Moon-Taek Park

Caspase-independent cell death by arsenic trioxide in human cervical cancer cells: reactive oxygen species-mediated poly(ADP-ribose) polymerase-1 activation signals apoptosis-inducing factor release from mitochondria.

Although mechanisms of arsenic trioxide (As2O3)-induced cell death have been studied extensively in hematologic cancers, those in solid cancers have yet to be clearly defined. In this study, we showed that the translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus is required for As2O3-induced cell death in human cervical cancer cells. We also showed that reactive oxygen species (ROS)-mediated poly(ADP-ribose) polymerase-1 (PARP-1) activation is necessary for AIF release from mitochondria. The treatment of human cervical cancer cells with As2O3 induces dissipation of mitochondrial membrane potential (Δψm), translocation of AIF from mitochondria to the nucleus, and subsequent cell death. Small interfering RNA targeting of AIF effectively protects cervical cancer cells against As2O3-induced cell death. As2O3 also induces an increase of intracellular ROS level and a marked activation of PARP-1. N-acetyl-l-cystein, a thiol-containing antioxidant, completely blocks As2O3-induced PARP-1 activation, Δψm loss, nuclear translocation of AIF from mitochondria, and the consequent cell death. Furthermore, pretreatment of 1,5-dihydroxyisoquinoline or 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone, PARP-1 inhibitors, effectively attenuates the loss of Δψm, AIF release, and cell death. These data support a notion that ROS-mediated PARP-1 activation signals AIF release from mitochondria, resulting in activation of a caspase-independent pathway of cell death in solid tumor cells by As2O3 treatment.

Endoplasmic Reticulum Stress-Induced JNK Activation Is a Critical Event Leading to Mitochondria-Mediated Cell Death Caused by β-Lapachone Treatment

Background β-lapachone (β-lap) is a bioreductive agent that is activated by the two-electron reductase NAD(P)H quinone oxidoreductase 1 (NQO1). Although β-lap has been reported to induce apoptosis in various cancer types in an NQO1-dependent manner, the signaling pathways by which β-lap causes apoptosis are poorly understood. Methodology/Principal Findings β-lap-induced apoptosis and related molecular signaling pathways in NQO1-negative and NQO1-overexpressing MDA-MB-231 cells were investigated. Pharmacological inhibitors or siRNAs against factors involved in β-lap-induced apoptosis were used to clarify the roles played by such factors in β-lap-activated apoptotic signaling pathways. β-lap leads to clonogenic cell death and apoptosis in an NQO1- dependent manner. Treatment of NQO1-overexpressing MDA-MB-231 cells with β-lap causes rapid disruption of mitochondrial membrane potential, nuclear translocation of AIF and Endo G from mitochondria, and subsequent caspase-independent apoptotic cell death. siRNAs targeting AIF and Endo G effectively attenuate β-lap-induced clonogenic and apoptotic cell death. Moreover, β-lap induces cleavage of Bax, which accumulates in mitochondria, coinciding with the observed changes in mitochondria membrane potential. Pretreatment with Salubrinal (Sal), an endoplasmic reticulum (ER) stress inhibitor, efficiently attenuates JNK activation caused by β-lap, and subsequent mitochondria-mediated cell death. In addition, β-lap-induced generation and mitochondrial translocation of cleaved Bax are efficiently blocked by JNK inhibition. Conclusions/Significance Our results indicate that β-lap triggers induction of endoplasmic reticulum (ER) stress, thereby leading to JNK activation and mitochondria-mediated apoptosis. The signaling pathways that we revealed in this study may significantly contribute to an improvement of NQO1-directed tumor therapies.

Hyperthermia improves therapeutic efficacy of doxorubicin carried by mesoporous silica nanocontainers in human lung cancer cells

Purpose: We investigated the use of hyperthermia to improve the anti-cancer efficacy of doxorubicin (DOX)-loaded mesoporous silica nanocontainer Si-SS-CD-PEG. The hypothesis was that heat stimulates glutathione-mediated degradation of cyclodextrin gatekeeper, thereby causing the release of DOX from the carrier and DOX-induced cell death. Materials and methods: The release of DOX from DOX-loaded Si-SS-CD-PEG suspended in PBS containing glutathione (GSH) was studied by assessing the changes in DOX fluorescence intensity. The effect of heating at 42°C on the release of DOX from the intracellular carriers was determined with confocal microscopy. The extents of clonogenic and apoptotic cell death caused by DOX-loaded Si-SS-CD-PEG were determined. Results: The release of DOX from DOX-loaded Si-SS-CD-PEG in PBS occurred only when GSH presented in the suspension, and heating at 42°C slightly increased the release of DOX from the carriers. Heating significantly elevated the GSH content in A549 cells and increased the release of DOX from the internalised carriers. Heating the cancer cells treated with the carriers at 42°C markedly increased the clonogenic death and apoptosis. The GSH content in A549 cells was greater than that in L-132 cells, and A549 cells were far more sensitive than L-132 cells to DOX-loaded Si-SS-CD-PEG at both 37°C and 42°C. Conclusions: Hyperthermia increased the GSH-mediated release of DOX from DOX-loaded Si-SS-CD-PEG. Furthermore, hyperthermia markedly elevated the GSH content in cancer cells, thereby increasing the release of DOX from the internalised carriers and potentiating the DOX-induced clonogenic and apoptotic cell death.

Novel histone deacetylase inhibitor CG200745 induces clonogenic cell death by modulating acetylation of p53 in cancer cells

SummaryHistone deacetylase (HDAC) plays an important role in cancer onset and progression. Therefore, inhibition of HDAC offers potential as an effective cancer treatment regimen. CG200745, (E)-N1-(3-(dimethylamino)propyl)-N8-hydroxy-2-((naphthalene-1-loxy)methyl)oct-2-enediamide, is a novel HDAC inhibitor presently undergoing a phase I clinical trial. Enhancement of p53 acetylation by HDAC inhibitors induces cell cycle arrest, differentiation, and apoptosis in cancer cells. The purpose of the present study was to investigate the role of p53 acetylation in the cancer cell death caused by CG200745. CG200745-induced clonogenic cell death was 2-fold greater in RKO cells expressing wild-type p53 than in p53-deficient RC10.1 cells. CG200745 treatment was also cytotoxic to PC-3 human prostate cancer cells, which express wild-type p53. CG200745 increased acetylation of p53 lysine residues K320, K373, and K382. CG200745 induced the accumulation of p53, promoted p53-dependent transactivation, and enhanced the expression of MDM2 and p21Waf1/Cip1 proteins, which are encoded by p53 target genes. An examination of CG200745 effects on p53 acetylation using cells transfected with various p53 mutants showed that cells expressing p53 K382R mutants were significantly resistant to CG200745-induced clonogenic cell death compared with wild-type p53 cells. Moreover, p53 transactivation in response to CG200745 was suppressed in all cells carrying mutant forms of p53, especially K382R. Taken together, these results suggest that acetylation of p53 at K382 plays an important role in CG200745-induced p53 transactivation and clonogenic cell death.

β-Lapachone Significantly Increases the Effect of Ionizing Radiation to Cause Mitochondrial Apoptosis via JNK Activation in Cancer Cells

Background β-lapachone (β-lap), has been known to cause NQO1-dependnet death in cancer cells and sensitize cancer cells to ionizing radiation (IR). We investigated the mechanisms underlying the radiosensitization caused by β-lap. Methodology/Principal Findings β-lap enhanced the effect of IR to cause clonogenic cells in NQO1+-MDA-MB-231 cells but not in NQO1−-MDA-MB-231 cells. β-lap caused apoptosis only in NQO1+ cells and not in NQO1− cells and it markedly increased IR-induced apoptosis only in NQO1+ cells. Combined treatment of NQO1+ cells induced ROS generation, triggered ER stress and stimulated activation of ERK and JNK. Inhibition of ROS generation by NAC effectively attenuated the activation of ERK and JNK, induction of ER stress, and subsequent apoptosis. Importantly, inhibition of ERK abolished ROS generation and ER stress, whereas inhibition of JNK did not, indicating that positive feedback regulation between ERK activation and ROS generation triggers ER stress in response to combined treatment. Furthermore, prevention of ER stress completely blocked combination treatment-induced JNK activation and subsequent apoptotic cell death. In addition, combined treatment efficiently induced the mitochondrial translocation of cleaved Bax, disrupted mitochondrial membrane potential, and the nuclear translocation of AIF, all of which were efficiently blocked by a JNK inhibitor. Caspases 3, 8 and 9 were activated by combined treatment but inhibition of these caspases did not abolish apoptosis indicating caspase activation played a minor role in the induction of apoptosis. Conclusions/Significance β-lap causes NQO1-dependent radiosensitization of cancer cells. When NQO1+ cells are treated with combination of IR and β-lap, positive feedback regulation between ERK and ROS leads to ER stress causing JNK activation and mitochondrial translocation of cleaved Bax. The resultant decrease in mitochondrial membrane leads to translocation of AIF and apoptosis.

Cisplatin enhances the anticancer effect of β-lapachone by upregulating NQO1

NAD(P)H:quinone oxidoreductase (NQO1) has been reported to play an important role in cell death caused by &bgr;-lapachone (&bgr;-lap), 3,4-dihydro-22,2-dimethyl-2H-naphthol[1,22b]pyran-5,6-dione. This study investigated whether cisplatin (cis-diamminedichloroplatinum) sensitizes cancer cells to &bgr;-lap by upregulating NQO1. The cytotoxicity of cisplatin and &bgr;-lap alone or in combination against FSaII fibrosarcoma cells of C3H mice in vitro was determined with a clonogenic survival assay and assessment of &ggr;-H2AX foci formation, a hallmark of DNA double-strand breaks. The cellular sensitivity to &bgr;-lap progressively increased during the 24 h after cisplatin treatment. The expression and enzymatic activity of NQO1 also increased during the 24 h after cisplatin treatment, and dicoumarol, an inhibitor of NQO1, was found to nullify the cisplatin-induced increase in &bgr;-lap sensitivity. The role of NQO1 in the cell death caused by &bgr;-lap alone or in combination with cisplatin was further elucidated using NQO1-positive and NQO1-negative MDA-MB-231 human breast cancer cells. Cisplatin increased the sensitivity of the NQO1-positive but not the NQO1-negative MDA-MB-231 cells to &bgr;-lap treatment. Combined treatment with cisplatin and &bgr;-lap suppressed the growth of FSaII tumors in the legs of C3H mice in a manner greater than additive. It is concluded that cisplatin markedly increases the sensitivity of cancer to &bgr;-lap in vitro and in vivo by upregulating NQO1.

Radiation-induced angiogenic signaling pathway in endothelial cells obtained from normal and cancer tissue of human breast

Despite strong possibility that endothelial cells (ECs) of tumors and normal tissues may differ in various aspects, most previous studies on ECs have used normal cells. Here, we purified ECs from tumorous and normal human breast tissues, and studied the effect of radiation on angiogenesis and relevant molecular mechanisms in these cells. We found that in normal tissue-derived ECs (NECs), 4 Gy irradiation increased tube formation, matrix metalloproteinase 2 (MMP-2) expression and extracellular signal-regulated kinase (ERK) pathway activation. In cancer-derived ECs (CECs), however, 4 Gy irradiation significantly reduced tube formation, increased the production of angiostatin and interleukin-6 (IL-6), and upregulated AKT and c-Jun N-terminal kinase (JNK) pathway activation. Knockdown experiments showed that siMMP-2 efficiently inhibited tube formation by irradiated NECs, whereas siPlasminogen effectively attenuated the radiation-induced suppression of tube formation and the upregulation of angiostatin in CECs. Moreover, siIL-6 clearly inhibited the radiation-induced generation of angiostatin in CECs. Inhibition of ERK with a pharmacological inhibitor or small interfering RNAs (siRNAs) markedly suppressed the radiation-induced tube formation and MMP-2 upregulation in NECs, whereas the inhibition of either AKT or JNK with pharmacological inhibitor or siRNA treatment of CECs markedly attenuated the inhibition of tube formation and the upregulation of angiostatin and IL-6 caused by 4 Gy irradiation. These observations collectively demonstrate that there are distinct differences in the radiation responses of NECs and CECs, and might provide important clues for improving the efficacy of radiation therapy.

Radio-sensitivities and angiogenic signaling pathways of irradiated normal endothelial cells derived from diverse human organs

The purpose of the present investigation was to study the effects of ionizing radiation on endothelial cells derived from diverse normal tissues. We first compared the effects of radiation on clonogenic survival and tube formation of endothelial cells, and then investigated the molecular signaling pathways involved in endothelial cell survival and angiogenesis. Among the different endothelial cells studied, human hepatic sinusoidal endothelial cells (HHSECs) were the most radio-resistant and human dermal microvascular endothelial cells were the most radio-sensitive. The radio-resistance of HHSECs was related to adenosine monophosphate-activated protein kinase and p38 mitogen-activated protein kinase-mediated expression of MMP-2 and VEGFR-2, whereas the increased radio-sensitivity of HDMECs was related to extracellular signal-regulated kinase-mediated generation of angiostatin. These observations demonstrate that there are distinct differences in the radiation responses of normal endothelial cells obtained from diverse organs, which may provide important clues for protection of normal tissue from radiation exposure.

Combination treatment with arsenic trioxide and phytosphingosine enhances apoptotic cell death in arsenic trioxide–resistant cancer cells

Resistance to anticancer drugs can sometimes be overcome by combination treatment with other therapeutic drugs. Here, we showed that phytosphingosine treatment in combination with arsenic trioxide (As2O3) enhanced cell death of naturally As2O3-resistant human myeloid leukemia cells. The combination treatment induced an increase in intracellular reactive oxygen species level, mitochondrial relocalization of Bax, poly(ADP-ribose) polymerase-1 (PARP-1) activation, and cytochrome c release from the mitochondria. N-acetyl-l-cysteine, a thiol-containing antioxidant, completely blocked Bax relocalization, PARP-1 activation, and cytochrome c release. Pretreatment of 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone, a PARP-1 inhibitor, or PARP-1/small interfering RNA partially attenuated cytochrome c release, whereas the same treatment did not affect Bax relocalization. The combination treatment induced selective activation of p38 mitogen-activated protein kinase (MAPK). Inhibition of p38 MAPK by treatment of SB203580 or expression of dominant-negative forms of p38 MAPK suppressed the combination treatment–induced Bax relocalization but did not affect PARP-1 activation. In addition, antioxidant N-acetyl-l-cysteine completely blocked p38 MAPK activation. These results indicate that phytosphingosine in combination with As2O3 induces synergistic apoptosis in As2O3-resistant leukemia cells through the p38 MAPK–mediated mitochondrial translocation of Bax and the PARP-1 activation, and that p38 MAPK and PARP-1 activations are reactive oxygen species dependent. The molecular mechanism that we elucidated in this study may provide insight into the design of future combination cancer therapies to cells intrinsically less sensitive to As2O3 treatment. [Mol Cancer Ther 2007;6(1):82–92]

Heat shock increases expression of NAD(P)H:quinone oxidoreductase (NQO1), mediator of β-lapachone cytotoxicity, by increasing NQO1 gene activity and via Hsp70-mediated stabilisation of NQO1 protein

NAD(P)H:quinone oxidoreductase (NQO1) mediates cell death caused by the novel anti‐cancer drug β‐lapachone (β‐lap). Therefore, β‐lap sensitivity of cells is positively related to the level of cellular NQO1. Heat shock up‐regulates NQO1 expression in cancer cells, thereby enhancing the clonogenic cell death caused by β‐lap. The mechanisms by which heat shock elevates NQO1 expression were investigated in the present study using human A549 lung cancer cells and human MDA‐MB‐231 breast cancer cells. When MDA‐MB‐231(NQO1+) cells stably transfected with NQO1 were heated at 42°C for 1 h the expression of NQO1 and the sensitivity of the cells to β‐lap progressively increased during the 24–48 h post‐heating period. Heating increased NQO1 transcription by cis‐acting elements such as xenobiotic response element and antioxidant response element located in the NQO1 gene promoter region. The turnover of NQO1 protein in heated cells was much slower than in unheated cells. NQO1 and heat shock protein 70 (Hsp70) co‐precipitated and co‐localised in cells before and after heating, demonstrating the close association of these two proteins in the cells. These results suggest that NQO1 is stabilised by the Hsp70 molecular chaperone. It is concluded that the prolonged increase in NQO1 expression after heat shock is due to increased NQO1 transcription, and also increased Hsp70‐mediated NQO1 stabilisation.