[Show abstract][Hide abstract] ABSTRACT: Oxygen and glucose deprivation (OGD) due to insufficient blood circulation can decrease cancer cell survival and proliferation in solid tumors. OGD increases the intracellular [AMP]/[ATP] ratio, thereby activating the AMPK. In this study, we have investigated the involvement of NQO1 in OGD-mediated AMPK activation and cancer cell death. We found that OGD activates AMPK in an NQO1-dependent manner, suppressing the mTOR/S6K/4E-BP1 pathway, which is known to control cell survival. Thus, the depletion of NQO1 prevents AMPK-induced cancer cell death in OGD. When we blocked OGD-induced Ca(2+)/CaMKII signaling, the NQO1-induced activation of AMPK was attenuated. In addition, when we blocked the RyR signaling, the accumulation of intracellular Ca(2+) and subsequent activation of CaMKII/AMPK signaling was decreased in NQO1-expressing cells under OGD. Finally, siRNA-mediated knockdown of CD38 abrogated the OGD-induced activation of Ca(2+)/CaMKII/AMPK signaling. Taken together, we conclude that NQO1 plays a key role in the AMPK-induced cancer cell death in OGD through the CD38/cADPR/RyR/Ca(2+)/CaMKII signaling pathway.
[Show abstract][Hide abstract] ABSTRACT: Aneuploidy is the most common characteristic of human cancer cells. It also causes genomic instability, which is involved
in the initiation of cancer development. Various lines of evidence indicate that nicotinamide adenine dinucleotide(P)H quinone
oxidoreductase 1 (NQO1) plays an important role in cancer prevention, but the molecular mechanisms underlying this effect
have not yet been fully elucidated. Here, we report that ionizing radiation (IR) induces substantial aneuploidy and centrosome
amplification in NQO1-deficient cancer cells, suggesting that NQO1 plays a crucial role in preventing aneuploidy. NQO1 deficiency
markedly increased the protein stability of Aurora-A in irradiated cancer cells. Small interfering RNA targeting Aurora-A
effectively attenuated IR-induced centrosome amplification concerned with aneuploidy in NQO1-deficient cancer cells. Furthermore,
we found that NQO1 specifically binds to Aurora-A via competing with the microtubule-binding protein, TPX2 (targeting protein
for Xklp2), and contributes to the degradation of Aurora-A. Our results collectively demonstrate that NQO1 plays a key role
in suppressing IR-induced centrosome amplification and aneuploidy through a direct interaction with Aurora-A.
[Show abstract][Hide abstract] ABSTRACT: 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
Preview · Article · Jul 2012 · Journal of Radiation Research
[Show abstract][Hide abstract] ABSTRACT: We developed a novel method for harvesting endothelial cells from blood vessels of freshly obtained cancer and adjacent normal tissue of human breast, and compared the response of the cancer-derived endothelial cells (CECs) and normal tissue-derived endothelial cells (NECs) to ionizing radiation. In brief, when tissues were embedded in Matrigel and cultured in endothelial cell culture medium (ECM) containing growth factors, endothelial cells grew out of the tissues. The endothelial cells were harvested and cultured as monolayer cells in plates coated with gelatin, and the cells of 2nd-5th passages were used for experiments. Both CECs and NECs expressed almost the same levels of surface markers CD31, CD105 and TEM-8 (tumor endothelial marker-8), which are known to be expressed in angiogenic endothelial cells, i.e., mitotically active endothelial cells. Furthermore, both CECs and NECs were able to migrate into experimental wound in the monolayer culture, and also to form capillary-like tubes on Matrigel-coated plates. However, the radiation-induced suppressions of migration and capillary-like tube formations were greater for CECs than NECs from the same patients. In addition, in vitro clonogenic survival assays demonstrated that CECs were far more radiosensitive than NECs. In summary, we have developed a simple and efficient new method for isolating endothelial cells from cancer and normal tissue, and demonstrated for the first time that endothelial cells of human breast cancer are significantly more radiosensitive than their normal counterparts from the same patients.
No preview · Article · Jun 2012 · Microvascular Research
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
Preview · Article · Nov 2011 · International Journal of Hyperthermia
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone (RH1) is a bioreductive agent that is activated by the two-electron reductase NAD(P)H quinone oxidoreductase 1 (NQO1). Although the cytotoxic efficacy of RH1 against tumours has been studied extensively, the molecular mechanisms underlying this anti-cancer activity have not yet been fully elucidated.
2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone-induced apoptosis and related signalling pathways in NQO1-negative and NQO1-overexpressing cells were evaluated. The role of p53 in RH1-induced cell death was investigated using parental and p53-deficient RKO human colorectal cancer cells by assaying clonogenic cell survival. Specific inhibitors and siRNAs targeting factors involved in RH1-induced apoptosis were used to clarify the roles played by such factors in RH1-activated apoptotic signalling pathways.
2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone induced apoptosis and clonogenic death, dependent on NQO1 and p53. Treatment of NQO1-overexpressing cells with RH1 caused rapid disruption of mitochondrial membrane potential, nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G (Endo G) from mitochondria, and subsequent caspase-independent apoptotic cell death. siRNA targeting AIF and Endo G effectively attenuated RH1-induced apoptotic cell death. Moreover, RH1 induced cleavage of Bax, which targets mitochondria. RH1 significantly activated the c-Jun N-terminal kinase (JNK) pathway, and inhibition of this pathway suppressed RH1-induced mitochondria-mediated apoptosis. RH1-induced generation and mitochondrial translocation of cleaved Bax were blocked by the JNK inhibitor, SP600125. Inhibition of JNK with SP600125 attenuated the mitochondrial translocation of JNK.
2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone activated JNK, resulting in mitochondria-mediated apoptotic cell death that was NQO1-dependent.
Preview · Article · Jun 2011 · British Journal of Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Histone 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)-N(1)-(3-(dimethylamino)propyl)-N(8)-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 p21(Waf1/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.
No preview · Article · Oct 2010 · Investigational New Drugs
[Show abstract][Hide abstract] ABSTRACT: Aurora-A, a serine/threonine kinase that is overexpressed in certain human cancer cell lines, plays an important role in mitotic progression. Aurora-A has also been reported to be involved in the activation of nuclear factor kappa B (NF-kappaB). The purpose of the present study was to identify the role of Aurora-A in the radiation-induced activation pathway of NF-kappaB. Wild-type and Aurora-A knockdown (Aurora-A(KD)) HeLa cells were irradiated with 4 Gy of gamma rays and the EMSA, luciferase reporter gene assay and immunoblot analysis were performed. The siRNA-based gene knockdown and overexpression system was adopted to elucidate the role of Aurora-A in radiation-induced NF-kappaB pathway activation. The clonogenic survival study indicated that Aurora-A(KD) cells and the wild-type cells transfected with Aurora-A siRNA or RelA/p65 siRNA were more radiosensitive than the wild-type cells. In both the wild-type and Aurora-A(KD) cells, radiation caused IkappaB kinase-mediated phosphorylation, degradation of IkappaBalpha and phosphorylation of RelA/p65. The nuclear translocation of RelA/p65 was also similar in the wild-type and Aurora-A(KD) cells. However, RelA/p65-DNA binding was markedly suppressed in Aurora-A(KD) cells compared to that in wild-type cells. It was concluded that Aurora-A enhances the binding of NF-kappaB to DNA, thereby increasing the gene transcription by NF-kappaB and decreasing the radiosensitivity of the cells.
No preview · Article · Sep 2010 · Radiation Research
[Show abstract][Hide abstract] ABSTRACT: NAD(P)H:quinone oxidoreductase (NQO1) has been reported to play an important role in cell death caused by beta-lapachone (beta-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 beta-lap by upregulating NQO1. The cytotoxicity of cisplatin and beta-lap alone or in combination against FSaII fibrosarcoma cells of C3H mice in vitro was determined with a clonogenic survival assay and assessment of gamma-H2AX foci formation, a hallmark of DNA double-strand breaks. The cellular sensitivity to beta-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 beta-lap sensitivity. The role of NQO1 in the cell death caused by beta-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 beta-lap treatment. Combined treatment with cisplatin and beta-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 beta-lap in vitro and in vivo by upregulating NQO1.
[Show abstract][Hide abstract] ABSTRACT: NAD(P)H:quinone oxidoreductase (NQO1) mediates cell death caused by the novel anti-cancer drug beta-lapachone (beta-lap). Therefore, beta-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 beta-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 degrees C for 1 h the expression of NQO1 and the sensitivity of the cells to beta-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.
Full-text · Article · Aug 2009 · International Journal of Hyperthermia