Hyperthermotherapy enhances antitumor effect of 5-aminolevulinic acid-mediated sonodynamic therapy with activation of caspase-dependent apoptotic pathway in human glioma

Abstract

Sonodynamic therapy (SDT) has shown great potential as an approach for cancer treatment, and hyperthermotherapy (HT) is also a promising cancer therapy. Here, we investigate whether HT could improve the efficacy of SDT and to make a preliminary exploration on potential mechanism. Xenograft tumor was established in nude mice model, and SNB19 and U87MG glioma cell lines were utilized for in vitro experiment. Alamar blue assay was performed to assess cell viability. Optical microscope was used to characterize the morphology changes of the glioma cells induced by SDT and HT treatments. Apoptotic rate, mitochondrial membrane potential (MMP), and intracellular production of reactive oxygen species (ROS) were examined by flow cytometer. The cell apoptosis of tumor tissues were detected by TUNEL assay. Furthermore, the expression of apoptosis-related proteins was detected with Western blot in vitro and immunohistochemistry in vivo. SDT plus HT group could significantly reduce the cell viability with circular-cell morphological change, compared with SDT group, and cell viability was decreased depending on raise of 5-ALA concentration, ultrasound exposure time, and temperature. The results also indicate that HT increased a conspicuous apoptosis, ROS production, and a remarkable loss in MMP induced by 5-ALA-SDT in vitro. Meanwhile, our data also demonstrated that the combined treatment could significantly induce apoptosis and delay tumor growth in vivo. Furthermore, in both in vitro and in vivo experiments, SDT plus HT group expressed significantly higher protein levels of Bax and cleaved caspase-3, 8, and 9 compared to SDT, HT, and control groups and significantly lower protein level of bcl-2 than the other three groups, while the expression of these proteins was unchanged between HT and control groups. HT may provide an important promotion on 5-ALA-SDT and further propose that SDT in combination with HT is a new potential application for the treatment of human glioma.

Ultrasound Modulates Fluorescence Strength and ABCG2 mRNA Response to Aminolevulinic Acid in Glioma Cells

“…A sharp peak at 636 nm, indicating the PpIX characteristic fluorescence spectra, was detected in SNB19, U87MG and T98G cells in the study (Fig.3). As our previous report 15 showed, there was a time-dependent intracellular accumulation of PpIX in glioma cell lines which were exposed to exogenous 5-ALA. The level of accumulation of PpIX in each cell line is different.…”

“…The relative level of PpIX in the same number of cells at 4 hours after 5-ALA administration was highest in T98G cells. We also previously reported the difference of PpIX accumulation among cell lines 15 . This difference partly implies that the rate of PpIX production induced from exogenous 5-ALA is different.…”

“…Among these, the most hazardous effect on cells is cell lysis, however, the condition used in our experiments, 1MHz, 3W/cm 2 , Duty cycle 10%, is below the threshold for such cell damage. Our previous study showed no potential cell lysis that resulted in complete cellular disruption 15 .…”

“…In this study, the condition of ultrasound irradiation was set as in our previous study 15 . The best condition should be optimized for the best acceleration of fluorescence intensity.…”

Sonodynamic Therapy: Advances and Challenges in Clinical Translation

“…This process helped deprive the tumor of nutrients, favoring its treatment. In a different approach, a combined SDT treatment was administered based on 5‐ALA with hyperthermia induced by 15 minutes in a 42 °C water bath. Sonodynamic therapy exposures consisted of 1 W/cm 2 at 1 MHz during 10 minutes with a duty factor of 20%.…”