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.     

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 as a mediator of 1,25-dihydroxyvitamin D3-induced apoptosis

Cellular calcium has been implicated in induction of apoptosis. We have shown that 1,25(OH)(2)D(3)-induced apoptosis is associated with a sustained increase in concentration of intracellular Ca(2+) ([Ca(2+)](i)) resulting from depletion of the endoplasmic reticulum (ER) Ca(2+) stores and activation of the voltage-insensitive Ca(2+) entry pathway [1,25-Dihydroxyvitamin D(3), intracellular Ca(2+) and apoptosis in breast cancer cells, in: A.W. Norman, R. Bouillon, M. Thomasset (Eds.), Vitamin D: Chemistry, Biology and Clinical Applications of the Steroid Hormone, University of California, Riverside, 1997, pp. 473-474; Vitamin D and intracellular calcium, in: P. Quinn, V. Kagan (Eds.), Subcellular Biochemistry: Fat-Soluble Vitamins, Plenum Press, New York, 1998, pp. 271-297; 1,25-Dihydroxyvitamin D(3) and calcium signaling, in: A.W. Norman, R. Bouillon, M. Thomasset (Eds.), Vitamin D Endocrine System: Structural, Biological, Genetic and Clinical Aspects, University of California, Riverside, 2000, pp. 715-718; 1,25-Dihydroxyvitamin D(3) triggers calcium-mediated apoptosis in breast cancer cells, in: A.W. Norman, R. Bouillon, M. Thomasset (Eds.), Vitamin D Endocrine System: Structural, Biological, Genetic and Clinical Aspects, University of California, Riverside, 2000, pp. 399-402; Endocrine 9 (1998) 321]. This study was undertaken to investigate mechanism of 1,25(OH)(2)D(3)-induced apoptosis in breast cancer cells and compare effects of the hormone on Ca(2+) and apoptosis in cancer and normal human mammary epithelial cells. The treatment of MCF-7 breast cancer cells with 1,25(OH)(2)D(3) induced a sustained increase in [Ca(2+)](i) and activated the Ca(2+)-dependent proapoptotic proteases, micro-calpain and caspase-12, as evaluated with antibodies to active (cleaved) forms of the enzymes and the calpain substrate. The selective inhibition of Ca(2+) binding sites of micro-calpain decreased apoptotic indices in the 1,25(OH)(2)D(3)-treated cells. 1,25(OH)(2)D(3) did not induce apoptosis in normal human mammary epithelial cells (HMECs), as evaluated by DNA fragmentation (TUNEL), loss of the plasma membrane asymmetry (Annexin V assay) and morphological criteria. In these cells, 1,25(OH)(2)D(3) triggered a transient Ca(2+) response, which was not accompanied by the calpain and caspase activation. HMEC, but not MCF-7 cells expressed the Ca(2+) binding protein calbindin-D(28k) and buffered Ca(2+) increases induced by a Ca(2+) ionophore ionomycin. 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) -->micro-calpain activation --> caspase-12 activation --> apoptosis. Our findings also imply that differences of Ca(2+) regulatory mechanisms in breast cancer versus normal mammary epithelial cells underlay resistance of normal cells and susceptibility of cancer cells to 1,25(OH)(2)D(3)-induced Ca(2+)-mediated apoptosis.     

Caspase-3 status is a determinant of the differential responses to genistein between MDA-MB-231 and MCF-7 breast cancer cells

Genistein, a soy isoflavone with anti-tumor properties, has both estrogenic and non-estrogenic activities. Genistein sensitive/estrogen receptor negative (ER-) MDA-MB-231 cells and genistein resistant/ER+MCF-7 cells are frequently cited as examples of differential responses to genistein due to different ER status. Other factors that may affect genistein response, however, are largely unknown. Based on our finding that MCF-7 is caspase-3 deficient, we examined whether caspase-3 status plays a role in the differential responses between the two cell lines. We demonstrate that reconstitution of caspase-3 significantly sensitizes MCF-7 cells to genistein. Specific knockdown of caspase-3 in MDA-MB-231 cells renders the cells resistant to genistein. We also found that caspases-4 and -10 were downregulated in MCF-7 cells. Reconstitution of caspase-10 in MCF-7 cells, however, resulted in little sensitization. Moreover, we show that caspase-3 downregulation is very common in breast cancer cell lines and tumor tissues. Taken together, our data indicate that caspase-3 is a critical determinant of cellular response to genistein, which may have important implications in studying soy/genistein-mediated anti-tumor activities.     

mu-Calpain and calpain-3 are not autolyzed with exhaustive exercise in humans

mu-Calpain and calpain-3 are Ca2+-dependent proteases found in skeletal muscle. Autolysis of calpains is observed using Western blot analysis as the cleaving of the full-length proteins to shorter products. Biochemical assays suggest that mu-calpain becomes proteolytically active in the presence of 2-200 microM Ca2+. Although calpain-3 is poorly understood, autolysis is thought to result in its activation, which is widely thought to occur at lower intracellular Ca2+ concentration levels ([Ca2+]i; approximately 1 microM) than the levels at which mu-calpain activation occurs. We have demonstrated the Ca2+-dependent autolysis of the calpains in human muscle samples and rat extensor digitorum longus (EDL) muscles homogenized in solutions mimicking the intracellular environment at various [Ca2+] levels (0, 2.5, 10, and 25 microM). Autolysis of calpain-3 was found to occur across a [Ca2+] range similar to that for mu-calpain, and both calpains displayed a seemingly higher Ca2+ sensitivity in human than in rat muscle homogenates, with approximately 15% autolysis observed after 1-min exposure to 2.5 microM Ca2+ in human muscle and almost none after 1- to 2-min exposure to the same [Ca2+]i level in rat muscle. During muscle activity, [Ca2+]i may transiently peak in the range found to autolyze mu-calpain and calpain-3, so we examined the effect of two types of exhaustive cycling exercise (30-s "all-out" cycling, n = 8; and 70% VO2 peak until fatigue, n = 3) on the amount of autolyzed mu-calpain or calpain-3 in human muscle. No significant autolysis of mu-calpain or calpain-3 occurred as a result of the exercise. These findings have shown that the time- and concentration-dependent changes in [Ca2+]i that occurred during concentric exercise fall near but below the level necessary to cause autolysis of calpains in vivo.     

Mu-calpain binds to lipid bilayers via the exposed hydrophobic surface of its Ca2+-activated conformation

Mu- and m-calpain are cysteine proteases requiring micro- and millimolar Ca2+ concentrations for their activation in vitro. Among other mechanisms, interaction of calpains with membrane phospholipids has been proposed to facilitate their activation by nanomolar [Ca2+] in living cells. Here the interaction of non-autolysing, C115A active-site mutated heterodimeric human mu-calpain with phospholipid bilayers was studied in vitro using protein-to-lipid fluorescence resonance energy transfer and surface plasmon resonance. Binding to liposomes was Ca2+-dependent, but not selective for specific phospholipid head groups. [Ca2+]0.5 for association with lipid bilayers was not lower than that required for the exposure of hydrophobic surface (detected by TNS fluorescence) or for enzyme activity in the absence of lipids. Deletion of domain V reduced the lipid affinity of the isolated small subunit (600-fold) and of the heterodimer (10- to 15-fold), thus confirming the proposed role of domain V for membrane binding. Unexpectedly, mutations in the acidic loop of the 'C2-like' domain III, a putative Ca2+ and phospholipid-binding site, did not affect lipid affinity. Taken together, these results support the hypothesis that in vitro membrane binding of mu-calpain is due to the exposed hydrophobic surface of the active conformation and does not reduce the Ca2+ requirement for activation.     

The intracellular genistein metabolite 5,7,3',4'-tetrahydroxyisoflavone mediates G2-M cell cycle arrest in cancer cells via modulation of the p38 signaling pathway

The cellular actions of genistein are believed to mediate the decreased risk of breast cancer associated with high soy consumption. We have investigated the intracellular metabolism of genistein in T47D tumorigenic and MCF-10A nontumorigenic cells and assessed the cellular actions of resultant metabolites. Genistein selectively induced growth arrest and G2-M phase cell cycle block in T47D but not MCF10A breast epithelial cells. These antiproliferative effects were paralleled by significant differences in the association of genistein to cells and in particular its intracellular metabolism. Genistein was selectively taken up into T47D cells and was subject to metabolism by CYP450 enzymes leading to the formation of both 5,7,3',4'-tetrahydroxyisoflavone (THIF) and two glutathionyl conjugates of THIF. THIF inhibited cdc2 activation via the phosphorylation of p38 MAP kinase, suggesting that this species may mediate genistein's cellular actions. THIF exposure activated p38 and caused subsequent inhibition of cyclin B1 (Ser 147) and cdc2 (Thr 161) phosphorylation, two events critical for the correct functioning of the cdc2-cyclin B1 complex. We suggest that the formation of THIF may mediate the cellular actions of genistein in tumorigenic breast epithelial cells via the activation of signaling through p38.     

Genistein inhibits contractile force, intracellular Ca2+ increase and Ca2+ oscillations induced by serotonin in rat aortic smooth muscle

The soy-derived isoflavones genistein and daidzein affect the contractile state of different kinds of smooth muscle. We describe acute effects of genistein and daidzein on contractile force and intracellular Ca2+ concentration ([Ca2+]i) in in situ smooth muscle of rat aorta. Serotonin (5-HT) (2 microM) or a depolarizing high K+ solution produced the contraction of aortic rings, which were immediately relaxed by 20 microM genistein and by 20 microM daidzein. Accordingly, both 5-HT and a high K+ solution increased the [Ca2+]i in in situ smooth muscle cells. Genistein strongly inhibited the [Ca2+]i increase evoked by 5-HT (74.0 +/- 7.3%, n = 11, p < 0.05), and had a smaller effect on high K+ induced [Ca2+]i increase (19.9 +/- 4.0%, n = 7, p < 0.05). The K+ channels blocker tetraethylammonium (TEA) (0.5 mM) diminished genistein effects on 5-HT-induced [Ca2+]i increase. Interestingly, during prolonged application of 5-HT, the [Ca2+]i oscillated and a short (90 s) preincubation with genistein (20 microM) significantly diminished the frequency of the oscillations. This effect was totally abolished by TEA. In conclusion, in rat aortic smooth muscle, genistein is capable of diminishing the increase in [Ca2+]i and in force evoked by 5-HT and high K+ solution, and of decreasing the frequency of [Ca2+]i oscillations induced by 5-HT. The short time required by genistein, and the relaxing effect of daidzein suggest that tyrosine kinases inhibition is not involved. The small inhibiting effect of genistein on the [Ca2+]i increase evoked by high K+ and the effect of TEA point to the activation by genistein of calcium-activated K+ channels.     

Genistein inhibits lysosomal enzyme release by suppressing Ca2+ influx in HL-60 granulocytes

The tyrosine kinase inhibitor genistein (5-200 microM) suppressed Ca(2+)-dependent fMLP (1 microM) and ATP (100 microM)-induced release of the lysosomal enzyme, beta-glucuronidase from neutrophil-like HL-60 granulocytes. Agonist-induced Ca2+ mobilization resulted from the release of intracellular Ca2+ stores and the influx of extracellular Ca2+. Genistein (200 microM) suppressed fMLP (1 microM) and ATP (100 microM)-induced Ca2+ mobilization, by 30-40%. Ca2+ release from intracellular stores was unaffected by genistein, however, genistein abolished agonist-induced Ca2+ (Mn2+) influx. Consistent with these findings, genistein (200 microM) or removal of extracellular Ca2+ (EGTA 1 mM), inhibited Ca(2+)-dependent agonist-induced beta-glucuronidase release by similar extents (about 50%). In the absence of extracellular Ca2+, genistein had a small additional inhibitory effect on fMLP and ATP-induced beta-glucuronidase release, suggesting an additional inhibitory site of action. Genistein also abolished store-operated (thapsigargin-induced) Ca2+ (Mn2+) influx. Neither fMLP nor ATP increased the rate of Mn2+ influx induced by thapsigargin (0.5 microM). These data indicate that agonist-induced Ca2+ influx and store-operated Ca2+ influx occur via the same genistein-sensitive pathway. Activation of this pathway supports approximately 50% of lysosomal enzyme release induced by either fMLP or ATP from HL-60 granulocytes.     

1,25-Dihydroxyvitamin D3 induces Ca-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 Ca²⁺ signal, sustained increase in intracellular Ca²⁺, triggers apoptotic cell death via activation of Ca²⁺-dependent proteases and that the apoptosis-inducing effect of the hormone 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) is mediated through Ca²⁺ signaling. Here, we report that 1,25(OH)₂D₃ induces apoptosis in mature mouse 3T3-L1 adipocytes via activation of Ca²⁺-dependent calpain and Ca²⁺/calpain-dependent caspase-12. Treatment of adipocytes with 1,25(OH)₂D₃ induced, in concentration- and time-dependent fashion, a sustained increase in the basal level of intracellular Ca²⁺. The increase in Ca²⁺ was associated with induction of apoptosis and activation of μ-calpain and caspase-12. Our results demonstrate that Ca²⁺-mediated apoptosis can be induced in mature adipocytes and that the apoptotic molecular targets activated by 1,25(OH)₂D₃ in these cells are Ca²⁺-dependent calpain and caspase-12. These findings provide rationale for evaluating the role of vitamin D in prevention and treatment of obesity.     

Mechanism of UV-Induced Apoptosis in Human Leukemia Cells: Roles of Ca/Mg-Dependent Endonuclease, Caspase-3, and Stress-Activated Protein Kinases

Ultraviolet light (UV) induced rapid apoptosis of U937 leukemia cells, concurrent with DNA fragmentation and cleavage of poly(ADP-ribose)polymerase (PARP) by activated caspase-3. Thein vitroreconstitution of intact HeLa S3 nuclei and apoptotic U937 cytosolic extract (CE) revealed that (i) Ca²⁺/Mg²⁺-dependent, Zn²⁺-sensitive endonuclease activated in the apoptotic CE induced DNA ladder in HeLa nuclei at pH 6.8–7.4, (ii) activated caspase-3 cleaved PARP in HeLa nuclei, and (iii) when the apoptotic CE was treated with the caspase-3 inhibitor (1 μM Ac-DEVD-CHO) or the caspase-1 inhibitor (10 μM Ac-YVAD-CHO), the former, but not the latter, caused a 50% inhibition of DNA fragmentation and the complete inhibition of PARP cleavage in HeLa nuclei. Similarly, Ac-DEVD-CHO (100 μM) inhibited apoptosis and DNA ladder by 50% and PARP cleavage completely in UV-irradiated U937 cells, but Ac-YVAD-CHO (100 μM) did not. Thus, UV-induced apoptosis of U937 cells involves the Ca²⁺/Mg²⁺-dependent endonuclease pathway and the caspase-3–PARP cleavage–Ca²⁺/Mg²⁺-dependent endonuclease pathway. The former pathway produced directly 50% of apoptotic DNA ladder, and the latter involved activated caspase-3 and PARP cleavage, followed by formation of the remaining 50% DNA ladder by the activated endonuclease. In UV-irradiated B-cell lines, further, p53-dependent increase of Bax resulted in a greater caspase-3 activation compared to its absence. However, UV-induced activation of JNK1 and p38 was not affected by the caspase-1 and -3 inhibitors in U937 cells, so that caspases-1 and -3 do not function upstream of JNK1 and p38.     

Genistein ameliorates beta-amyloid peptide (25-35)-induced hippocampal neuronal apoptosis

beta-Amyloid protein (Abeta), a major component of senile plaques of Alzheimer's disease (AD) brain, causes elevation of the intracellular free Ca2+ level and the production of robust free radicals, both of which contribute greatly to the AD-associated cascade including severe neuronal loss in the hippocampus. Genistein, the most active molecule of soy isoflavones, protects diverse kinds of cells from damage caused by a variety of toxic stimuli. In the present study, we investigated the neuroprotective effect of genistein against Abeta25-35-induced apoptosis in cultured hippocampal neurons, as well as the underlying mechanism. Abeta25-35-induced apoptosis, characterized by decreased cell viability, neuronal DNA condensation, and fragmentation, is associated with an increase in intracellular free Ca2+ level, the accumulation of reactive oxygen species (ROS), and the activation of caspase-3. All these phenotypes induced by Abeta25-35 are reversed by genistein. Our results further show that at the nanomolar (100 nM) level, genistein protects neurons from Abeta25-35-induced damage largely via the estrogen receptor-mediated pathway, and at the micromolar (40 microM) level, the neuroprotective effect of genistein is mediated mainly by its antioxidative properties. Our data suggest that genistein attenuates neuronal apoptosis induced by Abeta25-35 via various mechanisms.     

Ca<Superscript>2+</Superscript>, Mg<Superscript>2+</Superscript>-dependent DNase Involvement in Apoptotic Effects in Spermatozoa of Sea Urchin <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis> Induced by Two-Headed Sphingolipid Rhizochalin

Previously, we have purified three distinct DNases from spermatozoa of sea urchin Strongylocentrotus intermedius and we suppose the role of Ca²⁺, Mg²⁺-dependent DNase (Ca, Mg-DNase) in apoptosis of spermatozoa. Two-headed sphingolipid rhizochalin (Rhz) induced characteristic apoptotic nuclear chromatin changes, internucleosomal DNA cleavage, and activation of caspase-9, caspase-8, and caspase-3 in spermatozoa as was shown by fluorescence Hoechst 33342/PI/FDA analysis, DNA fragmentation assay, and fluorescence caspase inhibitors FAM-LEHD-fmk, FAM-IETD-fmk, and FAM-DEVD-fmk, respectively. Inhibitor of caspase-3 z-DEVD-fmk subdued Rhz-induced internucleosomal ladder formation, which confirmed the major role of caspase-3 in apoptotic DNA cleavage probably through Ca, Mg-DNase activation. Participation of sea urchin Ca, Mg-DNase in apoptosis of spermatozoa was demonstrated by ions Zn²⁺ blocking of Rhz-induced DNA fragmentation due to direct inhibition of the Ca, Mg-DNase and internucleosomal cleavage of HeLa S and Vero E6 cell nuclei chromatin by highly purified Ca, Mg-DNase.     

Effective chemopreventive activity of genistein against human breast cancer cells

Chemopreventive and cytotoxic effect of genistein against human breast cancer cell lines was investigated. Genistein inhibited cell proliferation in estrogen receptor-positive (MCF-7) and estrogen receptor-negative (MDA-MB-231) human breast carcinoma cell lines. Cytochrome P450 (CYP) 1A1-mediated ethoxyresorufin O-deethylase (EROD)activity was inhibited by genistein in a concentration-dependent manner. Genistein significantly inhibited 12-Otetradecanoylphorbol-13-acetate (TPA)-induced cyclooxygenase-2 activity and protein expression at the concentrations of 10 (p < 0.05), 25 (p < 0.05) and 50 mM (p < 0.01). In addition, ornithine decarboxylase (ODC) activity was reduced to 53.8 % of the control after 6 h treatment with 50 mM genistein in MCF-7 breast cancer cells. These results suggest that genistein could be of therapeutic value in preventing human breast cancer.     

Chicken skeletal muscle has three Ca2+-dependent proteinases

Chicken breast muscle has three Ca2+-dependent proteinases, two requiring millimolar Ca2+ (m-calpain and high m-calpain) and one requiring micromolar Ca2+ (mu-calpain). High m-calpain co-purifies with mu-calpain through successive DEAE-cellulose (steep gradient), phenyl-Sepharose, octylamine agarose, and Sephacryl S-300 columns, but elutes after mu-calpain when using a shallow KCl gradient to elute a DEAE-cellulose column. The mu- and m-calpains have 80 and 28 kDa polypeptides and are analogous to the mu- and m-calpains that have been purified from bovine, porcine and rabbit skeletal muscle. High m-calpain, which seems to be a new Ca2+-dependent proteinase, is still heterogeneous after the DEAE-cellulose column eluted with a shallow KCl gradient. Additional purification through two successive HPLC-DEAE columns and one HPLC-SW-4000 gel permeation column produces a fraction having six major polypeptides and 6-8 minor polypeptides on SDS-PAGE. A 74-76 kDa polypeptide in this fraction reacts in Western blots with monospecific, polyclonal anti-calpain antibodies that react with both the 80 kDa and the 28 kDa polypeptides of mu- or m-calpain. High m-calpain also is related to mu- and m-calpain in that it causes the same limited digestion of skeletal muscle myofibrils, has a similar pH optimum near pH 7.9-8.4, requires Ca2+ for activity, and reacts with the calpain inhibitor, calpastatin, and a variety of serine and cysteine proteinase inhibitors in a manner identical to mu- and m-calpain. High m-calpain differs from mu- and m-calpain in its elution off DEAE-cellulose columns and its requirement of 3800 microM Ca2+ for one-half maximal activity compared with 5.35 microM Ca2+ for mu-calpain and 420 microM Ca2+ for m-calpain. The physiological significance of high m-calpain in unclear. The presence of mu-calpain in chicken breast muscle suggests that all skeletal muscles contain both mu- and m-calpain, although the relative proportions of these two proteinases may vary in different species.     

Genistein induces apoptosis and autophagy in human breast MCF-7 cells by modulating the expression of proapoptotic factors and oxidative stress enzymes

Breast cancer is one of the common tumors occurring in woman and despite treatment, the prognostic is poor. Genistein, a soy isoflavone, has been reported to have chemopreventive\chemotherapeutic potential in multiple tumor types. Here, we investigated the genistein antiproliferative effect in MCF-7 breast cancer, underlying the molecular mechanisms involved in this effect. MCF-7 cancer and CCD1059sK fibroblast cells were treated with estradiol (10 nM) or genistein (0.01–100 μM) for 24, 48, and 72 h and the cell proliferation was investigated by MTT; membrane cell permeability was evaluated by LDH and PI incorporation; apoptosis was investigated by externalization of phosphatidylserine by FACS; and presence of autophagy was detected by LC3A/B immunostaining. The expression of apoptotic proteins and antioxidant enzymes was evaluated by qPCR. The results demonstrate that genistein (100 μM) for 72 h of treatment selectively reduced MCF-7 cell proliferation independent of estrogen receptor activation, while no cytotoxicity was observed in fibroblast cells. Further experiments showed that genistein induced phosphatidylserine externalization and LC3A/B immunopositivity in MCF-7 cells, indicating apoptosis and autophagy cell death. Genistein increased in three times proapoptotic BAX/Bcl-2 ratio and promoted a parallel downregulation of 20 times of antiapoptotic survivin. In addition, genistein promoted a decrease of 5.5, 9.3, and 3.6 times of MnSOD, CuZnSOD, and TrxR mRNA expression, respectively, while the GPx expression was increased by 6.5 times. These results suggest that the antitumor effect of genistein involved the modulation of antioxidant enzyme and apoptotic signaling expression, which resulted in apoptosis and progression of autophagy.     

Role of calcium and calcium-activated proteases in CYP2E1-dependent toxicity in HEPG2 cells.

The objective of this work was to investigate whether CYP2E1- and oxidative stress-dependent toxicity in HepG2 cells is mediated by an increase of cytosolic Ca2+ and activation of Ca2+-modulated processes. HepG2 cells expressing CYP2E1 (E47 cells) or control cells not expressing CYP2E1 (C34 cells) were preloaded with arachidonic acid (AA, up to 10 microm) and, after washing, incubated with iron-nitrilotriacetic acid (up to 100 microm) for variable periods (up to 12 h). Toxicity was greater in E47 cells than in C34 cells at all times and combinations of iron/AA tested. Cytosolic calcium increased with incubation time in both cell lines, but the increase was higher in E47 cells than in C34 cells. The rise in calcium was an early event and preceded the developing toxicity. Toxicity in E47 cells and the increase in Ca2+ were inhibited by omission of Ca2+ from the extracellular medium, and toxicity was restored by reincorporation of Ca2+. An inhibitor of Ca2+ release from intracellular stores did not prevent the toxicity or the increase in Ca2+, reflecting a role for the influx of extracellular Ca2+ in the toxicity. Reactive oxygen production was similar in media with or without calcium, indicating that calcium was not modulating CYP2E1-dependent oxidative stress. Toxicity, lipid peroxidation, and the increase of Ca2+ in E47 cells exposed to iron-AA were inhibited by alpha-tocopherol. E47 cells (but not C34 cells) exposed to iron-AA showed increased calpain activity in situ (40-fold). The toxicity in E47 cells mirrored calpain activation and was inhibited by calpeptin, suggesting that calpain activation plays a causal role in toxicity. These results suggest that CYP2E1-dependent toxicity in this model depends on the activation of lipid peroxidation, followed by an increased influx of extracellular Ca2+ and activation of Ca2+-dependent proteases.     

Genistein Enhances the Cisplatin‐Induced Inhibition of Cell Growth and Apoptosis in Human Malignant Melanoma Cells

Genistein, a naturally occurring isoflavone found chiefly in soybeans, has been reported to be a potent antitumor agent. Genistein is presumed to exert multiple effects related to the inhibition of cancer growth. Metastatic melanoma is a chemotherapy‐refractory neoplasm. The present study was designed to explore the possible activity of genistein to inhibit the aberrant proliferation and to induce apoptosis of human malignant melanoma cells in cooperation with cisplatin treatment. Five human melanoma cell lines were utilized for these experiments. Genistein at physiologic concentrations (20 μM) did not induce apoptosis by itself but did enhance cisplatin‐induced apoptosis in all five human melanoma cell lines tested. The enhanced susceptibility among the cell lines was diverse. Changes in the expression of two anti‐apoptotic proteins, bcl‐2 and bcl‐xL, and one pro‐apoptotic protein, apoptotic protease activating factor‐1 (Apaf‐1), were examined. Genistein alone or cisplatin alone generally did not alter bcl‐2 expression or bcl‐xL expression, but slightly increased Apaf‐1 in some cell lines. The combined treatment with genistein and cisplatin significantly reduced bcl‐2 and bcl‐xL protein and increased Apaf‐1 protein expression. These data suggest that genistein therapy may enhance the chemosensitivity of melanoma patients.     

Protein tyrosine phosphorylation and calcium signaling in thyroid FRTL-5 cells

We examined the importance of tyrosine kinase(s) on the ATP-evoked Ca²⁺ entry and DNA synthesis of thyroid FRTL-5 cells. ATP rapidly and transiently tyrosine phosphorylated a 72-kDa protein(s). This phosphorylation was abolished by pertussis toxin and by the tyrosine kinase inhibitor genistein, and was dependent on Ca²⁺ entry. Pretreatment of the cells with genistein did not affect the release of sequestered Ca²⁺, but the capacitative Ca²⁺ or Ba²⁺ entry evoked by ATP or thapsigargin was attenuated. Pretreatment of the cells with orthovanadate enhanced the increase in intracellular free Ca²⁺ ([Ca²⁺]_i), whereas the Ba²⁺ entry was not increased. Phorbol 12-myristate 13-acetate (PMA) phosphorylated the same protein(s) as did ATP. Genistein inhibited the ATP-evoked phosphorylation of MAP kinase and attenuated both the ATP- and the PMA-evoked DNA synthesis. However, genistein did not inhibit the ATP-evoked expression of c-fos. Furthermore, genistein enhanced the ATP-evoked release of arachidonic acid. Thus, ATP activates a tyrosine kinase via a Ca²⁺-dependent mechanism. A genistein-sensitive mechanism participates, in part, in the ATP-evoked activation of DNA synthesis. Genistein inhibits only modestly capacitative Ca²⁺ entry in FRTL-5 cells. J. Cell. Physiol. 175:211–219, 1998. © 1998 Wiley-Liss, Inc.