Termination of pre-hospital resuscitation by anaesthesiologists - causes and consequences. A retrospective study.

AIM: Differentiating between a newly deceased patient and the lifeless patient in whom immediate resuscitation is required may be facilitated by a pre-hospital anaesthesiologist. The purpose of our study was to investigate to what extent and why the pre-hospital anaesthesiologist pronounced life extinct in situations where an emergency medical technician (EMT) would have been required to resuscitate. METHODS: All lifeless patients seen pre-hospitally by the anaesthesiologist-manned Mobile Emergency Care Unit in Odense, Denmark, from 2010 to 2014 were retrospectively studied. RESULTS: Of 17 035 contacts, 1275 patients were lifeless without reliable signs of death. In 642 of these patients (3.8%) resuscitation was initiated (median age 68 years). The remaining 633 patients (3.7%) were declared dead at the scene without any resuscitation attempt (median age 77 years). These latter patients would have been attempted resuscitated, had the anaesthesiologist not been present. In 54.5% of cases where documentation was available in the patient records, reasons for not resuscitating these patients included time elapsed from incident to contact with physician, 'overall assessment', chronic disease, or do-not-resuscitate order. CONCLUSION: In one patient in 30, the MECU refrained from futile resuscitation in cases where legislation required an EMT to initiate resuscitation. This practice reduced unethical attempts of resuscitation, reduced unnecessary emergency ambulance transports, and reduced the work load of the hospital resuscitation teams for one unnecessary alarm every third day. Differentiating between lifeless patients and dead patients not exhibiting reliable signs of death, however, is a complex task which is only sparsely documented.

Effects of Seocalcitol (EB1089) on nitrosomethyl urea-induced rat mammary tumors.

Although 1,25-dihydroxyvitamin D3 is a potent cell-differentiating agent, its use in cancer prevention or therapy is precluded because it induces hypercalcemia. Synthetic analogs have been developed which inhibit tumor progression in animal models of breast cancer. One analog, Seocalcitol (EB1089) has been shown to be effective in causing regression of N-methyl-nitrosourea-induced rat mammary tumors. However, at the most effective oral dose, a significant increase in serum and urinary calcium levels were observed. In order to compare the efficacy of different dosing schedules of Seocalcitol, rats were treated either 6 times weekly (1 microg/kg) or by intermittent dosing to achieve the same total weekly dose. All dosing schedules of Seocalcitol were effective in inhibiting tumor progression. Once daily dosing was significantly more effective than intermittent dosing but was associated with a greater rise in serum calcium concentration. In order to evaluate alternative treatment strategies to limit calcemic effects, we assessed the efficacy of limiting vitamin D-induced hypercalcemia using bisphosphonates. Seocalcitol (2.5 microg/kg daily p.o. for 4 weeks) alone and in combination with pamidronate (APD 0.4 mg/kg per day s.c.) or the same dose of the bisphosphonate EB 1053 caused substantial tumor regression. No statistically significant difference was seen between combination treatment and Seocalcitol treatment alone. Co-treatment with APD or EB 1053 did not limit the rise in serum calcium induced by Seocalcitol alone. Cessation of treatment or administration of a lower dose (1microg/kg twice weekly) reversed hypercalcemia, hypercalciuria and weight loss induced by high dose Seocalcitol. However, reduction in tumor volume was maintained in the majority of animals.

Activity of CHS 828 in primary cultures of human hematological and solid tumors in vitro.

CHS 828 is a pyridyl cyanoguanidine that has shown promising preclinical anticancer activity against various experimental tumor models and is presently being tested in a phase II trial in man. In the present study the fluorometric microculture cytotoxicity assay was used for in vitro evaluation of CHS 828 activity in primary cell cultures from hematological and solid tumors. In total, 156 samples from various diagnoses were tested with 72-h continuous drug exposure. CHS 828 showed high relative in vitro activity against tumor cells from chronic lymphocytic leukemia as well as from acute leukemia and high-grade lymphoma. Activity was also observed in several solid tumor cell samples, although the group as a whole appeared less responsive. CHS 828 was significantly more active against hematological malignancies compared to normal lymphocytes. Correlation analysis with standard drugs revealed low to moderate correlation coefficients. The results show that CHS 828 has potent antitumor activity against primary cultures of human tumor cells from patients and might have a unique mechanism of action.

Synthetic low-calcaemic vitamin D(3) analogues inhibit secretion of insulin-like growth factor II and stimulate production of insulin-like growth factor-binding protein-6 in conjunction with growth suppression of HT-29 colon cancer cells.

The aims of the present study were to compare the ability of various synthetic analogues of 1 alpha,25-dihydroxyvitamin D(3) [1 alpha,25-(OH)(2)D(3)] to inhibit proliferation of HT-29 cells, a human colon adenocarcinoma cell line. HT-29 cells were incubated for 144 h with various concentrations (0-100 nM) of 1 alpha,25-(OH)(2)D(3), or the analogues EB1089, CB1093 or 1 beta,25-(OH)(2)D(3). All these analogues except 1 beta,25-(OH)(2)D(3) inhibited cell proliferation, but relative potencies and efficacies of EB1089 and CB1093 were much greater than that of the native vitamin. Cells grew in serum-free medium, reaching a plateau density at day 10 of culture, and addition of 10 nM 1 alpha,25-(OH)(2)D(3) or 1 beta,25-(OH)(2)D(3) did not alter the long-term growth characteristics of HT-29 cells. However, cells treated with 10 nM EB1089 or CB1093 grew at a rate slower than control and reached final densities that were 53+/-1 and 36+/-2% lower than control, respectively. Immunoblot analysis of serum-free conditioned medium using a monoclonal anti-insulin-like growth factor-(IGF)-II antibody showed that both 10 nM EB1089 and CB1093 markedly inhibited secretion of both mature 7500 M(r) and higher M(r) forms of IGF-II. Ligand blot and immunoblot analyses of conditioned media revealed the presence of IGFBPs of M(r) 24,000 (IGFBP-4), 30,000 (glycosylated IGFBP-4), 35,000 (IGFBP-2) and 32,000-34,000 (IGFBP-6). The level of IGFBP-2 was decreased by 42+/-8 and 49+/-7% by 10 nM EB 1089 and CB1093, respectively, compared to controls. IGFBP-6 was increased approximately twofold by EB1089 and CB1093, and exogenously added IGFBP-6 inhibited HT-29 cell proliferation. These results suggest that inhibition of HT-29 cell proliferation by EB1089 and CB1093 may be attributed, at least in part, to the decreased secretion of IGF-II. The increase in IGFBP-6 concentration coupled with its high affinity for IGF-II may also contribute to decreased cellular proliferation by an indirect mechanism involving sequestration of endogenously produced IGF-II.

MCF-7/VD(R): a new vitamin D resistant cell line.

Several in vitro and in vivo experiments have demonstrated potent cell regulatory effects of vitamin D compounds in cancer cells. Moreover, a promising phase I study with the vitamin D analogue Seocalcitol (EB 1089) in patients with advanced breast and colon cancer has already been carried out and more clinical trials evaluating the clinical effectiveness of EB 1089 in other cancer types are in progress (Mørk Hansen et al. [2000a]). However, only little is known about the mechanisms underlying the actions of vitamin D or about the possible development of drug resistance in the patients. Therefore, in an attempt to gain more insight into these aspects, we have developed the MCF-7/VD(R) cell line, a stable subclone of the human MCF-7 breast cancer cell line, which is resistant to the growth inhibitory and apoptosis inducing effects of 1alpha,25(OH)(2)D(3). Despite this characteristic, receptor studies on the VDR have clearly demonstrated that the MCF-7/VD(R) cells contain fully functional VDRs, although in a lower number than seen with the parental MCF-7 cells. The regulation of the 24-hydroxylase enzyme appeared to be intact in the MCF-7/VD(R) cells and no differences with regard to growth rate and morphological appearance between the MCF-7/VD(R) cells and the parental MCF-7 cells were observed. Interestingly, however, the sensitivity of the MCF-7/VD(R) cells to the pure anti-estrogen ICI 182,780 was found to be increased. The MCF-7/VD(R) cell line shows characteristics different from those of previously described vitamin D resistant breast cancer cell lines but also some similarities. Together such vitamin D resistant cell lines therefore serve as a useful tool for studying the exact mechanism of action of vitamin D and the development of vitamin D resistance.

Inhibitory effects of 1alpha,25-dihydroxyvitamin D(3) on the G(1)-S phase-controlling machinery.

The nuclear hormone 1alpha,25-dihydroxyvitamin D(3) induces cell cycle arrest, differentiation, or apoptosis depending on target cell type and state. Although the antiproliferative effect of 1alpha,25-dihydroxyvitamin D(3) has been known for years, the molecular basis of the cell cycle blockade by 1alpha,25-dihydroxyvitamin D(3) remains largely unknown. Here we have investigated the mechanisms underlying the G(1) arrest induced upon 1alpha,25-dihydroxyvitamin D(3) treatment of the human breast cancer cell line MCF-7. Twenty-four-hour exposure of exponentially growing MCF-7 cells to 1alpha,25-dihydroxyvitamin D(3) impeded proliferation by preventing S phase entry, an effect that correlated with appearance of the growth-suppressing, hypophosphorylated form of the retinoblastoma protein (pRb), and modulation of cyclin-dependent kinase (cdk) activities of cdk-4, -6, and -2. Time course immunochemical and biochemical analyses of the cellular and molecular effects of 1alpha,25-dihydroxyvitamin D(3) treatment for up to 6 d revealed a dynamic chain of events, preventing activation of cyclin D1/cdk4, and loss of cyclin D3, which collectively lead to repression of the E2F transcription factors and thus negatively affected cyclin A protein expression. While the observed 10-fold inhibition of cyclin D1/cdk 4-associated kinase activity appeared independent of cdk inhibitors, the activity of cdk 2 decreased about 20-fold, reflecting joint effects of the lower abundance of its cyclin partners and a significant increase of the cdk inhibitor p21(CIP1/WAF1), which blocked the remaining cyclin A(E)/cdk 2 complexes. Together with a rapid down-modulation of the c-Myc oncoprotein in response to 1alpha,25-dihydroxyvitamin D(3), these results demonstrate that 1alpha,25-dihydroxyvitamin D(3) inhibits cell proliferation by targeting several key regulators governing the G(1)/S transition.

Vitamin D and cancer: effects of 1,25(OH)2D3 and its analogs on growth control and tumorigenesis.

Today, it is well established that besides playing a crucial role in the establishment and maintenance of the calcium homeostasis in the body, the active form of vitamin D, 1,25(OH)2D3, also acts an effective regulator of cell growth and differentiation in a number of different cell types, including cancer cells. This has led to an increased interest in using 1,25(OH)2D3 in the treatment or prevention of cancer patients and to a substantial number of studies investigating the effect of 1,25(OH)2D3 on cancer cells. The results are encouraging, but clearly demonstrate that the therapeutic window of 1,25(OH)2D3 is extremely narrow due to the calcemic adverse effects of this compound. Much effort has consequently been directed into identifying vitamin D analogs with potent cell regulatory effects but with weaker effects on the calcium metabolism than those of 1,25(OH)2D3. In an attempt to clarify the mechanisms implicated in the cell regulatory effects of 1,25(OH)2D3 and eventually facilitate the process of developing new specific vitamin D analogs, numerous investigations have been carried out with 1,25(OH)2D3 and its analogs. The present review will focus on the results obtained in these studies and describe some of the synthetic analogs, which have shown to be of particular interest in relation to cancer.

Vitamin D compounds exert anti-apoptotic effects in human osteosarcoma cells in vitro.

Several studies have demonstrated that vitamin D regulates growth and differentiation in bone cells in vitro. In addition, in vivo studies have shown that vitamin D stimulates bone formation, increases the number of osteoblast precursor cells and prevents bone mineral loss. These observations indicate that vitamin D may have anabolic effects on bone, and thus therapeutic potential in the treatment of osteoporosis. However, little is known about the effects of vitamin D on apoptosis in bone cells and about the contribution of this process to the effect of vitamin D on bone mineral loss. To investigate this aspect in more detail, we studied the effect of 1alpha,25(OH)(2)D(3) and a series of analogues on apoptosis in human osteosarcoma cells. No significant induction of apoptosis was observed with any of the compounds after a 5 day treatment period. In contrast, some of the analogues showed a tendency to protect the cells from undergoing apoptosis. This anti-apoptotic effect of vitamin D was further confirmed by the ability of 1alpha,25(OH)(2)D(3) to suppress camptothecin- and staurosporin-induced DNA fragmentation in the cells. In cultures treated simultaneously with 1alpha,25(OH)(2)D(3) in combination with camptothecin or staurosporin, the level of DNA fragmentation was markedly reduced compared with cultures treated with camptothecin or staurosporin alone. On the basis of the present results, it is therefore concluded that vitamin D displays anti-apoptotic effects in human osteoblast-like osteosarcoma cells in vitro. This observation suggests that besides regulating growth and differentiation, vitamin D exerts its anabolic effects on bone by protecting osteoblastic cells from undergoing apoptosis.

Molecular evaluation of vitamin D3 receptor agonists designed for topical treatment of skin diseases.

MC903 (calcipotriol) is a synthetic, low calcemic analog of the nuclear hormone 1alpha,25-dihydroxyvitamin D3 and used in the treatment of psoriasis. The beneficial effects of MC903 on psoriasis are based on gene regulatory events. The genomic actions of 1alpha,25-dihydroxyvitamin D3 and its analogs are primarily mediated by a complex of the vitamin D3 receptor and the retinoid X receptor bound to a 1alpha,25-dihydroxyvitamin D3 response element that can be considered as the molecular switch of 1alpha,25-dihydroxyvitamin D3 signaling. In this study, the interaction of MC903 and two new analogs, GS1500 and EB1213, with this molecular switch was compared with that of 1alpha,25-dihydroxyvitamin D3. In DNA-dependent limited protease digestion assays, ligand-dependent gel shift assays and mammalian-one-hybrid assays, all four ligands appeared to be equally sensitive VDR agonists that activated vitamin D3 receptor-retinoid X receptor-1alpha,25-dihydroxyvitamin D3 response element complexes at a concentration of approximately 0.2 nM. The analyzed VDR agonists, however, also showed individual molecular properties, such as a reduced sensitivity in HaCaT cells (MC903), a selectivity for DNA-bound vitamin D3 receptor-retinoid X receptor heterodimers (GS1500) and a long-lasting stabilization of vitamin D3 receptor-retinoid X receptor-1alpha,25-dihydroxyvitamin D3 response element complexes (EB1213). This molecular evaluation demonstrated that the sensitivity in activating the vitamin D3 receptor is already optimal for MC903, but the analog may not be ideal in keeping the receptor active and in selectively triggering 1alpha,25-dihydroxyvitamin D3 signaling pathways.

Cell death with atypical features induced by the novel antitumoral drug CHS 828, in human U-937 GTB cells.

N-(6-(4-chlorophenoxy)hexyl)-N'-cyano-N"-4-pyridylguanidine (CHS 828), with promising antitumoral effects in vitro and in vivo, is currently in clinical Phase I and II studies. Its exact mechanism of action is unclear, but previous studies indicate that CHS 828 induces a controlled, delayed mode of cell death. The characteristics of the cell death process were investigated in vitro in the apoptosis-prone cell line U-937 GTB. Mitochondria showed hyperpolarization at 24 to 32 h and a subsequent late disruption of mitochondria membrane potential (Deltapsi(m)). Between 44 and 72 h of CHS 828 exposure, there was an increasing frequency of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) positive cells indicative of apoptosis, but caspase-3 was only modestly increased and caspases-8 and -9 showed no activation upon CHS 828 exposure. Furthermore, the morphology of exposed cells did not conform to classical apoptosis, and viability and morphology were unaffected by inhibition of caspases. Thus, CHS 828 induces several unexpected features in this system, suggesting a potentially novel mechanism of action.

Cellular pharmacodynamics of the cytotoxic guanidino-containing drug CHS 828. Comparison with methylglyoxal-bis(guanylhydrazone).

N-(6-(4-chlorophenoxy)hexyl)-N'-cyano-N"-4-pyridylguanidine (CHS 828) is a new guanidino-containing compound with antitumoral activity both in vitro and in vivo. Its activity profile differs from those of standard cytotoxic drugs but the mechanism of action is not yet fully understood. CHS 828 is presently in early phase I and II clinical trials. In the present study, the pharmacodynamic effects at the cellular level of CHS 828 was compared to another compound containing two guanidino groups, methylglyoxal-bis(guanylhydrazone) (MGBG). MGBG is known to inhibit the synthesis of polyamines, which are important in, e.g., proliferation and macromolecular synthesis. The concentration-response relationship of CHS 828 closely resembled that of MGBG and the drugs were similar with respect to inhibition of DNA and protein synthesis. On the other hand, CHS 828 induced a significant increase in cellular metabolism while MGBG did not. The cytotoxic effect of MGBG was reversed by the addition of exogenous polyamines, while that of CHS 828 was unaffected. Unlike MGBG, there was also no effect of CHS 828 on the levels of decarboxylating enzymes in the polyamine biosynthesis. In conclusion, CHS 828 does not appear to share any major mechanisms of action with the polyamine synthesis inhibitor MGBG. Further studies will be required to define the exact mechanism of action of CHS 828.

Examination of structurally selective derivatization of vitamin D(3) analogues by electrospray mass spectrometry.

The structural specificity of vitamin D derivatization by PTAD (4-phenyl-1,2,4-triazoline-3,5-dione) was probed using synthetic analogues and ion trap mass spectrometry. EB 1089, a vitamin D(3) analogue which contains a second site for Diels--Alder cycloaddition on its side-chain, allowed the examination of derivatization modes and comparisons of ion fragment structures. The origins of a PTAD-vitamin D(3) ion fragment, commonly used in metabolite characterization and quantitation of vitamin D(3) analogues (m/z 314), were established; ion trap mass spectrometry revealed that the PTAD comprises a portion of this diagnostic fragment, and is not lost by a retro-Diels--Alder step. Furthermore, the unique structure of the EB 1089 side-chain also permits facile determination of its side-chain metabolism. Use of PTAD derivatization and detection of metabolite-specific ion fragments identify hydroxylation at the end of the EB 1089 sidechain. It is believed that the results from these studies provide a clearer understanding of the mass spectrometry of triazolinedione derivatives, not only in the specific case of EB 1089, but also in their application to other vitamin D compounds.

Temporal effects of the novel antitumour pyridyl cyanoguanidine (CHS 828) on human lymphoma cells.

CHS 828, a novel pyridyl cyanoguanidine, has shown potent antitumour activity both in vitro and in vivo and is currently undergoing phase I evaluation in humans in collaboration with the European Organization for Research and Treatment of Cancer (EORTC). Here we study the temporal effects of CHS 828 on cytotoxicity, protein and DNA synthesis, cellular morphology and ultra structure using the lymphoma cell line U-937 GTB as the primary tumour model. In vitro analysis of tumour cell survival in response to CHS 828 revealed a cytotoxic effect progressively increased as a function of exposure time with maximum efficacy observed after 72 h. Activity of CHS 828 on U-937 GTB cells grown in vivo was also found. CHS 828 induced-cell death was dependent on intact protein synthesis and most cells appeared to lose their membrane integrity in the presence of a relatively well preserved nuclear structure. The results indicate that CHS 828 induced active and delayed cell death with a non-apoptotic morphology.

In vivo activity of CHS 828 on hollow-fibre cultures of primary human tumour cells from patients.

Fresh human tumour cells from patients with ovarian cancer and chronic lymphocytic leukaemia were cultured in semipermeable hollow fibres. The fibres were implanted on immunocompetent rats, which were treated with the cyanoguanidine N-(4-chlorophenoxyhexyl)-N'-cyano-N"-4-pyridylguanidine (CHS 828). CHS 828 showed high antitumour activity against all eight tumour samples; the fibres from control animals had a mean net growth of 73% while CHS 828 treatment induced a 37% mean reduction of cell density, without observable haematological toxicity. The results show a feasibility of using tumour cells directly from patients in the hollow-fibre rat model.

The vitamin D analogue EB 1089 prevents skeletal metastasis and prolongs survival time in nude mice transplanted with human breast cancer cells.

1,25-Dihydroxyvitamin D has potent antiproliferative and anti-invasive properties in vitro in cancer cells. However, its calcemic effect in vivo limits its therapeutic applications. Here, we report the efficacy of EB 1089, a low calcemic analogue of vitamin D, on the development of osteolytic bone metastases after intracardiac injection of the human breast cancer cell line MDA-MB-231 in nude mice. Animals injected with tumor cells were implanted simultaneously with osmotic minipumps containing either EB 1089 or vehicle. Both groups remained normocalcemic for the duration of the experiment. The total number of bone metastases, the mean surface area of osteolytic lesions, and tumor burden within bone per animal were markedly decreased in EB1089-treated mice. Furthermore, longitudinal analysis revealed that mice treated with EB1089 displayed a marked increase in survival and developed fewer bone lesions and less hind limb paralysis over time as compared with untreated animals. These results suggest that EB1089 may be beneficial in the prevention of metastatic bone lesions associated with human breast cancer.

Early stimulation of acidification rate by novel cytotoxic pyridyl cyanoguanidines in human tumor cells: comparison with m-iodobenzylguanidine.

CHS 828, a newly recognized pyridyl cyanoguanidine, has shown promising antitumor activity both in vitro and in vivo and is presently in early phase I clinical trial in collaboration with EORTC. In this study, the effects of CHS 828 and a series of analogues on extracellular acidification and cytotoxicity were compared with those of m-iodobenzylguanidine (MIBG) in human tumor cells. The extracellular acidification rate was measured using the Cytosensor microphysiometer, and determination of cytotoxicity and proliferation was [(14)C] performed by the fluorometric microculture cytotoxicity assay (FMCA) and measurement of [(14)C]thymidine and leucine uptake. CHS 828 significantly increased the acidification rate during the first 15-24 hr in a concentration-dependent manner. This effect was abolished by removal of glucose from the medium, substituted with 10 mM of pyruvate, indicating stimulated glycolysis as the source of the increased acidification rate. However, CHS 828 induced cytotoxicity at concentrations well below those that affected the rate of acidification; when a series of closely related pyridylguanidine analogues were tested and compared, no apparent relationship between cytotoxicity and acidification could be discerned. Furthermore, comparable increases in the acidification rate were evident in one subline with high-grade resistance to the cytotoxic actions of CHS 828. The results indicate that CHS 828 may share the inhibitory actions of MIBG on mitochondrial respiration with a subsequent increase in glycolysis and acidification rate. However, this mechanism of action appears neither necessary nor sufficient to fully explain the cytotoxic actions of CHS 828 in human tumor cells, actions which remain to be mechanistically clarified.

Induction of apoptosis by vitamin D3 analogue EB1089 in NCI-H929 myeloma cells via activation of caspase 3 and p38 MAP kinase.

EB1089, a novel 1,25-dihydroxyvitamin D3 analogue, has been known to have potent antiproliferative properties in a variety of malignant cells both in vitro and in vivo. In the present study, we analysed the effect of EB1089 on NCI-H929 human myeloma cells. EB1089 inhibited cell growth of NCI-H929 and efficiently induced the G1 phase arrest of the cell cycle in a dose-dependent manner. We could also detect apoptosis in NCI-H929 cells exposed to EB1089 (1 x 10-7 M for 72 h) using the sub-G1 group of the cell cycle by FACS and annexin V binding assays. Induction of apoptosis by EB1089 was associated with down-regulation of the Bcl-2 protein without change of the Bax protein. Regarding caspase activity, which plays a crucial role in apoptosis, EB1089-treated NCI-H929 cells revealed an increased activity of caspase 3 protease accompanied by degradation of the PARP protein in a dose- and time-dependent manner. In addition, EB1089 caused the down-regulation of p44 extracellular signal-related kinase (ERK) activity and up-regulation of the p38 kinase activity during apoptosis of NCI-H929 cells. These results suggest that EB1089 inhibits growth of NCI-H929 cells via G1 cell cycle arrest as well as apoptosis by activating p38 kinase and suppressing ERK activity.

Seocalcitol (EB 1089): a vitamin D analogue of anti-cancer potential. Background, design, synthesis, pre-clinical and clinical evaluation.

It is well established that the metabolically active form of vitamin D, 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) plays a key role in the establishment and maintenance of the calcium metabolism in the body. In addition to this classic effect of 1alpha,25(OH)2D3, substantial evidence has emerged demonstrating that 1alpha,25(OH)2D3 is able to regulate cell growth and differentiation in a number of different cell types, including cancer cells. However, the clinical usefulness of 1alpha,25(OH)2D3 is limited by its tendency to cause hypercalcaemia. Much effort has therefore been directed to identifying new vitamin D analogues with potent cell regulatory effects, but with weaker effects on the calcium metabolism than those of 1alpha,25(OH)2D3. One of these new synthetic analogues is Seocalcitol (EB 1089). Despite being 50-200 times more potent than 1alpha,25(OH)2D3 with respect to regulation of cell growth and differentiation in vitro as well as in vivo, EB 1089 displays a reduced calcaemic activity in vivo compared to that of 1alpha, 25(OH)2D3. These characteristics make EB 1089 a potentially useful compound for the treatment of cancer. Recent clinical evaluation of EB 1089 has focused mainly on establishing a maximum tolerated dose in cancer patients. Early results confirm that the low calcaemic activity observed in animals can be reproduced in the clinic. Furthermore, EB 1089 has been shown to induce regression of tumours, especially in hepatocellular carcinoma where complete remission has been obtained. In conclusion, the development of EB 1089 as an anti-cancer drug holds promise. However, its final evaluation must await the completion of ongoing controlled clinical trials.

Apoptosis is induced by the active metabolite of vitamin D3 and its analogue EB1089 in colorectal adenoma and carcinoma cells: possible implications for prevention and therapy.

Vitamin D3 is believed to reduce the risk of colon cancer, and serum levels inversely correlate with colorectal cancer incidence. The active metabolite, 1alpha,25-dihydroxyvitamin D3, has previously been shown to inhibit growth and promote differentiation of colon cancer cells. The vitamin D analogue, EB1089, is currently under clinical trial in a variety of cancers because of its growth-inhibitory effects in vitro and reduced hypercalcemic effects in vivo. The mechanism of growth inhibition by EB1089, however, remained to be determined. In this study we examined the effects of alpha,25-dihydroxyvitamin D3 and EB1089 on five colorectal tumor cell lines (two adenoma and three carcinoma) to determine the mechanism of growth inhibition and to ascertain whether premalignant adenoma cells were responsive to these agents. 1alpha,25-Dihydroxyvitamin D3 and EB1089 induced p53-independent apoptosis in adenoma and carcinoma cell lines in a dose-dependent manner between 10(-10) and 10(-6) M. EB1089, as well as inducing apoptosis, increased the proportion of cells in the G1 phase, particularly in the adenoma cell lines. In two of the three carcinoma cell lines (SW620 and PC/JW), levels of apoptosis induced by EB1089 were similar or greater than those induced by 1alpha,25-dihydroxyvitamin D3. Although the carcinoma cell line HT29 was relatively resistant to apoptosis induced by EB1089 compared with 1alpha,25-dihydroxyvitamin D3, EB1089 markedly inhibited cell yields. These observations offer promise for the clinical use of EB1089. To determine whether the induction of apoptosis by 1alpha,25-dihydroxyvitamin D3 and EB1089 was potentially via a differentiation pathway, alkaline phosphatase activity was measured as a marker of differentiation. Induction of alkaline phosphatase was observed in the floating apoptotic cells as well as in the adherent population. A link between the induction of differentiation and apoptosis by 1alpha,25-dihydroxyvitamin D3 and EB1089 is suggested by the occurrence of apoptosis subsequent to the induction of differentiation. To investigate the molecular pathway to apoptosis induction, members of the Bcl-2 family of proteins were examined (Bcl-2, Bcl-x, Bax, and Bak). Decreased Bcl-2 was observed in some cell lines, particularly in response to EB1089, but was not essential for apoptosis. Levels of the proapoptotic protein Bak, however, were consistently increased in all of the five cell lines in association with apoptosis induced by either agent. The results implicate Bak protein in the induction of apoptosis by 1alpha,25-dihydroxyvitamin D3 or its analogue EB1089. The ability of EB1089 to induce apoptosis in colorectal carcinoma cells suggests that this or other vitamin D analogues may prove clinically effective for the treatment of colorectal cancer. Furthermore, the fact that it induces cell cycle arrest and apoptosis in the premalignant adenoma cells may suggest an application in colorectal cancer chemoprevention.

Effect of a novel vitamin D3 analog, EB1089, on G1 cell cycle regulatory proteins in HL-60 cells.

Progression of cell cycle in eukaryotes is regulated by a series of the cyclin-dependent kinases (CDKs) and cyclin-dependent kinase inhibitors (CDKIs). It has been shown that 1,25(OH)2D3 is able to arrest cell cycle at G1 phase in malignant cells including HL-60 cells. EB1089 is a novel 1,25(OH)2D3 analog that has more potent antileukemic properties with reduced hypercalcemic effect in vitro and in vivo than 1,25(OH)2D3. In the present study, we examined the effect of EB1089 on HL-60 cells at the protein levels of several G1 regulatory proteins. Exposure of HL-60 cells to EB1089 (1x10-8 M) for 3 days showed the G1 block by FACS analysis. The level of p21 was markedly induced in HL-60 cells treated with EB1089 at 24 h, and p27 were progressively increased in a time-dependent manner. The expressions of CDK2 and CDK6 were down-regulated during G1 block of HL-60 cells, and CDK4 is progressively elevated. In addition, level of cyclin D1 was increased in a time-dependent manner, however, no change of cyclin E was noted through the G1 to S traverse. Immunoprecipitation study demonstrated that p27 did not bind to CDK2, CDK4 and CDK6 in EB1089-treated HL-60 cell extracts. In contrast, complexes immunoprecipitated from EB1089-treated HL-60 cells with antibodies CDK2 and CDK6 contained higher amounts of immunodetectable p21 protein compared to untreated HL-60 cells, whereas no detectable change was noted with anti-CDK4 antibody. Furthermore, the kinase activities of CDK2 and CDK6 were decreased while little change was observed in CDK4 activity. These data indicated that p21 protein is a strong candidate for the control of G1 progression in EB1089-treated HL-60 cells, and its major target molecules are CDK2 and CDK6.