Overexpression of acid ceramidase protects from tumor necrosis factor-induced cell death

Tumor necrosis factor (TNF) signals cell death and simultaneously induces generation of ceramide. To evaluate the contribution of ceramide to TNF-dependent cell death, we generated clones of the TNF-sensitive cell line L929 that constitutively overexpress human acid ceramidase (AC). Ceramidase, in concert with sphingosine kinase, metabolizes ceramide to sphingosine-1-phosphate (SPP), an inducer of proliferation. In response to TNF, parental L929 cells display a significant increase in intracellular ceramide correlated with an "atypical apoptosis" characterized by membrane blebbing, DNA fragmentation and degradation of poly(ADP-ribose) polymerase despite a lack of caspase activity. These features are strongly reduced or absent in AC-overexpressing cells. Pharmacological suppression of AC with N-oleoylethanolamine restored the accumulation of intracellular ceramide as well as the sensitivity of the transfectants to TNF, implying that an enhanced metabolization of intracellular ceramide by AC shifts the balance between intracellular ceramide and SPP levels towards cell survival. Correspondingly, inhibition of ceramide production by acid sphingomyelinase also increased survival of TNF-treated L929 cells.     

First evidence of sphingosine 1-phosphate lyase protein expression and activity downregulation in human neoplasm: implication for resistance to therapeutics in prostate cancer

This is the first report of sphingosine 1-phosphate lyase (SPL) protein expression and enzymatic activity in human neoplasm. This enzyme drives irreversible degradation of sphingosine 1-phosphate (S1P), a bioactive lipid associated with resistance to therapeutics in various cancers, including prostate adenocarcinoma. In fresh human prostatectomy specimens, a remarkable decrease in SPL enzymatic activity was found in tumor samples, as compared with normal adjacent tissues. A significant relationship between loss of SPL expression and higher Gleason score was confirmed in tissue microarray (TMA) analysis. Moreover, SPL protein expression and activity were inversely correlated with those of sphingosine kinase-1 (SphK1), the enzyme producing S1P. SPL and SphK1 expressions were independently predictive of aggressive cancer on TMA, supporting the relevance of S1P in prostate cancer. In human C4-2B and PC-3 cell lines, silencing SPL enhanced survival after irradiation or chemotherapy by decreasing expression of proteins involved in sensing and repairing DNA damage or apoptosis, respectively. In contrast, enforced expression of SPL sensitized cancer cells to irradiation or docetaxel by tilting the ceramide/S1P balance toward cell death. Interestingly, the S1P degradation products failed to sensitize to chemo- and radiotherapy, supporting the crucial role of ceramide/S1P balance in cancer. Of note, the combination of SPL enforced expression with a SphK1 silencing strategy by further decreasing S1P content made prostate cancer cells even more sensitive to anticancer therapies, suggesting that a dual strategy aimed at stimulating SPL, and inhibiting SphK1 could represent a future approach to sensitize cancer cells to cancer treatments. Mol Cancer Ther; 11(9); 1841-51. ?2012 AACR.     

Involvement of sphingolipids in apoptin-induced cell killing.

The potential anti-tumor agent Apoptin activates apoptosis in many human cancers and transformed cell lines, but is believed to be less potent in primary cells. Although caspase 3 is activated during apoptin-induced apoptosis, the mechanism of tumor cell killing remains elusive. We now show that apoptin-mediated cell death involves modulation of the sphingomyelin-ceramide pathway. Treating cells with Ad-GFPApoptin resulted in increased ceramide accumulation and enhanced expression of acid sphingomyelinase (ASMase) with a concomitant increase in ASMase activity and decreased sphingomyelin. Using confocal microscopy, ASMase, normally present in the endosomal/lysosomal compartment, was observed to translocate to the cell's periphery. Cotreatment of Ad-GFPApoptin-infected cells with the ASMase inhibitor desipramine (2.5 muM) attenuated (30%; P<0.01) apoptin-induced cell death. Apoptin was also able to induce a significant decline in sphingosine content by inhibition of ceramide deacylation through down-regulation of acid ceramidase at the protein level. Supporting the role of ceramide in apoptin action, treatment of cells with the combination of an exogenous cell-permeable ceramide analog (C6-ceramide) and Ad-GFPApoptin infection yielded a significant increase (P<0.01) in apoptosis over either treatment modality alone. Together, these data suggest that apoptin modulates ceramide/sphingolipid metabolism as part of its mechanism of action.     

Chemosensitizing effects of sphingosine kinase-1 inhibition in prostate cancer cell and animal models

We have previously reported that, in prostate cancer, inhibition of the oncogenic sphingosine kinase-1/sphingosine 1-phosphate (SphK1/S1P) pathway is a key element in chemotherapy-induced apoptosis. Here, we show that selective pharmacologic inhibition of SphK1 triggers apoptosis in LNCaP and PC-3 prostate cancer cells, an effect that is reversed by SphK1 enforced expression. More importantly, we show for the first time that the up-regulation of the SphK1/S1P pathway plays a crucial role in the resistance of prostate cancer cells to chemotherapy. Importantly, pharmacologic SphK1 inhibition with the B-5354c compound sensitizes LNCaP and PC-3 cells to docetaxel and camptothecin, respectively. In vivo, camptothecin and B-5354c alone display a limited effect on tumor growth in PC-3 cells, whereas in combination there is a synergy of effect on tumor size with a significant increase in the ceramide to S1P sphingolipid ratio. To conclude, our study highlights the notion that drugs specifically designed to inhibit SphK1 could provide a means of enhancing the effects of conventional treatment through the prosurvival antiapoptotic SphK1/S1P pathway.     

Modulation of ceramide metabolism enhances viral protein apoptin's cytotoxicity in prostate cancer.

Despite local and systemic therapies, the National Cancer Institute estimates that prostate cancer will cause over 30,000 deaths in 2006. This suggests that additional therapeutic approaches are needed. The chicken anemia viral protein Apoptin causes tumor-selective apoptosis in human tumor lines independent of p53 and Bcl-2 status. Tet-regulated expression of Apoptin from an adenoviral vector showed cytotoxicity in DU145, PC-3, and LNCaP tumor cells regardless of expression of p53, Bcl-2, Bcl-xL, Bax, survivin, FLIP(S), XIAP, or CIAP. Apoptin expression caused an increase in the tumor suppressor lipid ceramide, which regulates the cellular stress response. Interestingly, 10 of 15 primary prostate cancers examined by Western blotting overexpressed acid ceramidase (AC), suggesting that ceramide deacylation might serve to negate elevated levels of ceramide, creating a more antiapoptotic phenotype. This was confirmed in AC-overexpressing cells in which we observed decreased sensitivity to apoptosis following treatment with Apoptin. Addition of the AC inhibitor LCL204, in combination with Apoptin, augmented cell killing. This effect was also demonstrated in vivo in that Apoptin and LCL204 cotreatment significantly reduced tumor growth in DU145 xenografts (P<0.05). Taken together, our data demonstrated that Apoptin is a promising therapeutic agent for prostate cancer and that its function is improved when combined with acid ceramidase inhibitors.     

Distribution and dynamic changes of sphingolipids in blood in response to platelet activation

BACKGROUND: Sphingolipids are signaling molecules in a range of biological processes. While sphingosine-1-phosphate (S1P) is thought to be abundantly stored in platelets and released upon stimulation, knowledge about the distribution and function of other sphingolipids in blood is lacking. OBJECTIVES: To analyze the sphingolipid content of blood components with special emphasis on dynamic changes in platelets. METHODS: Blood components from mice and humans were prepared by gradient centrifugation and analyzed by liquid chromatography-mass spectrometry. Additionally, murine platelets were activated in vitro and in vivo. RESULTS: Isolated non-activated platelets of mice were devoid of S1P, but instead contained dihydrosphingosine-1-phosphate (dhS1P), along with a high concentration of ceramide. Activation of platelets in vitro led to a loss of dhS1P and an increase in sphingosine, accompanied by a reduction of ceramide content. Platelet activation in vivo led to an immediate and continuous rise of dhS1P in plasma, while S1P remained stable. The sphingolipid distribution of human blood was markedly different from mice. Human platelets contained dhS1P in addition to S1P. CONCLUSIONS: Mouse platelets contain dhS1P instead of S1P. Platelet activation causes loss of dhS1P and breakdown of ceramide, implying ceramidase activation. Release of dhS1P from activated platelets might be a novel signaling pathway. Finally, the sphingolipid composition of mouse and human blood shows large differences, which must be considered when studying sphingolipid biology.     

A high-performance liquid chromatographic assay for acid ceramidase activity in cultured fibroblasts from patients with Farber's disease and from controls

The high-performance liquid chromatographic (HPLC) method we devised for assay of acid ceramidase activity involves coupling of a fluorescent probe to the enzymatically released sphingosine in the reaction mixture and detection of the fluorescent sphingosine derivative by reverse-phase HPLC. Using the method, acid ceramidase activity in fibroblast homogenates was accurately assayed, with or without the addition of exogenous ceramide, as the substrate, and the patients and carriers of Farber's disease could be readily diagnosed.     

线粒体神经酰胺酶上调K562细胞Bcl-2蛋白表达水平

最近，克隆了一种新的选择性定位于线粒体的神经酰胺酶，提示线粒体存在着神经酰胺代谢途径，可能对线粒体的功能，特别对凋亡产生影响。本研究将线粒体神经酰胺酶基因转染到K562细胞，以此了解线粒体神经酰胺酶的细胞生物学效应。以脂质体介导将含有线粒体神经酰胺酶cDNA的pcDNA3．1／His—CDase质粒转染到K562细胞，G418筛选阳性克隆，建立稳定表达线粒体神经酰胺酶的K562TC细胞株。用MTT法、annexin V／PI法、FCM及Westernblot分别检测K562与K562TC细胞在细胞毒药物抗性、血清剥夺耐受性及Bcl-2蛋白表达水平方面的差异。结果表明：虽然K562TC与K562细胞对阿霉素、足叶乙甙及亚砷酸的敏感性没有差异，但是K562TC细胞Bcl-2蛋白水平明显升高，抗血清剥夺能力明显增强。以硫代反义寡核苷酸特异性封闭K562TC细胞线粒体神经酰胺酶，下调Bcl-2蛋白表达水平；二甲基鞘氨醇(鞘氨醇激酶抑制剂，降低细胞内1-磷酸鞘氨醇水平)同样下调K562TC细胞Bcl-2蛋白表达水平，而1-磷酸鞘氨醇明显上调K562细胞Bcl-2表达水平。结论：线粒体神经酰胺酶将线粒体神经酰胺代谢为鞘氨醇，进而在鞘氨醇激酶作用下生成1-磷酸鞘氨醇，此代谢途径上调K562细胞Bcl-2蛋白表达水平。     

Macrophage TNF mRNA expression induced by LPS is regulated by sphingomyelin metabolites.

Metabolism of macrophage (MO) membrane phospholipids produces key mediators of inflammation and major second messengers that modulate inflammatory responses during sepsis. Sphingomyelin is a major class of phospholipid that releases ceramide and sphingosine. This study was designed to investigate the involvement of sphingomyelin metabolites in MO activation by lipopolysaccharide (LPS). Rabbit alveolar MO were obtained by bronchoalveolar lavage and exposed to C6-ceramide, a cell-permeable analogue of natural ceramide, or sphingosine in the presence of Escherichia coli LPS (100 ng/mL). Tumor necrosis factor (TNF) mRNA expression was measured by Northern blot assays. Total nuclear extract was harvested for the measurement of nuclear factor KB (NFkappaB) with electrophoretic mobility shift assays. MO TNF production was measured by L929 bioassays. C-6 ceramide did not have any effects on MO TNF production or TNF mRNA expression with or without LPS stimulation. Inhibition of ceramide metabolism with 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), or N-oleoyl-ethanolamine (NOE) also did not induce TNF mRNA or TNF production. In comparison, sphingosine inhibited TNF mRNA expression as well as TNF production of LPS-stimulated MO. LPS-induced MO NFkappaB activity was also reduced by sphingosine. Our data indicate that ceramide alone has no effect on macrophage activity, but its metabolite sphingosine down-regulates MO activation induced by LPS stimulation. Therefore, the sphingomyelin pathway is involved in the regulation of MO activation.     

Antitumor activity of sphingosine kinase inhibitors

Sphingosine kinase (SK) is an oncogenic sphingolipid-metabolizing enzyme that catalyzes the formation of the mitogenic second messenger sphingosine-1-phosphate (S1P) at the expense of proapoptotic ceramide. Thus, SK is an attractive target for cancer therapy because blockage of S1P formation leads to inhibition of proliferation, as well as the induction of apoptosis in cancer cells. We have recently identified novel SK inhibitors with nanomolar to low micromolar potencies toward recombinant human SK. This study describes the continuing analysis of these inhibitors through in vitro and in vivo experiments. All three structurally diverse SK inhibitors tested showed antitumor activity in mice without exhibiting toxicity. Blood and tumor inhibitor concentrations exceeded in vitro potency levels. Cell signaling analyses in vitro revealed mixed inhibition of mitogen-activated protein kinase kinase and Akt phosphorylation by the SK inhibitors. Importantly, 4-[4-(4-chloro-phenyl)-thiazol-2-ylamino]-phenol (SKI-II) is orally bioavailable, detected in the blood for at least 8 h, and showed a significant inhibition of tumor growth in mice. These compounds are the first examples of nonlipid selective inhibitors of SK with in vivo antitumor activity and provide leads for further development of inhibitors of this important molecular target.     

Preclinical characterization of OSI-027, a potent and selective inhibitor of mTORC1 and mTORC2: distinct from rapamycin

The phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway is frequently activated in human cancers, and mTOR is a clinically validated target. mTOR forms two distinct multiprotein complexes, mTORC1 and mTORC2, which regulate cell growth, metabolism, proliferation, and survival. Rapamycin and its analogues partially inhibit mTOR through allosteric binding to mTORC1, but not mTORC2, and have shown clinical utility in certain cancers. Here, we report the preclinical characterization of OSI-027, a selective and potent dual inhibitor of mTORC1 and mTORC2 with biochemical IC(50) values of 22 nmol/L and 65 nmol/L, respectively. OSI-027 shows more than 100-fold selectivity for mTOR relative to PI3Kα, PI3Kβ, PI3Kγ, and DNA-PK. OSI-027 inhibits phosphorylation of the mTORC1 substrates 4E-BP1 and S6K1 as well as the mTORC2 substrate AKT in diverse cancer models in vitro and in vivo. OSI-027 and OXA-01 (close analogue of OSI-027) potently inhibit proliferation of several rapamycin-sensitive and -insensitive nonengineered and engineered cancer cell lines and also, induce cell death in tumor cell lines with activated PI3K-AKT signaling. OSI-027 shows concentration-dependent pharmacodynamic effects on phosphorylation of 4E-BP1 and AKT in tumor tissue with resulting tumor growth inhibition. OSI-027 shows robust antitumor activity in several different human xenograft models representing various histologies. Furthermore, in COLO 205 and GEO colon cancer xenograft models, OSI-027 shows superior efficacy compared with rapamycin. Our results further support the important role of mTOR as a driver of tumor growth and establish OSI-027 as a potent anticancer agent. OSI-027 is currently in phase I clinical trials in cancer patients.     

Radiation-induced acid ceramidase confers prostate cancer resistance and tumor relapse

Escape of prostate cancer (PCa) cells from ionizing radiation–induced (IR-induced) killing leads to disease progression and cancer relapse. The influence of sphingolipids, such as ceramide and its metabolite sphingosine 1-phosphate, on signal transduction pathways under cell stress is important to survival adaptation responses. In this study, we demonstrate that ceramide-deacylating enzyme acid ceramidase (AC) was preferentially upregulated in irradiated PCa cells. Radiation-induced AC gene transactivation by activator protein 1 (AP-1) binding on the proximal promoter was sensitive to inhibition of de novo ceramide biosynthesis, as demonstrated by promoter reporter and ChIP-qPCR analyses. Our data indicate that a protective feedback mechanism mitigates the apoptotic effect of IR-induced ceramide generation. We found that deregulation of c-Jun induced marked radiosensitization in vivo and in vitro, which was rescued by ectopic AC overexpression. AC overexpression in PCa clonogens that survived a fractionated 80-Gy IR course was associated with increased radioresistance and proliferation, suggesting a role for AC in radiotherapy failure and relapse. Immunohistochemical analysis of human PCa tissues revealed higher levels of AC after radiotherapy failure than those in therapy-naive PCa, prostatic intraepithelial neoplasia, or benign tissues. Addition of an AC inhibitor to an animal model of xenograft irradiation produced radiosensitization and prevention of relapse. These data indicate that AC is a potentially tractable target for adjuvant radiotherapy.     

Acid Ceramidase Upregulation in Prostate Cancer Cells Confers Resistance to Radiation: AC Inhibition, a Potential Radiosensitizer

Radiation resistance in a subset of prostate tumors remains a challenge to prostate cancer radiotherapy. The current study on the effects of radiation on prostate cancer cells reveals that radiation programs an unpredicted resistance mechanism by upregulating acid ceramidase (AC). Irradiated cells demonstrated limited changes of ceramide levels while elevating levels of sphingosine and sphingosine-1-phosphate. By genetically downregulating AC with small interfering RNA (siRNA), we observed radiosensitization of cells using clonogenic and cytotoxicity assays. Conversely, AC overexpression further decreased sensitivity to radiation. We also observed that radiation-induced AC upregulation was sufficient to create cross-resistance to chemotherapy as demonstrated by decreased sensitivity to Taxol and C₆ ceramide compared to controls. Lower levels of caspase 3/7 activity were detected in cells pretreated with radiation, also indicating increased resistance. Finally, utilization of the small molecule AC inhibitor, LCL385, sensitized PPC-1 cells to radiation and significantly decreased tumor xenograft growth. These data suggest a new mechanism of cancer cell resistance to radiation, through upregulation of AC that is, in part, mediated by application of the therapy itself. An improved understanding of radiotherapy and the application of combination therapy achieved in this study offer new opportunities for the modulation of radiation effects in the treatment of cancer.     

Ionizing radiation and chemotherapeutic drugs induce apoptosis in lymphocytes in the absence of Fas or FADD/MORT1 signaling. Implications for cancer therapy

Ionizing radiation and cytotoxic drugs used in the treatment of cancer induce apoptosis in many cell types, including tumor cells. It has been reported that tumor cells treated with anticancer drugs increase surface expression of Fas ligand (FasL) and are killed by autocrine or paracrine apoptosis signaling through Fas (Friesen, C., I. Herr, P.H. Krammer, and K.-M. Debatin. 1996. Nat. Med. 2:574-577). We show that lymphocytes that cannot be killed by FasL, such as those from Fas-deficient lpr mice or transgenic mice expressing a dominant negative mutant of Fas-associated death domain protein (FADD/MORT1), are as sensitive as normal lymphocytes to killing by gamma radiation or the cytotoxic drugs cis-platin, doxorubicin, and etoposide. In contrast, p53 deficiency or constitutive expression of Bcl-2 markedly increased the resistance of lymphocytes to gamma radiation or anticancer drugs but had no effect on killing by FasL. Consistent with these observations, lpr and wild-type T cells both had a reduced capacity for mitogen-induced proliferation after drug treatment, whereas bcl-2 transgenic or p53-deficient T cells retained significant clonogenic potential. These results demonstrate that apoptosis induced by ionizing radiation or anticancer drugs requires p53 and is regulated by the Bcl-2 protein family but does not require signals transduced by Fas and FADD/MORT1.     

Novel antagonists of the thioesterase domain of human fatty acid synthase

Fatty acid synthase (FAS) is up-regulated in a wide range of cancers and has been recently identified as a potential therapeutic target. Indeed, previous research has shown that inhibition of FAS with active site-modifying agents can block tumor cell proliferation, elicit tumor cell death, and prevent tumor growth in animal models. Here, we use a high-throughput fluorogenic screen and identify a novel pharmacophore, 5-(furan-2-ylmethylene) pyrimidine-2,4,6-trione, which inhibits the thioesterase domain of FAS. The novel antagonists are competitive inhibitors of the thioesterase domain, inhibit de novo fatty acid synthesis, and elicit FAS-dependent tumor cell death. This set of novel FAS antagonists provides an important pharmacologic lead for further development of anticancer therapeutics.     

PUMA induction by FoxO3a mediates the anticancer activities of the broad-range kinase inhibitor UCN-01

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.     

Bioactive sphingolipids in docetaxel-induced apoptosis in human prostate cancer cells

In this study, we examined the possible roles of ceramide/sphingosine-1-phosphate and ceramide/glucosyleceramide signaling in docetaxel-induced apoptosis by examining expression levels of the glucosyleceramide synthase and sphingosine kinase-1 and ceramide synthase gene family. As confirmed by isobologram analysis, docetaxel in combination with agents that increase intracellular ceramide levels increased the cytotoxic and apoptotic effects of docetaxel synergistically. More importantly, RT-PCR results revealed that expression levels of glucosyleceramide synthase and sphingosine kinase-1 were downregulated and ceramide synthase genes were upregulated in response to docetaxel. This study identifies mechanisms underlying the involvement of ceramide metabolizing genes in docetaxel-induced apoptosis in prostate cancer cells.     

Cationic long-chain ceramide LCL-30 induces cell death by mitochondrial targeting in SW403 cells

Ceramides are sphingolipid second messengers that are involved in the mediation of cell death. There is accumulating evidence that mitochondria play a central role in ceramide-derived toxicity. We designed a novel cationic long-chain ceramide [omega-pyridinium bromide D-erythro-C16-ceramide (LCL-30)] targeting negatively charged mitochondria. Our results show that LCL-30 is highly cytotoxic to SW403 cells (and other cancer cell lines) and preferentially accumulates in mitochondria, resulting in a decrease of the mitochondrial membrane potential, release of mitochondrial cytochrome c, and activation of caspase-3 and caspase-9. Ultrastructural analyses support the concept of mitochondrial selectivity. Interestingly, levels of endogenous mitochondrial C16-ceramide decreased by more than half, whereas levels of sphingosine-1-phosphate increased dramatically and selectively in mitochondria after administration of LCL-30, suggesting the presence of a mitochondrial sphingosine kinase. Of note, intracellular long-chain ceramide levels and sphingosine-1-phosphate remained unaffected in the cytosolic and extramitochondrial (nuclei/cellular membranes) cellular fractions. Furthermore, a synergistic effect of cotreatment of LCL-30 and doxorubicin was observed, which was not related to alterations in endogenous ceramide levels. Cationic long-chain pyridinium ceramides might be promising new drugs for cancer therapy through their mitochondrial preference.     

1-磷酸鞘氨醇对T细胞功能的影响

1-磷酸鞘氨醇（S1P）是鞘磷脂代谢过程中产生的一种重要信号分子，可与多条信号通路交联而产生广泛的生物学效应。T细胞表达5种S1P受体，即S1P1—S1P5。S1P信号可以调节T细胞的多种免疫效应，包括T细胞增殖和凋亡、T细胞向Th2细胞分化、细胞因子分泌及T细胞迁移等。抑制S1P信号通路的药物FTY720可将T细胞阻滞于次级淋巴器官，造成外周血T细胞显著减少而发挥强烈的免疫抑制作用。本文对S1P的合成和降解、S1P受体及其介导的信号途径、T细胞S1P受体的表达、S1P对T细胞功能的调节及S1P信号通路的免疫抑制药物作了概述．     

A novel indolocarbazole,ICP-1, abrogates DNA damage-induced cell cycle arrest and enhances cytotoxicity: similarities and differences to the cell cycle checkpoint abrogator UCN-01

DNA damaging agents such as cisplatin arrest cell cycle progression at either G1, S, or G2 phase, although the G1 arrest is only seen in cells expressing the wild-type p53 tumor suppressor protein. We have reported that 7-hydroxystaurosporine (UCN-01) overcomes S and G2 phase arrest and enhances the cytotoxicity of cisplatin. Abrogation of arrest appears to be selective for cells defective in p53 and therefore provides a potential, tumor-targeted therapy. Unfortunately, UCN-01 binds avidly to human plasma proteins, limiting access to the tumor. A screen of related indolocarbazoles identified analogues with both beneficial and undesirable properties. This led to a synthetic program to develop a novel analogue rationally designed to overcome the obstacles observed with the other analogues. We report the synthesis and analysis of a novel analogue, ICP-1. This analogue abrogated S and G2 phase arrest and enhanced cytotoxicity induced by cisplatin only in p53 defective cells. ICP-1 also abrogated arrest and enhanced cell killing induced by the topoisomerase I inhibitor SN38. Analysis of proteins that regulate cell cycle arrest suggest both drugs inhibit checkpoint kinases Chk1 and/or Chk2. In contrast to UCN-01, checkpoint abrogation by ICP-1 was only slightly inhibited by human plasma. UCN-01 and ICP-1 differed significantly in other regards. UCN-01 potently enhanced the activity of 1-beta-D-arabinofuranosylcytosine in both p53 wild-type and mutant cells, whereas ICP-1 was inactive in this combination. This property of UCN-01 was independent of its ability to inhibit protein kinase C because more specific inhibitors of protein kinase C failed to enhance cell killing induced by 1-beta-D-arabinofuranosylcytosine. High concentrations of UCN-01 also inhibit C-TAK1 that results in S phase-arrested cells directly entering mitosis, but this property was not observed with ICP-1. Hence, ICP-1 appears to be a more selective inhibitor of the S and G2 cell cycle checkpoint than previously studied analogues and is worthy of study in preclinical tumor models.