Vitamin D and COVID-19: How Much Vitamin D Does a Man Need?

A number of observational studies and reviews on the potential role of vitamin D in COVID-19 have been published since the beginning of this ongoing global pandemic [...].

Probiotic-enriched milk and dairy products increase gut microbiota diversity: a comparative study.

Targeting gut microbiota with probiotics has emerged as a promising nutritional approach for the prevention of obesity and metabolic syndrome. Cultured dairy products can be effectively employed for the delivery of probiotics to the gut as well as for the support of growth and survival of probiotic bacteria. The purpose of this study was to characterize the effects of probiotic-enriched pasteurized milk and dairy products (Greek-style yogurt and cottage cheese) of different origins (cow, goat, and camel) on taxonomic composition of the mouse gut microbiota. We hypothesized that cultured dairy products can be an effective vector for the delivery of probiotics to the gut because of its nutritional value, acidic nature, and long shelf-life. Mice were fed a standard low fat, plant polysaccharide-rich (LF/PP) diet supplemented with the probiotic-enriched milk and dairy products for 5 weeks. Next generation sequencing of DNA from mouse fecal samples was used to characterize the bacterial relative abundance. Mice fed a diet supplemented with camel milk demonstrated characteristic changes in the gut microbiota, which included an increase in relative abundance of order Clostridiales and genus Anaerostipes. Mice fed a diet supplemented with the probiotic-enriched cow cheese exhibited an increase in the relative abundance of order Clostridiales, family Ruminococcaceae, and family Lachnospiraceae. The results obtained and their bioinformatics analysis support the conclusion that camel milk and the probiotic cow cheese induce changes in the mouse gut microbiota, which can be characterized as potentially beneficial to health compared to the changes associated with a standard diet. These findings imply that probiotic-enriched milk and dairy products can be highly effective for the delivery and support of probiotic bacteria of the gut.

Vitamin D Status and Vitamin D-Dependent Apoptosis in Obesity.

The role of vitamin D in obesity appears to be linked to vitamin D insufficient/deficient status. However, mechanistic understanding of the role of vitamin D in obesity is lacking. We have shown earlier that the vitamin D hormonal form, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), induces cell death by apoptosis in mature adipocytes. This effect of the hormone is mediated by the cellular Ca2+ signaling pathway: a sustained increase of intracellular (cytosolic) Ca2+ concentration followed by activation of Ca2+-dependent initiators and effectors of apoptosis. In recent animal studies, we demonstrated that low vitamin D status is observed in diet-induced obesity (DIO). High intake of vitamin D3 in DIO decreased the weight of white adipose tissue and improved biomarkers related to adiposity and Ca2+ regulation. The anti-obesity effect of vitamin D (1,25(OH)2D3) in DIO was determined by the induction of Ca2+-mediated apoptosis in mature adipocytes executed by Ca2+-dependent apoptotic proteases (calpains and caspases). Thus, a high intake of vitamin D in obesity increases vitamin D nutritional status and normalizes vitamin D hormonal status that is accompanied by the reduction of adiposity. Overall, our findings imply that vitamin D may contribute to the prevention of obesity and obesity-related diseases and that the mechanism of the anti-obesity effect of 1,25(OH)2D3 includes induction of Ca2+-mediated apoptosis in adipocytes.

Effects of Synbiotic Supplement on Human Gut Microbiota, Body Composition and Weight Loss in Obesity.

Targeting gut microbiota with synbiotics (probiotic supplements containing prebiotic components) is emerging as a promising intervention in the comprehensive nutritional approach to reducing obesity. Weight loss resulting from low-carbohydrate high-protein diets can be significant but has also been linked to potentially negative health effects due to increased bacterial fermentation of undigested protein within the colon and subsequent changes in gut microbiota composition. Correcting obesity-induced disruption of gut microbiota with synbiotics can be more effective than supplementation with probiotics alone because prebiotic components of synbiotics support the growth and survival of positive bacteria therein. The purpose of this placebo-controlled intervention clinical trial was to evaluate the effects of a synbiotic supplement on the composition, richness and diversity of gut microbiota and associations of microbial species with body composition parameters and biomarkers of obesity in human subjects participating in a weight loss program. The probiotic component of the synbiotic used in the study contained Lactobacillus acidophilus, Bifidobacterium lactis, Bifidobacterium longum, and Bifidobacterium bifidum and the prebiotic component was a galactooligosaccharide mixture. The results showed no statistically significant differences in body composition (body mass, BMI, body fat mass, body fat percentage, body lean mass, and bone mineral content) between the placebo and synbiotic groups at the end of the clinical trial (3-month intervention, 20 human subjects participating in weight loss intervention based on a low-carbohydrate, high-protein, reduced energy diet). Synbiotic supplementation increased the abundance of gut bacteria associated with positive health effects, especially Bifidobacterium and Lactobacillus, and it also appeared to increase the gut microbiota richness. A decreasing trend in the gut microbiota diversity in the placebo and synbiotic groups was observed at the end of trial, which may imply the effect of the high-protein low-carbohydrate diet used in the weight loss program. Regression analysis performed to correlate abundance of species following supplementation with body composition parameters and biomarkers of obesity found an association between a decrease over time in blood glucose and an increase in Lactobacillus abundance, particularly in the synbiotic group. However, the decrease over time in body mass, BMI, waist circumstance, and body fat mass was associated with a decrease in Bifidobacterium abundance. The results obtained support the conclusion that synbiotic supplement used in this clinical trial modulates human gut microbiota by increasing abundance of potentially beneficial microbial species.

1,25-Dihydroxyvitamin D3 and type 2 diabetes: Ca2+-dependent molecular mechanisms and the role of vitamin D status.

The hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] induces cellular Ca2+ signals which regulate insulin secretion, while low vitamin D status may be a risk factor for type 2 diabetes (T2D). In pancreatic ß-cells in vitro, 1,25(OH)2D3 induces, via multiple Ca2+ signaling pathways, synchronous Ca2+ oscillations, which quantitatively, temporally, and spatially pattern pulsatile insulin secretion from these cells. In animal studies employing a high fat diet-induced obesity model of pre-T2D, an increased intake of vitamin D delayed development of T2D and adiposity and was associated with the improved blood markers of diabetes and the vitamin D nutritional and hormonal status [plasma concentrations of glucose, insulin, adiponectin, 25-hydroxyvitamin D, and 1,25(OH)2D3]. Observational studies demonstrated associations between vitamin D status, insulin secretion and resistance to T2D, however, randomized controlled trials did not provide conclusive insights into the potential role of vitamin D in prevention of T2D. The 1,25(OH)2D3-dependent cellular Ca2+ signaling can be important for maintaining the normal level of insulin secretion from pancreatic ß-cells, and an increased intake of vitamin D may contribute to the prevention of T2D and metabolic disorders associated with this disease.

Vitamin D-Cellular Ca2+ link to obesity and diabetes.

The vitamin D hormone 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3)-induced cellular Ca2+ signals regulate apoptosis in adipocytes and insulin secretion from pancreatic ß-cells, and low vitamin D status is considered a risk factor for obesity and type 2 diabetes. The anti-obesity effects of 1,25(OH)2D3 in mature adipocytes are determined by its activity to generate, via multiple Ca2+ signaling pathways, a sustained increase in intracellular Ca2+ followed by activation of the Ca2+-dependent initiators and effectors of apoptosis. In pancreatic ß-cells, 1,25(OH)2D3 induces synchronous Ca2+ oscillations, which pattern pulsatile insulin secretion from these cells. An increased intake of vitamin D3 in a high fat diet-induced obesity mouse model is associated with a decreased weight of white adipose tissue due to induction of apoptosis and the improved blood markers related to adiposity, diabetes, and vitamin D status (plasma concentrations of glucose, insulin, adiponectin, 25-hydroxyvitamin D, and 1,25(OH)2D3). High vitamin D3 intake is also effective in increasing the mineral content of growing bone in obese mice via regulatory effects mediated by 1,25(OH)2D3-parathyroid hormone axis. The 1,25(OH)2D3-dependent cellular Ca2+ signaling can be important for maintaining the normal levels of apoptosis in adipose tissue and insulin secretion from pancreatic ß-cells. An increased intake of vitamin D may contribute to the prevention of obesity, type 2 diabetes, and bone disorders associated with these diseases.

Effects of vitamin D and quercetin, alone and in combination, on cardiorespiratory fitness and muscle function in physically active male adults.

INTRODUCTION: Vitamin D and the antioxidant quercetin, are promising agents for improving physical performance because of their possible beneficial effects on muscular strength and cardiorespiratory fitness. PURPOSE: The purpose of this study was to determine the effects of increased intakes of vitamin D, quercetin, and their combination on antioxidant status, the steroid hormone regulators of muscle function, and measures of physical performance in apparently healthy male adults engaged in moderate-to-vigorous-intensity exercise training. METHODS: A total of 40 adult male participants were randomized to either 4,000 IU vitamin D/d, 1,000 mg/d quercetin, vitamin D plus quercetin, or placebo for 8 weeks. Measures of cardiorespiratory fitness and muscle function, blood markers for antioxidant and vitamin D status, and hormones 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and testosterone were measured pre- and postsupplementation. RESULTS: At enrollment, 88.6% of participants were vitamin D sufficient (serum 25-hydroxyvitamin D >50 nmol/L) and had normal serum testosterone levels. Supplementation with vitamin D significantly increased serum 25(OH)D concentration (by 87.3% in the vitamin D group, P<0.001) and was associated with an increasing trend of testosterone concentration. There were no changes in concentration of 1,25(OH)2D3 and markers of antioxidant status associated with vitamin D or quercetin supplementation. No improvements in physical performance measures associated with vitamin D and quercetin supplementation were found. CONCLUSION: The findings obtained demonstrate that long-term vitamin D and quercetin supplementation, alone or in combination, does not improve physical performance in male adults with adequate vitamin D, testosterone, and antioxidant status.

High vitamin D and calcium intakes increase bone mineral (Ca and P) content in high-fat diet-induced obese mice.

Vitamin D and calcium are essential for bone formation, mineralization, and remodeling. Recent studies demonstrated that an increased body mass can be detrimental to bone health. However, whether an increase in dietary vitamin D and calcium intakes in obesity is beneficial to bone health has not been established. The aim of this study was to examine the effects of increased vitamin D and calcium intakes, alone or in combination, on bone status in a high-fat diet-induced obesity (DIO) mouse model. We hypothesized that DIO in growing mice affects bone mineral status and that high vitamin D and calcium intakes will promote mineralization of the growing bone in obesity via Ca(2+) regulatory hormones, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and parathyroid hormone (PTH). Male mice were fed high vitamin D3 (10 000 IU/kg), high calcium (1.2%), or high vitamin D3 plus high-calcium diets containing 60% energy as fat for 10 weeks. Bone weight, specific gravity, mineral (Ca and P), and collagen (hydroxyproline) content were measured in the femur and the tibia. Regulators of Ca(2+) metabolism and markers of bone status (PTH, 25-hydroxyvitamin D [25(OH)D], 1,25(OH)2D3, and osteocalcin) were measured in blood plasma. Diet-induced obese mice exhibited lower bone Ca and P content and relative bone weight compared with the normal-fat control mice, whereas collagen (hydroxyproline) content was not different between the two groups. High vitamin D3 and calcium intakes significantly increased bone Ca and P content and relative bone weight in DIO mice, which was accompanied by an increase in 1,25(OH)2D3 and a decrease in PTH and osteocalcin concentrations in blood. The findings obtained indicate that increased vitamin D and calcium intakes are effective in increasing mineral (Ca and P) content in the growing bone of obese mice and that the hormonal mechanism of this effect may involve the vitamin D-PTH axis.

Vitamin D-mediated apoptosis in cancer and obesity.

Low vitamin D status has been associated with increased risk of several cancers and obesity; concurrently, obesity and cancer have been linked to impaired vitamin D status. In both cancer and obesity, selective elimination of cancer cells and adipocytes can result in decreasing tumor size and a long-term reduction in adipose tissue mass. These effects can be achieved through induction of apoptotic cell death. The vitamin D-derived hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) triggers apoptosis in epithelial cancer cells and mature adipocytes via induction of apoptotic Ca2+ signal - a sustained, prolonged increase in concentration of intracellular Ca2+. This Ca2+ signal functions as an apoptotic initiator that directly recruits apoptotic effectors, Ca2+-dependent proteases, in cancer cells and adipocytes. The 1,25(OH)2D3 - cellular Ca2+ - apoptosis link in cancer and obesity supports the rationale to include vitamin D compounds modulating intracellular Ca2+ and Ca2+-dependent apoptotic proteases as promising targets for discovery of new therapeutic and preventive agents for cancer and obesity. The concept of maintaining an increased vitamin D status for protecting against cancer and decreasing adiposity also warrants further evaluation.

High vitamin D and calcium intakes reduce diet-induced obesity in mice by increasing adipose tissue apoptosis.

SCOPE: Modulation of apoptosis is emerging as a promising antiobesity strategy because removal of adipocytes through this process will result in reducing body fat. Effects of vitamin D on apoptosis are mediated via multiple signaling pathways that involve common regulators and effectors converging on cellular Ca(2+) . We have previously shown that 1,25-dihydroxyvitamin D3 induces the Ca(2+) signal associated with activation of Ca(2+) -dependent apoptotic proteases in mature adipocytes. In this study, a diet-induced obesity (DIO) mouse model was used to evaluate the role of vitamin D and calcium in adiposity. METHODS AND RESULTS: DIO mice fed high vitamin D3 , high Ca, and high D3 plus high Ca diets demonstrated a decreased body and fat weight gain, improved markers of adiposity and vitamin D status (plasma concentrations of glucose, insulin, adiponectin, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, parathyroid hormone (PTH)), but an increased plasma Ca(2+) . High D3 and Ca intakes were associated with induction of apoptosis and activation of Ca(2+) -dependent apoptotic proteases, calpain and caspase-12, in adipose tissue of DIO mice. The combination of D3 plus Ca was more effective than D3 or Ca alone in decreasing adiposity. CONCLUSION: The results imply that high vitamin D and Ca intakes activate the Ca(2+) -mediated apoptotic pathway in adipose tissue. Targeting this pathway with vitamin D and Ca supplementation could contribute to the prevention and treatment of obesity. However, this potentially effective and affordable approach needs to be evaluated from a safety point of view.

Vitamin D: beyond bone.

In recent years, vitamin D has been received increased attention due to the resurgence of vitamin D deficiency and rickets in developed countries and the identification of extraskeletal effects of vitamin D, suggesting unexpected benefits of vitamin D in health and disease, beyond bone health. The possibility of extraskeletal effects of vitamin D was first noted with the discovery of the vitamin D receptor (VDR) in tissues and cells that are not involved in maintaining mineral homeostasis and bone health, including skin, placenta, pancreas, breast, prostate and colon cancer cells, and activated T cells. However, the biological significance of the expression of the VDR in different tissues is not fully understood, and the role of vitamin D in extraskeletal health has been a matter of debate. This report summarizes recent research on the roles for vitamin D in cancer, immunity and autoimmune diseases, cardiovascular and respiratory health, pregnancy, obesity, erythropoiesis, diabetes, muscle function, and aging.

Regulation of apoptosis in adipocytes and breast cancer cells by 1,25-dihydroxyvitamin D3: a link between obesity and breast cancer.

Modulation of apoptosis is emerging as a promising strategy for prevention and treatment of breast cancer and obesity because removal of mammary cancer cells and mature adipocytes through this process will result in decreasing tumor size and produce long-term reduction in adipose tissue mass. The hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) triggers apoptosis in breast cancer cells and adipocytes via the induction of the apoptotic Ca2+ signal - a sustained increase in concentration of intracellular Ca2+. This signal acts as an apoptotic initiator that directly recruits Ca2+-dependent apoptotic effectors, calpain and caspase 12, in breast cancer cells and adipocytes. Normal mammary epithelial cells are resistant to 1,25(OH)2D3-induced, Ca2+-mediated apoptosis because the mechanisms regulating Ca2+ in these cells do not sustain Ca2+ increase at the apoptosis-inducing level. Induction of apoptosis with 1,25(OH)2D3 in adipose tissue, particularly in the tumor-surrounding adipose tissue involved in tumor progression, can contribute to the anticancer effects of the hormone. The 1,25(OH)2D3-Ca2+ link between obesity and breast cancer supports the rationale to include Ca2+-dependent apoptotic proteases as molecular targets for the discovery of new therapeutic and preventive agents for breast cancer and obesity; it also supports the recommendation to maintain adequate or increased vitamin D and calcium intakes as one of the possible ways to protect against breast cancer and decrease adiposity.

Long-term quercetin supplementation reduces lipid peroxidation but does not improve performance in endurance runners.

PURPOSE: To evaluate the effects of chronic quercetin supplementation on endurance performance and antioxidant status in long distance runners. We hypothesized that an improved antioxidant status can be associated with enhanced performance. METHODS: During 6 weeks of supplementation utilizing a double blind, randomized design, young male subjects received quercetin (1000 mg/day) or placebo while maintaining their current training schedules. RESULTS: Following the end of the supplementation period, there was a significant time × supplement interaction for serum malondialdehyde (MDA), an indicator of lipid peroxidation. There were no significant pre- to post-supplement changes in parameter values employed for measuring total antioxidant capacity, superoxide dismutase activity, and protein oxidation (protein carbonyl) in serum. There were also no significant pre- to post-supplement differences in VO2peak, running economy, heart rate, and rating of perceived exertion (RPE) during the 10 km time trial. CONCLUSION: The findings obtained indicate that there is a relationship between quercetin supplementation and the statistically significant decreasing trend in MDA levels following 6 weeks of supplementation and training. This evidence suggests that quercetin can reduce oxidative stress (lipid peroxidation). However, performance improvements were not significant (as measured by VO2peak, running economy, heart rate, and RPE).

Calcium and vitamin D in obesity.

New and more effective nutritional measures are urgently needed for the prevention of obesity. The role of Ca and vitamin D in obesity has been recently implicated. Low Ca intake and low vitamin D status have been linked with an increased risk of obesity in epidemiological studies; however, clinical intervention trials designed to test this association have produced controversial results. The suggested anti-obesity mechanisms of Ca and vitamin D include the regulation of adipocyte death (apoptosis), adipogenesis and lipid metabolism. Dietary Ca has been also shown to increase faecal fat excretion. The potential role of Ca and vitamin D in shifting energy balance towards a more negative state is an area of considerable interest. Ultimately, a review of recent research findings does not allow the reaching of a definitive conclusion that increasing Ca intake and rising vitamin D status will influence fat mass and body weight or decrease the risk of obesity and overweight.

Vitamin D and cellular Ca2+ signaling in breast cancer.

The hormone 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) regulates a variety of signaling pathways via intracellular Ca(2+). Modulation of apoptosis is emerging as a promising strategy for treatment and prevention of cancer. Cellular Ca(2+) has been implicated in triggering of apoptosis, however, the vitamin D/Ca(2+)-dependent targets involved in apoptotic signaling have not been identified. Here, we review our studies on mechanisms of 1,25(OH)(2)D(3)-induced Ca(2+) signaling and Ca(2+)-mediated apoptosis in breast cancer cells. The results obtained demonstrate that 1,25(OH)(2)D(3) regulates Ca(2+) entry from the extracellular space, Ca(2+) mobilization from the intracellular stores and intracellular Ca(2+) buffering. In breast cancer cells, 1,25(OH)(2)D(3) induces the apoptotic Ca(2+) signal, a sustained increase in concentration of intracellular Ca(2+) ([Ca(2+)](i)) reaching elevated, but not cytotoxic levels. This increase in [Ca(2+)](i) is associated with activation of Ca(2+)-dependent µ-calpain and Ca(2+)/calpain-dependent caspase-12. Activation of these proteases appears to be sufficient for the execution of apoptosis in cancer cells. Normal mammary epithelial cells resist induction of apoptosis with 1,25(OH)(2)D(3) due to their large Ca(2+)-buffering capacity. The results indicate that the 1,25(OH)(2)D(3)-induced cellular Ca(2+) signal can act as an apoptotic initiator that directly recruits Ca(2+)-dependent apoptotic effectors capable of executing apoptosis. These findings provide a novel rationale for evaluating the role of vitamin D in prevention and treatment of breast cancer.

Polymethoxyflavones activate Ca2+-dependent apoptotic targets in adipocytes.

Induction of apoptosis is an emerging strategy for the prevention and treatment of obesity because removal of adipocytes via apoptosis will result in reducing body fat and may help to maintain a long-lasting weight loss. Our previous studies have shown that a sustained increase in intracellular Ca(2+) triggers apoptosis in various cell types via activation of Ca(2+)-dependent proteases and that the apoptosis-inducing effect of polymethoxyflavones (PMFs) in cancer cells is mediated through Ca(2+) signaling. This paper reports that PMFs induce apoptosis in mature mouse 3T3-L1 adipocytes via activation of Ca(2+)-dependent calpain and Ca(2+)/calpain-dependent caspase-12. Treatment of adipocytes with PMFs evoked, in a concentration- and time-dependent fashion, sustained increase in the basal level of intracellular Ca(2+). The increase in Ca(2+) was associated with induction of apoptosis and activation of mu-calpain and caspase-12. Apoptosis-inducing activity of hydroxylated PMFs was significantly higher than that of the corresponding nonhydroxylated compounds. These results demonstrate that the apoptotic molecular targets activated by PMFs in adipocytes are Ca(2+)-dependent calpain and caspase-12. The findings obtained provide rationale for evaluating the role of PMFs in the prevention and treatment of obesity.

1,25-Dihydroxyvitamin D3 induces Ca2+-mediated apoptosis in adipocytes via activation of calpain and caspase-12.

Induction of apoptotic cell death is emerging as a promising strategy for prevention and treatment of obesity because removing of adipocytes via apoptosis may result in reducing body fat and a long-lasting maintenance of weight loss. However, the mechanisms controlling adipocyte apoptosis are unknown and even the ability of adipocytes to undergo apoptosis has not been conclusively demonstrated. We have shown previously that the specific Ca(2+) signal, sustained increase in intracellular Ca(2+), triggers apoptotic cell death via activation of Ca(2+)-dependent proteases and that the apoptosis-inducing effect of the hormone 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is mediated through Ca(2+) signaling. Here, we report that 1,25(OH)(2)D(3) induces apoptosis in mature mouse 3T3-L1 adipocytes via activation of Ca(2+)-dependent calpain and Ca(2+)/calpain-dependent caspase-12. Treatment of adipocytes with 1,25(OH)(2)D(3) induced, in concentration- and time-dependent fashion, a sustained increase in the basal level of intracellular Ca(2+). The increase in Ca(2+) was associated with induction of apoptosis and activation of mu-calpain and caspase-12. Our results demonstrate that Ca(2+)-mediated apoptosis can be induced in mature adipocytes and that the apoptotic molecular targets activated by 1,25(OH)(2)D(3) in these cells are Ca(2+)-dependent calpain and caspase-12. These findings provide rationale for evaluating the role of vitamin D in prevention and treatment of obesity.

Apoptosis-inducing activity of hydroxylated polymethoxyflavones and polymethoxyflavones from orange peel in human breast cancer cells.

Sweet orange (Citrus sinensis L.) peel is a rich resource of flavonoids, especially polymethoxyflavones (PMFs). Citrus flavonoids exert a broad spectrum of biological activity, including antiproliferative and proapoptotic effects in cancer cells. We have recently shown that individual PMFs from orange peel induce Ca(2+)-mediated apoptosis in human breast cancer cells and that hydroxylation of PMFs is critical for enhancing their proapoptotic activity. Here, we report that the fraction of orange peel extract containing a mixture of non-hydroxylated PMFs (75.1%) and hydroxylated PMFs (5.44%) and the fraction containing only hydroxylated PMFs (97.2%) induce apoptosis in those cells as well. Treatment of MCF-7 breast cancer cells with these fractions inhibited growth and induced apoptosis associated with an increase in the basal level of intracellular Ca(2+). Effective concentrations of the hydroxylated PMFs fraction in inhibiting growth, inducing apoptosis, and increasing intracellular Ca(2+) were lower than those of the non-hydroxylated PMFs fraction. Our results strongly imply that bioactive PMFs from orange peel exert proapoptotic activity in human breast cancer cells, which depends on their ability to induce an increase in intracellular Ca(2+ )and thus, activate Ca(2+)-dependent apoptotic proteases.

Polymethoxylated flavones induce Ca(2+)-mediated apoptosis in breast cancer cells.

Flavonoids, polyphenolic phytochemicals which include flavones and isoflavones, are present in the common human diet. It has been suggested that these compounds may exert anticancer activity; however, the mechanisms involved remain unknown. We have recently shown (Sergeev, 2004, Biochem Biophys Res Commun 321: 462-467) that isoflavones can activate the novel apoptotic pathway mediated by cellular Ca(2+). Here, we report that polymethoxyflavones (PMFs) derived from sweet orange (Citrus sinensis L.) inhibit growth of human breast cancer cells via Ca(2+)-dependent apoptotic mechanism. The treatment of MCF-7 breast cancer cells with 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5-OH-HxMF) and 3'-hydroxy-5,6,7,4'-tetramethoxyflavone (3'-OH-TtMF) induced a sustained increase in concentration of intracellular Ca(2+) ([Ca(2+)](i)) resulting from both depletion of the endoplasmic reticulum Ca(2+) stores and Ca(2+) influx from the extracellular space. This increase in [Ca(2+)](i) was associated with the activation of the Ca(2+)-dependent apoptotic proteases, mu-calpain and caspase-12, as evaluated with the calpain and caspase-12 peptide substrates and antibodies to active (cleaved) forms of the enzymes. Corresponding non-hydroxylated PMFs, 3,5,6,7,8,3',4'-heptamethoxyflavone (HpMF) and 5,6,7,3',4'-pentamethoxyflavone (PtMF), were dramatically less active in inducing Ca(2+)-mediated apoptosis. Our results strongly suggest that the cellular Ca(2+) modulating activity of flavonoids underlies their apoptotic mechanism and that hydroxylation of PMFs is critical for their ability to induce an increase in [Ca(2+)](i) and, thus, activate Ca(2+)-dependent apoptotic proteases.

Calcium signaling in cancer and vitamin D.

Calcium signals induced by the Ca(2+) regulatory hormone 1,25(OH)(2)D(3) may determine the fate of the cancer cell. We have shown that, in breast cancer cell lines, 1,25(OH)(2)D(3) induces a sustained increase in concentration of intracellular Ca(2+) ([Ca(2+)](i)) by depleting the endoplasmic reticulum (ER) Ca(2+) stores via inositol 1,4,5-trisphosphate receptor/Ca(2+) release channel and activating Ca(2+) entry from the extracellular space via voltage-insensitive Ca(2+) channels. In normal cells, 1,25(OH)(2)D(3) triggered a transient Ca(2+) response via activation of voltage-dependent Ca(2+) channels, which were absent in breast cancer cells. The normal cells, but not breast cancer cells, expressed the Ca(2+) binding/buffering protein calbindin-D(28k) and were capable of buffering [Ca(2+)](i) increases induced by a mobilizer of the ER Ca(2+) stores, thapsigargin, or a Ca(2+) ionophore, ionomycin. The 1,25(OH)(2)D(3)-induced sustained increase in [Ca(2+)](i) in breast cancer cells was associated with induction of apoptotic cell death, whereas the transient [Ca(2+)](i) increase in normal cells was not. The forced expression of calbindin-D(28k) in cytosol or increase in the cytosolic Ca(2+) buffering capacity with the cell-permeant Ca(2+) buffer BAPTA prevented induction of apoptosis with 1,25(OH)(2)D(3) in cancer cells. The sustained increase in [Ca(2+)](i) in breast cancer cells was associated with activation of the Ca(2+)-dependent apoptotic proteases, mu-calpain and caspase-12, as evaluated with antibodies to active (cleaved) forms of the enzymes and the fluorogenic peptide substrates. Selective inhibition of the Ca(2+) binding sites of mu-calpain decreased apoptotic indices in the cancer cells treated with 1,25(OH)(2)D(3), thapsigargin, or ionomycin. The mu-calpain activation preceded expression/activation of caspase-12, and calpain was required for activation/cleavage of caspase-12. Certain non-calcemic vitamin D analogs (e.g., EB 1089) triggered a sustained [Ca(2+)](i) increase, activated Ca(2+)-dependent apoptotic proteases, and induced apoptosis in breast cancer cells in a fashion similar to that of 1,25(OH)(2)D(3). The 1,25(OH)(2)D(3)-induced transient Ca(2+) response in normal mammary epithelial cells was not accompanied by activation of mu-calpain and caspase-12. In conclusion, we have identified the novel apoptotic pathway in breast carcinoma cells treated with 1,25(OH)(2)D(3): increase in [Ca(2+)](i)-->mu-calpain activation-->caspase-12 activation-->apoptosis. Our results support the hypothesis that 1,25(OH)(2)D(3) directly activates this apoptotic pathway by inducing a sustained increase in [Ca(2+)](i). Differences of Ca(2+) regulatory mechanisms in cancer versus normal cells seem to allow 1,25(OH)(2)D(3) and vitamin D analogs to induce Ca(2+)-mediated apoptosis selectively in breast cancer cells. Thus, deltanoids may prove to be useful in the treatment of tumors susceptible to induction of Ca(2+)-mediated apoptosis.