Qing Li Zhao

Apoptosis Induced by Cadmium in Human Lymphoma U937 Cells through Ca2+-calpain and Caspase-Mitochondria- dependent Pathways

Apoptosis induced by cadmium has been shown in many tissues in vivo and in cultured cells in vitro. However, its molecular mechanism is not fully understood. When the human histiocytic lymphoma cell line U937 was treated with cadmium for 12 h, evidence of apoptotic features, including change in nuclear morphology, DNA fragmentation, formation of DNA ladder in agarose gel electrophoresis, and phosphatidylserine externalization, were obtained. Moreover, loss of the mitochondrial membrane potential (Δψm) was observed in the cadmium-treated cells and was inhibited by a broad caspase inhibitor (Z-VAD-FMK). Caspase inhibitors suppressed the DNA fragmentation in the order of Z-VAD-FMK > caspase-8 inhibitor > caspase-3 inhibitor. Expression of Bcl-xL and Bid decreased significantly in the cadmium-treated cells, although no apparent change in Bcl-2 and Bax expression was found. Tetrakis-(2-pyridylmethyl) ethylendiamine, a cell-permeable heavy metal chelator, partially reversed the increase of fluorescence of Fura-2 in the cadmium-treated cells. In addition, verapamil (70 μm), a voltage-dependent Ca2+ channel blocker, inhibited the DNA fragmentation induced by cadmium less than 100 μm and decreased the fluorescence of Fura-2. Cadmium up-regulated the expression of type 1 inositol 1,4,5-trisphosphate receptor (IP3R) but not type 2 or type 3 IP3R. Calpain inhibitors I and II partially prevented DNA fragmentation. No effects of Z-VAD-FMK on the expression of type 1 IP3R or of calpain inhibitors on the loss of Δψm were observed. These results suggest that cadmium possibly induced apoptosis in U937 cells through two independent pathways, the Ca2+-calpain-dependent pathway and the caspase-mitochondria-dependent pathway.

DNA Double-Strand Breaks Induced by Cavitational Mechanical Effects of Ultrasound in Cancer Cell Lines

Ultrasonic technologies pervade the medical field: as a long established imaging modality in clinical diagnostics; and, with the emergence of targeted high intensity focused ultrasound, as a means of thermally ablating tumours. In parallel, the potential of [non-thermal] intermediate intensity ultrasound as a minimally invasive therapy is also being rigorously assessed. Here, induction of apoptosis in cancer cells has been observed, although definitive identification of the underlying mechanism has thus far remained elusive. A likely candidate process has been suggested to involve sonochemical activity, where reactive oxygen species (ROS) mediate the generation of DNA single-strand breaks. Here however, we provide compelling new evidence that strongly supports a purely mechanical mechanism. Moreover, by a combination of specific assays (neutral comet tail and staining for γH2AX foci formation) we demonstrate for the first time that US exposure at even moderate intensities exhibits genotoxic potential, through its facility to generate DNA damage across multiple cancer lines. Notably, colocalization assays highlight that ionizing radiation and ultrasound have distinctly different signatures to their respective γH2AX foci formation patterns, likely reflecting the different stress distributions that initiated damage formation. Furthermore, parallel immuno-blotting suggests that DNA-PKcs have a preferential role in the repair of ultrasound-induced damage.

Effects of dissolved gases and an echo contrast agent on ultrasound mediated in vitro gene transfection.

The effects of acoustic cavitation on in vitro transfection by ultrasound were investigated. HeLa cells were exposed to 1.0 MHz continuous ultrasound in culture media containing the luciferase gene. Transfection efficiency was elevated when an echo contrast agent, Levovist was added or air was dissolved in the medium. When cells were sonicated in medium saturated with Ar, N2 or N2O which have different gamma values (Cp/Cv), or were saturated with He, Ar or Ne with different thermal conductivities, the effectiveness for the dissolved gases in the ultrasound mediated transfection was Ar > N2 > N2O or Ar > Ne > He, respectively. When free radical formation in water by ultrasound was monitored as a measure of inertial cavitation, it was similarly affected by dissolved gases. These results indicate that the efficiency of ultrasound mediated transfection was significantly affected either by occurrence of or by modification of inertial cavitation due to various gases.

Enhancement of hyperthermia-induced apoptosis by a free radical initiator, 2,2′-azobis (2-amidinopropane) dihydrochloride, in human histiocytic lymphoma U937 cells

To elucidate the mechanism how a free radical initiator, 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH), induces cell death at hyperthermic temperatures, apoptosis in a human histiocytic lymphoma cell line, U937, was investigated. Free radical formation deriving from the thermal decomposition of AAPH was examined by spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). An assay for DNA fragmentation, observation of nuclear morphological changes, and flow cytometry for phosphatidylserine (PS) externalization were used to detect apoptosis and revealed enhancement of 44.0°C hyperthermia-induced apoptosis by free radicals due to AAPH. However, free radicals alone derived from AAPH did not induce apoptosis. Hyperthermia induced the production of lipid peroxidation (LPO), an increase in intracellular Ca2+ concentration ([Ca2+]i) and enhanced expression of the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1). The effects of hyperthermia on LPO and [Ca2+]i were enhanced markedly by the combination with AAPH. A significant decrease in Bcl-2 expression, increase in Bax expression, a loss of mitochondrial membrane potential (ΔΨm) and a marked increase in cytochrome c expression were found only in cells treated with hyperthermia and AAPH. Although an intracellular Ca2+ ion chelator, BAPTA-AM, completely inhibited DNA fragmentation, water-soluble vitamine E, Trolox, only partially suppressed DNA fragmentation and the increase in [Ca2+]i. In contrast, LPO was inhibited completely by Trolox, but no inhibition by BAPTA-AM was found. These results suggest that apoptosis induced by hyperthermia alone is due to the increase in [Ca2+]i arising from increased expression of IP3R1 and LPO. Additional increase in [Ca2+]i due to increased LPO and the activation of mitochondria-caspase dependent pathway play a major role in the enhancement of apoptosis by the combination with hyperthermia and AAPH.

Ultrasound-induced apoptosis in the presence of Sonazoid and associated alterations in gene expression levels: a possible therapeutic application.

Ultrasound (US) has been shown to induce apoptosis and cell lysis in cancer cells. In this study, we report on the potential of using Sonazoid, a new echo-contrast agent, that is chemically more stable and US-resistant in combination with US in cancer therapy. The biological effects and their mechanisms in the presence or absence of ultrasonic exposure in vitro were investigated. In addition, the impact of the agent on the expression levels of genes responsive to US was studied using global-scale microarrays and computational gene expression analysis tools. Our results show that the combination led to enhanced cell killing in the presence of 1 MHz acoustic field. The apoptosis induction was shown to be mediated by the mitochondrial pathway. The occurrence of US-induced DNA damage was also observed. Despite these findings, the agent at concentrations similar to those clinically used can be considered as well tolerated. Furthermore, Sonazoid enhanced expression of genes that related to apoptosis and are responsive to US, although it alone had almost no effect. These results indicate the potential of Sonazoid for US contrast enhancement as well as the possibility of its use in US-aided therapies.

Inhibition of checkpoint kinase 1 abrogates G2/M checkpoint activation and promotes apoptosis under heat stress

Hyperthermia induced by heat stress (HS) inhibits the proliferation of cancer cells and induces their apoptosis. However, the mechanism underlying HS-induced apoptosis remains elusive. Here, we demonstrated a novel evidence that checkpoint kinase 1 (Chk1) plays crucial roles in the apoptosis and regulation of cell cycle progression in cells under HS. In human leukemia Jurkat cells, interestingly, the ataxia telangiectasia and Rad-3 related (ATR)-Chk1 pathway was preferentially activated rather than the ataxia telangiectasia mutated (ATM)-checkpoint kinase 2 (Chk2) pathway under HS. The selective inhibitors of ATR or Chk1 abrogated HS-induced apoptosis in human leukemia Jurkat cells whereas the inhibition of ATM or Chk2 caused only marginal effects. Inhibition of ATR and Chk1 also abrogated G2/M checkpoint activation by HS in Jurkat cells. The effects of small interfering RNA targeting Chk1 were similar to those of the selective inhibitor of Chk1. In addition, the efficiencies of Chk1 inhibition on G2/M checkpoint abrogation and apoptosis induction were confirmed in the adherent cancer cell lines HeLa, HSC3, and PC3, suggesting that the targeting of Chk1 can be effective in solid tumors cells. In conclusion, these findings indicate a novel molecular basis of G2/M checkpoint activation and apoptosis in cells exposed to HS.

Apoptotic cell death by the novel natural compound, cinobufotalin

Cinobufotalin (CB), one of the bufadienolides prepared from toad venom, was investigated for its cytotoxicity, and the underneath mechanism involved. We primarily utilized DNA fragmentation assay and microscopic observation to assess the effect of various doses of CB in human lymphoma U937 cells. Following that, we investigated other parameters involved in cell death mechanism such as reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and apoptotic proteins activation. HeLa cells were concomitantly used to generalize the data observed. Our results show that CB caused significant DNA fragmentation, decrease of MMP, and an increase in the intracellular Ca(2+) ion and ROS production. In addition, CB induced upregulation of Fas protein, proteolytic activation of cytochrome c, caspase-2, -3, -8 and -9 together with the activation of Bid and Bax. Our findings were further validated using either Fas/FasL antagonist or pan-caspase inhibitor to significantly inhibit CB-induced DNA fragmentation. In our study, we suggest that CB induces caspase dependent cell death in U937 cells, and that Fas plays a role in CB-induced apoptosis. Altogether, our data provides novel insights of the mechanism of action of CB and its potential as a future chemotherapeutic agent.

Effects of therapeutic ultrasound on the nucleus and genomic DNA

In recent years, data have been accumulating on the ability of ultrasound to affect at a distance inside the cell. Previous conceptions about therapeutic ultrasound were mainly based on compromising membrane permeability and triggering some biochemical reactions. However, it was shown that ultrasound can access deep to the nuclear territory resulting in enhanced macromolecular localization as well as alterations in gene and protein expression. Recently, we have reported on the occurrence of DNA double-strand breaks in different human cell lines exposed to ultrasound in vitro with some insight into the subsequent DNA damage response and repair pathways. The impact of these observed effects again sways between extremes. It could be advantageous if employed in gene therapy, wound and bone fracture-accelerated healing to promote cellular proliferation, or in cancer eradication if the DNA lesions would culminate in cell death. However, it could be a worrying sign if they were penultimate to further cellular adaptations to stresses and thus shaking the safety of ultrasound application in diagnosis and therapy. In this review, an overview of the rationale of therapeutic ultrasound and the salient knowledge on ultrasound-induced effects on the nucleus and genomic DNA will be presented. The implications of the findings will be discussed hopefully to provide guidance to future ultrasound research.

Low-intensity ultrasound adjuvant therapy: enhancement of doxorubicin-induced cytotoxicity and the acoustic mechanisms involved.

PurposeIn this study, the effects of low-intensity pulsed ultrasound (LIU) as an adjuvant to doxorubicin (DOX) treatment was further investigated in comparison to hyperthermia as another widely used adjuvant. The effects were compared with respect to cell killing and apoptosis induction in U937 cells. Human primary liver cancer (PLC) cells were also used to evaluate the effects of the combinations. The use of an echo contrast agent was investigated for further enhancement of cytotoxicity. Finally, the acoustic mechanisms involved were investigated.MethodsThe effects of different treatment regimens on cell viability were determined using the Trypan blue dye-exclusion test. Apoptosis induction was detected by flow cytometry using fluorescein isothiocyanate-annexin V and propidium iodide staining. The mechanistic study involved electron paramagnetic spin trapping for detecting free radical formation as an indicator of the occurrence of inertial cavitation and spectrophotometry for sucrose hydrolysis as an indicator for noncavitational effects.ResultsThe combination treatments exerted synergistic effects on cytotoxicity depending on the acoustic conditions used. The use of LIU as an adjuvant to DOX treatment was shown to be superior to the use of hyperthermia as an adjuvant. Moreover, the combination seems to be promising for other cancer types provided that the acoustic conditions are properly selected with respect to drug concentration. The key ultrasound mechanism responsible for the synergism observed was shown to be the production of free radicals by inertial cavitation. Non-cavitational forces were also shown to contribute to the effect.ConclusionThis study is motivating to engage in in vivo research with various cancer types as a step toward clinical applicability and is emphasizing on the importance of developing therapeutic protocols for setting LIU parameters with respect to other therapeutic conditions.

Effect of platinum nanoparticles on cell death induced by ultrasound in human lymphoma U937 cells.

In this study, we report on the potential use of platinum nanoparticles (Pt-NPs), a superoxide dismutase (SOD)/catalase mimetic antioxidant, in combination with 1MHz ultrasound (US) at an intensity of 0.4 W/cm(2), 10% duty factor, 100 Hz PRF, for 2 min. Apoptosis induction was assessed by DNA fragmentation assay, cell cycle analysis and Annexin V-FITC/PI staining. Cell killing was confirmed by cell counting and microscopic examination. The mitochondrial and Ca(2+)-dependent pathways were investigated. Caspase-8 expression and autophagy-related proteins were detected by spectrophotometry and western blot analysis, respectively. Intracellular reactive oxygen species (ROS) elevation was detected by flow cytometry, while extracellular free radical formation was assessed by electron paramagnetic resonance spin trapping spectrometry. The results showed that Pt-NPs exerted differential effects depending on their internalization. Pt-NPs functioned as potent free radical scavengers when added immediately before sonication while pre-treatment with Pt-NPs suppressed the induction of apoptosis as well as autophagy (AP), and resulted in enhanced cell killing. Dead cells displayed the features of pyknosis. The exact mode of cell death is still unclear. In conclusion, the results indicate that US-induced AP may contribute to cell survival post sonication. To our knowledge this is the first study to discuss autophagy as a pro-survival pathway in the context of US. The combination of Pt-NPs and US might be effective in cancer eradication.