In Vitro and In Vivo Inhibitory Effects of α-Mangostin on Cholangiocarcinoma Cells and Allografts

Abstract

We investigated the anti-cholangiocarcinoma effect of α-mangostin from Garcinia mangostana pericarp extract (GM) in a human cholangiocarcinoma (CCA) cell line and a hamster CCA allograft model. In vitro, human CCA cells were treated with GM at various concentrations and for different time periods; then cell-cycle arrest and apoptosis were evaluated using flow cytometry, and metastatic potential with wound healing assays. In vivo, hamster allografts were treated with GM, gemcitabine (positive control) and a placebo (negative control) for 1 month; tumor weight and volume were then determined. Histopathological features and immunostaining (CK19 and PCNA) characteristics were examined by microscopy. The present study found that α-mangostin could: inhibit CCA cell proliferation by inducing apoptosis through the mitochondrial pathway; induce G1 cell-cycle arrest; and inhibit metastasis. Moreover, α-mangostin could inhibit CCA growth, i.e. reduce tumor mass (weight and size) and alter CCA pathology, as evidenced by reduced positive staining for CK19 and PCNA. The present study thus suggested that α-mangostin is a promising anti-CCA compound whose ready availability in tropical countries might indicate use for prevention and treatment of CCA.

Keywords: Inhibitory effect; cholangiocarcinoma; α-mangostin.

Figure 1

Crude Extracts of Garcinia mangostana Hull

Figure 2

Thin-Layer Chromatogram of G. mangostana Pericarp. Standard α-mangostin (lane 1), G. mangostana pericarp extracts with methanol (lane 2) and crude G. mangostana pericarp extracts (lane 3). Stationary phase: siliga aluminium plate. Mobile phase: dichloromethane: ethyl acetate: methanol; (16:2:1). Detector: 2% w/v vanilline and 10% v/v sulphuric acids heated at 60°C for 5 minute.

Figure 3

The Percentage of White Blood Cells Viability in Control and Treatment Groups (α-Mangostin 0.5, 1.5, 4, 30, 60 µg/mi) are no Significantly Different Compared with Control Group (p>0.05)

Figure 4

The Cytotoxic Effects of α-Mangostin (0.5, 1, 1.5, 2, 2.5 µg/ml) on M214- Human CCA Cell Lines (A) and cytotoxic effects of α-mangostin on M214- human CCA cell lines of each time points (B) resulted in a dose and time dependent. Morphological change post treatment (C). *p<0.05 compared to control, red arrow; indicated apoptotic cell

Figure 5

Wound Healing Assay of the M214- Human CCA Cell Lines Showed the Inhibition of Cell Migration in Response to Treatment 0.5, 1, 1.5 µg/ml of α-Mangostin at 18 and 24 h. The Results are Expressed as mean±SD are Significantly Different Compared with Control Group (at 24 h *p< 0.05, **p<0.001)

Figure 6

Analysis of Cell Cycle Phases, DNA Content Histogram Showing Distribution of M214 Cells at Various Phases of Cell Cycle after Treatment with α-Mangostin (A, B). The results are expressed as mean±SD are significantly different compared with control group (*p<0.05)

Figure 7

Apoptosis Induction was Increased in KKU-M214 Cells Compare with Control. Flow Cytometry of Phosphatidylserine Exposure for M214-CCA Cells. CCA cell were exposed DMSO (Control), α-mangostin (1, 1.5, 2, 4, 8 µg/ml) and gemcitabine 10 µM for 48 h (A). The experiment was performed in three independent experiments and results are expressed as mean±SD. The percentage of apoptotic cells at 48 h are indicated (B). KKU-M214 cells express caspase3, P53, Bax and Bcl-2 after treated with various concentration of α-mangostin at 24 and 48 h (C). density of the band was normalized with internal actins (D), *,**p<0.05 compared to control.

Figure 8

The Gross Anatomy of Tumor Tissue the Groups of Control, Administered with Gemcitabine 20 mg/kg Intraperitoneal Injection and Administered with G. Mangostana 100 mg/kg per Orally (A). Tumor volume (B) and tumor weight (C) were significantly decreased compare to control group (*; p<0.001)

Figure 9

In Vivo Anti-Tumor Effect on Tumor Allograft Model for 21 Days of Treatment. Garcinia mangostana extracts 100 mg/kg/day oral daily (Figure 9 C, F and I) and gemcitabine 20 mg/kg per intraperitoneal (Figure 9 B, E and H) for 3 times/ week. drinking water was given to control group as the same volume (Figure 9 A, D and G). Tumor tissues were stained by hematoxylin and eosin (Figure 9 A-C), PCNA (Figure 9 D-F) and CK19 (Figure 9 G-I)