Amiram Ravid

Effect of polar organic compounds on leukemic cells: Butyrate‐induced partial remission of acute myelogenous leukemia in a child

Polar organic compounds, such as dimethylsulfoxide and butyric acid, are known to induce differentiation in Friend erythroleukemia cells as well as in other cell types. It has been found that many of the compounds that induce cellular differentiation, inhibit 3H‐thymidine incorporation and induce cell damage when incubated with leukemic cells from patients with acute or chronic myelogenous or acute lymphocytic leukemia. These effects are time and dose dependent. Among the compounds tested, butyrate was the most potent. Parenteral administration of butyrate (500 mg/kg/day) for ten days to a child with acute myelogenous leukemia in relapse, and resistant to conventional therapy, resulted in elimination of myeloblasts from the peripheral blood, an increase in mature myeloid cells and a reduction in 3H‐thymidine uptake by the patients peripheral blood cells. Bone marrow myeloblasts were reduced from 70‐80% to 20% following the course of intravenous butyrate. No impairment of liver or renal function and no coagulation abnormalities were observed during butyrate treatment. Organic agents that induce cell differentiation may provide additional reagents for the clinical management of selected cases of leukemia.

Vitamin D: An innate antiviral agent suppressing hepatitis C virus in human hepatocytes†‡

Vitamin D supplementation was reported to improve the probability of achieving a sustained virological response when combined with antiviral treatment against hepatitis C virus (HCV). Our aim was to determine the in vitro potential of vitamin D to inhibit HCV infectious virus production and explore the mechanism(s) of inhibition. Here we show that vitamin D3 remarkably inhibits HCV production in Huh7.5 hepatoma cells. These cells express CYP27B1, the gene encoding for the enzyme responsible for the synthesis of the vitamin D hormonally active metabolite, calcitriol. Treatment with vitamin D3 resulted in calcitriol production and induction of calcitriol target gene CYP24A1, indicating that these cells contain the full machinery for vitamin D metabolism and activity. Notably, treatment with calcitriol resulted in HCV inhibition. Collectively, these findings suggest that vitamin D3 has an antiviral activity which is mediated by its active metabolite. This antiviral activity involves the induction of the interferon signaling pathway, resulting in expression of interferon‐β and the interferon‐stimulated gene, MxA. Intriguingly, HCV infection increased calcitriol production by inhibiting CYP24A1 induction, the enzyme responsible for the first step in calcitriol catabolism. Importantly, the combination of vitamin D3 or calcitriol and interferon‐α synergistically inhibited viral production. Conclusion: This study demonstrates for the first time a direct antiviral effect of vitamin D in an in vitro infectious virus production system. It proposes an interplay between the hepatic vitamin D endocrine system and HCV, suggesting that vitamin D has a role as a natural antiviral mediator. Importantly, our study implies that vitamin D might have an interferon‐sparing effect, thus improving antiviral treatment of HCV‐infected patients. (HEPATOLOGY 2011;)

Upregulation of MMP‐9 production by TNFα in keratinocytes and its attenuation by vitamin D

MMP‐9, a member of the matrix metalloproteinase family that degrades collagen IV and processes chemokines and cytokines, participates in epidermal remodeling in response to stress and injury. Limited activity of MMP‐9 is essential while excessive activity is deleterious to the healing process. Tumor necrosis factor (TNFα), a key mediator of cutaneous inflammation, is a powerful inducer of MMP‐9. Calcitriol, the hormonally active vitamin D metabolite, and its analogs are known to attenuate epidermal inflammation. We aimed to examine the modulation of MMP‐9 by calcitriol in TNFα‐treated keratinocytes. The immortalized HaCaT keratinocytes were treated with TNFα in the absence of exogenous growth factors or active ingredients. MMP‐9 production was quantified by gelatin zymography and real‐time RT‐PCR. Activation of signaling cascades was assessed by western blot analysis and DNA‐binding activity of transcription factors was determined by EMSA. Exposure to TNFα markedly increased the protein and mRNA levels of MMP‐9, while pretreatment with calcitriol dose dependently reduced this effect. Employing specific inhibitors we established that the induction of MMP‐9 by TNFα was dependent on the activity of the epidermal growth factor receptor, c‐Jun‐N‐terminal kinase (JNK), NFκB and extracellular signal‐regulated kinase‐1/2. The effect of calcitriol was associated with inhibition of JNK activation and reduction of DNA‐binding activities of the transcription factors activator protein‐1 (AP‐1) and NFκB following treatment with TNFα. By down‐regulating MMP‐9 levels active vitamin D derivatives may attenuate deleterious effects due to excessive TNFα‐induced proteolytic activity associated with cutaneous inflammation. J. Cell. Physiol. 222: 729–737, 2010.

1,25-Dihydroxyvitamin D3 inhibits selectively the mitogenic stimulation of mouse medullary thymocytes.

Mouse thymocytes were separated into cortical and medullary subpopulations by differential agglutination with peanut agglutinin. A high-affinity receptor for 1,25-dihydroxyvitamin D3 is present in medullary immunocompetent mouse thymocytes and is absent from cortical immature cells. 1,25-dihydroxyvitamin D3, at physiological concentrations, inhibits the mitogenic response of the medullary cells to phytohemagglutinin and interleukin-2, but has no effect on the cortical subpopulation. Other less active metabolites of vitamin D had little or no effect on medullary cell stimulation.

Vitamin D enhances caspase‐dependent and ‐independent TNFα‐induced breast cancer cell death: The role of reactive oxygen species and mitochondria

Calcitriol, the hormonal form of vitamin D, potentiates the activity of some common anticancer drugs and agents of the anticancer immune system, including tumor necrosis factor α (TNFα). TNFα‐induced cytotoxicity is due to both caspase‐dependent and ‐independent pathways. Cotreatment with calcitriol enhanced both modes of TNFα‐induced death in MCF‐7 breast cancer cells. It increased caspase‐3‐like activity as assayed by the cleavage of poly‐(ADP‐ribose)polymerase and of the fluorogenic substrate ac‐DEVD‐AMC. It also enhanced TNFα‐induced caspase‐independent cytotoxicity in the presence of the pan‐caspase inhibitor zD‐2,6‐dichlorobenzoyloxymethylketone. The antioxidants N‐acetylcysteine, reduced glutathione, lipoic acid and ascorbic acid markedly reduced the enhancing effect of the hormone on TNFα‐induced caspase activation. N‐acetylcysteine and reduced glutathione also decreased caspase‐independent cytotoxicity in the presence or absence of calcitriol, indicating that reactive oxygen species (ROS) have a key role in the cross talk between TNFα and calcitriol. Mitochondrial damage is common to both TNFα‐induced caspase‐dependent and ‐independent pathways and may underlie excessive production of ROS. Mitochondrial membrane potential (ΔΨ) was assessed by the specific potential‐sensitive fluorescent probe JC‐1. The hormone augmented the drop in ΔΨ and release of cytochrome c from mitochondria, induced by TNFα. The effect of calcitriol on ΔΨ was mimicked by rotenone, which increased both the drop in ΔΨ and caspase activation induced by TNFα. It is possible that the interaction of TNFα and calcitriol on the level of the mitochondria is the underlying mechanism responsible for the enhancement of TNFα‐induced, ROS‐mediated caspase‐dependent and ‐independent cell death.

The Role of Reactive Oxygen Species in the Anticancer Activity of Vitamin D

Calcitriol, the hormonal form of vitamin D, enhances the anticancer activity of the immune cytokine tumor necrosis factor, interleukin 1 and interleukin 6 in human breast and renal cell carcinoma cells without affecting the cytotoxic action of interferon-alpha or killer lymphocytes. It also enhances cytotoxicity induced by the anticancer drug doxorubicin, by the redox cycling quinone menadione, and by the reactive oxygen species hydrogen peroxide. The synergistic interaction was accompanied by increased oxidative stress, as manifested by glutathione depletion and was abolished by exposure to the thiol antioxidant N-acetylcysteine. The hormone on its own brought about an increase in the cellular redox state as reflected in the ratio between oxidized and reduced glutathione and glyceraldehyde-3-phosphate dehydrogenase, and a reduction in the expression of the antioxidant enzyme Cu/Zn superoxide dismutase. These results support the notion that the interplay between active vitamin D derivatives and other anticancer agents such as immune cytokines and anticancer drugs plays a role in the in vivo anticancer activity of vitamin D and that reactive oxygen species are involved in the anticancer activity of vitamin D on its own and in its cross-talk with other anticancer modalities.

Vitamin D Enhances Caspase‐Dependent and Independent TNF‐Induced Breast Cancer Cell Death

Abstract: Calcitriol, the hormonal form of vitamin D, enhanced TNF‐induced cytotoxicity in MCF‐7 breast cancer cells. It increased the induction of caspase‐3‐like activity and TNF‐induced caspase‐independent cytotoxicity in the presence of a pan‐caspase inhibitor. The antioxidants N‐acetylcysteine, glutathione, lipoic acid, and ascorbic acid markedly reduced the effect of the hormone on TNF‐induced caspase activation, attesting to the involvement of reactive oxygen species (ROS) in the cross‐talk between the hormone and the cytokine. Calcitriol augmented the drop in mitochondrial membrane potential induced by TNF as assessed by the fluorescent probe JC‐1. We postulate that the interaction of TNF and calcitriol on the level of the mitochondria underlies the enhancement of TNF‐induced, ROS‐mediated caspase‐dependent and ‐independent cell death.

Programmed cell death of stressed keratinocytes and its inhibition by vitamin D: the role of death and survival signaling pathways.

The epidermis is confronted with multiple environmental and pathophysiological stresses. This study shows that TNFα, oxidative stress, hyperosmotic and heat shock induced both caspase-dependent and independent cell death in human HaCaT keratinocytes. The hormonal form of vitamin D, 1,25(OH)2D3, which is an autocrine hormone in the epidermis, protected the cells from all the examined stresses and pathways leading to cell death. We aimed to define the signaling pathways that determine the life-death balance of stressed keratinocytes and participate in their protection by 1,25(OH)2D3. As assessed by employing specific inhibitors, the survival pathways mediated by the EGF receptor, ERK, PI-3K or Src kinase, or basal transcriptional activity are important for unstressed cell survival. However, only the EGF receptor, PI-3K and the Src kinase pathways mediate the survival of stressed cells in a stimulus-specific manner. Inhibition of the p38 and/or the JNK death pathways reduced caspase activation induced by oxidative stress, hyperosmotic shock and TNFα. The protective effect of 1,25(OH)2D3 was not mediated by the examined survival pathways. 1,25(OH)2D3 inhibited the stress-induced activation of p38 and JNK. Since mimicking this effect by pharmacological inhibition resulted in the attenuation of caspase activation, we infer that these pathways are involved in keratinocyte protection by 1,25(OH)2D3.

Oxidative stress causes telomere damage in Fanconi anaemia cells - a possible predisposition for malignant transformation.

Fanconi anaemia (FA) is an autosomal recessive and X‐linked disease characterized by severe genetic instability and increased incidence of cancer. One explanation for this instability may be the cellular hypersensitivity to oxidative stress leading to chromosomal breaks. This study explored the possible oxidative damage to telomeres of FA lymphocyte cell line, HSC536/N, and its possible effect on telomere function. We postulated that combination of oxidative damage with overexpression of telomerase may provide a possible model for malignant transformation in FA. The cells were grown in the presence of telomerase inhibitor and exposed for 1 month to H2O2 combined with various antioxidants. This exposure caused shortening of telomere length and damage to the telomere single stranded overhang, which was prevented by several oxidants. This shortening was associated with development of severe telomere dysfunction. Control cells did not exhibit this sensitivity to H2O2. Telomere dysfunction did not evoke damage response in FA cells, in contrast to normal P53 upregulation in control cells. Reconstitution of telomerase activity protected FA telomeres from further oxidative damage. These results suggest a scenario in which oxidative stress causes telomere shortening and ensuing telomere dysfunction may form the basis for malignant transformation in FA cells. Upregulation of telomerase activity in sporadic FA cells may perpetuate that process, thus explaining the malignant character of FA cells in vivo.

1,25-Dihydroxyvitamin D3 acts directly on human lymphocytes and interferes with the cellular response to interleukin-2

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) inhibits mitogen-induced lymphocyte proliferation in peripheral blood mononuclear cell preparations. Receptors for 1,25(OH)2D3 are present in monocytes and are acquired by lymphocytes upon activation. It is not clear whether the hormone inhibits lymphocyte mitogenesis by a direct action on the lymphocyte or only indirectly by affecting the regulatory monocytes. We addressed this question by using highly purified human lymphocyte preparations which contain less than 0.1% monocytes. 1,25(OH)2D3 inhibited the stimulation of purified lymphocytes by phytohemagglutinin and in the presence of fixed accessory cells, which cannot respond to 1,25(OH)2D3. The inhibitory effect attained 35% similar to that obtained in the presence of monocytes. 1,25(OH)2D3 inhibited interleukin 2 (IL-2)-driven stimulation of purified T cells in three different systems: cells treated with a submitogenic concentration of phytohemagglutinin, IL-2-dependent cells which bear receptors for IL-2 and naive cells in the absence of other mitogens. We conclude: (1) the human T lymphocyte is a direct target for 1,25(OH)2D3; (2) 1,25(OH)2D3 inhibits the response of activated, IL-2-receptor-bearing human T cells to interleukin-2.