CGK733

CGK733 is an inhibitor of kinase ATM and ATR with IC50 value of ~200 nM [1].
Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase which plays pivotal role in DNA repair and cell cycle checkpoints. And when in hypoxia circumstance, loss of ATM-related (ATR) kinase induces the decreasing of HIF-1 DNA binding, which consequently affects GLUT-1 and CAIX expression in the protein level. The components of ATM and ATR regulated signaling pathways which enhances cellular sensitivity to chemo- and radiotherapy thus provide attractive pharmacological targets [2, 3].
CGK733 selectively inhibited ATM and ATR kinase activities and blocked their checkpoint signaling pathways. Treated HEK-293 cells with GSK733 would increase the sensitivity of cells to radiotherapy [1]. When tested with HBV-positive HCC cell line HepG2.2.15, CGK733 treatment significantly increased the cells sensitivity to taxol via inducing the formation of multinucleated cells [2]. Treated with CGK733, senescent breast, lung, and colon carcinoma cells were induced to undergo cell death [4]. CGK733 treated tumor cells could enhance the sensitivity to radio therapy via inhibiting DNA repair and cell cycle checkpoints of tumor cells [5]. In MCF-7 breast cancer cells, CGK733 treatment induced detectable decline levels of cyclin D1 protein and reduced phosphorylated and total retinoblastoma protein (RB) which inhibited cell proliferation [3].
References:
[1].    Williams, T.M., et al., Molecular imaging of the ATM kinase activity. Int J Radiat Oncol Biol Phys, 2013. 86(5): p. 969-77.
[2].    Wang, H., et al., CGK733 enhances multinucleated cell formation and cytotoxicity induced by taxol in Chk1-deficient HBV-positive hepatocellular carcinoma cells. Biochem Biophys Res Commun, 2012. 422(1): p. 103-8.
[3].    Alao, J.P. and P. Sunnerhagen, The ATM and ATR inhibitors CGK733 and caffeine suppress cyclin D1 levels and inhibit cell proliferation. Radiat Oncol, 2009. 4(51): p. 4-51.
[4].    Crescenzi, E., et al., Ataxia telangiectasia mutated and p21CIP1 modulate cell survival of drug-induced senescent tumor cells: implications for chemotherapy. Clin Cancer Res, 2008. 14(6): p. 1877-87.
[5].    Kuroda, S., Y. Urata, and T. Fujiwara, Ataxia-telangiectasia mutated and the Mre11-Rad50-NBS1 complex: promising targets for radiosensitization. Acta Med Okayama, 2012. 66(2): p. 83-92.

PERK/CHOP contributes to the CGK733-induced vesicular calcium sequestration which is accompanied by non-apoptotic cell death.[Pubmed:26259235]

Oncotarget. 2015 Sep 22;6(28):25252-65.

Calcium ions (Ca(2+)) are indispensable for the physiology of organisms and the molecular regulation of cells. We observed that CGK733, a synthetic chemical substance, induced non-apoptotic cell death and stimulated reversible calcium sequestration by vesicles in pancreatic cancer cells. The endoplasmic reticulum (ER) stress eukaryotic translation initiation factor 2-alpha kinase 3/C/EBP homologous protein (PERK/CHOP) signaling pathway was shown to be activated by treatment with CGK733. Ionomycin, an ER stress drug and calcium ionophore, can activate PERK/CHOP signaling and accelerate CGK733-induced calcium sequestration. Knockdown of CHOP diminished CGK733-induced vesicular calcium sequestration, but had no effects on the cell death. Proteomic analysis demonstrated that the ER-located calcium-binding proteins, calumenin and protein S100-A11, were altered in CGK733-treated cells compared to non-treated controls. Our study reveals that CGK733-induced intracellular calcium sequestration is correlated with the PERK/CHOP signaling pathway and may also be involved in the dysregulations of calcium-binding proteins.

CGK733-induced LC3 II formation is positively associated with the expression of cyclin-dependent kinase inhibitor p21Waf1/Cip1 through modulation of the AMPK and PERK/CHOP signaling pathways.[Pubmed:26486079]

Oncotarget. 2015 Nov 24;6(37):39692-701.

Microtubule-associated protein 1A/1B-light chain 3 (LC3)-II is essential for autophagosome formation and is widely used to monitor autophagic activity. We show that CGK733 induces LC3 II and LC3-puncta accumulation, which are not involved in the activation of autophagy. The treatment of CGK733 did not alter the autophagic flux and was unrelated to p62 degradation. Treatment with CGK733 activated the AMP-activated protein kinase (AMPK) and protein kinase RNA-like endoplasmic reticulum kinase/CCAAT-enhancer-binding protein homologous protein (PERK/CHOP) pathways and elevated the expression of p21Waf1/Cip1. Inhibition of both AMPK and PERK/CHOP pathways by siRNA or chemical inhibitor could block CGK733-induced p21Waf1/Cip1 expression as well as caspase-3 cleavage. Knockdown of LC3 B (but not LC3 A) abolished CGK733-triggered LC3 II accumulation and consequently diminished AMPK and PERK/CHOP activity as well as p21Waf1/Cip1 expression. Our results demonstrate that CGK733-triggered LC3 II formation is an initial event upstream of the AMPK and PERK/CHOP pathways, both of which control p21Waf1/Cip1 expression.

CGK733 enhances multinucleated cell formation and cytotoxicity induced by taxol in Chk1-deficient HBV-positive hepatocellular carcinoma cells.[Pubmed:22564734]

Biochem Biophys Res Commun. 2012 May 25;422(1):103-8.

Hepatocellular carcinoma (HCC) is one of the most deadly human cancers. Chronic hepatitis B virus (HBV) infection is one of the predominant risk factors associated with the development of HCC and complicates the treatment of HCC. In this study, we demonstrate that a HBV-positive HCC cell line HepG2.2.15, was more resistant to chemotherapy agents than its parental HBV-negative cell line HepG2. HBV-positive HCC cells exhibited defective Chk1 phosphorylation and increased chromosomal instability. CGK733, a small molecule inhibitor reportedly targeting the kinase activities of ATM and ATR, significantly enhanced taxol-induced cytotoxicity in HBV-positive HepG2.2.15 cells. The mechanism lies in CGK733 triggers the formation of multinucleated cells thus promotes the premature mitotic exit of taxol-induced mitotic-damaged cells through multinucleation and mitotic catastrophe in HBV-positive HepG2.2.15 cells. These results suggest that CGK733 could potentially reverse the taxol resistance in HBV-positive HCC cells and may suggest a novel strategy to treat HBV-infected HCC patients.

The ATM and ATR inhibitors CGK733 and caffeine suppress cyclin D1 levels and inhibit cell proliferation.[Pubmed:19903334]

Radiat Oncol. 2009 Nov 10;4:51.

The ataxia telangiectasia mutated (ATM) and the ATM- related (ATR) kinases play a central role in facilitating the resistance of cancer cells to genotoxic treatment regimens. The components of the ATM and ATR regulated signaling pathways thus provide attractive pharmacological targets, since their inhibition enhances cellular sensitivity to chemo- and radiotherapy. Caffeine as well as more specific inhibitors of ATM (KU55933) or ATM and ATR (CGK733) have recently been shown to induce cell death in drug-induced senescent tumor cells. Addition of these agents to cancer cells previously rendered senescent by exposure to genotoxins suppressed the ATM mediated p21 expression required for the survival of these cells. The precise molecular pharmacology of these agents however, is not well characterized. Herein, we report that caffeine, CGK733, and to a lesser extent KU55933, inhibit the proliferation of otherwise untreated human cancer and non-transformed mouse fibroblast cell lines. Exposure of human cancer cell lines to caffeine and CGK733 was associated with a rapid decline in cyclin D1 protein levels and a reduction in the levels of both phosphorylated and total retinoblastoma protein (RB). Our studies suggest that observations based on the effects of these compounds on cell proliferation and survival must be interpreted with caution. The differential effects of caffeine/CGK733 and KU55933 on cyclin D1 protein levels suggest that these agents will exhibit dissimilar molecular pharmacological profiles.

Role of polyamines in p53-dependent apoptosis of intestinal epithelial cells.[Pubmed:19136059]

Cell Signal. 2009 Apr;21(4):509-22.

Although p53 is known to play a critical role in the proliferation of gastrointestinal epithelia, the role of the Mdm2/p53 pathway in response to inducers of apoptosis in intestinal epithelial cells is unknown. Our data show that camptothecin (CPT)-induced apoptosis correlated with increased p53, p21Cip1, and Mdm2 protein levels, with a simultaneous increase in ATR Ser428, p53 Ser15 and Mdm2 Ser166 phosphorylation in IEC-6 cells. Increased p53 levels and its phosphorylation increased Bax protein, caspase-9, -3 activation and apoptosis. However, TNF-alpha/CHX-mediated apoptosis was independent of p53 protein levels and phosphorylation. The translation inhibitor, cycloheximide (CHX), prevented CPT-induced apoptosis. CHX completely prevented CPT-induced p53 phosphorylation and synthesis of p21Cip1, Bax and Bcl-xL proteins without altering p53 levels. The p53 activator, RITA, augmented CPT-induced apoptosis. The Mdm2 antagonist, Nutlin-3, significantly increased apoptosis, which was accompanied by increased p53, Mdm2 and p21Cip1 protein levels. The ATM/ATR kinase inhibitor, CGK733, blocked CPT-induced p53 Ser15 phosphorylation and protected cells from CPT-induced apoptosis. Inhibition of ornithine decarboxylase (ODC) with alpha-difluromethylornithine (DFMO) and subsequent depletion of intracellular polyamines increased p53 protein, Mdm2 Ser166 phosphorylation and conferred resistance to CPT-induced apoptosis. However, polyamine depletion had no effect on p53 phosphorylation. Nutlin-3 reversed the protective effect of DFMO and sensitized cells to CPT-induced apoptosis. These results suggest that p53 stabilization and accumulation in response to polyamine depletion predominantly modulate cell cycle checkpoints via p21Cip1 expression and inhibit transcription of target genes responsible for apoptosis. In contrast, phosphorylation and stabilization of p53 in response to DNA-damage lead to apoptosis, which indicates different roles of p53 during DNA damage and polyamine depletion.

Ataxia telangiectasia mutated and p21CIP1 modulate cell survival of drug-induced senescent tumor cells: implications for chemotherapy.[Pubmed:18347191]

Clin Cancer Res. 2008 Mar 15;14(6):1877-87.

PURPOSE: Premature or stress-induced senescence is a major cellular response to chemotherapy in solid tumors and contributes to successful treatment. However, senescent tumor cells are resistant to apoptosis and may also reenter the cell cycle. We set out to find a means to specifically induce senescent tumor cells to undergo cell death and not to reenter the cell cycle that may have general application in cancer therapy. EXPERIMENTAL DESIGN: We investigated the mechanisms regulating cell survival in drug-induced senescent tumor cells. Using immunofluorescence and flow cytometry-based techniques, we established the status of the ataxia telangiectasia mutated (ATM) signaling pathway in these cells. We assayed the requirement of ATM signaling and p21(CIP1) expression for survival in premature senescent tumor cells using pharmacologic inhibitors and antisense oligonucleotides. RESULTS: The ATM/ATR (ATM- and Rad3-related) signaling pathway was found to be constitutively active in drug-induced senescent tumor cells. We found that blocking ATM/ATR signaling with pharmacologic inhibitors, including the novel ATM inhibitors KU55933 and CGK733, induced senescent breast, lung, and colon carcinoma cells to undergo cell death. We show that the mechanism of action of this effect is directly via p21(CIP1), which acts downstream of ATM. This is in contrast to the effects of ATM inhibitors on normal, untransformed senescent cells. CONCLUSIONS: Blocking ATM and/or p21(CIP1) following initial treatment with a low dose of senescence-inducing chemotherapy is a potentially less toxic and highly specific treatment for carcinomas.

Enhanced DNA-PK-mediated RPA2 hyperphosphorylation in DNA polymerase eta-deficient human cells treated with cisplatin and oxaliplatin.[Pubmed:18289945]

DNA Repair (Amst). 2008 Apr 2;7(4):582-96.

The chemotherapeutic drugs cisplatin and oxaliplatin act by induction of DNA damage, including monoadducts, intrastrand and interstrand crosslinks. An increased understanding of the repair and replication of platinum-damaged DNA is required to improve the effectiveness of these drugs in killing cancer cells. We have investigated the effect of expression of DNA polymerase eta (poleta), a translesion synthesis (TLS) enzyme, on the response of human cell lines to cisplatin and oxaliplatin. Poleta-deficient cells are more sensitive to both drugs than are normal cells. In poleta-deficient cells, drug treatment leads to prolonged S-phase arrest, and increased phosphorylation of the phosphatidylinositol-3-kinase-related protein kinase (PIKK) substrates Chk1, p95/Nbs1 and RPA2, the 34kDa subunit of replication protein A. Cisplatin- and oxaliplatin-induced hyperphosphorylation of RPA2, and association of the hyperphosphorylated protein with chromatin, is elevated in poleta-deficient cells. Cisplatin-induced phosphorylation of RPA2 on serine 4/serine 8, but not on serine 33, is inhibited by the DNA-PK inhibitor, NU7441, but not by the ATM inhibitor, KU-55933. Cisplatin-induced DNA-PK-dependent hyperphosphorylation of RPA2 on serine 4/serine 8 occurs after recruitment of RPA to chromatin, as determined by immunofluorescence and by subcellular fractionation. ATR is required both for recruitment of RPA2 to chromatin and its subsequent hyperphosphorylation on serine 4/serine 8 by DNA-PK, since CGK733, an inhibitor of ATM and ATR, blocked both recruitment and hyperphosphorylation. Thus, increased sensitivity to cisplatin and oxaliplatin in DNA poleta-deficient cells is associated with prolonged S-phase arrest, and enhanced PIKK-signalling, in particular activation of DNA-PK-dependent hyperphosphorylation of RPA2 on serines 4 and 8.

Apoptotic death induced by the cyclophosphamide analogue mafosfamide in human lymphoblastoid cells: contribution of DNA replication, transcription inhibition and Chk/p53 signaling.[Pubmed:18289623]

Toxicol Appl Pharmacol. 2008 May 15;229(1):20-32.

Cyclophosphamide is one of the most often used anticancer drugs. Although DNA interstrand cross-links are considered responsible for its cytotoxicity, the mechanism of initiation and execution of cell death is largely unknown. Using the cyclophosphamide analogue mafosfamide, which does not need metabolic activation, we show that mafosfamide induces apoptosis dose and time dependently in lymphoblastoid cells, with clearly more apoptosis in p53(wt) cells. We identified two upstream processes that initiate apoptosis, DNA replication blockage and transcriptional inhibition. In lymphoblastoid cells, wherein DNA replication can be switched off by tetracycline, proliferation is required for inducing apoptosis at low dose mafosfamide. At high dose, transcriptional inhibition also contributes to cell death. The RNA synthesis inhibitor alpha-amanitin induced similar to mafosfamide more apoptosis in p53(wt) than in p53(mt) cells. In combination with mafosfamide, however, alpha-amanitin had no additive effect. Mafosfamide caused p53 stabilization by phosphorylation of Ser15, 20 and 37, and activation of ATM/ATR and Chk1/Chk2. Inhibition of ATM/ATR, PI3-kinase and Chk1/Chk2 by CGK733, wortmannin and DBH, respectively, attenuated the apoptotic response in p53(wt) but not p53(mt) cells. Mafosfamide induced caspase dependent apoptosis and, for low dose treated cells, caspases were preferentially activated in the S-phase, whereas at high dose caspases were activated in all cell cycle stages. These data support the conclusion that at low dose level of mafosfamide, DNA replication blockage is the dominant apoptosis-inducing event, while at high dose, transcriptional inhibition comes into play. The data provide a mechanistic explanation of why cyclophosphamide applied at therapeutic doses preferentially kills replicating tumor cells.