Characterized mechanism of α-mangostin-induced cell death: Caspase-independent apoptosis with release of endonuclease-G from mitochondria and increased miR-143 expression in human colorectal cancer DLD-1 cells

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Abstract

α-Mangostin, a xanthone from the pericarps of mangosteen (Garcinia mangostana Linn.), was evaluated for in vitro cytotoxicity against human colon cancer DLD-1 cells. The number of viable cells was consistently decreased by the treatment with α-mangostin at more than 20 μM. The cytotoxic effect of 20 μM α-mangostin was found to be mainly due to apoptosis, as indicated by morphological findings. Western blotting, the results of an apoptosis inhibition assay using caspase inhibitors, and the examination of caspase activity did not demonstrate the activation of any of the caspases tested. However, endonuclease-G released from mitochondria with the decreased mitochondrial membrane potential was shown. The levels of phospho-Erk1/2 were increased in the early phase until 1 h after the start of treatment and thereafter decreased, and increased again in the late phase. On the other hand, the level of phospho-Akt was sharply reduced with the process of apoptosis after 6 h of treatment. Interestingly, the level of microRNA-143, which negatively regulates Erk5 at translation, gradually increased until 24 h following the start of treatment. We also examined the synergistic growth suppression in DLD-1 cells by the combined treatment of the cells with α-mangostin and 5-FU which is one of the most effective chemotherapeutic agents for colorectal adenocarcinoma. The co-treatment with α-mangostin and 5-FU, both at 2.5 μM, augmented growth inhibition compared with the treatment with 5 μM of α-mangostin or 5 μM 5-FU alone. These findings indicate unique mechanisms of α-mangostin-induced apoptosis and its action as an effective chemosensitizer.

Introduction

The pericarps of mangosteen, Garcinia mangostana Linn, have been used as traditional medicine for treatment of skin infection and wounds in Southeast Asia for many years. Based on a previous study, we reported a potent cytotoxic activity of six xanthones from the pericarps of mangosteen against human leukemia HL60 cells and among them, α-mangostin-induced mitochondrial dysfunction.1 Moreover, we reported that α-mangostin-induced cell cycle arrest and apoptosis which was determined by morphological findings and DNA ladder formation in human colon cancer DLD-1 cells.2

Many serine/threonine protein kinases control cell growth, proliferation, differentiation, the cell cycle, survival, and death. Mitogen-activated protein kinases (MAPKs) and Akt are key regulatory proteins in cells. MAPKs are a widely conserved family of serine/threonine protein kinases involved in many cellular programs such as cell proliferation, differentiation, motility, and death.3 Akt, another serine/threonine protein kinase, is associated with cell survival, growth, and glycogen metabolism.4 Various phytochemicals, including epigallocatechin-3-gallate,5 resveratrol,6 arucanolide7, etc., have been shown to modulate the signaling pathways of MAPKs and/or Akt, leading to growth inhibition and cell death. MicroRNAs (miRNAs) are recently discovered short regulatory RNA molecules representing a new layer in post-transcriptional regulation of gene expression. Although more than 450 human miRNAs have been identified, only a very few of them have been characterized in detail. A large amount of miR-143 is expressed specifically in normal colon cells; however, its expression in human colon cancer tumors is markedly decreased.8, 9 We previously determined its target mRNA to be ERK5 by introducing miRNA-143 into DLD-1 cells.8

Here, using human colon cancer DLD-1 cells we show that 20 μM α-mangostin-induced caspase-independent apoptosis mediated by endonuclease-G released from mitochondria. The signaling pathways including Erk1/2, Erk5, and Akt were demonstrated to contribute to this α-mangostin-induced apoptosis. We also discovered synergistic growth suppression of these cells by the combined treatment with α-mangostin and 5-FU at certain concentrations. These findings suggest the potential therapeutic applicability of the phytochemical α-mangostin, especially when employed with 5-FU.

Section snippets

Cytotoxicity of α-mangostin in human colon cancer DLD-1 cells

We examined α-mangostin at various concentrations for its cytotoxic effect on DLD-1 cells, as judged from the results of the Trypan blue-exclusion test (Fig. 1a). The growth of DLD-1 cells was suppressed by the treatment with α-mangostin at the concentration of more than 2 μM, and the concentration for 50% inhibition (IC50) was 7.5 ± 0.3 μM. Apoptotic cell death was observed at more than 15 μM. At 20 μM, the viable cell number was consistently decreased (Fig. 1b) during the treatment; and typical

Discussion

The cytotoxic activity of α-mangostin has been shown to result from cell cycle arrest at G1/S and the subsequent apoptosis to be mediated by the intrinsic pathway in DLD-1 cells.2 In this study, we intended to disclose the mechanism of α-mangostin-induced apoptosis in DLD-1 cells.2 We previously reported that α-mangostin-induced apoptosis was mediated by the caspase-3 pathway in HL60 leukemia cells.14 However, our present results with DLD-1 cells indicate that α-mangostin-induced apoptosis was

Conclusion

The present study showed that the cytotoxic effect of 20 μM α-mangostin was found to be mainly due to apoptosis which was found to be caspase-independent. However, endonuclease-G released from mitochondria with the decreased mitochondrial membrane potential was shown. At the same time, the level of phospho-Akt was sharply reduced with the process of apoptosis after 6 h of treatment. Interestingly, the level of microRNA-143, which negatively regulates Erk5 at translation, gradually increased until

Agents

α-Mangostin was purified to greater than 98% as described in our previous studies.1, 2 5-FU, a commonly used anti-cancer drug for the treatment of colorectal adenocarcinoma, was purchased from a commercial source (Sigma–Aldrich Co., MO). Both were dissolved in DMSO at the concentration of 20 mM and further diluted to the desired working concentration before use.

Cell culture, morphological study, and cell viability

Human colon cancer DLD-1 cells were grown in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Sigma,

Acknowledgment

This work was supported in part by a grant from the program Grant-in-Aid for Young Scientists (B) of the Japan Society for the Promotion of Science.

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