Protease-activated receptor-1 activation of endothelial cells induces protein kinase Calpha-dependent phosphorylation of syntaxin 4 and Munc18c: role in signaling p-selectin expression

Endothelial cells exhibit regulated exocytosis in response to inflammatory mediators such as thrombin and histamine. The exocytosis of Weibel-Palade bodies (WPBs) containing von Willebrand factor, P-selectin, and interleukin-8 within minutes after stimulation is important for vascular homeostasis. SNARE proteins are key components of the exocytic machinery in neurons and some secretory cells, but their role in regulating exocytosis in endothelial cells is not well understood. We examined the function of SNARE proteins in mediating exocytosis of WPBs in endothelial cells. We identified the presence of syntaxin 4, syntaxin 3, and the high affinity syntaxin 4-regulatory protein Munc18c in human lung microvascular endothelial cells. Small interfering RNA-induced knockdown of syntaxin 4 (but not of syntaxin 3) inhibited exocytosis of WPBs as detected by the reduction in thrombin-induced cell surface P-selectin expression. Thrombin ligation of protease-activated receptor-1 activated the phosphorylation of syntaxin 4 and Munc18c, which, in turn, disrupted the interaction between syntaxin 4 and Munc18. Protein kinase Calpha activation was required for the phosphorylation of syntaxin 4 and Munc18c as well as the cell surface expression of P-selectin. We also observed that syntaxin 4 knockdown inhibited the adhesion of neutrophils to thrombin-activated endothelial cells, demonstrating the functional role of syntaxin 4 in promoting endothelial adhesivity. Thus, protease-activated receptor-1-induced protein kinase Calpha activation and phosphorylation of syntaxin 4 and Munc18c are required for the cell surface expression of P-selectin and the consequent binding of neutrophils to endothelial cells.     

Munc18-1 is critical for plasma membrane localization of syntaxin1 but not of SNAP-25 in PC12 cells

Although Munc18-1 was originally identified as a syntaxin1–interacting protein, the physiological significance of this interaction remains unclear. In fact, recent studies of Munc18-1 mutants have suggested that Munc18-1 plays a critical role for docking of secretory vesicles, independent of syntaxin1 regulation. Here we investigated the role of Munc18-1 in syntaxin1 localization by generating stable neuroendocrine cell lines in which Munc18-1 was strongly down-regulated. In these cells, the secretion capability, as well as the docking of dense-core vesicles, was significantly reduced. More importantly, not only was the expression level of syntaxin1 reduced, but the localization of syntaxin1 at the plasma membrane was also severely perturbed. The mislocalized syntaxin1 resided primarily in the perinuclear region of the cells, in which it was highly colocalized with Secretogranin II, a marker protein for dense-core vesicles. In contrast, the expression level and the plasma membrane localization of SNAP-25 were not affected. Furthermore, the syntaxin1 localization and the secretion capability were restored upon transfection-mediated reintroduction of Munc18-1. Our results indicate that endogenous Munc18-1 plays a critical role for the plasma membrane localization of syntaxin1 in neuroendocrine cells and therefore necessitates the interpretation of Munc18-1 mutant phenotypes to be in terms of mislocalized syntaxin1.     

Radiosensitizing effect of gold nanoparticle loaded with small interfering RNA-SP1 on lung cancer: AuNPs-si-SP1 regulates GZMB for radiosensitivity

Radioresistance is a major challenge that largely limits the efficacy of radiotherapy in lung cancer. Gold nanoparticles (AuNPs) are emerging as novel radiosensitizers for cancer patients. Therefore, this study was designed to explore the radiosensitizing effect and mechanism of AuNPs loaded with small interfering RNA (siRNA)-SP1 (AuNPs-si-SP1) on lung cancer. AuNPs-si-SP1 was prepared by the noncovalent binding between AuNPs and siRNA-SP1. The adsorption capacity of AuNPs to siRNA-SP1 was analyzed by gel electrophoresis. The cell uptake of AuNPs-si-SP1 was observed under a laser confocal microscopy. Silencing efficacy of AuNPs-si-SP1 was validated by RT-qPCR and Western blot analysis. Cell viability was determined by CCK-8 assay, radiosensitization by plate colony formation assay, cell apoptosis and cell cycle by flow cytometry, and DNA double strand breaks by immunofluorescence in the presence or absence of AuNPs-si-SP1 or GZMB. The downstream mechanism of SP1 was predicted by bioinformatics analysis, followed by verification by Western blot analysis. Subcutaneous tumorigenesis in nude mice was established to verify the radiosensitization of AuNPs-si-SP1 and GZMB in vivo. AuNPs-si-SP1 effectively absorbed SP1 siRNA and was highly internalized by A549 cells to reduce SP1 protein expression. AuNPs-si-SP1 or GZMB overexpression promoted cells to G2/M phase, DNA double strand breaks, and enhanced radiosensitivity. SP1 could repress GZMB expression in lung cancer cells. In vivo experiments manifested that AuNPs-si-SP1 could inhibit the growth of solid tumor in nude mice to achieve radiosensitization by inhibiting SP1 to upregulate GZMB. AuNPs-si-SP1 might increase the radiosensitivity of lung cancer by inhibiting SP1 to upregulate GZMB.     

Radiosensitizing effect of gold nanoparticle loaded with small interfering RNA-SP1 on lung cancer: AuNPs-si-SP1 regulates GZMB for radiosensitivity

Radioresistance is a major challenge that largely limits the efficacy of radiotherapy in lung cancer. Gold nanoparticles (AuNPs) are emerging as novel radiosensitizers for cancer patients. Therefore, this study was designed to explore the radiosensitizing effect and mechanism of AuNPs loaded with small interfering RNA (siRNA)-SP1 (AuNPs-si-SP1) on lung cancer. AuNPs-si-SP1 was prepared by the noncovalent binding between AuNPs and siRNA-SP1. The adsorption capacity of AuNPs to siRNA-SP1 was analyzed by gel electrophoresis. The cell uptake of AuNPs-si-SP1 was observed under a laser confocal microscopy. Silencing efficacy of AuNPs-si-SP1 was validated by RT-qPCR and Western blot analysis. Cell viability was determined by CCK-8 assay, radiosensitization by plate colony formation assay, cell apoptosis and cell cycle by flow cytometry, and DNA double strand breaks by immunofluorescence in the presence or absence of AuNPs-si-SP1 or GZMB. The downstream mechanism of SP1 was predicted by bioinformatics analysis, followed by verification by Western blot analysis. Subcutaneous tumorigenesis in nude mice was established to verify the radiosensitization of AuNPs-si-SP1 and GZMB in vivo. AuNPs-si-SP1 effectively absorbed SP1 siRNA and was highly internalized by A549 cells to reduce SP1 protein expression. AuNPs-si-SP1 or GZMB overexpression promoted cells to G2/M phase, DNA double strand breaks, and enhanced radiosensitivity. SP1 could repress GZMB expression in lung cancer cells. In vivo experiments manifested that AuNPs-si-SP1 could inhibit the growth of solid tumor in nude mice to achieve radiosensitization by inhibiting SP1 to upregulate GZMB. AuNPs-si-SP1 might increase the radiosensitivity of lung cancer by inhibiting SP1 to upregulate GZMB.     

Perspectives and mechanisms for targeting mitotic catastrophe in cancer treatment

Mitotic catastrophe is distinct from other cell death modes due to unique nuclear alterations characterized as multi and/or micronucleation. Mitotic catastrophe is a common and virtually unavoidable consequence during cancer therapy. However, a comprehensive understanding of mitotic catastrophe remains lacking. Herein, we summarize the anticancer drugs that induce mitotic catastrophe, including microtubule-targeting agents, spindle assembly checkpoint kinase inhibitors, DNA damage agents and DNA damage response inhibitors. Based on the relationships between mitotic catastrophe and other cell death modes, we thoroughly evaluated the roles played by mitotic catastrophe in cancer treatment as well as its advantages and disadvantages. Some strategies for overcoming its shortcomings while fully utilizing its advantages are summarized and proposed in this review. We also review how mitotic catastrophe regulates cancer immunotherapy. These summarized findings suggest that the induction of mitotic catastrophe can serve as a promising new therapeutic approach for overcoming apoptosis resistance and strengthening cancer immunotherapy.     

Targeting of cancer cell death mechanisms by resveratrol: a review

Cancer cell death is the utmost aim in cancer therapy. Anti-cancer agents can induce apoptosis, mitotic catastrophe, senescence, or autophagy through the production of free radicals and induction of DNA damage. However, cancer cells can acquire some new properties to adapt to anti-cancer agents. An increase in the incidence of apoptosis, mitotic catastrophe, senescence, and necrosis is in favor of overcoming tumor resistance to therapy. Although an increase in the autophagy process may help the survival of cancer cells, some studies indicated that stimulation of autophagy cell death may be useful for cancer therapy. Using some low toxic agents to amplify cancer cell death is interesting for the eradication of clonogenic cancer cells. Resveratrol (a polyphenol agent) may affect various signaling pathways related to cell death. It can induce death signals and also downregulate the expression of anti-apoptotic genes. Resveratrol has also been shown to modulate autophagy and induce mitotic catastrophe and senescence in some cancer cells. This review focuses on the important targets and mechanisms for the modulation of cancer cell death by resveratrol.

     

Inhibitors of phosphatidylinositol 3'-kinases promote mitotic cell death in HeLa cells

The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in many biological processes, including cell cycle progression, cell growth, survival, actin rearrangement and migration, and intracellular vesicular transport. However, the involvement of the PI3K pathway in the regulation of mitotic cell death remains unclear. In this study, we treated HeLa cells with the PI3K inhibitors, 3-methyladenine (3-MA, as well as a widely used autophagy inhibitor) and wortmannin to examine their effects on cell fates using live cell imaging. Treatment with 3-MA decreased cell viability in a time- and dose-dependent manner and was associated with caspase-3 activation. Interestingly, 3-MA-induced cell death was not affected by RNA interference-mediated knockdown (KD) of beclin1 (an essential protein for autophagy) in HeLa cells, or by deletion of atg5 (an essential autophagy gene) in mouse embryonic fibroblasts (MEFs). These data indicate that cell death induced by 3-MA occurs independently of its ability to inhibit autophagy. The results from live cell imaging studies showed that the inhibition of PI3Ks increased the occurrence of lagging chromosomes and cell cycle arrest and cell death in prometaphase. Furthermore, PI3K inhibitors promoted nocodazole-induced mitotic cell death and reduced mitotic slippage. Overexpression of Akt (the downstream target of PI3K) antagonized PI3K inhibitor-induced mitotic cell death and promoted nocodazole-induced mitotic slippage. These results suggest a novel role for the PI3K pathway in regulating mitotic progression and preventing mitotic cell death and provide justification for the use of PI3K inhibitors in combination with anti-mitotic drugs to combat cancer.     

Radiosensitization of glioma cells by modulation of Met signalling with the hepatocyte growth factor neutralizing antibody,AMG102

The hepatocyte growth factor (HGF)/Met signalling pathway is up‐regulated in many cancers, with downstream mediators playing a role in DNA double strand break repair. Previous studies have shown increased radiosensitization of tumours through modulation of Met signalling by genetic methods. We investigated the effects of the anti‐HGF monoclonal antibody, AMG102, on the response to ionizing radiation in a model of glioblastoma multiforme in vitro and in vivo. Radiosensitivity was evaluated in vitro in the U‐87 MG human glioma cell line. Met activation was measured by Western blot, and the effect on survival following radiation was evaluated by clonogenic assay. Mechanism of cell death was evaluated by apoptosis and mitotic catastrophe assays. DNA damage was quantitated by γH2AX foci and neutral comet assay. Growth kinetics of subcutaneous tumours was used to assess the effects of AMG102 on in vivo tumour radiosensitivity. AMG102 inhibited Met activation after irradiation. An enhancement of radiation cell killing was shown with no toxicity using drug alone. Retention of γH2AX foci at 6 and 24 hrs following the drug/radiation combination indicated an inhibition of DNA repair following radiation, and comet assay confirmed DNA damage persisting over the same duration. At 48 and 72 hrs following radiation, a significant increase of cells undergoing mitotic catastrophe was seen in the drug/radiation treated cells. Growth of subcutaneous tumours was slowed in combination treated mice, with an effect that was greater than additive for each modality individually. Modulation of Met signalling with AMG102 may prove a novel radiation sensitizing strategy. Our data indicate that DNA repair processes downstream of Met are impaired leading to increased cell death through mitotic catastrophe.     

Peroxiredoxin II restrains DNA damage-induced death in cancer cells by positively regulating JNK-dependent DNA repair

The 2-Cys peroxiredoxins (Prx) belong to a family of antioxidant enzymes that detoxify reactive oxygen and nitrogen species and are distributed throughout the intracellular and extracellular compartments. However, the presence and role of 2-Cys Prxs in the nucleus have not been studied. This study demonstrates that the PrxII located in the nucleus protects cancer cells from DNA damage-induced cell death. Although the two cytosolic 2-Cys Prxs, PrxI and PrxII, were found in the nucleus, only PrxII knockdown selectively and markedly increased cell death in the cancer cells treated with DNA-damaging agents. The increased death was completely reverted by the nuclearly targeted expression of PrxII in an activity-independent manner. Furthermore, the antioxidant butylated hydroxyanisole did not influence the etoposide-induced cell death. Mechanistically, the knockdown of Prx II expression impaired the DNA repair process by reducing the activation of the JNK/c-Jun pathway. These results suggest that PrxII is likely to be attributed to a tumor survival factor positively regulating JNK-dependent DNA repair with its inhibition possibly sensitizing cancer cells to chemotherapeutic agents.     

Targeting Chk2 improves gastric cancer chemotherapy by impairing DNA damage repair

Our results demonstrate that the addition of cisplatin after paclitaxel-induced mitotic arrest was more effective than individual treatment on gastric adenocarcinoma cells (MKN45). However, the treatment did not induce benefits in cells derived from lymph node metastasis (ST2957). Time-lapse microscopy revealed that cell death was caused by mitotic catastrophe and apoptosis induction, as the use of the caspase inhibitor z-VAD-fmk decreased cell death. We propose that the molecular mechanism mediating this cell fate is a slippage suffered by these cells, given that our Western blot (WB) analysis revealed premature cyclin B degradation. This resulted in the cell exiting from mitosis without undergoing DNA damage repair, as demonstrated by the strong phosphorylation of H2AX. A comet assay indicated that DNA repair was impaired, and Western blotting showed that the Chk2 protein was degraded after sequential treatment (paclitaxel-cisplatin). Based on these results, the modulation of cell death during mitosis may be an effective strategy for gastric cancer therapy.     

The red wine component ellagic acid induces autophagy and exhibits anti‐lung cancer activity in vitro and in vivo

Red wine consists of a large amount of compounds such as resveratrol, which exhibits chemopreventive and therapeutic effects against several types of cancers by targeting cancer driver molecules. In this study, we tested the anti‐lung cancer activity of 11 red wine components and reported that a natural polyphenol compound ellagic acid (EA) inhibited lung cancer cell proliferation at an efficacy approximately equal to that of resveratrol. EA markedly increased the expression of the autophagosomal marker LC3‐II as well as inactivation of the mechanistic target of rapamycin signalling pathway. EA elevated autophagy‐associated cell death by down‐regulating the expression of cancerous inhibitor of protein phosphatase 2A (CIP2A), and CIP2A overexpression attenuated EA‐induced autophagy of lung cancer cells. Treating tumour‐bearing mice with EA resulted in significant inhibition of tumour growth with suppression of CIP2A levels and increased autophagy. In addition, EA potentiated the inhibitory effects of the natural compound celastrol on lung cancer cells in vitro and in vivo by enhancing autophagy and down‐regulating CIP2A. These findings indicate that EA may be a promising chemotherapeutic agent for lung cancer, and that the combination of EA and celastrol may have applicability for the treatment of this disease.     

Effects of redox modulation by inhibition of thioredoxin reductase on radiosensitivity and gene expression

The thioredoxin system is a promising target when aiming to overcome the problem of clinical radiation resistance. Altered cellular redox status and redox sensitive thiols contributing to induction of resistance strongly connect the ubiquitous redox enzyme thioredoxin reductase (TrxR) to the cellular response to ionizing radiation. To further investigate possible strategies in combating clinical radiation resistance, human radio-resistant lung cancer cells were subjected to a combination of single fractions of γ-radiation at clinically relevant doses and non-toxic levels of a well-characterized thioredoxin reductase inhibitor, the phosphine gold(I) compound [Au(SCN)(PEt(3))]. The combination of the TrxR-inhibitor and ionizing radiation reduced the surviving fractions and impaired the ability of the U1810 cells to repopulate by approximately 50%. In addition, inhibition of thioredoxin reductase caused changes in the cell cycle distribution, suggesting a disturbance of the mitotic process. Global gene expression analysis also revealed clustered genetic expression changes connected to several major cellular pathways such as cell cycle, cellular response to stress and DNA damage. Specific TrxR-inhibition as a factor behind the achieved results was confirmed by correlation of gene expression patterns between gold and siRNA treatment. These results clearly demonstrate TrxR as an important factor conferring resistance to irradiation and the use of [Au(SCN)(PEt(3))] as a promising radiosensitizing agent.     

Identification of differentially expressed genes contributing to radioresistance in lung cancer cells using microarray analysis

Radiotherapy has played a key role in the control of tumor growth in many cancer patients. It is usually difficult to determine what fraction of the tumor cell population is radioresistant after a course of radiotherapy. The response of tumor cells to radiation is believed to be accompanied by complex changes in the gene expression pattern. It may be possible to use these to sensitize radioresistant tumor cells and improve radiocurability. Based on the biological effects of ionizing radiation, in the present study, we developed one oligonucleotide microarray to analyze the expression of 143 genes in cells of two lung cancer cell lines with different radiosensitivities. Compared to NCI-H446 cells, expression of 18 genes significantly increased the basal levels in the radioresistant A549 cells, in which eight genes were up-regulated and 10 genes were down-regulated. In A549 cells irradiated with 5 Gy, 22 (19 up-regulated and three down-regulated) and 26 (eight up-regulated and 18 down-regulated) differentially expressed genes were found 6 and 24 h after irradiation, respectively. In NCI-H446 cells, the expression of 17 (nine up-regulated and eight down-regulated) and 18 (six up-regulated and 12 down-regulated) genes was altered 6 and 24 h after irradiation, respectively. RT-PCR was performed, and we found that MDM2, BCL2, PKCZ and PIM2 expression levels were increased in A549 cells and decreased in NCI-H446 cells after irradiation. Genes involved in DNA repair, such as XRCC5, ERCC5, ERCC1, RAD9A, ERCC4 and the gene encoding DNA-PK, were found to be increased to a higher level in A549 cells than in NCI-H446 cells. Antisense suppression of MDM2 resulted in increased radiosensitivity of A549 cells. Taken together, these results demonstrate the possibility that a group of genes involved in DNA repair, regulation of the cell cycle, cell proliferation and apoptosis is responsible for the different radioresistance of these two lung cancer cells. This list of genes may be useful in attempts to sensitize the radioresistant lung cancer cells.     

Effect of separase depletion on ionizing radiation-induced cell cycle checkpoints and survival in human lung cancer cell lines

Abstract.   Objectives : This study is to evaluate the effect of separase depletion on cell cycle progression of irradiated and non-irradiated cells through the G₂/M phases and consecutive cell survival. Materials and methods : Separase was depleted with siRNA in two human non-small cell lung carcinoma (NSCLC) cell lines. Cell cycle progression, mitotic fraction, DNA repair, apoptotic and clonogenic cell death were determined. Results : By depletion of endogenous separase with siRNA in NSCLCs, we showed that separase affects progression through the G₂ phase. In non-irradiated exponentially growing cells, separase depletion led to an increased G₂ accumulation from 17.2% to 29.1% in H460 and from 15.7% to 30.9% in A549 cells and a decrease in mitotic cells. Depletion of separase significantly ( P <  0.01) increased the fraction of radiation-induced G₂ arrested cells 30–56 h after irradiation and led to decrease in the mitotic fraction. This was associated with increased double-strand break repair as measured by γ-H2AX foci kinetics in H460 cells and to a lesser extent in A549 cells. In addition, a decrease in the expression of mitotic linked cell death after irradiation was found. Conclusions : These results indicate that separase has additional targets involved in regulation of G₂ to M progression after DNA damage. Prolonged G₂ phase arrest in the absence of separase has consequences on repair of damaged DNA and cell death.     

PUL21a-Cyclin A2 Interaction is Required to Protect Human Cytomegalovirus-Infected Cells from the Deleterious Consequences of Mitotic Entry

Entry into mitosis is accompanied by dramatic changes in cellular architecture, metabolism and gene expression. Many viruses have evolved cell cycle arrest strategies to prevent mitotic entry, presumably to ensure sustained, uninterrupted viral replication. Here we show for human cytomegalovirus (HCMV) what happens if the viral cell cycle arrest mechanism is disabled and cells engaged in viral replication enter into unscheduled mitosis. We made use of an HCMV mutant that, due to a defective Cyclin A2 binding motif in its UL21a gene product (pUL21a), has lost its ability to down-regulate Cyclin A2 and, therefore, to arrest cells at the G1/S transition. Cyclin A2 up-regulation in infected cells not only triggered the onset of cellular DNA synthesis, but also promoted the accumulation and nuclear translocation of Cyclin B1-CDK1, premature chromatin condensation and mitotic entry. The infected cells were able to enter metaphase as shown by nuclear lamina disassembly and, often irregular, metaphase spindle formation. However, anaphase onset was blocked by the still intact anaphase promoting complex/cyclosome (APC/C) inhibitory function of pUL21a. Remarkably, the essential viral IE2, but not the related chromosome-associated IE1 protein, disappeared upon mitotic entry, suggesting an inherent instability of IE2 under mitotic conditions. Viral DNA synthesis was impaired in mitosis, as demonstrated by the abnormal morphology and strongly reduced BrdU incorporation rates of viral replication compartments. The prolonged metaphase arrest in infected cells coincided with precocious sister chromatid separation and progressive fragmentation of the chromosomal material. We conclude that the Cyclin A2-binding function of pUL21a contributes to the maintenance of a cell cycle state conducive for the completion of the HCMV replication cycle. Unscheduled mitotic entry during the course of the HCMV replication has fatal consequences, leading to abortive infection and cell death.     

Adenovirus E4orf4 Protein-Induced Death of p53?/? H1299 Human Cancer Cells Follows a G1 Arrest of both Tetraploid and Diploid Cells due to a Failure To Initiate DNA Synthesis

The adenovirus E4orf4 protein selectively kills human cancer cells independently of p53 and thus represents a potentially promising tool for the development of novel antitumor therapies. Previous studies suggested that E4orf4 induces an arrest or a delay in mitosis and that both this effect and subsequent cell death rely largely on an interaction with the B55 regulatory subunit of protein phosphatase 2A. In the present report, we show that the death of human H1299 lung carcinoma cells induced by expression of E4orf4 is typified not by an accumulation of cells arrested in mitosis but rather by the presence of both tetraploid and diploid cells that are arrested in G₁ because they are unable to initiate DNA synthesis. We believe that these E4orf4-expressing cells eventually die by various processes, including those resulting from mitotic catastrophe.     

Curcumin affects components of the chromosomal passenger complex and induces mitotic catastrophe in apoptosis-resistant Bcr-Abl-expressing cells

The Bcr-Abl oncoprotein plays a major role in the development and progression of chronic myeloid leukemia and is a determinant of chemotherapy resistance occurring during the blast crisis phase of the disease. The aim of this article was to investigate the possibility of combating the resistance to apoptosis caused by Bcr-Abl by inducing an alternative cell death process. As a model of chronic myeloid leukemia, we employed Bcr-Abl-transfected mouse progenitor 32D cells with low and high Bcr-Abl expression levels corresponding to drug-sensitive and drug-resistant cells, respectively. The drug curcumin (diferuloylmethane), a known potent inducer of cell death in many cancer cells, was investigated for efficacy with Bcr-Abl-expressing cells. Curcumin strongly inhibited cell proliferation and affected cell viability by inducing apoptotic symptoms in all tested cells; however, apoptosis was a relatively late event. G(2)-M cell cycle arrest, together with increased mitotic index and cellular and nuclear morphology resembling those described for mitotic catastrophe, was observed and preceded caspase-3 activation and DNA fragmentation. Mitosis-arrested cells displayed abnormal chromatin organization, multipolar chromosome segregation, aberrant cytokinesis, and multinucleated cells-morphologic changes typical of mitotic catastrophe. We found that the mitotic cell death symptoms correlated with attenuated expression of survivin, a member of the chromosomal passenger complex, and mislocalization of Aurora B, the partner of survivin in the chromosomal passenger complex. Inhibition of survivin expression with small interfering RNA exhibited similar mitotic disturbances, thus implicating survivin as a major, albeit not the only, target for curcumin action. This study shows that curcumin can overcome the broad resistance to cell death caused by expression of Bcr-Abl and suggests that curcumin may be a promising agent for new combination regimens for drug-resistant chronic myeloid leukemia.     

MK-8776, a novel Chk1 inhibitor,exhibits an improved radiosensitizing effect compared to UCN-01 by exacerbating radiation-induced aberrant mitosis

Checkpoint kinase 1 (Chk1) is an evolutionarily conserved serine/threonine kinase that plays an important role in G₂/M checkpoint signaling. Here, we evaluate the radiosensitizing effects of a novel selective Chk1 inhibitor MK-8776, comparing its efficacy with a first-generation Chk1 inhibitor UCN-01, and attempt to elucidate the mechanism of radiosensitization. In a clonogenic survival assay, MK-8776 demonstrated a more pronounced radiosensitizing effect than UCN-01, with lower cytotoxicity. Importantly, radiosensitization by MK-8776 can be achieved at doses as low as 2.5 Gy, which is a clinically applicable irradiation dose. MK-8776, but not UCN-01, exacerbated mitotic catastrophe (MC) and centrosome abnormalities, without affecting repair kinetics of DNA double strand breaks. Furthermore, live-cell imaging revealed that MK-8776 significantly abrogated the radiation-induced G₂/M checkpoint, prolonged the mitotic phase, and enhanced aberrant mitosis. This suggests that Chk1 inhibition by MK-8776 activates a spindle assembly checkpoint and increases mitotic defects in irradiated EMT6 cells. In conclusion, we have shown that, at minimally toxic concentrations, MK-8776 enhances radiation-induced cell death through the enhancement of aberrant mitosis and MC, without affecting DNA damage repair.