Shingo Dan

ASK1 mediates apoptotic cell death induced by genotoxic stress

Genotoxic stress-induced apoptosis is mediated by caspase family proteases as triggered by other stimuli. In this study, we found that the DNA-damaging agent cisplatin (cDDP) activated MAP kinase kinase kinase ASK1 and subsequent downstream subgroups of MAP kinase kinase, SEK1 (or MKK4) and MKK3/MKK6, which in turn activated c-Jun N-terminal kinase 1/stress-activated protein kinase (JNK1/SAPK) and p38 MAP kinase prior to caspase family protease activation and the onset of apoptosis in human ovarian carcinoma (OVCAR-3) and human kidney (293T) cells. As reported previously, benzyloxy carbonyl-Asp-CH2OC(O)-2, 6-dichlorobenzene (Z-Asp), a preferential inhibitor of caspase family proteases, blocked the apoptosis of OVCAR-3 cells induced by the genotoxic stress cDDP. Z-Asp, however, did not inhibit ASK1 activation and the subsequent kinase cascades. Overexpression of kinase-negative ASK1 (K709R), which inhibited ASK1 activation and the downstream MKK3-p38 and MKK4-JNK1 pathways, also suppressed the caspase protease activation and apoptosis induced by cDDP. These results indicate that the ASK1 pathway is involved in genotoxic stress-induced apoptosis and mediates apoptosis at a step upstream of caspase protease activation.

Selective induction of apoptosis in Philadelphia chromosome-positive chronic myelogenous leukemia cells by an inhibitor of BCR–ABL tyrosine kinase, CGP 57148

The BCR–ABL tyrosine kinase has been implicated as the cause of Philadelphia chromosome (Ph1)-positive leukemias. We report herein that CGP 57148, a selective inhibitor of the ABL tyrosine kinase, caused apoptosis specifically in bcr–abl-positive chronic myelogenous leukemia (CML) cells, K562 and KYO-1. Upon treatment with CGP 57148, CRKL, a specific substrate for BCR–ABL that propagates signals via phosphatidylinositol-3′ kinase (PI3K), was dephosphorylated, indicating inhibition of BCR–ABL tyrosine kinase at the cellular level. Likewise, MAPK/ERK, a downstream mediator of Ras, was also dephosphorylated. Caspase activation and cleavage of retinoblastoma protein (pRB) accompanied the development of CGP 57148-induced apoptosis. Inhibition of caspase suppressed apoptosis and the cleavage of pRB, and in turn arrested cells in the G1 phase. These results indicate that CGP 57148 shows apoptogenic and anti-proliferative effects on bcr–abl-positive cells by blocking BCR–ABL-initiated signaling pathways.

Correlating Phosphatidylinositol 3-Kinase Inhibitor Efficacy with Signaling Pathway Status: In silico and Biological Evaluations

The phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in human cancers, and several agents targeting this pathway including PI3K/Akt/mammalian target of rapamycin inhibitors have recently entered clinical trials. One question is whether the efficacy of a PI3K pathway inhibitor can be predicted based on the activation status of pathway members. In this study, we examined the mutation, expression, and phosphorylation status of PI3K and Ras pathway members in a panel of 39 pharmacologically well-characterized human cancer cell lines (JFCR39). Additionally, we evaluated the in vitro efficacy of 25 PI3K pathway inhibitors in addition to conventional anticancer drugs, combining these data to construct an integrated database of pathway activation status and drug efficacies (JFCR39-DB). In silico analysis of JFCR39-DB enabled us to evaluate correlations between the status of pathway members and the efficacy of PI3K inhibitors. For example, phospho-Akt and KRAS/BRAF mutations prominently correlated with the efficacy and the inefficacy of PI3K inhibitors, respectively, whereas PIK3CA mutation and PTEN loss did not. These correlations were confirmed in human tumor xenografts in vivo, consistent with their ability to serve as predictive biomarkers. Our findings show that JFCR39-DB is a useful tool to identify predictive biomarkers and to study the molecular pharmacology of the PI3K pathway in cancer.

Chemosensitivity profile of cancer cell lines and identification of genes determining chemosensitivity by an integrated bioinformatical approach using cDNA arrays

We have established a panel of 45 human cancer cell lines (JFCR-45) to explore genes that determine the chemosensitivity of these cell lines to anticancer drugs. JFCR-45 comprises cancer cell lines derived from tumors of three different organs: breast, liver, and stomach. The inclusion of cell lines derived from gastric and hepatic cancers is a major point of novelty of this study. We determined the concentration of 53 anticancer drugs that could induce 50% growth inhibition (GI50) in each cell line. Cluster analysis using the GI50s indicated that JFCR-45 could allow classification of the drugs based on their modes of action, which coincides with previous findings in NCI-60 and JFCR-39. We next investigated gene expression in JFCR-45 and developed an integrated database of chemosensitivity and gene expression in this panel of cell lines. We applied a correlation analysis between gene expression profiles and chemosensitivity profiles, which revealed many candidate genes related to the sensitivity of cancer cells to anticancer drugs. To identify genes that directly determine chemosensitivity, we further tested the ability of these candidate genes to alter sensitivity to anticancer drugs after individually overexpressing each gene in human fibrosarcoma HT1080. We observed that transfection of HT1080 cells with the HSPA1A and JUN genes actually enhanced the sensitivity to mitomycin C, suggesting the direct participation of these genes in mitomycin C sensitivity. These results suggest that an integrated bioinformatical approach using chemosensitivity and gene expression profiling is useful for the identification of genes determining chemosensitivity of cancer cells.

Inhibition profiles of phosphatidylinositol 3-kinase inhibitors against PI3K superfamily and human cancer cell line panel JFCR39.

As accumulating evidences suggest close involvement of phosphatidylinositol 3-kinase (PI3K) in various diseases particularly cancer, considerable competition occurs in development of PI3K inhibitors. Consequently, novel PI3K inhibitors such as ZSTK474, GDC-0941 and NVP-BEZ235 have been developed. Even though all these inhibitors were reported to inhibit class I PI3K but not dozens of protein kinases, whether they have different molecular targets remained unknown. To investigate such molecular target specificity, we have determined the inhibitory effects of these novel inhibitors together with classical PI3K inhibitor LY294002 on PI3K superfamily (including classes I, II, and III PI3Ks, PI4K and PI3K-related kinases) by using several novel non-radioactive biochemical assays. As a result, ZSTK474 and GDC-0941 indicated highly similar inhibition profiles for PI3K superfamily, with class I PI3K specificity much higher than NVP-BEZ235 and LY294002. We further investigated their growth inhibition effects on JFCR39, a human cancer cell line panel which we established for molecular target identification, and analysed their cell growth inhibition profiles (fingerprints) by using COMPARE analysis programme. Interestingly, we found ZSTK474 exhibited a highly similar fingerprint with GDC-0941 (r=0.863), more similar than with that of either NVP-BEZ235 or LY294002, suggesting that ZSTK474 shares more in molecular targets with GDC-0941 than with either of the other two PI3K inhibitors, consistent with the biochemical assay result. The biological implication of the difference in molecular target specificity of these PI3K inhibitors is under investigation.

AMF-26, a Novel Inhibitor of the Golgi System, Targeting ADP-ribosylation Factor 1 (Arf1) with Potential for Cancer Therapy

Background: Golgi is a potential target for cancer treatment, but no inhibitor became an anticancer drug. Results: Using a unique bioinformatics approach, we identified a novel Golgi inhibitor, AMF-26, targeting Arf1 activation and possessing potent antitumor activity. Conclusion: AMF-26 is a promising new anticancer drug lead. Significance: Our data indicate that Arf1 activation is a promising target for cancer treatment. ADP-ribosylation factor 1 (Arf1) plays a major role in mediating vesicular transport. Brefeldin A (BFA), a known inhibitor of the Arf1-guanine nucleotide exchange factor (GEF) interaction, is highly cytotoxic. Therefore, interaction of Arf1 with ArfGEF is an attractive target for cancer treatment. However, BFA and its derivatives have not progressed beyond the pre-clinical stage of drug development because of their poor bioavailability. Here, we aimed to identify novel inhibitors of the Arf1-ArfGEF interaction that display potent antitumor activity in vivo but with a chemical structure distinct from that of BFA. We exploited a panel of 39 cell lines (termed JFCR39) coupled with a drug sensitivity data base and COMPARE algorithm, resulting in the identification of a possible novel Arf1-ArfGEF inhibitor AMF-26, which differed structurally from BFA. By using a pulldown assay with GGA3-conjugated beads, we demonstrated that AMF-26 inhibited Arf1 activation. Subsequently, AMF-26 induced Golgi disruption, apoptosis, and cell growth inhibition. Computer modeling/molecular dynamics (MD) simulation suggested that AMF-26 bound to the contact surface of the Arf1-Sec7 domain where BFA bound. AMF-26 affected membrane traffic, including the cis-Golgi and trans-Golgi networks, and the endosomal systems. Furthermore, using AMF-26 and its derivatives, we demonstrated that there was a significant correlation between cell growth inhibition and Golgi disruption. In addition, orally administrated AMF-26 (83 mg/kg of body weight; 5 days) induced complete regression of human breast cancer BSY-1 xenografts in vivo, suggesting that AMF-26 is a novel anticancer drug candidate that inhibits the Golgi system, targeting Arf1 activation.

Activation of c-Abl tyrosine kinase requires caspase activation and is not involved in JNK/SAPK activation during apoptosis of human monocytic leukemia U937 cells.

Genotoxic stress triggers the activation of several sensor molecules, such as p53, JNK1/SAPK and c-Abl, and occasionally promotes the cells to apoptosis. We previously reported that JNK1/SAPK regulates genotoxic stress-induced apoptosis in p53-negative U937 cells by activating caspases. c-Abl is expected to act upstream of JNK1/SAPK activation upon treatment with genotoxic stressors, but its involvement in apoptosis development is still unclear. We herein investigated the kinase activities of c-Abl and JNK1/SAPK during apoptosis elicited by genotoxic anticancer drugs and tumor necrosis factor (TNF) in U937 cells and their apoptosis-resistant variant UK711 cells. We found that the activation of JNK1/SAPK and c-Abl correlated well with apoptosis development in these cell lines. Unexpectedly, however, the JNK1/SAPK activation preceded the c-Abl activation. Moreover, the caspase inhibitor Z-Asp suppressed c-Abl activation and the onset of apoptosis but not the JNK1/SAPK activation. Interestingly, c-Abl tyrosine kinase inhibition by CGP 57148 reduced apoptosis without interfering with JNK1/SAPK activation. These results indicate that c-Abl acts not upstream of JNK1/SAPK but downstream of caspases during the development of p53-independent apoptosis and is possibly involved in accelerating execution of the cell death pathway.

Inhibition of PI3K by ZSTK474 suppressed tumor growth not via apoptosis but G0/G1 arrest.

Phosphoinositide 3-kinase (PI3K) is a potential target in cancer therapy. Inhibition of PI3K is believed to induce apoptosis. We recently developed a novel PI3K inhibitor ZSTK474 with antitumor efficacy. In this study, we have examined the underlying mode of action by which ZSTK474 exerts its antitumor efficacy. In vivo, ZSTK474 effectively inhibited the growth of human cancer xenografts. In parallel, ZSTK474 treatment suppressed the expression of phospho-Akt, suggesting effective PI3K inhibition, and also suppressed the expression of nuclear cyclin D1 and Ki67, both of which are hallmarks of proliferation. However, ZSTK474 treatment did not increase TUNEL-positive apoptotic cells. In vitro, ZSTK474 induced marked G(0)/G(1) arrest, but did not increase the subdiploid cells or activate caspase, both of which are hallmarks of apoptosis. These results clearly indicated that inhibition of PI3K by ZSTK474 did not induce apoptosis but rather induced strong G(0)/G(1) arrest, which might cause its efficacy in tumor cells.

ZSTK474, a specific phosphatidylinositol 3-kinase inhibitor, induces G1 arrest of the cell cycle in vivo

Phosphatidylinositol 3-kinase (PI3K) is regarded as a promising therapeutic target because it is often activated in cancer. We previously reported that ZSTK474, a specific PI3K inhibitor, inhibits tumour cell proliferation via G1 arrest of the cell cycle without inducing apoptosis in vitro. However, it remained unclear whether ZSTK474 induces G1 arrest to exert antitumour efficacy in vivo. We recently developed a live imaging system, named Fluorescent Ubiquitination-based Cell Cycle Indicator (Fucci), to visualise cell cycle distribution. Here, by using this system, we tested whether ZSTK474 induces G1 arrest in tumour cells in vivo, as well as in vitro. Fucci-introduced human breast cancer MCF-7 cells and cervical cancer HeLa cells were subcutaneously xenografted in nude mice. ZSTK474 was administered to the tumour-bearing mice for 5 days, and the cell cycle distribution in the xenografted tumours were analysed by monitoring fluorescence in live mice. We demonstrate that ZSTK474 induces G1arrest along with tumour suppression in vivo. Moreover, we show that ZSTK474 suppresses the tumour growth without inducing apoptosis. Interestingly, such increase in G1 cells and tumour suppression was maintained during long-term (3-month) administration of ZSTK474. These results suggest that ZSTK474 exerts its in vivo antitumour efficacy via G1 arrest but not via apoptosis as long as it is administered, and could be used for months as maintenance therapy for patients with advanced cancers.

IVSE, isolated from Inula japonica,suppresses LPS-induced NO production via NF-κB and MAPK inactivation in RAW264.7 cells.

AIMS Our previous study showed that the extract of Inula japonica Thunb. (I. japonica) has anti-inflammatory and anti-asthmatic activities. In an attempt to find anti-inflammatory compounds from I. japonica, we recently isolated 1,6α-dihydroxy-4αH-1,10-secoeudesma-5(10),11(13)-dien-12,8β-olide (SE), 6α-isobutyryloxy-1-hydroxy-4αH-1,10-secoeudesma-5(10),11(13)-dien-12,8β-olide (IBSE), and 6α-isovaleryloxy-1-hydroxy-4αH-1,10-secoeudesma-5(10),11(13)-dien-12,8β-olide (IVSE) from the extract of I. japonica, and investigated their inhibitory effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. MAIN METHODS The inhibitory effect of IVSE, SE and IBSE on NO production in LPS-induced RAW264.7 cells was examined using Griess reagent, and the effects of IVSE on the expressions of inducible nitric oxide synthase (iNOS) and its upstream signal proteins including IκB kinase (IKK)/inhibitor kappa B (IκB)-α/nuclear factor κB (NF-κB) and mitogen-activated protein kinases (MAPKs) were investigated by Western blot. KEY FINDINGS Among the 3 compounds isolated, SE, IBSE, and IVSE inhibited NO production at 2.5 μM with 5.1%, 40.4%, and 52.8%, respectively. IVSE displayed the most potent inhibition of NO production. Mechanism analysis indicated that IVSE dramatically decreased the expression of iNOS, reduced the translocation of the NF-κB subunit p65 into the nucleus by interrupting the phosphorylation and degradation of IκB-α, and inhibited the activation of the upstream mediator IKK α/β. Furthermore, our results showed that IVSE inhibited the phosphorylation of MAPKs including extracellular regulated kinases (ERK1/2), c-Jun N-terminal kinases (JNK) and p38. SIGNIFICANCE IVSE exhibited anti-inflammatory activity by inhibiting NO production, in which inactivation of NF-κB and MAPKs might be involved. Our results suggest that IVSE might become an anti-inflammatory drug candidate.