Loreto B. Feril

Ultrasound activation of TiO2 in melanoma tumors

Sonodynamic therapy (SDT) is a new modality using ultrasound (US) to activate certain chemical sensitizers for cancer therapy. In this study, the effect of US combined with a nanoparticle titanium dioxide (TiO(2)) on melanoma cell was investigated in vitro and in vivo. Melanoma cells (C32) were irradiated with US in the presence and/or absence of TiO(2). Cell viability was measured immediately after US irradiation (1MHz, 0.5 and 1.0W/cm(2) for 10s). The effect of the combination of TiO(2) and US exposure (1MHz, 1.0W/cm(2), 2 min duration) on subcutaneously implanted C32 solid tumors in mice were investigated by measuring tumor volume regression. The cell viability was significantly decreased only after US irradiation in the presence of TiO(2). In vivo results showed significant inhibition of tumor growth in groups treated with TiO(2) and US. To our knowledge, this is the first report to demonstrate the cell killing effect of TiO(2) nanoparticles under the irradiation US in vitro and in vivo.

Evaluation and comparison of three novel microbubbles: enhancement of ultrasound-induced cell death and free radicals production.

Three novel lipid-shell-type microbubbles (MBs), AS-0100, BG6356A and BG6356B, have been evaluated for their impact on ultrasound (US)-induced cell death and free radicals production. Previously studied and well-characterized US exposure conditions were employed in which human myelomonocytic lymphoma U937 cells were exposed to 1MHz pulsed US beam (0.3W/cm(2), 10% duty factor) for 1min with or without MBs. Three different concentrations of each MB were used. Apoptosis and cell lysis were assessed by examining phosphatidylserine externalization and by counting viable cells, respectively, 6h post-exposure. Free radicals production and scavenging activities were evaluated using electron paramagnetic resonance (EPR)-spin trapping. The results showed that only AS-0100 and BG6356A were able to enhance the US-induced apoptosis, mainly by increasing the secondary necrosis. Apoptosis and cell lysis seemed to depend more on mechanical forces exerted by oscillating MBs while free radicals played a trivial role. BG series MBs exhibited pronounced scavenging activities. Generally, despite the need for further optimization, AS-0100 and BG6356A appear to be promising as adjuncts in cases where US-induced cell death is required.

Dose-dependent inhibition of ultrasound-induced cell killing and free radical production by carbon dioxide

Previous studies have shown that if a solution for cell suspension is saturated with CO(2), ultrasound-induced in vitro cell killing and free-radical production are inhibited. However, the dose dependency of this observation has not been explored. Here, we used NaHCO(3) and HCl to produce a predictable concentration of CO(2) within the culture medium. Using 1 MHz continuous wave 4 W/cm(2) ultrasound, we sonicated U937 cells suspension for 1 min at 37 degrees C with CO(2) at different concentrations. At 2 mM, reduced cell killing was observed that further decreased with increasing CO(2) concentration until 100% protection was attained at 20 mM. Ultrasound-induced free-radical production was significantly decreased at 1 mM and became undetectable at 2 mM CO(2). This finding shows that CO(2)-mediated inhibition is concentration dependent and that the threshold for free-radical production is one order of magnitude higher than the threshold for cell killing induced by ultrasound. In addition, it also cautions researchers when adding acids and acid-based agents to a culture medium, which almost always contains NaHCO(3), in experiments related to the bioeffects of ultrasound.

Use of ultrasound in drug delivery systems: emphasis on experimental methodology and mechanisms

Recent studies have shown that ultrasound energy could be applied for targeting or controlling drug release. This new concept of therapeutic ultrasound combined with drugs has induced a great amount of interest in various medical fields. In this paper, several experimental systems are cited in which ultrasound is being utilized to evaluate new application of this modality. The mechanisms of ultrasound-mediated drug delivery are discussed in addition to the review of current advances in the use of ultrasound in systems involving research in cancer therapy, gene therapy, microbubbles and other drug delivery in vitro and in vivo experiments.

Synergistic inhibition of malignant melanoma proliferation by melphalan combined with ultrasound and microbubbles.

The cavitational effects of ultrasound (US) exposure induce transient pores on the cell membrane (sonoporation). Sonoporation have been applied in the field of cancer therapy by promoting delivery of extracellular molecules such as drugs and genes into cytoplasm. In addition, it is known that using US together with microbubbles (MB) elevates permeability of these agents. In this study, by applying the US-MB strategy for melanoma chemotherapy, we evaluated the antitumor effect of melphalan combined with US-MB on a melanoma cell line (C32) in vitro and in vivo. The in vitro cytotoxic effect of the melphalan with US-MB was greater than that of melphalan alone or melphalan in combination with US. In vivo experiments using xenografts, intratumoral injection of melphalan and MB with US exposure led to a greater degree of tumor regression than did the intratumoral injection of the melphalan alone or melphalan in combination with US. These results suggest that US-MB promotes the antitumor effect of melphalan by increasing delivery of molecules into cells and that this strategy may become an effective method of adjuvant therapy against malignant melanoma.

Molecular mechanisms of hyperthermia-induced apoptosis enhanced by docosahexaenoic acid: Implication for cancer therapy

To develop a non-toxic enhancer for hyperthermia-induced cell death as a potential cancer treatment, we studied the effect and mechanism of docosahexaenoic acid (DHA) on hyperthermia-induced apoptosis. Treatment with 20μM DHA and 44°C for 10min induced significant apoptosis, increased intracellular reactive oxygen species (ROS), and caspase-3 activation in U937 cells, but heat or DHA alone did not induce notable apoptosis. Decreased mitochondrial transmembrane potentials were dramatically increased by the combined treatment, accompanied by increased pro-apoptotic Bcl-2 family protein tBid, and decreased anti-apoptotic Bcl-2 and Bcl-xL. Combined hyperthermia-DHA treatment induced significant phosphorylation of protein kinase C (PKC)-δ (p-PKC-δ), and apoptosis in a DHA dose-dependent manner. Using both 20μM DHA and 44°C for 10min induced significant PKC-δ cleavage and its translocation to mitochondria. These results were also seen in HeLa cells. However, MAPKs and Akt were not affected by the treatment. In conclusion, DHA enhances hyperthermia-induced apoptosis significantly via a mitochondria-caspase-dependent pathway; its underlying mechanism involves elevated intracellular ROS, mitochondria dysfunction, and PKC-δ activation.

Synergistic effect of ultrasound and antibiotics against Chlamydia trachomatis-infected human epithelial cells in vitro

To investigate whether or not the combined ultrasound and antibiotic treatment is effective against chlamydial infection, a new ultrasound exposure system was designed to treat chlamydia-infected cells. First, the minimum inhibitory concentrations of antibiotics against Chlamydia trachomatis were determined. Infected cultures were treated with antibiotics then sonicated at intensity of 0.15 or 0.44 W/cm(2) with or without Bubble liposomes. After 48 or 72 h after infection, chlamydial inclusions were stained and examined by fluorescence microscopy. The internalization of dextran-fluorescein conjugates by ultrasound irradiation with Bubble liposomes was observed by fluorescence microscopy. The results showed that application of nanobubble-enhanced ultrasound caused no significant effect on cell viability and chlamydial infectivity. However, Doxycycline (1/2 MIC) or CZX (1.0 μg/ml) in combination with nanobubble-enhanced ultrasound dramatically reduced the number of inclusions compared with that administered with antibiotics only. Bubble dose-dependent synergy was also observed. After ultrasound irradiation at intensity of 0.44 W/cm(2) on the presence of Bubble liposomes, 10% of HeLa cells were observed to have internalized the dextran molecules. This study suggests the possibility of using nanobubble-enhanced ultrasound to deliver antibiotic molecules into cells to eradiate intracellular bacteria, such as chlamydiae, without causing much damage to the cells itself.

Molecular mechanisms involved in the adaptive response to cadmium-induced apoptosis in human myelomonocytic lymphoma U937 cells.

We examined the molecular mechanisms involved in the adaptive response to cadmium (Cd)-induced apoptosis in human myelomonocytic lymphoma U937 cells. When U937 cells were treated with 50 μM cadmium chloride (CdCl2) for 12 h, significant apoptosis occurred. This was associated with an increase in intracellular reactive oxygen species (ROS), sustained phosphorylation of JNK, activation of caspase-3, a decrease in Mcl-1 (anti-apoptotic Bcl-2 proteins), and increases in Bim, Noxa and tBid (a pro-apoptotic protein under the Bcl-2 family). No apoptosis occurred when the cells were treated with 1 μM CdCl2 for 72 h. However, pretreatment with low-dose CdCl2 dramatically altered the sensitivity of the cells to 50 μM CdCl2 with inhibition of apoptosis. Concomitantly, there were significant decreases in the generation of intracellular ROS and the activation of JNK. Pretreatment with 1 μM CdCl2 also attenuated the decrease in Mcl-1 and the increases in Bim, Noxa and tBid induced by 50 μM CdCl2. In conclusion, pretreatment with low-dose Cd inhibited apoptosis induced by high-dose Cd. The mechanism involves inhibition of intracellular ROS generation and JNK activation, and modulating the balance between the expression of Mcl-1 and its binding partners, Bim, Noxa and tBid.

Ultrasound-mediated interferon β gene transfection inhibits growth of malignant melanoma

We investigated the effects of ultrasound-mediated transfection (sonotransfection) of interferon β (IFN-β) gene on melanoma (C32) both in vitro and in vivo. C32 cells were sonotransfected with IFN-β in vitro. Subcutaneous C32 tumors in mice were sonicated weekly immediately after intra-tumor injection with IFN-β genes mixed with microbubbles. Successful sonotransfection with IFN-β gene in vitro was confirmed by ELISA, which resulted in C32 growth inhibition. In vivo, the growth ratio of tumors transfected with IFN-β gene was significantly lower than the other experimental groups. These results may lead to a new method of treatment against melanoma and other hard-to-treat cancers.

Low-intensity pulsed ultrasound enhances cell killing induced by X-irradiation.

To determine the effect of pulsed ultrasound (US) on radiation-induced cell killing, U937 and Molt-4 cell lines were exposed to 1.0 MHz US with 50% of duty factor at 0.3 W/cm(2) and pulsed at 1 Hz immediately after exposure to X-rays at 0, 0.5, 2.5 and 5 Gy. The cells were assayed 24 h after the treatments. The result showed significant enhancement of cell killing in the combined treatments. However, the ratio of apoptotic cells induced either by X-rays or US alone did not significantly change. These findings suggest that pulsed US can enhance the anticancer effect of X-irradiation due to US streaming under non-inertial cavitational condition. This combined treatment can potentially enhance the therapeutic effect of radiation therapy.