Roles of tumor necrosis factor-α and interleukin-6 in regulating bone cancer pain via TRPA1 signal pathway and beneficial effects of inhibition of neuro-inflammation and TRPA1

Abstract

Background: Pain is one of the most common and distressing symptoms suffered by patients with progression of bone cancer; however, the mechanisms responsible for hyperalgesia are not well understood. The purpose of our current study was to determine contributions of the sensory signaling pathways of inflammatory tumor necrosis factor-α and interleukin-6 and downstream transient receptor potential ankyrin 1 (TRPA1) to neuropathic pain induced by bone cancer. We further determined whether influencing these pathways can improve bone cancer pain.

Methods: Breast sarcocarcinoma Walker 256 cells were implanted into the tibia bone cavity of rats to induce mechanical and thermal hyperalgesia. ELISA and western blot analysis were used to examine (1) the levels of tumor necrosis factor-α and interleukin-6 in dorsal root ganglion and (2) protein expression of tumor necrosis factor-α and interleukin-6 receptors (TNFR1 and IL-6R) and TRPA1 as well as intracellular signals (p38-MAPK and JNK).

Results: Tumor necrosis factor-α and interleukin-6 were elevated in the dorsal root ganglion of bone cancer rats, and expression of TNFR1, IL-6R, and TRPA1 was upregulated. In addition, inhibition of TNFR1 and IL-6R alleviated mechanical and thermal hyperalgesia in bone cancer rats, accompanied with downregulated TRPA1 and p38-MAPK and JNK.

Conclusions: We revealed specific signaling pathways leading to neuropathic pain during the development of bone cancer, including tumor necrosis factor-α-TRPA1 and interleukin-6-TRPA1 signal pathways. Overall, our data suggest that blocking these signals is beneficial to alleviate bone cancer pain.

Keywords: Bone cancer; TRPA1; cytokines; mechanical hyperalgesia; thermal hyperalgesia.

Figure 1.

(a) Mechanical and thermal sensitivity in control rats and bone cancer rats. With the development of bone cancer, PWT and PWL were decreased in cancer rats as compared with control rats. Significant mechanical and thermal hyperalgesia appeared one week after inoculation of cancer cells (*P < 0.05 vs. control rats, n = 12 in each group). (b) The levels of TNF-α and IL-6 in the DRG were amplified in bone cancer rats after two weeks of inoculation of cancer cells as compared with control rats (P < 0.05, cancer rats vs. control rats, n = 15 in each group). (c) Bone cancer also increased protein expression of TNFR1, IL-6R, and TRPA1 as compared with control rats (P < 0.05, cancer rats vs. control rats, n = 6–8 in each group). Top panel is typical bands and bottom panels are averaged data. IL-6: interleukin-6; IL-6R: IL-6 receptor; TNF-α: tumor necrosis factor-α; TNFR1: TNF-α receptor; TRPA1: transient receptor potential ankyrin 1.

Figure 2. Effects of blocking TNF-α on mechanical and thermal sensitivity. TNF-α was inhibited by PTX (10, 20, and 40 mg/kg body weight; i.p. each day over three consecutive days). PWT and PWL were smaller in bone cancer rats without treatment. As PTX was given, PWT and PWL were increased in a dose-dependent way. ^#P < 0.05 versus control rats. *P < 0.05 versus no treatment and other dosages. **P < 0.05, indicated as the same dose of PTX among different days. The number of animals is 8 in control rats and 12 in bone cancer rats without treatment. The number of bone cancer rats with injection of PTX is 8 (10 mg/kg), 6 (20 mg/kg), and 9 (40 mg/kg). PTX: pentoxifylline.

Figure 3.

Effects of blocking IL-6 signal on mechanical and thermal sensitivity. IL-6R was inhibited by SC144 (5, 10, and 20 mg/kg body weight; i.p. each day over three consecutive days). PWT and PWL were smaller in bone cancer rats without treatment than in control rats. SC144 increased PWT and PWL in bone cancer rats as compared with no treatment. The effects of SC144 appeared in a dose-dependent way. ^#P < 0.05 versus control rats. *P < 0.05 versus no treatment and other dosages. **P < 0.05, indicated as the same dose of SC144 among different days. The number of animals is 10 in control rats and 12 in bone cancer rats without treatment. The number of bone cancer rats with injection of SC144 is 6 (5 mg/kg), 8 (10 mg/kg), and 8 (20 mg/kg).

Figure 4.

Effects of blocking TRPA1 on mechanical and thermal sensitivity. TRPA1 was blocked by administration of HC030031 (1, 3, and 10 mg/kg body weight; i.p. each day over three consecutive days). PWT and PWL decreased in bone cancer rats with no treatment. Injection of HC030031 increased PWT and PWL in bone cancer rats as compared to the group with no treatment. ^#P < 0.05 versus control rats. *P < 0.05 versus no treatment and other dosages. **P < 0.05, indicated as the same dose of HC030031 among different days. The number of animals is 12 in each group for control rats and for bone cancer rats without treatment. The number of bone cancer rats with injection of HC030031 is 8 in each group for three dosages (1 mg/kg, 3 mg/kg, and 10 mg/kg).

Figure 5.

The effects of TNF-α and IL-6 inhibition on signal pathways leading to neuropathic pain. PTX (40 mg/kg body weight, i.p. each day for three consecutive days) was given to inhibit TNF-α. SC144 (20 mg/kg body weight, i.p. each day for three consecutive days) was given to inhibit IL-6 signal pathways. Three days after the beginning of respective injection of PTX and SC144, DRG tissues were removed for examination of TRPA1 and p38-MAPK and p-JNK signal pathways. (a)Averaged data and typical bands, without treatment bone cancer increased TRPA1 as well as intracellular signal p-p38-MAPK and p-JNK (phosphorylated form) in the DRG as compared with control rats. Furthermore, administration of PTX attenuated increases of TRPA1 and signal pathways in bone cancer rats. Note that the total protein levels of p38-MAPK and JNK were not elevated significantly by bone cancer. *P < 0.05 versus control rats and bone cancer rats with PTX. n = 6–8 in each group. (b) Averaged data and typical bands showing the effects of SC144. Bone cancer increased the protein levels of TRPA1 and intracellular signal p-p38-MAPK and p-JNK (phosphorylated form) in the DRG as compared with control animals. SC144 attenuated amplification of TRPA1 and these signal pathways in bone cancer rats. Total protein levels of p38-MAPK and JNK were not elevated significantly by bone cancer. *P < 0.05 versus control rats and bone cancer rats with SC144. n = 8–10 in each group. PTX: pentoxifylline; TRPA1: transient receptor potential ankyrin 1.