Grisha Pirianov

Potentiation by vitamin D analogs of TNFalpha and ceramide-induced apoptosis in MCF-7 cells is associated with activation of cytosolic phospholipase A2.

Synthetic analogs of vitamin D induce apoptosis in cultured breast cancer cells and cause regression of experimentally-induced rat mammary tumors. To further elucidate the mechanisms involved, we have examined interactions between two vitamin D analogs (CB1093 and EB1089) and known mediators of apoptosis, TNFα and ceramide. Pretreatment of MCF-7 breast cancer cells with CB1093 and EB1089 substantially potentiated cytotoxic effects of TNFα as assessed by cell viability assay, DNA fragmentation and videomicroscopy. No significant changes in the levels of TNFα or TNF-RI transcripts were detected. CB1093 primed cells demonstrated enhanced responsiveness to cell permeable C2-ceramide in terms of increased DNA fragmentation and loss of cell viability. Activation of cytosolic phospholipase A2 (cPLA2) has been implicated in TNFα-mediated apoptosis. As assessed by [3H]-arachidonic acid release, cells primed for 48 h with CB1093 (50 nM) showed enhanced cPLA2 activation in response to TNFα or ceramide. CB1093 treatment alone led to cPLA2 activation and loss of cell viability which was inhibited by the specific inhibitor AACOCF3. These results suggest that TNFα and vitamin D analogs share a common pathway leading to apoptosis involving cPLA2 activation and/or ceramide generation.

Interaction of vitamin D analogs with signaling pathways leading to active cell death in breast cancer cells

Induction of apoptosis is a feature of the anti-tumor effects of certain vitamin D analogs. The aim of this study was to identify if common effectors are involved in cell death mediated by serum starvation, vitamin D analogs and tumor necrosis factor (TNF) alpha in 3 human breast cancer cell lines: MCF-7, T47-D and Hs578T. Incubation of cells in serum-free medium induced apoptosis as assessed by loss of cell viability and increased DNA fragmentation. Addition of IGF-I (30 ng/ml) protected against loss of cell viability in MCF-7 cells and co-treatment with two synthetic analogs (CB1093 and EB1089, 50 nM for 4 days) prevented these anti-apoptotic effects of IGF-I. Pretreatment of MCF-7 and Hs578T cells with the vitamin D analogs substantially potentiated the cytotoxic effects of TNFalpha. This cytokine was not cytotoxic for T47-D cells but co-incubation with CB1093 led to loss of cell viability. Potentiation by CB1093 of TNFalpha-induced apoptosis in MCF-7 cells was accompanied by increased activation of cytosolic phospholipase A2 and arachidonic acid release, which was partially inhibited by AACOCF3, a specific cPLA2 inhibitor. The broad-spectrum caspase inhibitor z-VAD-fmk prevented TNFalpha but not CB1093 mediated cell death and activation of cPLA2. Serum starvation induced apoptosis was accompanied by cPLA2 activation, which was inhibited by IGF-I and by z-VAD-fmk. However, the ability of these agents to suppress cPLA2 activation was abrogated by co-treatment with CB1093, suggesting a role for arachidonic acid release in the caspase-independent mechanism by which vitamin D analogs prevent the protective effects of IGF-I on breast cancer cell survival.

MCF‐7/VDR: 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/VDR 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 1α,25(OH)2D3. Despite this characteristic, receptor studies on the VDR have clearly demonstrated that the MCF‐7/VDR 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/VDR cells and no differences with regard to growth rate and morphological appearance between the MCF‐7/VDR cells and the parental MCF‐7 cells were observed. Interestingly, however, the sensitivity of the MCF‐7/VDR cells to the pure anti‐estrogen ICI 182,780 was found to be increased. The MCF‐7/VDR 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. J. Cell. Biochem. 82:422–436, 2001.

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 µg/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 µg/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 EB1053 caused substantial tumor regression. No statistically significant difference was seen between combination treatment and Seocalcitol treatment alone. Co-treatment with APD or EB1053 did not limit the rise in serum calcium induced by Seocalcitol alone. Cessation of treatment or administration of a lower dose (1 µg/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.

Elevation of Plasma High-Density Lipoproteins Inhibits Development of Experimental Abdominal Aortic Aneurysms

Objective—Patients with abdominal aortic aneurysms have lower concentrations of high-density lipoproteins (HDLs), leading us to investigate whether increasing plasma HDLs could influence aneurysm formation. Methods and Results—Using the angiotensin II−induced hypercholesterolemic and the CaCl2-induced normocholesterolemic mouse model of AAA, we investigated the hypothesis that elevation of HDLs inhibits AAA. HDLs elevated before or at the time of AAA induction reduced AAA formation in both models but had no effect on early ruptures. Analysis of protein lysates from specific aortic segments demonstrated site-specific effects of HDLs on early signal transduction and cellular attrition. We found that HDLs reduced extracellular signal related kinases 1/2 activation in the suprarenal segment, while having no effect on p38 mitogen-associated protein kinase activation in any aortic segment and inhibiting c-Jun N-terminal kinase activation in all aortic segments. In addition, HDL elevation inhibited angiotensin II−induced apoptosis while inducing autophagy in the suprarenal segment of the aorta. Using Illumina gene array profiling we investigated the ability of HDL to modulate basal suprarenal aortic gene expression. Conclusion—Increasing plasma HDLs inhibit experimental AAA formation, independent of hypercholesterolemia via reduced extracellular signal related kinases 1/2 activation and alteration of the balance of cellular attrition. HDLs modulate genes involved in matrix remodelling, cell migration, and proliferation.

Diabetes as a Negative Risk Factor for Abdominal Aortic Aneurysm – Does the Disease Aetiology or the Treatment Provide the Mechanism of Protection?

There is strong epidemiological evidence that patients with diabetes have a lower incidence of abdominal aortic aneurysm. The precise mechanism of this negative association is unknown. Whilst a number of studies have supported the hypothesis that protection is a function of diabetes-mediated changes in the vascular extracellular matrix biology, there is also support for the idea that the treatment regimens used in diabetes may afford protection against AAA. In particular the pleiotropic drug family, the thiazolidinediones have been examined as candidates to ameliorate aneurysm formation. Both the thiazolidinediones, and the structurally related family, fibrates, have been shown to have anti-inflammatory and antioxidative effects, in addition to ability to modulatate glucose and lipid homeostasis. In this brief review we present the current data exploring the use of thiazolidinediones in experimental aneurysm development. Despite the fact that both thiazolidinediones Rosiglitazone and Pioglitazone are no longer prescribed in Europe and the US, they have provided important insights into the mechanism of action, and the application of other pleiotropic drugs in the treatment of AAA. One such pleiotropic drug is high-density lipoproteins (HDLs), which have been shown to have a broad spectrum of effects, including activation of PPARs, which may favour their use as a new drug target for protection against AAA development.

Involvement of stress activated protein kinases (JNK and p38) in 1,25 dihydroxyvitamin D3-induced breast cell death.

It has been previously demonstrated that 1,25 dihydroxyvitamin D(3) (1,25-D(3)) exerts inhibitory effects in breast cancer cells. The aim of this study was to determine whether mitogen-activated protein kinase (MAPK) pathways are associated with 1,25-D(3)-induced cell death in breast cancer. We used three breast cell lines which have different sensitivities to 1,25-D(3) treatment. Non-malignant MCF-12A cells were more sensitive to 1,25-D(3) treatment than malignant MCF-7 cells (growth inhibition IC(50) 75 nM vs. 100 nM, p<0.001) while malignant MDA-MB-231 cells were resistant. Moreover, 1,25-D(3)-induced apoptosis was caspase-dependent in MCF-12A cells and caspase-independent in MCF-7 cells. Following MAPK activation analysis, we found a significant activation of JNK in MCF-12A cells and malignant MCF-7 cells in response to 1,25-D(3) treatment. Furthermore, 1,25-D(3) treatment stimulated p38 activity in MCF-12A cells and in MCF-7 cells. ERK1/2 activity was unaffected by 1,25-D(3) treatment in all breast cells. Importantly, no increased MAPK activity was observed in MDA-MB-231 breast cancer cells which displayed resistance to 1,25-D(3)-induced apoptosis. Utilising specific pharmacological inhibitors of JNK and p38, it was demonstrated that MCF-12A and MCF-7 cells were protected from death induced by 1,25-D(3). These results implicate JNK and p38 signalling in 1,25-D(3)-induced cancer breast cell death.

Role of the eIF4E binding protein 4E‐BP1 in regulation of the sensitivity of human pancreatic cancer cells to TRAIL and celastrol‐induced apoptosis

Tumour cells can be induced to undergo apoptosis after treatment with the tumour necrosis factor α‐related death‐inducing ligand (TRAIL). Although human pancreatic cancer cells show varying degrees of response they can be sensitised to the pro‐apoptotic effects of TRAIL in the presence of celastrol, a natural compound extracted from the plant Tripterygium wilfordii Hook F. One important aspect of the cellular response to TRAIL is the control of protein synthesis, a key regulator of which is the eukaryotic initiation factor 4E‐binding protein, 4E‐BP1.

Comparative proteomics reveals a systemic vulnerability in the vasculature of patients with abdominal aortic aneurysms

INTRODUCTION Abdominal aortic aneurysms (AAA) are associated with inflammation, apoptosis, and matrix degradation. AAA tissue represents the end stage of disease, limiting its utility in identification of factors culpable for initiation of aneurysm development. Recent evidence suggests that AAAs are a local representation of a systemic disease of the vasculature. Morphologic and molecular changes, comparable to those found in the aneurysm wall, have been demonstrated in veins from patients with AAAs. Changes in the vascular tissue proteome of patients with AAAs were investigated, using inferior mesenteric vein (IMV), to gain insight into early molecular changes contributing to AAA development. METHODS IMV was harvested from 16 patients with AAA and 16 matched controls. Whole IMV lysates were subjected to 2-D difference in gel electrophoresis (2D-DIGE) with quantitative densitometry. Protein spots differentially expressed in AAA were identified using mass spectrometry. Differential protein expression was validated by Western blotting and localized to cell type by immunohistochemistry (IHC). RESULTS Decreased levels of prohibitin (AAA, 2.00 ± 1.37; controls, 3.81 ± 1.39; 1.9-fold change; P = .02) AAA (7.33 ± 3.9; controls, 14.5 ± 5.6; 2-fold change; P = .001), along with relative increases in a cleaved fragment of vimentin (AAA, 12.9 ± 9; controls, 6.9 ± 4.7; 2-fold change; P = .11) were identified in AAA patients. All proteins were localized to the vascular smooth muscle cells. CONCLUSIONS Proteins important in combating the injurious effects of oxidative stress and modulating the response to inflammation appear reduced in the vasculature of patients with AAA. These changes may represent early events in AAA formation. Enhancing expression of these proteins might offer a novel therapeutic avenue to inhibit AAA development.

Pioglitazone Identifies a New Target for Aneurysm Treatment: Role of Egr1 in an Experimental Murine Model of Aortic Aneurysm

Peroxisome proliferator-activated receptor γ agonists have been shown to inhibit angiotensin II (AngII)-induced experimental abdominal aortic aneurysms. Macrophage infiltration to the vascular wall is an early event in this pathology, and therefore we explored the effects of the peroxisome proliferator-activated receptor γ agonist pioglitazone on AngII-treated macrophages. Using microarray-based expression profiling of phorbol ester-stimulated THP-1 cells, we found that a number of aneurysm-related gene changes effected by AngII were modulated following the addition of pioglitazone. Among those genes, polycystic kidney disease 1 (PKD1) was significantly up-regulated (multiple testing corrected p < 0.05). The analysis of the PKD1 proximal promoter revealed a putative early growth response 1 (EGR1) binding site, which was confirmed by chromatin immunoprecipitation (ChIP) and quantitative PCR. Further analysis of publicly available ChIP-sequencing data revealed that this putative binding site overlapped with a conserved EGR1 binding peak present in 5 other cell lines. Quantitative real-time PCR showed that EGR1 suppressed PKD1, while AngII significantly up-regulated PKD1, an effect counteracted by pioglitazone. Conversely, in EGR1 short hairpin RNA lentivirally transduced THP-1 cells, reduced EGR1 led to a significant up-regulation of PKD1, especially after treatment with pioglitazone. In vivo, deficiency of Egr1 in the haematopoietic compartment of mice completely abolished the incidence of CaCl2-induced aneurysm formation.