Federica I. Wolf

Mechanism of Activation of Caspase Cascade During β-Carotene-Induced Apoptosis in Human Tumor Cells

In this study, we examined possible mechanisms of caspase activation during carotenoid-induced apoptosis in tumor cells. We found that β-carotene induces apoptosis by the activation of caspase-3 in human leukemia (HL-60), colon adenocarcinoma (HT-29) as well as melanoma (SK-MEL-2) cell lines. This activation is dose dependent and follows that of caspase-8 and caspase-9. Although caspase-8 cleavage is an early event, reaching its maximum activation at 3 h, caspase-9 reaches its maximum activation only at 6 h. The addition of IETD-CHO, a caspase-8-specific inhibitor, completely prevents β-carotene-induced apoptosis, whereas only a partial prevention was observed in the presence of LEHD-CHO, a caspase-9-specific inhibitor. β-Carotene activates caspase-9 via cytochrome c release from mitochondria and loss of mitochondrial membrane potential (Dym). Concomitantly, a dose-dependent decrease in the antiapoptotic protein Bcl-2 and a dose-dependent increase in the cleaved form of BID (t-BID) are observed. Moreover, NF-κB activation is involved in β-carotene-induced caspase cascade. These results support a pharmacological role for β-carotene as a candidate antitumor agent and show a possible sequence of molecular events by which this molecule may induce apoptosis in tumor cells.

Regulation of cell-cycle progression and apoptosis by beta carotene in undifferentiated and differentiated HL-60 leukemia cells: possible involvement of a redox mechanism.

Although epidemiologic studies have demonstrated that a high intake of vegetables containing β‐carotene lowers the risk of cancer, recent intervention studies have revealed that β‐carotene supplementation to smokers resulted in a high incidence of lung cancer. We hypothesized that β‐carotene may act as a pro‐ or anticancerogenic agent by modulating pathways involved in cell growth and that such a modulation may involve a redox mechanism. To test this hypothesis, cell proliferation, apoptosis and redox status were evaluated in undifferentiated and dimethylsulfoxide‐differentiated HL‐60 cells exposed to β‐carotene. The carotenoid modified cell cycle progression and induced apoptosis in a dose‐dependent manner. These effects were more remarkable in undifferentiated cells than in differentiated cells. In accord with these findings, in undifferentiated cells, β‐carotene was more effective in decreasing cyclin A and Bcl‐2 expression and in increasing p21 and p27 expression. Neither Bcl‐xL nor Bax expression were significantly modified by the carotenoid. From a mechanistic point of view, the delay in cell growth by β‐carotene was highly coincident with the increased intracellular reactive oxygen species production and oxidized glutathione content induced by the carotenoid. Moreover, α‐tocopherol minimized the effects of β‐carotene on cell growth. These data provide evidence that β‐carotene modulates molecular pathways involved in cell cycle progression and apoptosis and support the hypothesis that a redox mechanism may be implicated. They also suggest that differentiated cells may be less susceptible to the carotenoid than highly neoplastic undifferentiated cells.

Magnesium depletion causes growth inhibition, reduced expression of cyclin D1, and increased expression of P27KIP1 in normal but not in transformed mammary epithelial cells

In this study, we have evaluated the effects of extracellular magnesium restriction on the growth and cell cycle parameters of normal (HC11) and transformed (MCF‐7) breast epithelial cell lines. Cells were incubated in medium with different concentrations of Mg2+ (from 0.5 to 0 mM) and the growth rates were determined by [3H]‐thymidine incorporation and cell counting. The growth of the HC11 cells was drastically inhibited by Mg2+ depletion whereas the MCF‐7 cells were only slightly inhibited (about 50% and 15%, respectively, after incubation in 0.05 mM Mg for 48 h). Cell cycle analyses showed a decrease in the percentage of cells in the S phase when both cell lines were incubated at low Mg2+ concentration. However, while the percentage of cells in both the G0/G1 and G2/M phases was increased in the HC11 cells, only the percentage of cells in the G2/M phase was increased in the MCF‐7 cell line. Extracellular magnesium depletion was associated with increased expression of the cyclin‐dependent kinase inhibitor p27Kip1 and decreased expression of cyclin D1 in the HC11 but not in the MCF‐7 cells. We also demonstrated that Mg2+ depletion does not inhibit kinase activities in the normal HC11 cells and that Mg2+‐restricted HC11 cells are still responsive to the epidermal growth factor (EGF)‐ and insulin‐mediated stimulation of cell growth. These data suggest that normal but not transformed mammary epithelial cells are inhibited by extracellular Mg2+ restriction and that this effect might be mediated by changes in the levels of expression of both cyclin D1 and p27Kip1. J. Cell. Physiol. 180:245–254, 1999.

Magnesium and tumors: Ally or foe?

Magnesium plays a crucial role in many cell functions such as energy metabolism, protein and DNA syntheses, and cytoskeleton activation. Proliferating cells have long been known to contain more magnesium than quiescent cells, and experimental conditions that decreased magnesium availability affected cell proliferation rate. There is little information about how tumor growth influenced systemic availability of magnesium in a patient, nor is it clear whether treatment-associated changes of magnesaemia influenced tumor growth and dissemination. Hypomagnesaemia is observed during multi-agent therapies with cisplatin or the anti-EGFR antibody, cetuximab. The latter was shown to cause hypomagnesaemia by impeding EGF-dependent activation of TRPM6, the main cation channel responsible for Mg transcellular absorption in the intestine and kidney. Limited observations also suggest that hypomagnesaemia could favorably influence tumor response to cetuximab. All such findings brought magnesium into the arena of clinical oncology, but potential caveats should be kept in mind before considering practical implications. We briefly review that magnesium causes pleiotropic, often diverging effects on tumor growth, vascularization, and metastatization, such that both favorable and unfavorable effects can be identified. Inflammatory responses to hypomagnesaemia should also be considered. Translating biology into clinical facts will therefore require a deeper understanding of such a complexity.

Peripheral lymphocyte 8-OHdG levels correlate with age-associated increase of tissue oxidative DNA damage in Sprague-Dawley rats. Protective effects of caloric restriction.

8-hydroxy-deoxyguanosine adducts (8-OHdG), indices of oxidative DNA damage, were measured by immunohystochemistry with diaminobenzidine detection in the brain, skeletal muscle, heart, liver, tenuum mucosa and lymphocytes from young (4 months) and aged (24 months) Sprague-Dawley rats fed ad libitum or held on two different caloric restriction diets (alternate day ad libitum feeding or daily feeding with 40% reduced calories). In the absence of caloric restriction the levels of oxidative DNA damage increased as a function of age in all tissues examined, with a maximum approximately 3-fold increase being detected in the peripheral lymphocytes and the heart and a minimum approximately 2-fold increase being detected in the liver and brain tissues. Caloric restriction regimens effectively reduced age-dependent increase of oxidative DNA damage in all tissues examined; in particular, the brain and small intestine did not exhibit any age-related increase of oxidative DNA damage. We propose that the levels of 8-OHdG in peripheral lymphocytes may serve a biochemical index of age-related whole organism oxidative DNA damage. Immunohistochemistry might be exploited as a rapid and simple techniques for measuring lymphocytes oxidative DNA damage in large scale studies.

Regulation of Magnesium content during proliferation of mammary epithelial cells (HC-11).

To study the role of Magnesium in the regulation of cell proliferation we characterized the proliferation behaviour of HC-11 mammary epithelial cells that were grown in media containing low to high Mg concentrations. Cells grown under control conditions (0.5 mM Mg in the medium) or in the presence of high (H) Mg (45 mM) displayed similar log-phases and reached confluence in 72h. In the presence of low (L) Mg (0.025 mM) the cells exhibited a reduced growth rate and did not reach confluence at 72h. Intra cellular total Mg increased from 12 to 36h of culture in all cells examined but returned to basal levels in those cells which reached confluence (i.e., control and H-Mg cells). Intra cellular Mg increased independent of mitosis-induced changes of volume and adenine nucleotides pools but correlated with an increased percentage of cells in the S phase and with total nucleic acid contents. These bell-shaped changes of intra cellular Mg were less evident in L-Mg cells, likely due to a combination of low Mg levels in the medium and decreased growth rate. Changes in membrane potential and pH were important factors that contributed to maintaining intra cellular Mg at physiologic levels in the face of increased or decreased availability of extra cellular Mg. H-Mg cells were depolarised and more acidic than control cells; conversely, L-Mg cells showed a pattern of hyperpolarization and alkalinization. These results lend support to the concept that Mg may be involved in regulating cell proliferation, and show that cells maintain adequate levels of intra cellular Mg, and hence their proliferation potential, even under conditions of extreme changes of extra cellular Mg.

Magnesium restriction induces granulocytic differentiation and expression of P27Kip1 in human leukemic HL‐60 cells

When cultured in Mg restricted medium, human leukemic HL‐60 cells develop morphological and functional granulocytic differentiation. In 0.03 mM Mg, cells display the distinctive features of differentiation, without appreciable inhibition of proliferation. In 0.01 mM Mg, cells show terminal differentiation, accompanied by clear inhibition of proliferation. Such cells accumulate in the G0/G1 phase and subsequently die via apoptosis, similar to HL‐60 cells that have been induced to differentiate by DMSO. These phenotypic changes are associated with a marked increase in the expression level of the cyclin dependent kinase inhibitor p27Kip1. Cyclin E expression is also slightly increased in Mg restricted cells, whereas no changes are observed in the expression level of cyclin D1. We also show that during differentiation cell total Mg decreases, whereas [Mg2+]i increases in both Mg‐depleted and DMSO‐treated cells. These data suggest that the maturation process is paralleled by a redistribution of intracellular Mg, leading to a shift from the bound to the free form. These changes could modulate the kinetics of Mg‐dependent enzyme(s) that are involved in the control of the differentiation pathway. We propose that this model may represent an useful tool for the study of the mechanisms of cell differentiation and related events, such as aging and death. J. Cell. Biochem. 70:313–322, 1998.

Vascular endothelial growth factor (VEGF) expression in noise-induced hearing loss.

Noise-induced hearing loss has been associated with alterations in cochlear blood flow. Our study analyzed the expression of Vascular Endothelial Growth Factor (VEGF) and its functional receptors, Flt-1 and Flk-1, in the cochlear structures of noise-exposed and unexposed guinea pigs. VEGF is a prototypical angiogenic agent, with multiple functions on vascular biology, ranging from vascular permeability to endothelial cell migration, proliferation, differentiation, and survival. Acoustic trauma was induced by a continuous pure tone of 6 kHz, at 120 dB SPL for 30 min. Auditory function was evaluated by electrocochleographic recordings at 2-20 kHz for 7 days. Noise-induced cochlear morphological changes were studied by immunohistochemistry and scanning electron microscopy. The expression of VEGF and its receptors was examined by immunohistochemistry and western blotting analysis. The hearing threshold shift reached a level of 60 dB SPL on day 1 after trauma and underwent a partial recovery over time, reaching a value of about 20 dB SPL on day 7. Outer hair cell loss was more prominent in the area located 14-16 mm from the apex. Increased cochlear VEGF expression was observed in noise-exposed animals, in particular at the level of stria vascularis, spiral ligament, and spiral ganglion cells. No changes were observed in the expression of VEGF-receptors. Our data suggest a role for VEGF in the regulation of the vascular network in the inner ear after acoustic trauma and during auditory recovery, with potentially important clinical and therapeutic implications.

Age-dependent modifications of expression level of VEGF and its receptors in the inner ear

The mechanisms responsible for age-associated hearing loss are still incompletely characterized. In this study, we used a murine model of age-dependent hearing loss and evaluated whether this condition is associated with vascular modifications of the structures of the inner ear. We used old C57BL/6J mice that are affected by rapid and severe age-related hearing loss, and analyzed the expression pattern of vascular endothelial growth factor (VEGF), a prototypical angiogenic cytokine, and its receptors Flt-1 and Flk-1 in the inner ear. We report for the first time morphological and quantitative data about the expression of these crucial angiogenic molecules in the murine cochlea. We also show that in this animal model, cochlear VEGF expression is significantly reduced as a function of age. Our findings provide new evidence of possible interdependent relationships between aging, VEGF, and presbycusis, suggesting that vascular abnormalities might play a role in aging-associated hearing loss, with potentially important fundamental and clinical implications.

Magnesium and its transporters in cancer: a novel paradigm in tumour development.

The relationship between magnesium and cancer is not as simple as could be assumed from the well-established requirement of magnesium for cell proliferation. Basic and pre-clinical studies indicate that magnesium deficiency can have both anti- and pro-tumour effects. In the present review, we briefly outline the new findings on the role of magnesium in angiogenesis and metastatization, and focus on the relationship between tumour cell proliferation and metabolic reprogramming, discussing how magnesium and its transporters are involved in these processes. The role of magnesium in cancer is also critically examined with regard to mitochondrial function, apoptosis and resistance to treatment. Finally, we bring together the latest experimental evidence indicating that alteration in the expression and/or activity of magnesium channels is a frequent finding in cancer cells and human tumour tissues examined to date, and we discuss the potential implications for developing novel diagnostic and therapeutic strategies.