Cetuximab enhances radiosensitivity of esophageal squamous cell carcinoma cells by G2/M cycle arrest and DNA repair delay through inhibiting p-EGFR and p-ERK.

BACKGROUND: Although radiotherapy has improved local control in esophageal squamous cell carcinoma (ESCC), a considerable number of patients still experience relapse due to resistance. In this study, we aimed to evaluate the effects of cetuximab on radiosensitivity in two ESCC cell lines (ECA109 and TE-13) and to investigate their underlying mechanisms. METHODS: Cells were pretreated with or without cetuximab before irradiation. The MTT assay and clonogenic survival assay were performed to evaluate cell viability and radiosensitivity. Flow cytometry was performed to analyze cell cycle distribution and apoptosis. The γH2AX foci were counted to determine cellular DNA-repairing capacity via immunofluorescence assay. The phosphorylation of key molecules involved in the epidermal growth factor receptor (EGFR) signaling pathway and DNA double-strand break (DSB) repair were measured by western blot. RESULTS: Cetuximab alone was not sufficient to suppress cell viability, but significantly enhanced radiation-induced inhibition of clonogenic survival in ECA109 and TE-13. The radiation sensitivity enhancement ratio (SER) was 1.341 and 1.237 for ECA109 and TE-13, respectively. ESCC cells treated with cetuximab were arrested at the G2/M phase in response to radiation. No significant increase in apoptotic rate was observed in irradiated cells that were treated with cetuximab. The average number of γH2AX foci increased in the combination group (cetuximab and radiation). Cetuximab suppressed phosphorylation of EGFR and downstream ERK, but had no significant effect on AKT. CONCLUSIONS: These results indicate the potential for use of cetuximab as an effective radiosensitizer in ESCC. Cetuximab promotes G2/M cycle arrest and reduces DSB repair, as well as inhibiting EGFR and downstream ERK pathways in ESCC.

Xiaoaiping improves the general state of rats with malignant ascites secondary to gastric cancer by blocking the TGF-ß1 signaling pathway.

OBJECTIVE: To investigate the effect of Xiaoaiping on the general state of rats with malignant ascites secondary to gastric cancer by blocking the transforming growth factor-ß1 (TGF-ß1) signaling pathway. METHODS: Fifty healthy Wistar rats were randomly divided into 5 groups, with 10 rats in each group. After successful modeling, 0.2 g/(20 g∙d), 0.4 g/(20 g∙d) and 0.8 g/(20 g∙d) of Xiaoaiping injections were administrated at the low dose group (LDG), medium dose group (MDG) and high dose group (HDG), respectively. The model group (MG) was injected intraperitoneally with the same amount of sterile normal saline for 8 d. Rats in the control group (CG) were healthy without any treatment. The general states (mental state, dietary habits, reactions and body shape) of rats in each group were observed. The abdominal circumference, platelet (PLT) count, white blood cell (WBC) count, serum ferritin (SF), ascites volume, cell survival rate, and expression levels of TGF-ß1 signaling pathway (TGF-ß1, Smad2) were compared among the groups. RESULTS: PLT and WBC counts in the MG were lower than those in the CG. Ascites volume, tumor cell survival rate, and SF in the MG were higher than those in the LDG, MDG and HDG (P<0.05). Thymus index in the LDG, MDG and HDG were significantly higher than that in the MG (P<0.05). There was no significant difference in the spleen indices among the groups (P>0.05). The kidney index, serum creatinine and urea nitrogen levels in LDG, MDG and HDG were significantly lower than those of MG (P<0.05). The LDG, MDG and HDG exhibited significantly higher peripheral blood CD4+ cells and CD4+/CD8+, and lower CD8+ level, as compared with the MG (P<0.05). The levels of serum interferon γ (IFN-γ), interleukin (IL)-1α, IL-1ß, IL-2, IL-4 and tumor necrosis factor-α (TNF-α) in the LDG, MDG and HDG were higher than those in the MG (P<0.05). The ascites volume and tumor cell survival rate were decreased sequentially in the LDG, MDG and HDG (P<0.05). The MG had higher mRNA levels of TGF-ß1 and Smad2 than the CG (P<0.05). No statistically significant difference was found in TGF-ß1 and Smad2 between the LDG and MG (P>0.05). CONCLUSION: Xiaoaiping could significantly reduce the ascites volume in rats with gastric cancer, inhibit the production of tumor cells in the abdominal cavity, and improve the general state of rats in a dose-dependent manner. The mechanism may be related to the down-regulation of the mRNA level of TGF-ß1 and Smad2.

Activation and expression of µ-calpain in dorsal root contributes to RTX-induced mechanical allodynia.

Background Calpain is a calcium-dependent cysteine protease, and inhibition of calpain by pre-treatment with MDL28170 attenuated the rat mechanical allodynia in a variety of pain models. Postherpetic neuralgia (Shingles) is a neuropathic pain conditioned with the presence of profound mechanical allodynia. Systemic injection of resiniferatoxin can reproduce the clinical symptoms of postherpetic neuralgia. In this study, we determined to study whether activation of calpain contributes to cleave the myelin basic protein of dorsal root and is involved in resiniferatoxin-induced mechanical allodynia of postherpetic neuralgia animal model. Results Resiniferatoxin up-regulated the expression and activation of µ-calpain in dorsal root. The expression of µ-calpain was located in Schwann cell of dorsal root, and resiniferatoxin increased the expression of µ-calpain in Schwann cell in L4-L6 dorsal root at six weeks after injection. Resiniferatoxin also induced myelin basic protein degradation in L4-L6 dorsal root at six weeks after injection. Moreover, intraperitoneal injection of calpain inhibitor MDL28170 prevented the degradation of myelin basic protein and then reduced the sprouting of myelinated afferent fibers into spinal lamina II, thus relieving resiniferatoxin-induced mechanical allodynia. Conclusions Up-regulation and activation of µ-calpain located in Schwann cell may be the mechanism underlying resiniferatoxin-mediated proteolysis of myelin basic protein in dorsal root. Calpain inhibitor MDL28170 prevents resiniferatoxin-induced sprouting of myelinated afferent fibers and mechanical allodynia through inhibition of degradation of the myelin basic protein in dorsal root. Our results indicate that inhibition of pathological µ-calpain activation may present an interesting novel drug target in the treatment of postherpetic neuralgia.

Netrin-1 Contributes to Myelinated Afferent Fiber Sprouting and Neuropathic Pain.

Netrin-1 is a neuronal guidance molecule implicated in the development of spinal cord neurons and cortical neurons. In the adult spinal cord, UNC5H (repulsive receptor of netrin-1), but not deleted in colorectal cancer (DCC) (attractive receptor of netrin-1), constitutes a major mode of netrin-1 signal transduction, which may be involved in axon repulsion and inhibits neurite outgrowth. Abnormal sprouting of myelinated afferent fibers in the spinal dorsal horn can cause mechanical allodynia associated with postherpetic neuralgia (PHN, Shingles) and other neuropathic pains. However, whether netrin-1 participates in sprouting of myelinated afferent fibers and mechanical allodynia remains unknown. In an ultropotent TRPV1 agonist resiniferatoxin (RTX)-induced PHN-like model, RTX treatment for 6 weeks increased netrin-1 expression in dorsal horn neurons, including NK-1-positive projection neurons. In human neuroblastoma SH-SY5Y cells, we found that TRPV1 antagonist capsazepine antagonized RTX-induced upregulation of netrin-1. After RTX treatment, UNC5H2 expression was gradually decreased, whereas DCC expression was significantly increased. Silencing netrin-1 in the spinal dorsal horn significantly attenuated RTX-induced mechanical allodynia and sprouting of myelinated fibers into the spinal lamina II. Our results suggest that RTX treatment upregulates netrin-1 expression through activation of TRPV1 receptors and change UNC5H2-rich spinal dorsal horn into a growth-permissive environment by increasing DCC expression, thus enhancing the sprouting of myelinated afferent nerves. Netrin-1 may be targeted for reducing primary afferent sprouting and mechanical allodynia in PHN and other neuropathic pain conditions.