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Combined exposure of mammary and prostate carcinoma cells to UCN-01 and MEK1/2 inhibitors enhances apoptosis. Cells were either treated with vehicle or with ZVAD (20 µM). In parallel, cells were incubated with matched vehicle control (DMSO), with 25 µM PD98059 alone, with 150 nM UCN-01 alone, or with 25 µM PD98059 and 150 nM UCN-01. Cells were either exposed to radiation (2 Gy) or mock irradiated. Portions of cells were taken 24 hours (1440 min) post irradiation and fixed onto slides by cytocentrifuge followed by staining for double stranded DNA breaks as in Methods. Panel A. MDA-MB-231 cells; Panel B. MCF7 cells; Panel C. DU145 cells; Panel D. T47D cells; Panel E. LNCaP cells; Panel F. Time course of apoptosis for MDA-MB-231 and MCF7 cells treated with 25 µM PD98059 and 150 nM UCN-01 (cf Panels A and B). Data shown are the mean number of staining cells from randomly selected fields of fixed cells (n=5 per slide, 3 parallel individual experiments ± SEM) which were examined and counted via flourescent light microscopy. * p < 0.05 greater than control value.
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Recent studies have suggested that inhibition of the mitogen activated protein kinase (MAPK) pathway as well as abrogation of cell cycle check-point control can potentiate the lethal actions of chemotherapeutic drugs and radiation. We therefore investigated the impact of combined exposure to the check-point abrogator (UCN-01) in conjunction with ME...
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... Nonetheless, the combination of UCN-01 with topotecan or cisplatin has shown some preliminary evidence of patient activity (Hotte et al., 2006;Perez et al., 2006). We have noted in a wide variety of tumor cell types that UCN-01 activates the ERK1/2 pathway and that pharmacological or genetic inhibition of the ERK1/2 pathway dramatically potentiates apoptosis and suppresses tumor growth in vivo (Dai et al., 2001McKinstry et al., 2002;Hawkins et al., 2005;Hamed et al., 2008). We have reported previously that the novel CHK1 inhibitor AZD7762 interacts with MEK1/2 inhibitors and farnesyltransferase inhibitors in a manner similar to that of UCN-01 to kill malignant hematopoietic cells in vitro . ...
... Nonetheless, the combination of UCN-01 with topotecan or cisplatin has shown some preliminary evidence of patient activity (Hotte et al., 2006;Perez et al., 2006). We have noted in a wide variety of tumor cell types that UCN-01 activates the ERK1/2 pathway and that pharmacological or genetic inhibition of the ERK1/2 pathway dramatically potentiates apoptosis and suppresses tumor growth in vivo (Dai et al., 2001McKinstry et al., 2002;Hawkins et al., 2005;Hamed et al., 2008). We have reported previously that the novel CHK1 inhibitor AZD7762 interacts with MEK1/2 inhibitors and farnesyltransferase inhibitors in a manner similar to that of UCN-01 to kill malignant hematopoietic cells in vitro . ...
Prior studies have demonstrated that inhibition of CHK1 can promote the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and phosphorylation of histone H2AX and that inhibition of poly(ADP-ribose) polymerase 1 (PARP1) can affect growth factor-induced ERK1/2 activation. The present studies were initiated to determine whether CHK1 inhibitors interacted with PARP1 inhibition to facilitate apoptosis. Transient expression of dominant-negative CHK1 raised basal ERK1/2 activity and prevented CHK1 inhibitors from activating ERK1/2. CHK1 inhibitors modestly increased the levels of PARP1 ADP ribosylation and molecular or small-molecule inhibition of PARP1 blocked CHK1 inhibitor-stimulated histone H2AX phosphorylation and activation of ERK1/2. Stimulated histone H2AX phosphorylation was ataxia telangiectasia-mutated protein-dependent. Multiple CHK1 inhibitors interacted in a greater than additive fashion with multiple PARP1 inhibitors to cause transformed cell-killing in short-term viability assays and synergistically killed tumor cells in colony-formation assays. Overexpression of BCL-xL or loss of BAX/BAK function, but not the function of BID, suppressed CHK1 inhibitor + PARP1 inhibitor lethality. Inhibition of BCL-2 family protein function enhanced CHK1 inhibitor + PARP1 inhibitor lethality and restored drug-induced cell-killing in cells overexpressing BCL-xL. Thus, PARP1 plays an important role in regulating the ability of CHK1 inhibitors to activate ERK1/2 and the DNA damage response. An inability of PARP1 to modulate this response results in transformed cell death mediated through the intrinsic apoptosis pathway.
... Most of the previous studies have focused on the effect of UCN-01 on cell cycle checkpoints, which is widely believed to be its major mode of action in anticancer therapy. Several reports suggest UCN-01 can potentiate apoptosis via the mitochondrial pathway (24)(25)(26). However, the exact mechanism underlying this activity is unclear, and the role of apoptosis in the therapeutic response to UCN-01 remains conjecture. ...
Most targeted anticancer drugs are inhibitors of kinases that are aberrantly activated in cancer cells. However, the mechanisms by which kinase inhibitors suppress tumor growth remain unclear. In this study, we found that UCN-01, a staurosporine analogue and broad-range kinase inhibitor used in clinical trials, inhibits colon cancer cell growth by inducing apoptosis via PUMA, a BH3-only Bcl-2 family member and a p53 target. PUMA expression was markedly elevated in a p53-independent fashion following UCN-01 treatment. The induction of PUMA by UCN-01 was mediated by direct binding of FoxO3a to the PUMA promoter following inhibition of AKT signaling. Deficiency in PUMA abrogated UCN-01-induced apoptosis, caspase activation, and mitochondrial dysfunction, and rendered UCN-01 resistance in a clonogenic assay, whereas elevated PUMA expression or a BH3 mimetic sensitized UCN-01 induced apoptosis. Chemosensitization by UCN-01 seemed to involve simultaneous PUMA induction through both p53-dependent and p53-independent mechanisms. Furthermore, deficiency in PUMA suppressed the antitumor effects of UCN-01 in a xenograft model, concurrent with reduced apoptosis and caspase activation in vivo. These results suggest that PUMA-mediated apoptosis is pivotal for the anticancer activities of UCN-01, and possibly other clinically used kinase inhibitor drugs, and that PUMA manipulation may be useful for improving their anticancer activities.
... Deletion of Chk1 is embryonically lethal and Chk1 activation downstream of ATR/ATM plays a key role in regulating Cdc25C and Cdc2 activity, and thus cell cycle progression, after DNA damage. We discovered that in addition to its established cell cycle regulatory targets; inhibition of Chk1 rapidly lead to activation of ERK1/2 in multiple solid and blood tumor cell types, but not in their nontransformed cell counterparts (Dai et al., 2002;McKinstry et al., 2002). Activation of ERK1/2 was in part dependent on Chk1 inhibitor –induced DNA damage, as inhibition of PARP1 blocked γH2AX and ERK1/2 phosphorylation. ...
... In the Tol et al. study, the addition of the inhibitory ERBB1 antibody cetuximab to bevacizumab + capecitabine + oxaliplatin therapy led to shorter survival of colorectal cancer patients. We have also noted that inhibition of ERBB1 suppresses the toxicity of single agent Chk1 inhibitor treatment (McKinstry et al., 2002). One reason for these findings could be the possibility that ERBB1 signaling not only generates cyto-protective signals but has also been linked to the tyrosine phosphorylation and activation of the CD95 death receptor (Reinehr et al., 2003). ...
Cancer cells contain multiple signal transduction pathways whose activities are frequently elevated due to their transformation, and that are often activated following exposure to established cytotoxic therapies including ionizing radiation and chemical DNA damaging agents. Many pathways activated in response to transformation or toxic stresses promote cell growth and invasion and counteract the processes of cell death. As a result of these findings many drugs, predominantly protein and lipid kinase inhibitors, of varying specificities, have been developed to block signaling by cell survival pathways in the hope of killing tumor cells and sensitizing them to toxic therapies. Unfortunately, due to the plasticity of signaling processes within a tumor cell, inhibition of any one growth factor receptor or signaling pathway frequently has only modest long-term effects on cancer cell viability, tumor growth, and patient survival. As a result of this realization, a greater emphasis has begun to be placed on rational combinations of drugs that simultaneously inhibit multiple inter-linked signal transduction/survival pathways. This, it is hoped, will limit the ability of tumor cells to adapt and survive because the activity within multiple parallel survival signaling pathways has been reduced. This review will discuss some of the approaches that have been taken to combine signal transduction modulatory agents to achieve enhanced tumor cell killing.
... The plasmids to express GFP-tagged human LC3, (his) 6 tagged hamster Grp78/BiP, dominant-negative PERK (Myc-tagged PERKΔC), and human HSP70 promoter linked to luciferase were kindly provided by Dr. S. Spiegel (Virginia Commonwealth University, Richmond, VA), Dr. A. S. Lee (USC/Norris Cancer Center, Los Angeles, CA), Dr. J. A. Diehl (University of Pennsylvania, Philadelphia, PA), and by author S.K.C., respectively. Other reagents and techniques were as described by McKinstry et al. (2002), Fehrenbacher et al. (2004; Carón et al. (2005), and Yacoub et al. (2004). ...
We have further defined mechanism(s) by which 2-amino-N-{4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-phenyl}acetamide [OSU-03012 (OSU)], a derivative of the cyclooxygenase-2 (COX2) inhibitor celecoxib but lacking COX2 inhibitory activity, kills transformed cells. In cells lacking expression of protein kinase R-like endoplasmic reticulum kinase (PERK(-/-)), the lethality of OSU was attenuated. OSU enhanced the expression of Beclin 1 and ATG5 and cleavage of pro-caspase 4 in a PERK-dependent fashion and promoted the Beclin 1- and ATG5-dependent formation of vacuoles containing LC3, followed by a subsequent caspase 4-dependent cleavage of cathepsin B and a cathepsin B-dependent formation of low pH intracellular vesicles; cathepsin B was activated and released into the cytosol and genetic suppression of caspase 4, cathepsin B, or apoptosis-inducing factor function significantly suppressed cell killing. In parallel, OSU caused PERK-dependent increases in 70-kDa heat shock protein (HSP70) expression and decreases in 90-kDa heat shock protein (HSP90) and Grp78/BiP expression. Changes in HSP70 expression were post-transcriptional. Knock-down or small-molecule inhibition of HSP70 expression enhanced OSU toxicity, and overexpression of HSP70 suppressed OSU-induced low pH vesicle formation and lethality. Our data demonstrate that OSU-03012 causes cell killing that is dependent on PERK-induced activation of multiple toxic proteases. OSU-03012 also increased expression of HSP70 in a PERK-dependent fashion, providing support for the contention that OSU-03012-induced PERK signaling promotes both cell survival and cell death processes.
... Many studies have supported the hypothesis that activation of the ERK 1/2 pathway is anti-apoptotic and promotes survival and growth. 42,43 In contrast, some studies have shown pro-apoptotic and growth inhibitory effects that were dependent on MEK/ERK 1/2 (hyper-)activation. 44,45 We used a specific MEK-inhibitor to effectively suppress ERK 1/2 activation and found that the anti-proliferative effects of bilirubin on the HRT-18 cells were abolished, showing that ERK 1/2 was required for growth inhibition. ...
Bilirubin for decades was considered a potentially toxic waste product of heme degradation until the discovery that it is a potent antioxidant. Accumulating data from observations in humans and experimental studies indicate that the bile pigment may be protective against certain diseases. Based on our own observations that bilirubin induces cell cycle arrest in abnormally proliferating vascular smooth muscle cells and clinical observations describing a lesser incidence of cancer in healthy individuals with high normal or slightly elevated serum bilirubin levels, we hypothesized that bilirubin might suppress tumor cell proliferation in vitro and in vivo. As possible effectors we analyzed key proteins that are involved in cell cycle progression and apoptosis. In vivo, tumor growth was assessed in BALB/c nude mice bearing HRT-18 colon cancer xenografts that were treated with bilirubin. In vitro, we investigated the effect of bilirubin on various cell lines and the signaling pathways involved in bilirubin action on tumor cell proliferation in HRT-18 cells using western blots. Bilirubin potently inhibited tumor cell proliferation in vivo and acted cytostatic and pro-apoptotic in vitro. The signaling cascades responsible for this action involved induction of p53, p27, hypophosphorylation of the retinoblastoma tumor suppressor protein as well as caspase activation. These effects were dependent on ERK 1/2. Our study demonstrates that bilirubin may play a role in the defense against cancer by interfering with pro-cancerogenic signaling pathways.
... Recent evidence has also suggested that inhibition of the MAPK pathway sensitizes a broad range of tumor cells to the toxic effects of ionizing radiation and chemotherapeutic drugs (66). Thus, interference with MAPK signaling, and the cell cycle check points in particular, using small-molecule compounds such as the UCN-01, might represent promising strategies for activation of the cell death pathway in prostate cancer (67). ...
Prostate cancer is the most frequently diagnosed cancer among men and the second leading cause of male cancer deaths. Initially, tumor growth is androgen dependent and thus responsive to pharmacologic androgen deprivation, but there is a high rate of treatment failure because the disease evolves in an androgen-independent state. Growing evidence suggests that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade represents a pivotal molecular circuitry participating directly or indirectly in prostate cancer evolution. The crucial role of the protein elements comprising this complex signal transduction network makes them potential targets for pharmacologic interference. Here, we will delineate the current knowledge regarding the involvement of the Ras/MAPK pathway in prostate carcinogenesis, spotlight ongoing research concerning the development of novel targeted agents such as the Ras/MAPK inhibitors in prostate cancer, and discuss the future perspectives of their therapeutic efficacy.
... In previous studies, our group reported that exposure of human leukemia (Dai et al., 2001) and myeloma cells to UCN-01 resulted in a marked activation of MEK1/2/ERK1/2. This phenomenon has also been observed in cell lines derived from human solid tumors including breast, prostate (McKinstry et al., 2002), head and neck squamous cancer (Facchinetti et al., 2004), and recently in an in vivo mouse tumor model independently of tumor p53 status (Hawkins et al., 2005). Furthermore, prevention of MEK1/2/ERK1/2 activation (e.g., by pharmacological MEK1/2 inhibitors) dramatically increases apoptosis. ...
... Our group has reported that exposure of diverse hematopoietic (e.g., leukemia and myeloma) and nonhematopoietic malignant cells to UCN-01 resulted in Chk1 inhibition and a striking induction of the MEK1/2/ERK1/2 cascade. Pharmacological interruption of the former event triggered a pronounced apoptotic response, accompanied by increased Cdc2 dephosphorylation and activation (Dai et al., 2001McKinstry et al., 2002). These findings strongly support the notion that MEK1/2/ERK1/2 activation is a protective compensatory response in cells exposed to UCN-01 and plays a critical role in diminishing UCN-01-related lethality. ...
The functional roles of Cdc2 and checkpoint kinase 1 (Chk1) in synergistic interactions between 7-hydroxystaurosporine (UCN-01) and mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitors [e.g., 2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide (PD184352)] were examined in human multiple myeloma cells in relation to MEK1/2/ERK1/2 activation and lethality. Time course studies revealed that MEK1/2/extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation preceded Cdc2 dephosphorylation (Tyr15) after UCN-01 exposure. Furthermore, enforced expression of Cdc2 or small inducible RNA (siRNA)-mediated Cdc2 knockdown failed to modify ERK1/2 activation status in either the presence or absence of UCN-01, arguing against a causal relationship between these events. However, ectopic expression of Cdc2 sensitized cells to the lethality of UCN-01/MEK inhibitor regimen, whereas Cdc2 knockdown by siRNA significantly diminished the lethal effects of this combination. Conversely, Chk1 knockdown by siRNA enhanced lethality mediated by UCN-01/PD184352. It is interesting that Chk1 knockdown reduced basal ERK1/2 activation and antagonized the ability of UCN-01 to activate ERK1/2. Finally, ectopic expression of constitutively active MEK1 significantly protected cells from the UCN-01/MEK1/2 inhibitor regimen without modifying Cdc2 activation status. Together, these findings indicate that although UCN-01-mediated Chk1 inhibition and Cdc2 activation are unlikely to be responsible for MEK1/2/ERK1/2 activation, both of these events contribute functionally to enhanced lethality in cells coexposed to MEK inhibitors. They also suggest a role for Chk1 in UCN-01-induced ERK1/2 activation, implying the existence of a heretofore unrecognized link between Chk1 and ERK1/2 signaling.
... The present studies demonstrate that in mammary carcinoma cells grown in vitro and in vivo, subsequent exposure of Taxoteretreated cells to a pharmacologic MEK1/2 inhibitor (PD184352 (CI-1040)) potentiates taxane-induced cell killing. 34,35 Similar data were also obtained in vitro using LNCaP prostate and A498 renal carcinoma cells. However, the relationship between ERK1/2 activity and Taxotere exposure is complex and may be cell-type-specific as the survival of PC-3 prostate carcinoma cells was not altered by this drug combination. ...
Taxol (paclitaxel) and Taxotere (docetaxel) are considered as two of the most important anti-cancer chemotherapy drugs. The cytotoxic action of these drugs has been linked to their ability to inhibit microtubule depolymerization, causing growth arrest and subsequent cell death. Studies by a number of laboratories have also linked suppression of MEK1/2 signaling to enhanced Taxol toxicity in vitro and in vivo. The present study examined the interactions of the semi-synthetic taxane Taxotere with MEK1/2 inhibitors in epithelial tumor cells. In vitro colony formation studies demonstrated that Taxotere and the MEK1/2 inhibitor PD184352 interacted in a sequence dependent fashion to synergistically kill human mammary carcinoma cells (MDA-MB-231, MCF7) as well as in other tumor cell types; e.g. prostate and renal cell carcinoma. Athymic mice were implanted in the rear flank with either MDA-MB-231 or MCF7 cells and tumors permitted to form to a volume of approximately 100 mm3 prior to a two day exposure of either Vehicle, PD184352 (25 mg/kg), Taxotere (15 mg/kg) or the drug combination. Tumor volume was measured every other day and tumor growth determined over the following approximately 30 days. Transient exposure of MDA-MB-231 tumors or MCF7 tumors to PD184352 did not significantly alter tumor growth rate or the mean tumor volume in vivo approximately 15-30 days after drug administration. Transient Taxotere exposure of MDA-MB-231 or to a lesser extent MCF7, tumors modestly reduced the mean tumor volume in vivo approximately 15-30 days after drug administration. In contrast, combined treatment with PD184352 and Taxotere significantly reduced MDA-MB-231 and MCF7 tumor growth. The tumor control values for MDA-MB-231 cells and MCF7 cells were 0.43 and 0.71, respectively. Fractionated irradiation of MDA-MB-231 tumors during drug exposure or single dose irradiation prior to drug administration did not significantly further suppress tumor growth beyond that of cells exposed to Taxotere and MEK1/2 inhibitor. Single dose irradiation of tumors after drug exposure, however, caused a significant further suppression of tumor growth below that caused by drug exposure. These findings were also reflected in ex vivo colony formation analyses of isolated tumor cells. Collectively, these findings argue that Taxotere and MEK1/2 inhibitors have the potential to suppress mammary tumor growth in vivo which is enhanced by sequence-dependent exposure to ionizing radiation. Based on the cell lines used in these studies, our findings argue that the interaction of Taxotere and PD184352 is independent of p53 status, estrogen dependency, caspase 3 levels or oncogenic K-RAS expression.
... Numerous other micronutrients have shown cell growth inhibition primarily through negative regulation of the ERK pathway [51]. MEK1/2 inhibitors potentiate apoptosis caused by Tyrphostin AG825 and a check point abrogator UCN-01 in breast and prostate cancer cells [18,52]. Also, ERK inhibition is able to increase radiation induced apoptosis in DU145 cells [53-55]. ...
The biochemistry of the mitogen activated protein kinases ERK, JNK, and p38 have been studied in prostate physiology in an attempt to elucidate novel mechanisms and pathways for the treatment of prostatic disease. We reviewed articles examining mitogen-activated protein kinases using prostate tissue or cell lines. As with other tissue types, these signaling modules are links/transmitters for important pathways in prostate cells that can result in cellular survival or apoptosis. While the activation of the ERK pathway appears to primarily result in survival, the roles of JNK and p38 are less clear. Manipulation of these pathways could have important implications for the treatment of prostate cancer and benign prostatic hypertrophy.
