Apoptosis induced by the sonomechanical effects of low intensity pulsed ultrasound in a human leukemia cell line

Abstract

To obtain an optimal condition for ultrasound (US)-induced apoptosis that could be useful for cancer therapy, we applied low intensity pulsed US to sonicate U937 cells in vitro. Cells were then incubated at different time intervals before measuring apoptosis. The apoptosis was assessed by DNA fragmentation and phosphatidylserine externalization. The pattern of the decrease in mitochondrial membrane potential was determined by flow cytometry. Optimal apoptosis (70.0±13.8%) with minimal lysis was attained with 1 MHz ultrasound 0.3 W/cm², 10% duty factor at 100 Hz for 1 min) at 12 h after sonication. Lack of US-induced free radical detection and absence of Heme oxygenase-1, an intracellular oxidative stress marker, up-regulation in cells, suggest that sonomechanical, not sonochemical, effects are the main mechanism involved.

Introduction

Apoptosis is a programmed cell death necessary in the homeostatic elimination of aged, unwanted or sub-lethally damaged cells. In a living body, it is a bio-energy saving mechanism by which the contents of the cells are being utilized again by other cells especially by the macrophages or other phagocytes. The advantage of apoptosis over the other mode of cell death is implied here, other advantages also include less immune reaction by the body if cells die by apoptosis [1]. In cancer therapy, induction of apoptosis is a preferred mode of killing the cancer cells [2], [3]. Radiation therapy, hyperthermia treatment, and chemotherapy are modalities being used in the treatment of cancer that makes use of the level of apoptosis as an indicator of therapeutic effectiveness, and somehow on the other hand, therapeutic safety. More recently, ultrasound (US) started to emerge as a tool in cancer therapy, not only due to the advances in diagnostic US but also due to its therapeutic potential. It has been shown that US is very effective in generating heat for hyperthermia treatment and high intensity focused ultrasound (HIFU) has been found very effective for tumor ablation [4], [5]. Undoubtedly, US-induced hyperthermia can induce apoptosis, while it has also been shown that non-thermal US can also induce apoptosis [6].

The ability of non-thermal low intensity US to induce apoptosis has been investigated [7], [8], [9], [10], [11], [12], [13]. In our previous study, we have shown that apoptosis can be induced on cancer cells in vitro even at intensities sub-threshold for cell lysis, and initially increases with intensities that can induce cell lysis. At much higher intensities however, cell lysis always predominates over apoptosis. Using such in vitro set up, we also have shown that apoptosis at lower intensities can be enhanced by hyperthermia [7], by hypotonia [9] and by a temperature dependent free radical generator [11]. Despite the enhanced level of apoptosis in those combined treatment, the portion of cells that went apoptotic remained low and were always associated with enhanced cell lysis and necrosis.

In our previous studies, we have hypothesized that the mechanism involved in the bioeffects of low intensity US is mechanical in nature, which we previously termed ‘sonomechanical’ effects [9], and that the degree of membrane damage and how effective cells institute the membrane repair are pivotal in understanding the shift of cell killing from lysis to apoptosis and vice versa. From this concept, we hypothesized that inducing certain degree of membrane damage and allowing the repair mechanism to deal with the damage will promote cell killing that does not instantly lyse the cells, particularly apoptosis. To verify this hypothesis, it is the aim of this present study to search for an ‘ideal condition’ to attain such an optimal apoptosis induction by low intensity pulsed US. Furthermore, roles of free radicals and signal transduction [14] will also be investigated to elucidate the mechanism involved.