Elsevier

Phytomedicine

Volume 19, Issue 11, 15 August 2012, Pages 1007-1015
Phytomedicine

Involvement of NF-κB and Bcl2/Bax signaling pathways in the apoptosis of MCF7 cells induced by a xanthone compound Pyranocycloartobiloxanthone A

https://doi.org/10.1016/j.phymed.2012.05.012Get rights and content

Abstract

The plant Artocarpus obtusus is a tropical plant that belongs to the family Moraceae. In the present study a xanthone compound Pyranocycloartobiloxanthone A (PA) was isolated from this plant and the apoptosis mechanism was investigated. PA induced cytotoxicity was observed using MTT assay. High content screening (HCS) was used to observe the nuclear condensation, cell permeability, mitochondrial membrane potential (MMP) and cytochrome c release. Reactive oxygen species formation was investigated on treated cells by using fluorescent analysis. Human apoptosis proteome profiler assays were performed to investigate the mechanism of cell death. In addition mRNA levels of Bax and Bcl2 were also checked using RT-PCR. Caspase 3/7, 8 and 9 were measured for their induction while treatment. The involvement of NF-κB was analyzed using HCS assay. The results showed that PA possesses the characteristics of selectively inducing cell death of tumor cells as no inhibition was observed in non-tumorigenic cells even at 30 μg/ml. Treatment of MCF7 cells with PA induced apoptosis with cell death-transducing signals, that regulate the MMP by down-regulation of Bcl2 and up-regulation of Bax, triggering the cytochrome c release from mitochondria to cytosol. The release of cytochrome c triggered the activation of caspases-9, then activates downstream executioner caspase-3/7 and consequently cleaved specific substrates leading to apoptotic changes. This form of apoptosis was found closely associated with the extrinsic pathway caspase (caspase-8) and inhibition of translocation of NF-κB from cytoplasm to nucleus. The results demonstrated that PA induced apoptosis of MCF7 cells through NF-κB and Bcl2/Bax signaling pathways with the involvement of caspases.

Introduction

Traditional medicinal herbs are widely known to be effective in the treatment of many diseases, particularly those that could not be cured by modern medicine. In case of cancer, phytochemicals from these herbs has been proven to decrease the risk of cancer and increase the survival of patients (Issa et al., 2006, Yoon et al., 2011). Several phytochemicals from the nature have exhibited significant anticancer as well as apoptosis effects by targeting various molecular and cellular mechanisms towards breast cancer (Awad et al., 2007, Choi and Kim, 2008).

Apoptosis is a vital physiological process essential for normal development and maintenance of tissue homeostasis (Desagher and Martinou 2000). This mode of cell death is widely studied, since the importance of regulation of apoptosis contributes to the key factor in the anticancer drug development. Among the various targets for cancer research, reactive oxygen species (ROS) is considered as an important one for anticancer drug research, since accumulation of excessive ROS will leads to oxidative DNA damage (Evans et al., 2004, Schumacker, 2006) followed by disruption of the mitochondrial membrane potential (MMP) and release of cytochrome c into the cytosol, to triggers caspase-9 activation and initiates the executioner caspases which leads cell to apoptosis (Simon et al. 2000). In addition, the susceptibility of tumor cells to the induction of apoptosis by chemotherapeutic agents is controlled by the ratio of Bcl2/Bax proteins in the mitochondria (Mohan et al. 2010).

Subsequent to Bcl2 family proteins, heat shock proteins (HSP) also considered as promote tumorigenesis (Jaattela 1999). HSPs are also known to protect cells from stress by preventing the protein aggregation and promote the refolding of denatured proteins (Bukau and Horwich 1998). Increased expression of HSP70 has been reported in high-grade malignant tumors (Knorr et al., 2010, Yang et al., 2011). As HSPs have the ability to prevent the drug induced apoptosis, inhibitors to HSP could be a target of right drug candidate identification. Not only HSPs, but nuclear factor-kappa B (NF-κB), a ubiquitous transcription factor also plays an important role in governing apoptosis and inflammation (Su et al. 1999).

The plant Artocarpus obtusus is tropical plant belongs to the family Moraceae. Recently Hashim et al. have reported that a xanthone compound Pyranocycloartobiloxanthone A (PA) (Fig. 1) exert antiproliferative activity and apoptotic mode of cell death in MCF7 cells (Hashim et al. 2012). Now, we have further found that PA activates a complex signaling pathway required for cell death induction. In particular, an early downregulation of Bcl2, upregulation of Bax, release of cytochrome c from mitochondria into cytosol and the sequential activation of caspases were found to occur in PA-induced apoptosis. The production of ROS also was present in the cells after treatment. In addition, treatment with PA resulted in significant inhibition of NF-κB translocation from cytoplasm to nuclei activated by tumor necrosis factor alpha (TNF-α).

Section snippets

Plant materials

The stem bark of Artocarpus obtusus was collected from Sarawak, identified by Dr. Rusea Go, and a voucher specimen (S94402) has been deposited at the Herbarium, Department of Biology, Faculty of Science, Universiti Putra Malaysia.

Extraction and isolation of Pyranocycloartobiloxanthone A (PA)

Pyranocycloartobiloxanthone A as yellow needle-shaped crystals was purified from the dried and ground stem bark in our lab. Their chemical and physical data as obtained in our work were in agreement with those reported previously (Hashim et al. 2010).

Cell viability assay

All cells that are

PA inhibited the growth of MCF7 cells selectively in vitro

The cytotoxic effects of PA on MCF7 cells were assessed using the MTT assay. As shown in Table 1, PA inhibited the growth of MCF7 cells and exhibited significant inhibition at concentrations of 3.5 ± 0.50 and 2.4 ± 0.21 μg/ml at 24 and 48 h respectively. Meanwhile, the normal cells (MCF10A and WRL 68) used in this study did not died significantly even at the highest concentrations (30 μg/ml) of PA.

PA-induced apoptosis in MCF7 cells

To confirm the presence of apoptosis, we examined nuclear morphological changes of MCF7 cells by

Discussion

Most approaches used in cancer treatment, such as chemotherapy and radiation therapy are likely to be affected by the apoptotic properties of cells; therefore, it has apparent therapeutic implications. Apoptosis is associated with many biochemical changes in the cells, which includes nuclear fragmentation, mitochondrial potential change, regulations in caspases, etc. (Hunter et al. 2007). In the present study, we analyzed the in vitro effect of PA against MCF7 cell line, and release of

Conflict of interest

No conflict of interest.

Acknowledgement

The authors would like to express their utmost gratitude and appreciation to University of Malaya and Ministry of Higher Education (HIR grant F00009-21001) for providing grant to conduct this study.

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