Apoptosis induced by sonodynamic treatment by protoporphyrin IX on MDA-MB-231 cells

doi:10.1016/j.ultras.2011.10.013

Ultrasonics

Volume 52, Issue 4, April 2012, Pages 490-496

https://doi.org/10.1016/j.ultras.2011.10.013 Get rights and content

Abstract

Sonodynamic therapy (SDT) is a promising modality for cancer treatment, involving the synergistic interaction of ultrasound and some chemical compounds termed as sono-sensitizers. It has been found that SDT can lead to apoptotic cell death because of the induction of direct sonochemical and subsequent redox reactions. However, the detailed mechanisms are not clear. This study was to identify the cytotoxic effects of ultrasound-activated protoporphyrin IX (PpIX) on MDA-MB-231 cells. The fluorescence microscope was used to detect the sub-cellular localization of PpIX. Several distinct sonochemical effects were found after SDT treatment, including the decrease of cell viability, generation of intracellular ROS, the loss of mitochondrial membrane potential. The activation of some special apoptosis-associated proteins [Caspase-9, Caspase-3 and polypeptide poly (ADP-robose) polymerase] was evaluated by western blotting. The results show that PpIX mediated SDT (PpIX-SDT) treatment could obviously inhibit the proliferation of MDA-MB-231 cells, and which was significantly reduced by the pan-Caspase inhibitor z-VAD-fmk and the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC). Further, SDT induced a conspicuous loss of mitochondrial membrane potential (MMP) and a mass of ROS accumulation in MDA-MB-231 cells at 1 h post-treatment and the SDT-treated cells showed obvious Caspase-3 and Caspase-9 activation, and PARP cleavage at 6 h after treatment. And, the general apoptosis marker-Caspase-3 activation-was also greatly relieved by NAC. These findings primarily indicate a Caspase-depended apoptosis could be induced by PpIX-SDT in MDA-MB-231 cells, and the intracellular ROS was involved during the apoptotic process.

Highlights

► The ultrasonically induced cytotoxicity of MDA-MB-231 cells was significantly enhanced by PpIX. ► Mitochondria may be the main target of SDT because PpIX co-localized well with MTG in MDA-MB-231. ► SDT could induce apoptotic cell death of MDA-MB-231 cells, and which might be Caspase dependent. ► Intracellular ROS was involved in SDT-induced cytotoxic effect in MDA-MB-231 cells.

Introduction

Breast cancer is one of the most fatal cancer types in many countries, especially industrialized countries [1]. Until now, there is no effective cure method. Traditional therapies, such as radiotherapy and chemotherapy, are not the best ways of treatment for breast cancer. Surgical treatment can be the relatively effective method for the sufferer, but with higher recurrence rate, and which will cause great injury for the physical body and beauty, and produce psychological shadow for the patients. The reasons for this situation are the development of drug resistance and severe side effects of these therapies, which are still unresolved problems in clinical oncology [2]. Therefore, the search for novel anti-tumor treatment is extremely urgent.

Sonodynamic therapy (SDT) is a relatively new and promising approach for cancer treatment, firstly proposed by Umemura et al. [3]. Ultrasound has an appropriate tissue attenuation coefficient for penetrating the ability to focus energy into small volumes and activate sonosensitizer, which is tumor-specific accumulation. This makes it possible to damage the pathological site with minimal damage to peripheral healthy tissues [4], indicating SDT has a valuable application for targeted cancer therapy.

The most commonly used sonosensitizers are hematoporphyrin and its derivatives [5]. Protoporphyrin IX (PpIX) is known to be one component of hematoporphyrin derivative. Previous reports have shown that PpIX can accumulate specially in proliferative tumors [6]. PpIX has a high affinity for peripheral benzodiazepine receptor on the outer membrane of mitochondrial [7], and mainly mediates mitochondria stress during ultrasound irradiation [8], [9]. The in vivo studies also show PpIX has a good tumor enriching property and a long retention time in tumors [10], and the in vitro ultrasound activating PpIX can obviously induce immediate sonolysis and delayed cell apoptosis [8], [11]. These studies suggest that PpIX might be a nicer sonosensitizer in SDT treatment.

Recently, SDT focused on the mechanisms of killing effects by using different ultrasonic parameters and different sonosensitizers [5], [12], [13], [14], [15]. A series of theories have been proposed, such as singlet oxygen, alkoxyl radicals, lipid peroxidation (LPO), apoptosis, and so on [16], [17], [18]. However, the mechanism of SDT is influenced by multiple factors, including the nature of the biological model, the sonosensitizer, and the ultrasonic parameters.

Apoptosis induction is arguably the most potent defense against cancer [19]. Many studies have documented the sonodynamically induced apoptosis in a variety of cancer cell types, by an array of different sensitizers. But until now, SDT is still in the experimental study for leukemia and skin cancer treatment, little information concerning of the responses of breast cancer to SDT is known. According to the advantages of SDT mediated anti-cancer therapy, it will fully play its merit for treatment of breast cancer. Therefore, the present study was to investigate the ultrasonically induced cell damaging effect in human breast cancer MDA-MB-231 cells with the presence of 1 μM PpIX, at the ultrasound frequency of 1.1 MHz and an intensity of 1 W/cm². This paper evidenced the apoptotic response of MDA-MB-231 cells to SDT treatment and evaluated some possible mechanism for the action. The findings may shed new light on human breast cancer treatment.

Section snippets

Cell culture

Human breast cancer MDA-MB-231 cells were obtained from the cell bank of the Chinese Academy of Science, Shanghai, China. The cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Life Technologies, Inc., USA) supplemented with 10% fetal bovine serum (FBS, Hyclone, USA), 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C in a humidified atmosphere of 5% CO₂ atmosphere. Cells in the exponential phase of growth were used in each experiment.

Ultrasonic exposure system

The ultrasound exposure set-up was

The intracellular localization of Pp IX

After 2 h incubation of PpIX, cells were co-loaded with mitochondria probe MT-G (Green) and nuclear probe Ho (Blue). As shown in Fig. 1, PpIX fluorescence (Red) corresponded well with MT-G green fluorescence but did not overlap with Ho blue fluorescence, suggesting PpIX diffused in the cytoplasm and mainly accumulated in the mitochondria of MDA-MB-231 cells.

Enhancement of ultrasound induced cell killing by PpIX

Cell survival was assessed by MTT assay. As shown in Fig. 2, the cell survival of 1 μM PpIX alone was 95.24% compared with control, showing

Discussion

Apoptosis is an essential biologic process and is characterized by a number of well-defined features including the activation of Caspase and so on. Apoptosis is a complex mechanism that can be induced and regulated by many signal stimulus pathways. Apoptosis induction is arguably the most potent defense against cancer [23]. Recent investigations have revealed that combination of low-energy ultrasound with sonosensitizers could induce apoptosis in many cancer cells. Most of those studies were

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grant No. 81000999 and No. 10904087), the Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20100202110006) and the Natural Science Foundation of Shaanxi Province, China (Grant No. 2011JQ4012).

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Apoptosis induced by sonodynamic treatment by protoporphyrin IX on MDA-MB-231 cells

Abstract

Highlights

Introduction

Section snippets

Cell culture

Ultrasonic exposure system

The intracellular localization of Pp IX

Enhancement of ultrasound induced cell killing by PpIX

Discussion

Acknowledgments

Clin. Breast Cancer

Ultrasound Med. Biol.

J. Photochem. Photobiol. B, Biol.

J. Biol. Chem.

Ultrasound Med. Biol.

Ultrasonics

Ultrasound Med. Biol.

Ultrason. Sonochem.

Cell Biol. Int.

J. Biol. Chem.

Ultrasound Med. Biol.

J. Pharm. Sci.

Mitochondrion

Ultrasonics

Viability analysis and apoptosis induction of breast cancer cells in a microfluidic device: effect of cytostatic drugs

Biomed. Microdevices