The role of ERα36 in cell type-specific functions of estrogen and cancer development.

Exploring the regulatory effects of estrogen on different body organs via its receptors is largely of interest. Recently, the expression, signaling and the clinical significance of ERα36, the newly identified isoform of ERα, mediating non-genomic signaling of estrogen, have been studied in a wide range of organs and tumors. ERα36 is expressed highly in the CNS and actively involved in neuroprotection. It is also suggested to be an important estrogen receptor involved in preserving bone in postmenopausal women. On the oncological side, although ERα36 has usually been considered to be an oncogenic molecule, results from some studies paradoxically imply its protective role in certain tumors. Collectively, it seems that ERα36 is highly involved in cell type-specific functions of estrogen through its MAPK/ERK signaling, which is dependent on ERα36 expression levels, ligand concentrations and disease stage. The response is also dependent on the levels of ERα66 and ERß. These factors influence the ERK kinetic and determine the ultimate mitogenic or antimitogenic signaling of estrogen, leading to cell survival or cell death. In this review, we summarize the recent organ-specific, cellular and molecular events and the mechanisms involved in estrogen effects mediated through the ERα36/ ERα66 with a particular focus on carcinomas where more clinical information has recently emerged.

Expression patterns of ERα66 and its novel variant isoform ERα36 in lactotroph pituitary adenomas and associations with clinicopathological characteristics.

PURPOSE: The regulatory effects of estradiol on pituitary homeostasis have been well documented. However, the expression patterns of ERα66 and ERα36 and their correlations with the clinical course of postoperative prolactinoma tumors remain unclear. METHODS: The expression of ERα36, ERα66, Ki67, p53, and CD31 were determined by immunohistochemistry in 62 prolactinoma patients. Snap-frozen tumors and normal pituitaries were also examined by western blotting for estrogen receptor detection. RESULTS: A broad expression of ERα36 was identified in normal pituitaries. The median scores of ERα36 and ERα66 expression were 8 and 6 in normal pituitaries and 4 and 0 in tumors, respectively. Four phenotypes of ERα36 and ERα66 expression were explored in tumors with regard to sex, invasiveness, dopamine resistance, and recurrence. Low ERα36 expression was associated with tumor invasion and increased Ki67. Low ERα66 expression was associated with tumor invasion, dopamine-agonist resistance, and enhanced tumor size. Multivariable logistic regression analysis showed that low ERα36 expression is an independent risk factor for invasiveness. The significant inverse association of ERα66 with invasiveness, dopamine resistance, and tumor size remained significant after adjustment for sex as a potential confounder. After controlling for sex, the low ERα66/low ERα36 phenotype was 6.24 times more prevalent in invasive tumors than in noninvasive tumors. Although the decreasing trend of CD31 expression from surrounding nontumoral lactotroph adenomas to tumors was similar to that of the estrogen receptors, a significant correlation was not observed here. CONCLUSION: The decreasing trends of ERα36 and ERα66 expression from normal pituitaries to tumors are associated with aggressive clinical behavior.

Albumin nanoparticles increase the anticancer efficacy of albendazole in ovarian cancer xenograft model.

BACKGROUND: The poor prognosis of patients with drug resistant ovarian cancer and the lack of targeted therapy have raised the need for alternative treatments. Albendazole (ABZ) is an anti-parasite compound capable of impairing microtubule formation. We hypothesized that ABZ could be repurposed as a potential anti-angiogenic drug due to its potent inhibition of vascular endothelial growth factor (VEGF) in ovarian cancer with ascites. However, the poor aqueous solubility of ABZ limits its potential for cancer therapy. In this study, we have assembled ABZ with bovine serum albumin into nanoparticles with a size range of 7-10 nm (BSA-ABZ) and 200-250 nm (Nab-ABZ). We further examined the anticancer effects of ABZ carrying nanoparticles in ovarian cancer cells, in both in vitro and in vivo models. RESULTS: Drug release studies demonstrated that about 93% of ABZ was released from BSA-ABZ 10 nm in comparison to 83% from Nab-ABZ 200 nm at pH 7.4 in 8 days. In vitro cell proliferation studies showed that the BSA-ABZ 10 nm exhibited the highest killing efficacy of ovarian cancer cells with surprisingly least toxicity to healthy ovarian epithelial cells. Confocal microscopy and fluorescence activated cell sorting analysis (FACS) revealed more efficient internalization of the BSA-ABZ 10 nm by cancer cells. For in vivo studies, we examined the tumor growth, ascites formation and the expression of VEGF and secreted protein acidic and rich in cysteine (SPARC) in tumor samples and only VEGF in plasma samples. The BSA-ABZ 10 nm reduced the tumor burden significantly (p < 0.02) at a much lower drug dose (10 µg/ml) compare to free drug. Both formulations were capable of suppressing the ascites volume significantly (p < 0.05) and reducing the number of ascites cells. The expression of VEGF and SPARC was also reduced, which indicates the underlying therapeutic mechanism of the ABZ. CONCLUSION: Our data suggest that the BSA-ABZ may hold promise for the treatment and control of progression of ovarian cancer with ascites. However further studies are required to examine the efficacy of both the formulations in aggressive models of recurrent ovarian cancer with respect to particle size and dosing parameters.

Involvement of urokinase-type plasminogen activator system in cancer: an overview.

Currently, there are several studies supporting the role of urokinase-type plasminogen activator (uPA) system in cancer. The association of uPA to its receptor triggers the conversion of plasminogen into plasmin. This process is regulated by the uPA inhibitors (PAI-1 and PAI-2). Plasmin promotes degradation of basement membrane and extracellular matrix (ECM) components as well as activation of ECM latent matrix metalloproteases. Degradation and remodeling of the surrounding tissues is crucial in the early steps of tumor progression by facilitating expansion of the tumor mass, release of tumor growth factors, activation of cytokines as well as induction of tumor cell proliferation, migration, and invasion. Hence, many tumors showed a correlation between uPA system component levels and tumor aggressiveness and survival. Therefore, this review summarizes the structure of the uPA system, its contribution to cancer progression, and the clinical relevance of uPA family members in cancer diagnosis. In addition, the review evaluates the significance of uPA system in the development of cancer-targeted therapies.

Human Sprouty1 suppresses growth, migration, and invasion in human breast cancer cells.

Breast cancer is the most common cancer and the leading cause of cancer death in women worldwide. Expression of human Sprouty1 (hSpry1) gene is downregulated in most breast cancer patients, implicating it as an important tumor suppressor gene. So, we hypothesized that overexpression of hSpry1 gene may suppress breast cancer cell growth, migration, and invasion. Here, we demonstrate that in breast cancer cell lines, MDA-MB-231 and T47D, transfection-induced overexpression of hSpry1 reduced cell population, proliferation, and colony formation in vitro without affecting cell apoptosis. Adhesion molecules act as both positive and negative modulators of cellular migration and invasion. Here, we found that overexpression of hSpry1 enhances the initial establishment events in breast cancer cell adhesion to type IV collagen and vitronectin. Moreover, the overexpression of hSpry1 in the highly invasive MDA-MB-231 breast cancer cells causes a significant reduction in cellular migration and invasion through Matrigel membranes. In addition, we showed that hSpry1 overexpression prevents VEGF secretion. VEGF is essential for primary tumor growth, migration, and invasion. Thus, our study provides a novel mechanism of tumor suppression activity of hSpry1.

Effect of minocycline on pentylenetetrazol-induced chemical kindled seizures in mice.

Inflammation is one of the mechanisms involved in seizure induction. In this study, the effect of minocycline, an anti-inflammatory drug, was investigated on kindling acquisition. Chemical kindling was induced by injection of a subthreshold dose of pentylenetetrazol (PTZ; 37.5 mg/kg) in mice on every other day. Two groups of animals received minocycline (25 mg/kg) at 1 h before or 1 h after PTZ injection. Following the last PTZ injection, the changes in gene expression of TNF-α receptor, γ2 subunit of GABAA receptor and NR2A subunit of NMDA receptor were assessed in the hippocampus and piriform cortex. Injection of minocycline before PTZ increased the latency to stage 4 seizure, and decreased the duration of stages 4 and 5 seizure. It also prevented the increase in the mRNA of NR2A subunit of NMDA receptor in the hippocampus and removed the PTZ-induced increase in mRNA of γ2 subunit of GABAA receptor in piriform cortex of PTZ kindled mice. Minocycline also prevented the increase in TNF-α receptor gene expression in both hippocampus and piriform cortex. Injection of minocycline after PTZ had no significant effect on measured parameters. Therefore, it can be concluded that minocycline may exert an anticonvulsant effect through preventing the increase in GABAA and NMDA receptor subunits. These effects are accompanied by a reduction in an important inflammation index, TNF-α receptor.

Significance of vascular endothelial growth factor in growth and peritoneal dissemination of ovarian cancer.

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis which drives endothelial cell survival, proliferation, and migration while increasing vascular permeability. Playing an important role in the physiology of normal ovaries, VEGF has also been implicated in the pathogenesis of ovarian cancer. Essentially by promoting tumor angiogenesis and enhancing vascular permeability, VEGF contributes to the development of peritoneal carcinomatosis associated with malignant ascites formation, the characteristic feature of advanced ovarian cancer at diagnosis. In both experimental and clinical studies, VEGF levels have been inversely correlated with survival. Moreover, VEGF inhibition has been shown to inhibit tumor growth and ascites production and to suppress tumor invasion and metastasis. These findings have laid the basis for the clinical evaluation of agents targeting VEGF signaling pathway in patients with ovarian cancer. In this review, we will focus on VEGF involvement in the pathophysiology of ovarian cancer and its contribution to the disease progression and dissemination.

Minocycline inhibits growth of epithelial ovarian cancer.

OBJECTIVE: These studies were designed to determine whether minocycline inhibits ovarian cancer growth in vitro and in vivo and the molecular mechanisms involved. MATERIALS AND METHODS: The effect of minocycline on ovarian cancer cell proliferation, cell cycle progression and apoptosis was assessed using human ovarian cancer cell lines OVCAR-3, SKOV-3 and A2780. Then, the capacity of minocycline to inhibit growth of OVCAR-3 xenografts in female nude mice was examined. RESULTS: Minocycline inhibited cell proliferation and colony formation, down-regulated cyclins A, B and E leading to arrest of cells in the G(0) phase of the cycle and suppression of DNA synthesis. Furthermore, exposure of these cells to minocycline led to DNA laddering, activation of caspase-3 and cleavage of PARP-1. In nude mice bearing sub-cutaneous tumors, minocycline suppressed tumor proliferation index, angiogenesis and tumor growth. CONCLUSION: These findings provide the initial basis for further evaluation of minocycline in the treatment of ovarian cancer.

Effect of cross-linking on the performance of micelles as drug delivery carriers: a cell uptake study.

Poly(polyethylene glycol methyl ether methacrylate-co-methacrylic acid)-block-poly(methyl methacrylate) P(PEGMEMA-co-MAA)-b-PMMA block copolymer were prepared via RAFT (reversible addition-fragmentation chain transfer) polymerization and subsequently self-assembled into micelles as a drug delivery carrier for albendazole (ABZ). For comparison, the micelles were additionally cross-linked to study the effect of shell-cross-linking on the biological activity. The hydrodynamic diameter of cross-linked and un-cross-linked micelles was approximately 40 nm in both cases. While the cross-linked micelle was stable even in good solvents for both blocks, the un-cross-linked micelle was found to lose its integrity in cell growth media. Crosslinking had a major effect on the rate of drug release reducing it dramatically from 50% (uncrosslinked) to around 20% (crosslinked) over a 30 h incubation period. Both drug delivery systems were tested on human prostate cancer cells (PC-3, DU-145) and human ovarian cancer cells (OVCAR-3, A-2780). No toxic effects were measured with the unloaded micelle while the ABZ loaded un-cross-linked micelle lead to IC(50) values between 0.2 and 0.9 µM depending on the cell line. The IC(50) dropped to values between 0.006 and 0.06 µM, depending on cell line, once the micelles were stabilized by cross-linking. Three treatment cycles with ABZ for one day, followed by two days incubation in media using ABZ-loaded drug carriers led to complete cell death even at low concentrations in the case of the cross-linked micelle only. Cellular uptake has been studied using fluorescently labeled micelles and Nile red as model drug, showing cell uptake above the CMC but no micelle uptake below the CMC. Additional biological studies, such as colony formation assay and tubulin disorganization tests, were also performed to gain more insight into the effect of cross-linking of the shell of the micelle. In conclusion, shell-cross-linking is highly recommended, even for glassy micelles, for an efficient cellular uptake at low concentrations.

Estrogen as a key regulator of energy homeostasis and metabolic health.

Over the last two decades, it has become evident that estrogens preserve the integrity of energy homeostasis at central and peripheral levels. Estrogen deficiency, such as that caused by menopause or ovariectomy, has been linked to obesity and metabolic disorders that can be resolved or reversed by estrogen therapy. 17ß-estradiol (E2), as the major estrogen in the body, primarily regulates energy balance via estrogen receptor alpha (ERα). At the central level, E2 plays its catabolic role predominantly by interacting with hypothalamic arcuate neurons and sending signals via ventromedial hypothalamic neurons to control brown adipose tissue-mediated thermogenesis. In peripheral tissues, several organs, particularly the liver, brown and white adipose tissues, and pancreatic ß cells, have attracted considerable attention. In this review, we focused on the current state of knowledge of "central and peripheral" estrogen signaling in regulating energy balance via "nuclear and extranuclear pathways" in both "females and males". In this context, according to an exploratory approach, we tried to determine the principal estrogen receptor subtype/isoform in each section, the importance of extranuclear-initiated estrogen signaling on metabolic functions, and how sex differences related to ER signaling affect the prevalence of some of the metabolic disorders. Moreover, we discussed the data from a third viewpoint, understanding the clinical significance of estrogen signaling in abnormal metabolic conditions such as obesity or being on a high-fat diet. Collectively, this review exposes novel and important research gaps in our current understanding of dysmetabolic diseases and can facilitate finding more effective treatment options for these disorders.

Monepantel antitumor activity is mediated through inhibition of major cell cycle and tumor growth signaling pathways.

In women, epithelial ovarian cancer is the leading cause of gynaecological malignancy-related deaths. Development of resistance to standard platinum and taxane based chemotherapy and recurrence of the disease necessitate development of novel drugs to halt disease progression. An established concept is to target molecular and signaling pathways that substantially contribute to development of drug resistance and disease progression. We have previously shown that, monepantel (MPL) a novel small molecule acetonitrile derivative is highly effective in suppressing growth, proliferation and colony formation of ovarian cancer cells. These effects are achieved through inhibition of the mTOR/p70S6K pathway in cancer cells. The present study was conducted to find in vivo corroboration and explore the effect of MPL om other growth stimulating putative signaling pathways. Here, female nude mice with subcutaneous OVCAR-3 xenografts were treated with 25 and 50 mg/kg doses of MPL administered (IP) three times weekly for 2 weeks. At the doses employed, MPL was modestly effective at suppressing tumor growth, but highly effective in inhibiting, mTOR, P70S6K and 4EBP1. There were also modest reductions in tumor cyclin D1 and retinoblastoma protein expression. Furthermore, it was found that MPL treatment causes down-regulation of IGF-1R, and c-MYC thus unveiling new dimensions to the growing antitumor actions of this potential anticancer drug. MPL treatment led to reduced tumor volume and weights without causing any detectable side effects. Coupled with the recent human safety data published on this molecule, expanded future trials are highly anticipated.

Novel protein interactors of urokinase-type plasminogen activator receptor.

The urokinase-type plasminogen activator receptor (uPAR) has been implicated in tumor growth and metastasis. The crystal structure of uPAR revealed that the external surface is largely free to interact with a number of proteins. Additionally, due to absence of an intracellular cytoplasmic protein domain, many of the biological functions of uPAR necessitate interactions with other proteins. Here, we used yeast two-hybrid screening of breast cancer cDNA library to identify hSpry1 and HAX1 proteins as putative candidate proteins that interact with uPAR bait constructs. Interaction between these two candidates and uPAR was confirmed by GST-pull down, co-immunoprecipitation assays and confocal microscopy. These novel interactions that have been identified may also provide further evidence that uPAR can interact with a number of other proteins which may influence a range of biological functions.

Albendazole inhibits endothelial cell migration, tube formation, vasopermeability, VEGF receptor-2 expression and suppresses retinal neovascularization in ROP model of angiogenesis.

The angiogenic process begins with the cell proliferation and migration into the primary vascular network, and leads to vascularization of previously avascular tissues and organs as well to growth and remodeling of the initially homogeneous capillary plexus to form a new microcirculation. Additionally, an increase in microvascular permeability is a crucial step in angiogenesis. Vascular endothelial growth factor (VEGF) plays a central role in angiogenesis. We have previously reported that albendazole suppresses VEGF levels and inhibits malignant ascites formation, suggesting a possible effect on angiogenesis. This study was therefore designed to investigate the antiangiogenic effect of albendazole in non-cancerous models of angiogenesis. In vitro, treatment of human umbilical vein endothelial cells (HUVECs) with albendazole led to inhibition of tube formation, migration, permeability and down-regulation of the VEGF type 2 receptor (VEGFR-2). In vivo albendazole profoundly inhibited hyperoxia-induced retinal angiogenesis in mice. These results provide new insights into the antiangiogenic effects of albendazole.

Potent inhibition of tumoral hypoxia-inducible factor 1alpha by albendazole.

BACKGROUND: Emerging reports suggest resistance, increased tumor invasiveness and metastasis arising from treatment with drugs targeting vascular endothelial growth factor (VEGF). It is believed that increased tumoral hypoxia plays a prominent role in the development of these phenomena. Inhibition of tumoral hypoxia inducible factor (HIF-1alpha) is thus becoming an increasingly attractive therapeutic target in the treatment of cancer. We hypothesized that the anti-VEGF effect of albendazole (ABZ) could be mediated through inhibition of tumoral HIF-1alpha. METHOD: In vitro, the effects of ABZ on HIF-1alpha levels in human ovarian cancer cells (OVCAR-3) were investigated using hypoxic chamber or desferrioxamine (DFO) induced-hypoxia. In vivo, the effects of ABZ (150 mg/kg, i.p., single dose) on the tumor levels of HIF-1alpha and VEGF protein and mRNA were investigated by western blotting, RT-PCR and real time-PCR. RESULTS: In vitro, ABZ inhibited cellular HIF-1alpha protein accumulation resulting from placement of cells under hypoxic chamber or exposure to DFO. In vivo, tumors excised from vehicle treated mice showed high levels of both HIF-1alpha and VEGF. Whereas, tumoral HIF-1alpha and VEGF protein levels were highly suppressed in ABZ treated mice. Tumoral VEGFmRNA (but not HIF-1alphamRNA) was also found to be highly suppressed by ABZ. CONCLUSION: These results demonstrate for the first time the effects of an acute dose of ABZ in profoundly suppressing both HIF-1alpha and VEGF within the tumor. This dual inhibition may provide additional value in inhibiting angiogenesis and be at least partially effective in inhibiting tumoral HIF-1alpha surge, tumor invasiveness and metastasis.

Enhanced cytotoxicity of benzimidazole carbamate derivatives and solubilisation by encapsulation in cucurbit[n]uril.

The albendazole derivatives (2-methoxyethyl) 5-propylthio-1H-benzimidazole-2-yl carbamate (MEABZ), N1-(2-methoxyethoxycarbonyl)-2-amino-5-propylthiobenzimidazole and N1-(2-methoxyethoxycarbonyl)-2-amino-6-propylthiobenzimidazole (MEABZ isomers A and B) and (2-hydroxyethyl) 5-propylthio-1H-benzimidazole-2-yl carbamate (HEABZ) have been synthesised. The cytotoxicity of these compounds was evaluated against a human colorectal cancer cell line (HT-29) and a human prostate cancer cell line (PC-3). The results demonstrate MEABZ, a new benzimidazole, is up to ten times more cytotoxic than the parent drug albendazole, whereas the MEABZ isomers A and B and HEABZ show no activity. A comparison of the cytotoxicity of these compounds, relative to ABZ, provides structure-activity data for this important class of anticancer agents. The aqueous solubilities of MEABZ encapsulated in Q[n] have been determined by (1)H NMR spectroscopy. The aqueous solubility of MEABZ at a physiologically relevant pH increased by 1200-fold by encapsulation in Q[8], from 8 microM to 9.4 mM, while Q[6,7] encapsulation substantially increased the solubility to more than 2 mM. Encapsulation in Q[7] and Q[8] induced significant upfield shifts for the MEABZ propyl and benzimidazole resonances. The upfield shifts indicate that the propyl and benzimidazole protons are located within the Q[7] and Q[8] cavity upon encapsulation. By contrast, encapsulation in Q[6] induced large upfield shifts for the (1)H resonances from the carbamate functional group, indicating that MEABZ associates with Q[6] at its portals, with only the carbamate group binding within the cavity.

Urokinase plasminogen activator system as a potential target for cancer therapy.

Proteolysis of extracellular matrix (ECM) and basement membrane is an essential mechanism used by cancer cells for their invasion and metastasis. The ECM proteinases are divided into three groups: metalloproteinases, cysteine proteinases and serine proteinases. The urokinase plasminogen activator (uPA) system is one of the serine proteinase systems involved in ECM degradation. Members of this system, including uPA and its receptor (uPAR), are overexpressed in several malignant tumors. This system plays a major role in adhesion, migration, invasion and metastasis of cancer cells, thus making it an important target for anticancer drug therapy. Several strategies, including the use of antisense oligodeoxynucleotides, ribozymes, DNAzyme, RNAi, uPA inhibitors, soluble uPAR, catalytically inactive uPA fragments, synthetic peptides and synthetic hybrids are under study, as they interfere with the expression and/or activity of uPA or uPAR in tumor cells. Herein, we discuss the various pharmaceutical strategies under investigation to combat the uPA activity in cancer.

Solubilisation and cytotoxicity of albendazole encapsulated in cucurbit[n]uril.

The aqueous solubilities of albendazole encapsulated in cucurbit[6, 7 and 8]urils (Q[6], Q[7] and Q[8]) have been determined by (1)H NMR spectroscopy, and the effect of encapsulation on their cytotoxicities evaluated. Encapsulation in Q[6] and Q[7] increased the aqueous solubility of albendazole by 2000-fold, from 3 microM to 6 mM at pH 6.6, while Q[8]-encapsulation increased the solubility to over 2 mM. Encapsulation in Q[7] and Q[8] induced significant upfield shifts for the albendazole propyl and benzimidazole resonances, compared to those observed for Q[6]-binding and what would normally be expected for the respective functional groups. The upfield shifts indicate that the albendazole propyl and benzimidazole protons are located within the Q[7] and Q[8] cavity upon encapsulation. Alternatively, encapsulation in Q[6] only induced a large upfield shift for the albendazole carbamate methyl resonance, indicating that the drug associates with Q[6] at its portals, with only the carbamate group within the cavity. Simple molecular models based on the observed relative changes in chemical shift could be constructed that were consistent with the conclusions from the NMR experiments. Cytotoxicity assays against human colorectal cells (HT-29), human ovarian cancer cells (1A9) and the human T-cell acute lymphoblastic leukaemia cells (CEM) indicated that encapsulation in Q[7] did not significantly reduce the in vitro anti-cancer activity of albendazole.

Albendazole-cyclodextrin complex: enhanced cytotoxicity in ovarian cancer cells.

BACKGROUND: Over recent years, we have identified a potentially new indication for albendazole (ABZ) namely that of an anticancer agent. Our recent data indicate that besides regional use, the drug is quite likely to be useful as a systemic anticancer agent. However, with extremely low solubility, ABZ has to be prepared in a biocompatible solubilized form before any systemic evaluation is possible. The present study aimed at preparing soluble ABZ and evaluating its in vitro antiproliferative efficacy and toxicity. EXPERIMENTAL DESIGN: Using beta-cyclodextrins (CDs), various formulations of ABZ were prepared and tested in cell culture for antiproliferative efficacy, cell integrity and cell toxicity against human ovarian cancer cell lines 1A9, OVCAR-3 and SKOV-3. Hepatocytes isolated from patients undergoing liver tumor resection were used for toxicity evaluations. RESULTS: Treatment of tumor cells with ABZ-CD + citric acid (CA) solution led to dose-dependent inhibition of cell proliferation. Compared to an ethanolic solution of ABZ, ABZ-CD + CA increased the antiproliferative efficacy of ABZ. Furthermore, in contrast to the ethanolic solution, ABZ-CD-CA complex profoundly (p<0.001) reduced the number of OVCAR-3 colonies formed. Fresh human hepatocytes exposed for 3 days to the highest ABZ concentration used in the study (1 microM), revealed no drug toxicity. CONCLUSION: Complexation of ABZ with beta-cyclodextrin leads to the formation of an ABZ solution with potent antiproliferative effects. This solution may find clinical value as an intravenous anticancer agent.

Inhibition of hepatocellular cancer by EB1089: in vitro and in vive study.

BACKGROUND: Despite significant antiproliferative potential, clinical application of 1,25 dihydroxy vitamin D3 [1,25(OH)2VD3] in the treatment of cancer has been hindered due to the development of hypercalcemia. Various derivatives of 1,25(OH)2VD3 have been synthesized to overcome this side-effect. Seocalcitol (EB1089) is a vitamin D analog that has been extensively studied and shown to have profoundly reduced hypercalcemic effects. Here the effects of EB1089 were evaluated in Hep 3B, SKHEP-1, PLC/PRF/5, HTC and Novikoff hepatocellular cancer (HCC) cell lines. MATERIALS AND METHODS: In vitro, cells were treated with different concentrations of EB1089 (1-1000 nM). Analytical tests were then performed including cell count and 3H thymidine assay. For in vivo analysis, SKHEP-1 cells were xenografted into nude male mice. Twenty-four hours after inoculation, mice were randomly assigned to a control group (n=10) or one of the treatment groups (3 groups of 10 mice) receiving 0.02, 0.1 or 0.5 microg/kg/day of EB1089. Control animals received the vehicle (propylene glycol). To minimize the number of intraperitoneal injections, oral and intraperitoneal routes were used on alternate days. Tumor size was measured every third day and the volumes were estimated using the formula 0.5 x length x (width)2. RESULTS: In vitro: Proliferation of Hep 3B, PLC/PRF/5 and SKHEP-1 HCC cells was significantly inhibited at all EB1089 concentrations tested, while HTC cells only responded to 1000 nM concentration of EB1089. Proliferation of Novikoff cells was unaffected by the drug at all concentrations examined. In vivo: EB1089 effectively inhibited SKHEP-1 tumor growth without inducing hypercalcemia (p<0.05). CONCLUSION: Results of the present study indicate that EB1089 is an effective growth inhibitor of HCC tumors.