Apoptosis of THP-1 Derived Macrophages Induced by Sonodynamic Therapy Using a New Sonosensitizer Hydroxyl Acetylated Curcumin

Abstract

Curcumin is extracted from the rhizomes of the traditional Chinese herb Curcuma longa. Our previous study indicated curcumin was able to function as a sonosensitizer. Hydroxyl acylated curcumin was synthesized from curcumin to eliminate the unstable hydroxy perssad in our group. The potential use of Hydroxyl acylated curcumin as a sonosensitizer for sonodynamic therapy (SDT) requires further exploration. This study investigated the sonodynamic effect of Hydroxyl acylated curcumin on THP-1 macrophage. THP-1 macrophages were cultured with Hydroxyl acylated curcumin at a concentration of 5.0 μg/mL for 4 hours and then exposed to pulse ultrasound irradiation (0.5 W/cm2 with 1.0 MHz ) for 5 min, 10 min and 15 min. Six hours later, cell viability decreased significantly by CCK-8 assay. After ultrasound irradiation, the ratio of apoptosis and necrosis in SDT group was higher than that in control, Hydroxyl acylated curcumin alone and ultrasound alone. Moreover, the apoptotic rate was higher than necrotic rate with the flow cytometry analysis. Furthermore, Hydroxyl acylated curcumin-SDT induced reactive oxygen species (ROS) generation in THP-1 macrophages immediately after the ultrasound treatment while ROS generation was reduced significantly with the scavenger of singlet oxygen Sodium azide (NaN3). Hydroxyl acylated curcumin-SDT led to a conspicuous loss of mitochondrial membrane potential (MMP) compared with other groups, while MMP was increased significantly with the scavenger of singlet oxygen Sodium azide (NaN3), ROS inhibitor N-acetyl cysteine (NAC) and Mitochondrial Permeability Transition Pore (MPTP) inhibitor Cyclosporin A (CsA). The cytochrome C, cleaved-Caspase-9, cleaved-Caspase-3 and cleaved-PARP upregulated after SDT through Western blotting. These findings suggested that Hydroxyl acylated curcumin under low-intensity ultrasound had sonodynamic effect on THP-1 macrophages via generation of intracellular singlet oxygen and mitochondria-caspase signaling pathway, indicating that Hydroxyl acylated curcumin could be used as a novel sonosensitizer in SDT for atherosclerosis.

The efficacy and mechanism of apoptosis induction by hypericin-mediated sonodynamic therapy in THP-1 macrophages

“…To determine the total protein fraction, after 6 hours of incubation following SDT, the cells were washed twice with PBS and lysed with RIPA buffer 19 on ice for 30 minutes. Then, the cells were harvested and collected via centrifugation at 12,000 rpm for 30 minutes at 4°C.…”

“…[17][18][19] Most sonosensitizers originate from porphyrin-derivative photosensitizers, such as hematoporphyrin (HP), which also causes tumor cell death when irradiated by ultrasound. However, this class of sonosensitizers has several disadvantages, including high costs and high dosage requirements when used for SDT.…”

“…Our group demonstrated that emodin, curcumin, and 5-aminolevolinic acid (ALA) have sonodynamic effects in macrophages when combined with ultrasound by inducing apoptosis in vitro. 1,19,21 The polycyclic dione hypericin (HY), which is one of the active ingredients of St John's wort, is a photosensitizer that accumulates in dark glands found in flowers and leaves. [22][23][24] HY has been proposed as a highly promising photosensitizer due to its minimal toxicity, tumor high selectivity, high clearance from the host body, much singlet oxygen production and anti-inflammatory properties, as HY is able to induce the apoptosis of human lens epithelial cells, nasopharyngeal carcinoma cells, and U87 glioma cells 20,23,[25][26][27][28] during the rapid healing process after PDT.…”

“…The present findings clearly indicate that HY is a more efficient sonosensitizer than those employed previously. 1,19,21 The effects of SDT depend not only on the sonosensitizer concentration and the ultrasound activating time but also on the localization within THP-1 macrophages. Most radical products have very short lifetimes and diffusion ranges; those derived from sonosensitizers determine the localization of the primary damage and apoptotic triggers during the SDT process.…”

“…1,21 A critical step in apoptosis is mitochondrial membrane permeabilization, indicating the loss of mitochondrial membrane integrity. 19 This step results in the release of proteins that are critical for downstream events and constitutes a no return point in cell death. We confirmed that HY primarily accumulated in the mitochondria of THP-1 macrophages.…”