Potassium usnate, a water-soluble usnic acid salt, shows enhanced bioavailability and inhibits invasion and metastasis in colorectal cancer

Abstract

Usnic acid (UA), a lichen secondary substance, has considerable anticancer activity in vitro, whereas its effect in vivo is limited. Here, potassium usnate (KU) was prepared by the salinization of UA to enhance its water solubility. KU showed increased bioavailability compared with UA in the tumor, liver, and plasma of a CT26 syngeneic mouse tumor xenograft model after oral administration, as determined by LC-MS/MS analysis. KU exhibited potent anticancer effects on colorectal cancer cells and inhibited liver metastasis in an orthotopic murine colorectal cancer model. KU treatment downregulated the epithelial-mesenchymal markers Twist, Snail, and Slug and the metastasis-related genes CAPN1, CDC42, CFL1, IGF1, WASF1, and WASL in cells and tumor tissues. The present results suggest the potential application of the water-soluble form of UA, KU, in anticancer therapy.

Figure 1

Usnic acid showed in vitro anticancer activity in colorectal cancer cells. (A) Relative cell viability of HCT116, DLD1, SW480, HT29, SW620, Caco2, CT26, and COLO320 cells treated with usnic acid (UA). (B,C) Invasion assays in Caco2, HCT116, and CT26 cells treated with 5 μM UA (B), and quantification of invaded cell numbers in each group (C). (D) Quantitative analysis of metastasis score in isolated mouse liver tissues from orthotopic liver metastasis models (n = 4 each group). “10x” denote that UA was administered ten times within 2 weeks, while without indication denotes that of six times. Results are reported as the mean ± standard error of the mean.

Figure 2

Usnate distribution in the tumor, liver, and plasma after oral administration of potassium usnate in a CT26 syngeneic mouse tumor xenograft model. (A) Chemical structure of UA and potassium usnate (KU). (B–D) Quantitative LC-MS/MS analysis of usnate in tumor tissues (B), liver tissues (C), and plasma (D) in a CT26 syngeneic mouse tumor xenograft model after oral administration of UA or KU. Results are reported as the mean ± standard error of the mean. ***P < 0.001.

Figure 3

KU shows potent anticancer activity in colorectal cancer cells. (A) Relative viability of HCT116, DLD1, SW480, HT29, SW620, Caco2, CT26, and COLO320 cells treated with KU. (B,C) Invasion assays in Caco2, HCT116, and CT26 cells treated with 5 μM KU (B), and quantification of invaded cell numbers in each group (C). (D) Comparison of IC₅₀ values between UA and KU. (E) Comparison of the relative invasive abilities of Caco2, HCT116, and CT26 cells treated with UA and KU. Results are reported as the mean ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

KU inhibits liver metastasis in an orthotopic murine colorectal cancer model. (A) Representative images of liver tissues isolated from four mice treated with DW or KU. (B) Quantitative analysis of metastasis score in isolated mouse liver tissues from the orthotopic liver metastasis model (n = 4 each group). (C) Hematoxylin and eosin staining and immunohistochemical analysis of phosphor-Histone H3 (pHH3) of isolated liver tissues from the mouse liver metastasis model. Scale bars, 500 μm. (D) Representative images of IVIS luciferase results in mice inoculated with colorectal cancer cells. (E) Quantitative analysis of signals from the IVIS luciferase images. On day 3 after tumor establishment, mice were analyzed by optical bioluminescence imaging at 2, 9, and 16 days after intraperitoneal KU administration (5, 10, and 20 mg/kg/mouse, three times a week). Control groups received DW instead of treatment. The average signal intensity of 20 mg/kg KU-treated mice was weaker than that of control mice (P = 0.105) at 16 days after tumor cell inoculation. (F) Liver function test results of AST and ALT levels in tumor tissue. Results are reported as the mean ± standard error of the mean, n = 4, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

KU downregulates epithelial-mesenchymal transition markers and the expression of genes involved in cell motility. (A) Gene expression of EMT markers in Caco2 cells treated with UA and KU. (B) Protein levels of EMT markers in Caco2 cells treated with UA and KU. (C–E) Immunohistochemical analysis of Twist (C), Snail (D), and Slug (E) in isolated liver tissues from the mouse liver metastasis model administered with 20 mg/kg KU. Scale bar, 50 μm. (F) The levels of apoptosis and EMT markers in isolated liver tissues from the mouse liver metastasis model administered with 20 mg/kg KU (n = 2 each group). (G) mRNA expression levels of cell motility-related genes in Caco2 cells treated with UA and KU. Results are reported as the mean ± standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001.