CGX, a standardized herbal syrup, inhibits colon-liver metastasis by regulating the hepatic microenvironments in a splenic injection mouse model

Abstract

Background: Colon-liver metastasis is observed in approximately 50% of patients with colorectal cancer and is a critical risk factor for a low survival rate. Several clinical studies have reported that colon-liver metastasis is accelerated by pathological hepatic microenvironments such as hepatic steatosis or fibrosis. Chunggan syrup (CGX), a standardized 13-herbal mixture, has been prescribed to patients with chronic liver diseases, including fatty liver, inflammation and fibrotic change, based on preclinical and clinical evidence. Aim of the study: In the present study, we investigated anti-liver metastatic the effects of CGX in a murine colon carcinoma (MC38)-splenic injection mouse model. Materials and methods: C57BL/6N mice were administered with CGX (100, 200 or 400 mg/kg) for 14 days before or after MC38-splenic injection under normal and high-fat diet (HFD) fed conditions. Also, above experiment was repeated without MC38-splenic injection to explore underlying mechanism. Results: The number of tumor nodules and liver weight with tumors were sup-pressed by preadministration of CGX in both normal and HFD fed mice. Regarding its mechanisms, we found that CGX administration significantly activated epithelial-cadherin (E-cadherin), but decreased vascular endothelial-cadherin (VE-cadherin) in hepatic tissues under MC38-free conditions. In addition, CGX administration significantly reduced hepatic steatosis, via modulation of lipolytic and lipogenic molecules, including activated adenosine monophosphate activated protein kinase (AMPK) and peroxisome proliferator activated receptor-alpha (PPARα). Conclusion: The present data indicate that CGX exerts an anti-colon-liver metastatic property via modulation of hepatic lipid related microenvironments.

Keywords: chuggan syrup (CGX); colon-liver metastasis; hepatic microenvironment; hepatic steatosis; herbal medicine.

FIGURE 1

Fingerprinting of CGX and animal experimental designs. Eight reference compounds (A) in CGX (B) were analyzed using HPLC, and semiquantitative analysis was performed using their standard curves (C). The mice (99 mice in total) were used according to four different experimental designs (D).

FIGURE 2

Anti-metastatic effects of pre- and postadministration of CGX. Mice were given pre- (A–C) and (G) or postadministered (D–F) CGX (100, 200 or 400 mg/kg, 14 days) via MC38-splenic injection. After 21 days of MC38-splenic injection (1 × 10⁵/mouse), the numbers of tumor nodules and liver weight were measured. H&E staining was performed in liver tissue and visualized at ×100 magnifications (G–H). The data are expressed as the means ± SD (n = 7). *p < 0.05 or **p < 0.01 indicates statistical significance compared to the control group. T; Tumor nodule.

FIGURE 3

Effects of CGX on lipid components in body and hepatic tissue. Under MC38-free conditions, mice were administered with CGX (100, 200 or 400 mg/kg, 14 days). Body weight (A) was measured once a week, and the three abdominal fats (epididymal, retroperitoneal and visceral fat, (B) and liver (D) were weighed after sacrifice. The hepatic fat composition was measured using a DEXA analyzer and expressed as a relative value compared to the control group (C,E). Hepatic TG and TC were determined using commercial kits (F,G). The data are expressed as the means ± SD (n = 5 or 7). *p < 0.05 or **p < 0.01 indicates statistical significance compared to the control group. DEXA; dual energy X-ray absorptiometry, TC; total cholesterol, TG; triglyceride.

FIGURE 4

Effects of CGX on cell adhesion- and lipid metabolism-related molecules. The protein (A,B) and mRNA levels (C) were determined using western blotting and PCR, and normalized to β-actin, respectively. The data are expressed as the means ± SD (n = 3). *p < 0.05 or **p < 0.01 indicates statistical significance compared to the control group. E-cadherin; epithelial cadherin, VE-cadherin; vascular endothelial cadherin, pAMPK; phospho adenosine monophosphate kinase, PPARα; peroxisome proliferator-activated receptor alpha, DGAT1 and two; diacylglycerol acyltransferase 1 and 2, HMGCR; hydroxy-methyl-glutaryl coenzyme A reductase, LIPE; hormone-sensitive lipase, MGLL; monoglyceride lipase, ACAA2; acetyl-coenzyme A acyltransferase 2.

FIGURE 5

Effects of anti-metastatic effects of CGX in the HFD-induced metastasis model. Beginning 2 weeks after HFD feeding, the mice were orally administered water or CGX (400 mg/kg) for 14 days. A splenic injection of MC38 cells (1 × 10⁵/mouse) was performed on the day of the final administration of CGX. The liver weights and numbers of nodules were evaluated by two researchers who were blinded to group allocations (A–C). Oil red O staining was performed in the liver tissue and visualized at ×200 magnifications (D). Steatosis area was scored according to the nonalcoholic steatohepatitis (NASH) clinical research network scoring system (E). Hepatic TG and TC were determined using common kits (F) and (G). The data are expressed as the means ± SD (n = 5). *p < 0.05 or **p < 0.01 indicates statistical significance compared to each group. T; Tumor nodule.