Quercetin reduces Ehrlich tumor-induced cancer pain in mice

Abstract

Cancer pain directly affects the patient's quality of life. We have previously demonstrated that the subcutaneous administration of the mammary adenocarcinoma known as Ehrlich tumor induces pain in mice. Several studies have shown that the flavonoid quercetin presents important biological effects, including anti-inflammatory, antioxidant, analgesic, and antitumor activity. Therefore, the analgesic effect and mechanisms of quercetin were evaluated in Ehrlich tumor-induced cancer pain in mice. Intraperitoneal (i.p.) treatments with quercetin reduced Ehrlich tumor-induced mechanical and thermal hyperalgesia, but not paw thickness or histological alterations, indicating an analgesic effect without affecting tumor growth. Regarding the analgesic mechanisms of quercetin, it inhibited the production of hyperalgesic cytokines IL-1β and TNFα and decreased neutrophil recruitment (myeloperoxidase activity) and oxidative stress. Naloxone (opioid receptor antagonist) inhibited quercetin analgesia without interfering with neutrophil recruitment, cytokine production, and oxidative stress. Importantly, cotreatment with morphine and quercetin at doses that were ineffective as single treatment reduced the nociceptive responses. Concluding, quercetin reduces the Ehrlich tumor-induced cancer pain by reducing the production of hyperalgesic cytokines, neutrophil recruitment, and oxidative stress as well as by activating an opioid-dependent analgesic pathway and potentiation of morphine analgesia. Thus, quercetin treatment seems a suitable therapeutic approach for cancer pain that merits further investigation.

Figure 1

Acute treatment with quercetin inhibits Ehrlich tumor-induced pain-like behavior in mice. Mice received the intraplantar (i.pl.) administration of Ehrlich tumor cells 1 × 10⁶ (a–c), and in the 8th day after injection, the tumor cells mice received the acute treatment with quercetin (10, 30, and 100 mg/kg i.p.). Mechanical hyperalgesia (a), thermal hyperalgesia (b), and paw thickness (c) were accessed at 1, 3, 5, and 7 hours after the treatment. Data are presented as means ± SEM of six mice per group per experiment and are representative of two separated experiments: ^∗ p < 0.05 compared to the saline group and ^# p < 0.05 compared to the tumor group. One-way ANOVA followed by Tukey's test.

Figure 2

The chronic treatment with quercetin inhibits in a dose-dependent manner Ehrlich tumor-induced pain-like behavior in mice. Mice received the intraplantar (i.pl.) administration of Ehrlich tumor cells (1 × 10⁶ (a–c) or 1 × 10⁷ (d)) and were treated daily with quercetin (10, 30, and 100 mg/kg i.p.) during 12 days (a–c) or 8 days (d) starting 10 min after tumor injection. The control group of Ehrlich tumor vehicle was saline and saline plus quercetin group was a control of possible per se effects of quercetin. Mechanical hyperalgesia (a), thermal hyperalgesia (b), paw thickness (c), and overt pain-like behavior (d) were evaluated 3 h after the treatment. Data are presented as means ± SEM of six mice per group per experiment and representative of two separated experiments: ^∗ p < 0.05  compared to the saline group and ^# p < 0.05 compared to the tumor group. One-way ANOVA followed by Tukey's test.

Figure 3

Quercetin does not alter Ehrlich tumor-induced histological modifications. Mice received saline (25 μL) or Ehrlich tumor cells (1 × 10⁶/25 μL) and were treated i.p. with quercetin (100 mg/kg, 2% DMSO diluted in saline) or vehicle (2% DMSO) 10 min after the i.pl. injection. The treatment continued daily during 12 days. In the 12th day, mice were euthanized and the paw was collected for histological analysis performed with hematoxylin/eosin staining. Panel (a) shows the histology of saline i.pl. plus quercetin vehicle group, (b) saline i.pl. plus quercetin (100 mg/kg i.p.), (c and e) tumor animal treated with vehicle, and (d and f) tumor animal treated with quercetin (100 mg/kg i.p.). Arrows indicate intact bone cartilage, presence of skeletal muscle fibers, dermis and epidermis: (a-b) bone/cartilage destruction (c-d), tissue necrosis (e-f), and presence of tumor cells (c–f).

Figure 4

Quercetin inhibits neutrophil recruitment induced by Ehrlich tumor cells. Mice were treated i.p. with quercetin (100 mg/kg, 2% DMSO diluted in saline) or vehicle (2% DMSO) 10 min after the injection of Ehrlich tumor cell (1 × 10⁶/25 μL) or saline (25 μL). The neutrophil recruitment was evaluated in samples of paw skin collected after 12 days of treatment using the myeloperoxidase (MPO) activity assay. Data are presented as means ± SEM of six mice per group per experiment and representative of two separated experiments: ^∗ p < 0.05 compared to the saline group and ^# p < 0.05 compared to the tumor group. One-way ANOVA followed by Tukey's test.

Figure 5

Quercetin inhibits IL-1β and TNFα production induced by Ehrlich tumor cells in the spinal cord and paw skin. Mice were treated i.p. with quercetin (100 mg/kg, 2% DMSO diluted in saline) or vehicle (2% DMSO) 10 min after the injection of Ehrlich tumor cell (1 × 10⁶/25 μL) or saline (25 μL). The treatment continued daily. In the 12th day after injection, the Ehrlich tumor cells, the spinal cord, and the paw skin samples were collected for cytokine measurement. IL-1β in spinal cord (a) or paw skin (b) and TNFα in spinal cord (c) or paw skin (d) were determined by ELISA. Data are presented as means ± SEM of six mice per group per experiment and representative of two separated experiments: ^∗ p < 0.05 compared to the saline group and ^# p < 0.05 compared to the tumor group. One-way ANOVA followed by Tukey's test.

Figure 6

Quercetin inhibits the oxidative stress induced by Ehrlich tumor cells. Mice were treated with quercetin (100 mg/kg, i.p.) or vehicle 10 min after the injection of Ehrlich tumor cells (1 × 10⁶/25 μL) or saline. The treatment continued daily during 12 days, and, in the 12th day, 3 h after the treatment, samples of spinal cord and paw skin were collected for the oxidative stress assays. The FRAP and ABTS ability of spinal cord ((a) and (c), resp.) and paw skin ((b) and (d), resp.) tissues and GSH levels in spinal cord (e) and paw skin (f) were accessed. Data are presented as means ± SEM of six mice per group per experiment and representative of two separated experiments: ^∗ p < 0.05 compared to the saline group and ^# p < 0.05 compared to the tumor group. One-way ANOVA followed by Tukey's test.

Figure 7

The opioid receptor antagonist, naloxone, inhibits quercetin analgesia in the Ehrlich tumor-induced pain model. Mice were treated with quercetin (100 mg/kg, i.p., starting 10 min after tumor administration) during 8 days after the injection of Ehrlich tumor cells (1 × 10⁶ or 1 × 10⁷ cells/25 μL) or saline and, in the 8th day, one group of mice that received quercetin was also treated with naloxone (1 mg/kg i.p. diluted in saline) 1 h before the treatment with quercetin. The evaluation of mechanical hyperalgesia (a), thermal hyperalgesia (b), and paw thickness (c) was performed 1, 3, 5, and 7 h after the treatments, and the overt pain-like behavior (d) was evaluated 1 h after the treatment. Data are presented as means ± SEM of six mice per group per experiment and representative of two separated experiments: ^∗ p < 0.05 compared to the saline group, ^# p < 0.05 compared to the tumor group, and ^∗∗ p < 0.05 compared to the quercetin group. One-way ANOVA followed by Tukey's test.

Figure 8

Naloxone did not reverse the effect of quercetin in reducing Ehrlich tumor cells-induced neutrophil recruitment. Mice were treated with quercetin (100 mg/kg, i.p., starting 10 min after tumor administration) during 8 days after the injection of Ehrlich tumor cells (1 × 10⁶ or 1 × 10⁷ cells/25 μL) or saline and, in the 8th day, one group of mice that received quercetin was also treated with naloxone (1 mg/kg i.p. diluted in saline) or its vehicle 1 h before the treatment with quercetin. The neutrophil recruitment was evaluated in samples of paw skin collected after 3 h of the treatment with quercetin by the myeloperoxidase (MPO) activity assay. Data are presented as means ± SEM of six mice per group per experiment and representative of two separated experiments: ^∗ p < 0.05 compared to the saline group and ^# p < 0.05 compared to the tumor group. One-way ANOVA followed by Tukey's test.

Figure 9

Naloxone did not reverse the effect of quercetin in reducing Ehrlich tumor cells-induced cytokine production. Mice were treated with quercetin (100 mg/kg, i.p., starting 10 min after tumor administration) during 8 days after the injection of Ehrlich tumor cells (1 × 10⁶ or 1 × 10⁷ cells/25 μL) or saline and, in the 8th day, one group of mice that received quercetin was also treated with naloxone (1 mg/kg i.p. diluted in saline) or its vehicle 1 h before the treatment with quercetin. IL-1β concentration in spinal cord (a) or paw skin (b) and TNFα concentration in spinal cord (c) or paw skin (d) were determined by ELISA 3 h after the treatment with quercetin. Data are presented as means ± SEM of six mice per group per experiment and representative of two separated experiments: ^∗ p < 0.05 compared to the saline group and ^# p < 0.05 compared to the tumor group. One-way ANOVA followed by Tukey's test.

Figure 10

Naloxone did not reverse the effect of quercetin in reducing Ehrlich tumor cells-induced oxidative stress. Mice were treated with quercetin (100 mg/kg, i.p., starting 10 min after tumor administration) during 8 days after the injection of Ehrlich tumor cells (1 × 10⁶ or 1 × 10⁷ cells/25 μL) or saline and, in the 8th day, one group of mice that received quercetin was also treated with naloxone (1 mg/kg i.p. diluted in saline) or its vehicle 1 h before the treatment with quercetin. Three hours after the treatment with quercetin, samples of spinal cord and paw skin were collected for the oxidative stress assays. The FRAP and ABTS ability of spinal cord ((a) and (c), resp.) and paw skin ((b) and (d), resp.) tissues and GSH levels in the spinal cord (e) and paw skin (f) were accessed. Data are presented as means ± SEM of six mice per group per experiment and representative of two separated experiments: ^∗ p < 0.05 compared to the saline group and ^# p < 0.05 compared to the tumor group. One-way ANOVA followed by Tukey's test.

Figure 11

Combined treatment with quercetin and morphine at doses that are ineffective as single treatment reduces Ehrlich tumor-induced pain-like responses. Mice were treated with quercetin (10 mg/kg i.p., a dose without significant analgesic effect per se), before the injection of Ehrlich tumor cells (1 × 10⁶ or 1 × 10⁷ cells, i.pl.). Mice were treated daily during 8 days and, in the 8th day, mice were treated with quercetin and after 2 h and 15 min received morphine (1 mg/kg i.p., a dose without significant analgesic effect per se). Mechanical (a) and thermal hyperalgesia (b), paw thickness (c), and overt pain-like behavior (d) were evaluated 3 h after the last quercetin treatment. Data are presented as means ± SEM of six mice per group per experiment and representative of two separated experiments: ^∗ p < 0.05 compared to the saline group, ^# p < 0.05 compared to the tumor group, and ^∗∗ p < 0.05 compared to the quercetin 10 mg/kg and morphine 1 mg/kg. One-way ANOVA followed by Tukey's test.