceeding 25 μM VSports手机版. Furthermore, cell death and ROS production were significantly reduced by co-treatment with N-acetylcysteine, suggesting that CPZ toxicity is associated with ROS-induced cell death. These data suggest that CPZ can modulate system xc- activity in vitro and this translates into antinociception in an in vivo model of CIBP where systemic administration of CPZ successfully delayed the onset and reversed CIBP-induced nociceptive behaviors resulting from intrafemoral MDA-MB-231 tumors. .

Keywords: breast cancer; cancer-induced bone pain; glutamate; system xc−. V体育安卓版.

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Conflict of interest statement

Disclosure The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Quantification of cystine acquisition in MDA-MB-231 cells in the presence of capsazepine (CPZ). Notes: CPZ inhibits the uptake of 14C-cystine when incubated with 0–50 μM of the compound. This effect is comparable to intracellular levels of 14C-cystine after incubation with sulfasalazine, a known system xc− inhibitor. Data are expressed as the fold change in counts per minute/mg protein (±standard error of the mean) relative to vehicle control (dimethyl sulfoxide). **P<0.01; ***P<0.001; ****P<0.0001.
Figure 2
Figure 2
Capsazepine treatment induces the production of reactive oxygen species. Notes: Quantification of intracellular levels of reactive oxygen species (ROS) as measured by DCFDA over 24 and 48 hours of treatment with capsazepine (CPZ) and 5′-iodoresiniferatoxin (IRTX; A). After 48 hours, 25 μM of CPZ induces a significant increase in ROS relative to dimethyl sulfoxide-treated cells (P<0.001). Treatment with IRTX does not result in significant changes in ROS over this time course. These data are represented as mean fold change ±standard error of mean relative to vehicle-treated cells. One-way analysis of variance (ANOVA) was used to measure significant increases in ROS relative to vehicle-treated control. CPZ-induced ROS production is abolished by co-treatment with 5 mM N-acetyl cysteine (NAC; B). An unpaired t-test was used to assess significance of NAC addition at each time point. Cell survival decreases in a dose-dependent manner after treatment for 48 hours with CPZ. Co-treatment with 5 mM NAC increases cell survival relative to treatment with CPZ alone (C). Cell survival at 100 μM CPZ is significantly higher in the presence of 5 mM NAC (P<0.001; one-way ANOVA comparing each concentration of CPZ without NAC to those with the addition of NAC). *P<0.05; **P<0.01; ***P<0.001. Abbreviations: DCFDA, dichlorofluorescein diacetate derivative; RFU, relative fluorescent units.
Figure 3
Figure 3
Treatment with 25 μM capsazepine significantly induces xCT expression by 24 and 48 hours relative to dimethyl sulfoxide (DMSO) treatment. Notes: These data are expressed as the fold change in xCT mRNA levels relative to DMSO ±standard error of mean and analyzed using a one-way analysis of variance (24 hours P<0.001, 48 hours P<0.05). *P<0.05; **P<0.01.
Figure 4
Figure 4
Histological analysis of tumor xenografts in bone represented by hematoxylin and eosin staining. Notes: (A) Sham-injected mice. Tumor-injected mice show extensive invasion of xenograft into the femur with destruction of growth plate (B, D) and in some cases breaching of the periosteum (C). Arrowheads indicate tumor tissue.
Figure 5
Figure 5
Dynamic Plantar Aesthesiometer (DPA) measurements of force required for withdrawal of the injected limb compared to the baseline results in capsazepine (CPZ)-treated and non-treated mice (100% on the y-axis is therefore equivalent to the animal’s behavior pre-tumor implantation surgery). A significant decrease in the force required to induce paw withdrawal relative to baseline is only seen in the vehicle-treated group beginning on day 27 as indicated by the asterisks. Both the 5 and 10 mg/kg CPZ-treated groups do not show any significant deviation from baseline measurements. A one-way analysis of variance (ANOVA) with a Dunnett post-test was used to show significant differences between time points relative to the baseline control. Paw withdrawal thresholds are increased in CPZ-treated mice increase in force required for paw withdrawal in the CPZ-treated mice relative to vehicle-treated mice (10 mg/kg) n=10; (5 mg/kg) n=11; (vehicle) n=13. One-way repeated measures ANOVA was used on the measurements past day 25 (marked by dotted line) showing significant differences between groups (P<0.0001). While both doses were significantly different from vehicle mice (P<0.05), differences between doses were not significant. Data are expressed as the mean required force as a percentage of the baseline score ±standard error of mean. *P<0.05; **P<0.01; ***P<0.001.
Figure 6
Figure 6
(A) Capsazepine (CPZ)-treated mice and non-treated mice. A significant decrease in weight applied to the tumor-bearing paw relative to baseline marked the onset of pain behavior in vehicle-treated mice on day 29 as indicated by the asterisks. This is delayed until day 36 for the 5 mg/kg CPZ-treated mice, and no significant changes from baseline are seen in the 10 mg/kg CPZ-treated group. A one-way analysis of variance (ANOVA) with a Dunnett post-test was used to show significant differences between time points relative to the baseline control. This graph shows a significant increase in weight distribution in the CPZ-treated mice, relative to vehicle-treated mice (10 mg/kg) n=10; (5 mg/kg) n=11; (vehicle) n=13. A one-way repeated measures ANOVA was used on the measurements past day 25 (marked by dotted line) showing significant differences between groups (P=0.0008). While both doses were significantly different from vehicle mice (P<0.05), differences between doses were not significant. Data are expressed as the mean weight bearing in the injected limb as a percentage of the baseline score ±SEM. (B) DWB measurements of time spent on the injected limb compared to the baseline results in CPZ-treated mice and non-treated mice. A decrease in the time spent on the tumor-bearing limb significantly deviates from baseline at day 32 with the CPZ-treated group not showing any deviation from baseline at any time point. Relative to the vehicle-treated group, an increase in time spent on injected limb is seen in the CPZ-treated mice (10 mg/kg) n=10; (5 mg/kg) n=11; (vehicle) n=13. A one-way repeated measures ANOVA was used on the measurements past day 25 (marked by dotted line) showing significant differences between groups (P<0.005). While both doses were significantly different from vehicle mice (P<0.05), differences between doses were not significant. Data are expressed as the mean time spent on the injected limb as a percentage of the baseline time ±SEM. *P<0.05; **P<0.01; ***P<0.001.
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