Tetramethylpyrazine-Rhein Derivative inhibits the migration of canine inflammatory mammary carcinoma cells by mitochondrial damage-mediated apoptosis and cadherins downregulation

Abstract

Background: Canine inflammatory mammary carcinoma (CIMC) has a high incidence of metastasis, high lethality, and poor prognosis, which needs novel adjuvant agents. Tetramethylpyrazine-Rhein Derivative (TRD) has been shown to have antitumor activity, which is a potential research direction for CIMC.

Purpose: This study evaluated the efficacy of TRD on CIMC in vitro and in vivo, and provided possibilities for the application of active compounds in traditional Chinese medicine.

Methods: In vitro, TRD cytotoxicity was measured with CCK-8. Flow cytometry and transmission electron microscope were used to detect the cell cycle, cell death, and changes in mitochondria. Wound-healing assay, cell invasion assay, and scanning electron microscope were used to evaluate the suppression of cell migration and invasion. Expression changes were detected by RT-qPCR and western blot assay. In vivo, the lung metastasis models were randomly divided into control, low-dose TRD, high-dose TRD, and positive groups. Each group was administered orally once a day for 18 days and took in vivo imaging photos.

Results: The IC₅₀ of TRD in CHMp and MDCK were 42.59 and 79.37 μM, respectively. TRD mediated cell apoptosis by mitochondrial damage and caused S and G₂/M phase arrest by downregulating cyclin B1. Moreover, TRD reduced filopodia and inhibited cell migration by downregulating cadherins. In CIMC lung metastasis models, TRD could effectively inhibit tumor growth (P < 0.001) in the lungs without significant toxicity.

Conclusion: TRD showed potential activity to inhibit CIMC lung metastasis with multi-target and low toxicity.

Keywords: Cadherin; Canine inflammatory mammary carcinoma; MAPK; TGF-β1/smad; Tetramethylpyrazine-Rhein Derivative.