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1.
Biochem Biophys Res Commun ; 549: 128-134, 2021 04 16.
Article in English | MEDLINE | ID: mdl-33676180

ABSTRACT

γ-Glutamylcyclotransferase (GGCT) is involved in glutathione homeostasis, in which it catalyzes the reaction that generates 5-oxoproline and free amino acids from γ-glutamyl peptides. Increasing evidence shows that GGCT has oncogenic functions and is overexpressed in various cancer tissues, and that inhibition of GGCT activity exerts anticancer effects in vitro and in vivo. Here, we demonstrate that U83836E ((2R)-2-[[4-(2,6-dipyrrolidin-1-ylpyrimidin-4-yl)piperazin-1-yl]methyl]-3,4-dihydro-2,5,7,8,-tetramethyl-2H-1-benzopyran-6-ol, dihydrochloride), a lazaroid that inhibits lipid peroxidation, inhibits GGCT enzymatic activity. U83836E was identified from a high-throughput screen of low molecular weight compounds using a fluorochrome-conjugated GGCT probe. We directly quantified that U83836E specifically inhibited GGCT by measuring the product of a fluorochrome-conjugated GGCT substrate assay, and showed that U83836E inhibited GGCT activity in extracts of NIH3T3 cells overexpressing GGCT. Moreover, U83836E significantly inhibited tumor growth in a xenograft model that used immunodeficient mice orthotopically inoculated with MCF7 human breast cancer cells. These results indicate that U83836E may be a useful GGCT inhibitor for the development of potential cancer therapeutics.


Subject(s)
Breast Neoplasms/pathology , Chromans/pharmacology , Enzyme Inhibitors/pharmacology , Piperazines/pharmacology , gamma-Glutamylcyclotransferase/antagonists & inhibitors , Animals , Cell Proliferation/drug effects , Cell Survival/drug effects , Female , Humans , MCF-7 Cells , Mice , Mice, SCID , NIH 3T3 Cells , Xenograft Model Antitumor Assays , gamma-Glutamylcyclotransferase/metabolism
2.
Oncotarget ; 8(43): 74006-74018, 2017 Sep 26.
Article in English | MEDLINE | ID: mdl-29088764

ABSTRACT

The anti-cancer agent CBP501 binds to calmodulin (CaM). Recent studies showed that migration and metastasis are inhibited by several CaM antagonists. However, there is no available evidence that CBP501 has similar effects. Here we found that CBP501 inhibits migration of non-small cell lung cancer (NSCLC) cells in vitro, even in the presence of migration inducing factors such as WNT, IL-6, and several growth factors. CBP501 also inhibited epidermal growth factor (EGF) enhanced invasion and the epithelial-to-mesenchymal transition (EMT), and this inhibition was accompanied by (i) suppression of Akt and ERK1/2 phosphorylation, and (ii) suppression of expression of transcription factor Zeb1 and the mesenchymal marker Vimentin. A pull down analysis performed using sepharose-immobilized CaM showed that CBP501 blocks the interaction between CaM and KRas. Furthermore, EGF induced Akt activation and cell migration was effectively suppressed by KRas down-regulation in NSCLC cells. Stable knockdown of KRas also made cells insensitive to CBP501's inhibition of growth factor-induced migration. Taken together, these results indicate that CBP501 inhibits binding of CaM with KRas and thereby suppresses the PI3K/AKT pathway, migration, invasion and EMT. These findings have identified a previously unrecognized effect of CBP501 on downstream KRas signaling mechanisms involving EMT and invasion, and provide support for the further clinical development of this agent.

3.
Oncotarget ; 8(45): 78277-78288, 2017 Oct 03.
Article in English | MEDLINE | ID: mdl-29108228

ABSTRACT

CBP501, a calmodulin-binding peptide, is an anti-cancer drug candidate and functions as an enhancer of platinum uptake into cancer cells. Here we show that CBP501 promotes immunogenic cell death (ICD) in combination with platinum agents. CBP501 enhanced a clinically relevant low dose of cisplatin (CDDP) in vitro as evidenced by upregulation of ICD markers, including cell surface calreticulin exposure and release of high-mobility group protein box-1. Synergistic induction of ICD by CDDP plus CBP501 as compared to CDDP alone was confirmed in the well-established vaccination assay. Furthermore, cotreatment of CDDP plus CBP501 significantly reduced the tumor growth and upregulated the percentage of tumor infiltrating CD8+ T cell in vivo. Importantly, the antitumor effect of CDDP plus CBP501 was significantly reduced by anti-CD8 antibody treatment. Based on this novel effect of CBP501, we analyzed the combination treatment with immune checkpoint inhibitors in vivo. Mice treated with CBP501 in combination with CDDP and anti-PD-1 or anti-PD-L1 showed an additive antitumor effect. These results support the conclusion that CBP501 enhances CDDP-induced ICD in vitro and in vivo. The findings also support the further clinical development of the CBP501 for enhancing the antitumor activity of immune checkpoint inhibitors in combination with CDDP.

4.
Oncotarget ; 8(38): 64015-64031, 2017 Sep 08.
Article in English | MEDLINE | ID: mdl-28969049

ABSTRACT

CBP501 is an anti-cancer drug candidate which has been shown to increase cis-diamminedichloro-platinum (II) (CDDP) uptake into cancer cell through calmodulin (CaM) inhibition. However, the effects of CBP501 on the cells in the tumor microenvironment have not been addressed. Here, we investigated new aspects of the potential anti-tumor mechanism of action of CBP501 by examining its effects on the macrophages. Macrophages contribute to cancer-related inflammation and sequential production of cytokines such as IL-6 and TNF-α which cause various biological processes that promote tumor initiation, growth and metastasis (1). These processes include the epithelial to mesenchymal transition (EMT) and cancer stem cell (CSC) formation, which are well-known, key events for metastasis. The present work demonstrates that CBP501 suppresses lipopolysaccharide (LPS)-induced production of IL-6, IL-10 and TNF-α by macrophages. CBP501 also suppressed formation of the tumor spheroids by culturing with conditioned medium from the LPS-stimulated macrophage cell line RAW264.7. Moreover, CBP501 suppressed expression of ABCG2, a marker for CSCs, by inhibiting the interaction between cancer cells expressing VCAM-1 and macrophages expressing VLA-4. Consistently with these results, CBP501 in vivo suppressed metastases of a tumor cell line, 4T1, one which is insensitive to combination treatment of CBP501 and CDDP in vitro. Taken together, these results offer potential new, unanticipated advantages of CBP501 treatment in anti-tumor therapy through a mechanism that entails the suppression of interactions between macrophages and cancer cells with suppression of sequential CSC-like cell formation in the tumor microenvironment.

5.
Mol Cancer Ther ; 13(12): 3013-23, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25253782

ABSTRACT

Chromosome region maintenance 1 (CRM1) mediates the nuclear export of proteins and mRNAs, and is overexpressed in various cancers. Recent studies have also reported that CRM1 protein expression is a negative prognostic factor in patients with cancer. Therefore, CRM1 is considered a potential target for anticancer therapy. Our previous study demonstrated that CBS9106, a synthetic small-molecular inhibitor of CRM1, decreases CRM1 protein through proteasomal degradation without affecting CRM1 mRNA levels. However, the mechanism by which CRM1 is degraded is not well understood. Here, we demonstrate a novel signaling pathway that plays an important role in CBS9106-induced CRM1 degradation. We found that MLN4924, a selective inhibitor of NEDD8-activating enzyme (NAE), effectively inhibits cullin neddylation and attenuates CBS9106-induced CRM1 degradation in a time- and dose-dependent manner. MLN4924 also attenuated CBS9106-induced nuclear accumulation of Ran-binding protein 1 (RanBP1), cell growth inhibition, and apoptosis. Furthermore, RNAi-mediated knockdown of neddylation pathway proteins (NEDD8 and UBA3) or cullin ring ligase (CRL) component protein (Rbx1) attenuated CRM1 protein degradation and G1 phase cell-cycle arrest by CBS9106. Knockdown of CSN5 or CAND1 also partially inhibited CBS9106-induced CRM1 degradation. These findings demonstrate that CBS9106-induced CRM1 degradation is conferred by CRL activity involving the neddylation pathway, and that this response to CBS9106 leads to cell growth inhibition and apoptosis.


Subject(s)
Aminopyridines/pharmacology , Cullin Proteins/metabolism , Karyopherins/metabolism , Pyrrolidinones/pharmacology , Receptors, Cytoplasmic and Nuclear/metabolism , Signal Transduction , Apoptosis/drug effects , COP9 Signalosome Complex , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Cycle/drug effects , Cell Line, Tumor , Cell Nucleus/metabolism , Cell Survival/drug effects , Cyclopentanes/pharmacology , Gene Knockdown Techniques , HCT116 Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Lysosomes/metabolism , NEDD8 Protein , Nuclear Proteins/metabolism , Peptide Hydrolases/genetics , Peptide Hydrolases/metabolism , Protein Transport , Proteolysis/drug effects , Pyrimidines/pharmacology , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Suppressor Protein p53/metabolism , Ubiquitin-Activating Enzymes/genetics , Ubiquitin-Activating Enzymes/metabolism , Ubiquitins/genetics , Ubiquitins/metabolism , Exportin 1 Protein
6.
Mol Cancer Ther ; 13(9): 2215-25, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25053821

ABSTRACT

CBP501 is an anticancer drug candidate that was investigated in two randomized phase II clinical trials for patients with nonsquamous non-small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MPM). CBP501 has been shown to have two mechanisms of action, namely calmodulin modulation and G2 checkpoint abrogation. Here, we searched for a biomarker to predict sensitivity to CBP501. Twenty-eight NSCLC cell lines were classified into two subgroups, CBP501-sensitive and -insensitive, by quantitatively analyzing the cis-diamminedichloro-platinum (II) (CDDP)-enhancing activity of CBP501 through treatments with short-term (1 hour) coexposure to CDDP and CBP501 or to either alone. Microarray analysis was performed on these cell lines to identify gene expression patterns that correlated with CBP501 sensitivity. We found that multiple nuclear factor erythroid-2-related factor 2 (Nrf2) target genes showed high expression in CBP501-insensitive cell lines. Western blot and immunocytochemical analysis for Nrf2 in NSCLC cell lines also indicated higher protein level in CBP501-insensitive cell lines. Moreover, CBP501 sensitivity is modulated by silencing or sulforaphane-induced overexpression of Nrf2. These results indicate that Nrf2 transcription factor is a potential candidate as a biomarker for resistance to CBP501. This study might help to identify those subpopulations of patients who would respond well to the CBP501 and CDDP combination treatment of NSCLC.


Subject(s)
Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/metabolism , Drug Resistance, Neoplasm , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , NF-E2-Related Factor 2/metabolism , Peptide Fragments/chemistry , cdc25 Phosphatases/chemistry , Biomarkers, Tumor/chemistry , Calmodulin/chemistry , Cell Cycle , Cell Line, Tumor , G2 Phase , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Lentivirus/metabolism , Microscopy, Fluorescence , Oligonucleotide Array Sequence Analysis , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism
7.
Bioorg Med Chem ; 19(23): 7049-56, 2011 Dec 01.
Article in English | MEDLINE | ID: mdl-22032894

ABSTRACT

CBP501 is a chemically modified peptide composed of twelve unnatural d-amino acids, which inhibits Chk kinase and abrogates G2 arrest induced by DNA-damaging agents. Here we identified an alphaC helix in 14-3-3 protein as a CBP501-binding site using T7 phage display technology. An affinity selection of T7 phage-displayed peptide using biotinylated CBP501 identified a 14-mer peptide NSDCIISRKIEQKE. This peptide sequence showed similarity to a portion of the alphaC helix of human 14-3-3ε, suggesting that CBP501 may bind to this region. Surface plasmon resonance (SPR) and ELISA demonstrated that CBP501 interacts with 14-3-3ε specifically at the screen-guided region. An avidin-agarose bead pull-down assay showed that CBP501 also binds to other 14-3-3 isoforms in Jurkat cells. Among the other known Chk kinase inhibitors tested, CBP501 showed the strongest affinity for 14-3-3ε. Thus, we conclude that in addition to the direct inhibition of Chk kinase activity, CBP501 directly binds to cellular 14-3-3 proteins through alphaC helix.


Subject(s)
14-3-3 Proteins/metabolism , Bacteriophage T7/metabolism , Peptide Fragments/metabolism , Peptides/metabolism , cdc25 Phosphatases/metabolism , 14-3-3 Proteins/genetics , Amino Acid Sequence , Bacteriophage T7/genetics , Binding Sites , Circular Dichroism , Humans , Jurkat Cells , Models, Molecular , Molecular Sequence Data , Peptide Library , Peptides/chemistry , Peptides/genetics , Protein Structure, Secondary
8.
Blood ; 118(14): 3922-31, 2011 Oct 06.
Article in English | MEDLINE | ID: mdl-21841164

ABSTRACT

CRM1 plays an important role in the nuclear export of cargo proteins bearing nuclear exporting signal sequences. Leptomycin B (LMB), a well-known CRM1 inhibitor, possesses strong antitumor properties. However, its toxicity prevents it from being clinically useful. In this study, we demonstrate that a novel compound, CBS9106, inhibits CRM1-dependent nuclear export, causing arrest of the cell cycle and inducing apoptosis in a time- and dose-dependent manner for a broad spectrum of cancer cells, including multiple myeloma cells. CBS9106 reduces CRM1 protein levels significantly without affecting CRM1 mRNA expression. This effect could be reversed by adding bortezomib or LMB. Moreover, CBS9106-biotin allows capture of CRM1 protein by streptavidin beads in a competitive manner with LMB and vice versa. Mass spectrometric analysis shows that CBS9106 reacts with a synthetic CRM1 peptide that contains Cys528 but not with a Cys528 mutant peptide. Oral administration of CBS9106 significantly suppresses tumor growth and prolongs survival in mice bearing tumor xenograft without a significant loss in body weight. A reduced level of CRM1 protein is also observed in tumor xenografts isolated from mice treated with CBS9106. Taken together, these results indicate that CBS9106 is a novel reversible CRM1 inhibitor and a promising clinical candidate.


Subject(s)
Aminopyridines/therapeutic use , Antineoplastic Agents/therapeutic use , Karyopherins/antagonists & inhibitors , Karyopherins/metabolism , Multiple Myeloma/drug therapy , Pyrrolidinones/therapeutic use , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Receptors, Cytoplasmic and Nuclear/metabolism , Active Transport, Cell Nucleus/drug effects , Aminopyridines/pharmacology , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Karyopherins/genetics , Male , Mice , Mice, SCID , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Proteasome Endopeptidase Complex/metabolism , Pyrrolidinones/pharmacology , Receptors, Cytoplasmic and Nuclear/genetics , Ubiquitin/metabolism , Exportin 1 Protein
9.
Mol Cancer Ther ; 10(10): 1929-38, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21831962

ABSTRACT

CBP501 is an anticancer drug currently in randomized phase II clinical trials for patients with non-small cell lung cancer and malignant pleural mesothelioma. CBP501 was originally described as a unique G(2) checkpoint-directed agent that binds to 14-3-3, inhibiting the actions of Chk1, Chk2, mitogen-activated protein kinase-activated protein kinase 2, and C-Tak1. However, unlike a G(2) checkpoint inhibitor, CBP501 clearly enhances the accumulation of tumor cells at G(2)-M phase that is induced by cisplatin or bleomycin at low doses and short exposure. By contrast, CBP501 does not similarly affect the accumulation of tumor cells at G(2)-M that is induced by radiation, doxorubicin, or 5-fluorouracil treatment. Our recent findings point to an additional mechanism of action for CBP501. The enhanced accumulation of tumor cells at G(2)-M upon combined treatment with cisplatin and CBP501 results from an increase in intracellular platinum concentrations, which leads to increased binding of platinum to DNA. The observed CBP501-enhanced platinum accumulation is negated in the presence of excess Ca(2+). Some calmodulin inhibitors behave similarly to, although less potently than, CBP501. Furthermore, analysis by surface plasmon resonance reveals a direct, high-affinity molecular interaction between CBP501 and CaM (K(d) = 4.62 × 10(-8) mol/L) that is reversed by Ca(2+), whereas the K(d) for the complex between CBP501 and 14-3-3 is approximately 10-fold weaker and is Ca(2+) independent. We conclude that CaM inhibition contributes to CBP501's activity in sensitizing cancer cells to cisplatin or bleomycin. This article presents an additional mechanism of action which might explain the clinical activity of the CBP501-cisplatin combination.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Bleomycin/pharmacology , Calmodulin/metabolism , Cisplatin/pharmacology , Neoplasms/drug therapy , Peptide Fragments/pharmacology , cdc25 Phosphatases/pharmacology , Bleomycin/administration & dosage , Calcium Chloride/pharmacology , Calmodulin/antagonists & inhibitors , Cell Line, Tumor , Cell Survival/drug effects , Cisplatin/administration & dosage , Cisplatin/pharmacokinetics , DNA Adducts/biosynthesis , Drug Synergism , Humans , Neoplasms/metabolism , Neoplasms/pathology , Peptide Fragments/pharmacokinetics , cdc25 Phosphatases/pharmacokinetics
10.
Clin Cancer Res ; 17(10): 3431-42, 2011 May 15.
Article in English | MEDLINE | ID: mdl-21220472

ABSTRACT

PURPOSE: Two phase I dose-escalation studies were conducted to determine the maximum tolerated dose (MTD) and safety profile of the G(2) checkpoint abrogator CBP501, as a single agent and in combination with cisplatin. EXPERIMENTAL DESIGN: Patients with advanced solid tumors were treated with CBP501 alone (D1/D8/D15, q4w, from 0.9 mg/m(2)), or with cisplatin (both on D1, q3w, from 3.6 mg/m(2) CBP501, 50 mg/m(2) cisplatin). Dose escalation proceeded if dose-limiting toxicity (DLT) was observed in 1 or less of 3 to 6 patients; CBP501 dose increments were implemented according to the incidence of toxicity. MTD was determined from DLTs occurring during the first two cycles. RESULTS: In the combination study, the DLT was a histamine-release syndrome (HRS) occurring 10 to 60 minutes after initiating infusion that was attenuated by prophylaxis comprising dexamethasone, diphenhydramine, ranitidine, and loratadine. The MTD was 25 mg/m(2) CBP501 and 75 mg/m(2) cisplatin, with two patients at the highest dose (36.4 mg/m(2) CBP501, 75 mg/m(2) cisplatin) experiencing grade 3 HRS. The only DLT with monotherapy was transient G(3) rise of troponin in one patient. Grade 3 to 4 treatment-related events were rare. Promising activity was observed with CBP501/cisplatin, mainly in ovarian and mesothelioma patients who had previously progressed on platinum-containing regimens. Among ovarian cancer patients, low expression of DNA repair proteins was associated with partial response or stable disease. CONCLUSIONS: CBP501 is well tolerated in patients as monotherapy and with cisplatin. At the recommended phase II dose (RP2D), the combination is feasible and HRS manageable with prophylaxis. Evidence of antitumor activity was observed in platinum-resistant patients.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cisplatin/administration & dosage , Neoplasms/drug therapy , Peptide Fragments/administration & dosage , Peptide Fragments/therapeutic use , cdc25 Phosphatases/administration & dosage , cdc25 Phosphatases/therapeutic use , Adult , Aged , Aged, 80 and over , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/adverse effects , Antineoplastic Agents/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Cisplatin/adverse effects , Disease Progression , Female , G2 Phase/drug effects , Genes, cdc/drug effects , Humans , Male , Maximum Tolerated Dose , Middle Aged , Neoplasms/pathology , Peptide Fragments/adverse effects , cdc25 Phosphatases/adverse effects
11.
Mol Cancer Ther ; 6(1): 147-53, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17237275

ABSTRACT

Cell cycle G(2) checkpoint abrogation is an attractive strategy for sensitizing cancer cells to DNA-damaging anticancer agent without increasing adverse effects on normal cells. However, there is no single proven molecular target for this therapeutic approach. High-throughput screening for molecules inhibiting CHK1, a kinase that is essential for the G(2) checkpoint, has not yet yielded therapeutic G(2) checkpoint inhibitors, and the tumor suppressor phenotypes of ATM and CHK2 suggest they may not be ideal targets. Here, we optimized two G(2) checkpoint-abrogating peptides, TAT-S216 and TAT-S216A, based on their ability to reduce G(2) phase accumulation of DNA-damaged cells without affecting M phase accumulation of cells treated with a microtubule-disrupting compound. This approach yielded a peptide CBP501, which has a unique, focused activity against molecules that phosphorylate Ser(216) of CDC25C, including MAPKAP-K2, C-Tak1, and CHK1. CBP501 is >100-fold more potent than TAT-S216A and retains its selectivity for cancer cells. CBP501 is unusually stable, enters cells rapidly, and increases the cytotoxicity of DNA-damaging anticancer drugs against cancer cells without increasing adverse effects. These findings highlight the potency of CBP501 as a G(2)-abrogating drug candidate. This report also shows the usefulness of the cell cycle phenotype-based protocol for identifying G(2) checkpoint-abrogating compounds as well as the potential of peptide-based compounds as focused multitarget inhibitors.


Subject(s)
Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , G2 Phase/drug effects , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Peptides/pharmacology , cdc25 Phosphatases/metabolism , cdc25 Phosphatases/pharmacology , Amino Acid Sequence , Animals , Antineoplastic Agents/chemistry , Bleomycin/adverse effects , Bleomycin/pharmacology , Cell Cycle Proteins/metabolism , Cell Death/drug effects , Cisplatin/adverse effects , Cisplatin/pharmacology , HCT116 Cells , Humans , Jurkat Cells , Male , Mice , Mice, SCID , Models, Molecular , Molecular Sequence Data , Neoplasms/pathology , Peptide Fragments/chemistry , Peptides/chemistry , Phenotype , Phosphorylation/drug effects , Xenograft Model Antitumor Assays , cdc25 Phosphatases/chemistry
12.
Mol Cancer Ther ; 3(4): 513-9, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15078995

ABSTRACT

Many conventional anticancer treatments kill cells irrespective of whether they are normal or cancerous, so patients suffer from adverse side effects due to the loss of healthy cells. Anticancer insights derived from cell cycle research has given birth to the idea of cell cycle G2 checkpoint abrogation as a cancer cell specific therapy, based on the discovery that many cancer cells have a defective G1 checkpoint resulting in a dependence on the G2 checkpoint during cell replication. Damaged DNA in humans is detected by sensor proteins (such as hHUS1, hRAD1, hRAD9, hRAD17, and hRAD26) that transmit a signal via ATR to CHK1, or by another sensor complex (that may include gammaH2AX, 53BP1, BRCA1, NBS1, hMRE11, and hRAD50), the signal of which is relayed by ATM to CHK2. Most of the damage signals originated by the sensor complexes for the G2 checkpoint are conducted to CDC25C, the activity of which is modulated by 14-3-3. There are also less extensively explored pathways involving p53, p38, PCNA, HDAC, PP2A, PLK1, WEE1, CDC25B, and CDC25A. This review will examine the available inhibitors of CHK1 (Staurosporin, UCN-01, Go6976, SB-218078, ICP-1, and CEP-3891), both CHK1 and CHK2 (TAT-S216A and debromohymenialdisine), CHK2 (CEP-6367), WEE1 (PD0166285), and PP2A (okadaic acid and fostriecin), as well as the unknown checkpoint inhibitors 13-hydroxy-15-ozoapathin and the isogranulatimides. Among these targets, CHK1 seems to be the most suitable target for therapeutic G2 abrogation to date, although an unexplored target such as 14-3-3 or the strategy of targeting multiple proteins at once may be of interest in the future.


Subject(s)
Antineoplastic Agents/pharmacology , G2 Phase/drug effects , Neoplasms/drug therapy , Neoplasms/pathology , Animals , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/metabolism , G1 Phase/physiology , G2 Phase/physiology , Humans
13.
Biochem Biophys Res Commun ; 317(3): 817-22, 2004 May 07.
Article in English | MEDLINE | ID: mdl-15081413

ABSTRACT

Human T-cell leukemia-derived Jurkat cells are known to be defective in the G1 checkpoint. DNA-damaging agent bleomycin arrests the cell cycle at G2 phase of Jurkat cells, and microtubule-acting colchicine arrests it at the M phase. Simaomicin alpha, an actinomycete metabolite, itself showed no effect on the cell cycle status of Jurkat cells at least up to 6.0 nM. However, the compound (0.6-6.0 nM) was found to abrogate the bleomycin-induced G2 arrest, yielding a drastic decrease in cells at the G2 phase and increase in cells at the subG1 and G1 phases. On the other hand, the compound did not show any effect on the colchicine-induced M phase arrest in Jurkat cells. Furthermore, the compound showed almost no effect on the cell cycle status of the bleomycin-treated or -untreated normal cell line HUVEC. These data suggested that simaomicin alpha disrupts the cell cycle G2 checkpoint of cancer cells selectively, leading to sensitization of cancer cells to anti-cancer reagents.


Subject(s)
Bleomycin/antagonists & inhibitors , G2 Phase/drug effects , Isoquinolines/pharmacology , Bleomycin/pharmacology , Humans , Jurkat Cells
14.
J Antibiot (Tokyo) ; 57(10): 662-8, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15638327

ABSTRACT

The DNA-damaging agent bleomycin arrests the cell cycle at the G2 phase of Jurkat cells defective in the G1 checkpoint, and microtubule-acting colchicine arrests it at the M phase. Boromycin itself, an actinomycete metabolite, showed no effect on the cell cycle status of Jurkat cells at least up to 340 nM. However, the compound (3.4-340 nM) was found to abrogate bleomycin-induced G2 arrest even at 3.4 nM, resulting in a drastic decrease in cells at the G2 phase and increase in cells at the subG1 phase. On the other hand, boromycin did not show any effect on the colchicine-induced M phase arrest in Jurkat cells, nor on the cell cycle status of the bleomycin-treated or -untreated HUVEC, normal cells conserving both G1 and G2 checkpoints. Furthermore, boromycin potentiated anti-tumor activity of bleomycin in scid mice inoculated with Jurkat cells. These data suggest that boromycin disrupts the cell cycle at the G2 checkpoint of cancer cells selectively, leading to sensitization of cancer cells to anti-cancer reagents.


Subject(s)
Bleomycin/pharmacology , Borates/pharmacology , G2 Phase/drug effects , Animals , Humans , Jurkat Cells , Male , Mice , Mice, SCID
15.
Oncogene ; 21(11): 1717-26, 2002 Mar 07.
Article in English | MEDLINE | ID: mdl-11896603

ABSTRACT

Cdc25 activates maturation promoting factor (MPF) and promotes mitosis by removing the inhibitory phosphate from the Tyr-15 of Cdc2 in human cells. In this study, we searched the interacting protein(s) of human Cdc25C using the yeast two-hybrid screen and identified proliferating cell nuclear antigen (PCNA) as an interacting partner of Cdc25C. The interaction between Cdc25C and PCNA was confirmed in vitro and in vivo. Co-immunoprecipitation analyses using human T cell line, Jurkat, further revealed that Cdc25C interacted with PCNA transiently when cells began to enter mitosis. Immunofluorescence analysis also showed that Cdc25C and PCNA were transiently co-localized in the nucleus at the beginning of M phase. Together with the previous observations of the interaction between various cdc/cyclin and PCNA, our findings strongly suggested a potential role of PCNA at the G2 to M phase transition of cell cycle.


Subject(s)
Cell Cycle Proteins/physiology , G2 Phase , Mitosis , Proliferating Cell Nuclear Antigen/physiology , cdc25 Phosphatases/physiology , Cell Cycle Proteins/analysis , Humans , Jurkat Cells , Proliferating Cell Nuclear Antigen/analysis , Two-Hybrid System Techniques , cdc25 Phosphatases/analysis
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