Selenium Lessens Osteoarthritis by Protecting Articular Chondrocytes from Oxidative Damage through Nrf2 and NF-κB Pathways.

Osteoarthritis (OA) causes joint pain and disability due to the abnormal production of inflammatory cytokines and reactive oxygen species (ROS) in chondrocytes, leading to cell death and cartilage matrix destruction. Selenium (Se) intake can protect cells against oxidative damage. It is still unknown whether Se supplementation is beneficial for OA. This study investigated the effects of Se on sodium iodoacetate (MIA)-imitated OA progress in human chondrocyte cell line (SW1353 cells) and rats. The results showed that 0.3 µM of Se treatment could protect SW1353 cells from MIA-induced damage by the Nrf2 pathway by promoting the gene expression of glutathione-synthesis-related enzymes such as the glutamate-cysteine ligase catalytic subunit, the glutamate-cysteine ligase modifier subunit, and glutathione synthetase. In addition, glutathione, superoxide dismutase, glutathione peroxidase, and glutathione reductase expressions are also elevated to eliminate excessive ROS production. Moreover, Se could downregulate NF-κB, leading to a decrease in cytokines, matrix proteases, and glycosaminoglycans. In the rats, MIA-induced cartilage loss was lessened after 2 weeks of Se supplementation by oral gavage; meanwhile, glutathione synthesis was increased, and the expressions of pro-inflammatory cytokines were decreased. These results suggest that Se intake is beneficial for OA due to its effects of decreasing cartilage loss by enhancing antioxidant capacity and reducing inflammation.

Development of validated sandwich ELISA for detecting peanut allergen Ara h 3 in food.

Peanut is an important food that can cause food allergies, often leading to moderate and severe allergic symptoms such as skin rashes, asthma, and even anaphylactic shock.Research indicates that Ara h 3 is one of the major peanut allergen. In order to establish a simple analytical method for detecting Ara h 3, we developed a sandwich enzyme-linked immunosorbent assay (ELISA) with antibodies that were induced from purified Ara h 3. The experimental results showed that the purified Ara h 3 had good purity, and we successfully prepared capture and detection antibodies. The method established in this study exhibited high specificity and did not cross-react with soybeans, cashew nuts, and sesame. For validation, including precision, recovery and sensitivity were in good condition. We also detected the Ara h 3 in peanut related foods. Overall, the ELISA developed in this study is a reliable method for Ara h 3 detection.

S-Equol Protects Chondrocytes against Sodium Nitroprusside-Caused Matrix Loss and Apoptosis through Activating PI3K/Akt Pathway.

Osteoarthritis (OA) is a common chronic disease with increasing prevalence in societies with more aging populations, therefore, it is causing more concern. S-Equol, a kind of isoflavones, was reported to be bioavailable and beneficial to humans in many aspects, such as improving menopausal symptoms, osteoporosis and prevention of cardiovascular disease. This study investigated the effects of S-Equol on OA progress in which rat primary chondrocytes were treated with sodium nitroprusside (SNP) to mimic OA progress with or without the co-addition of S-Equol for the evaluation of S-Equol's efficacy on OA. Results showed treatment of 0.8 mM SNP caused cell death, and increased oxidative stress (NO and H2O2), apoptosis, and proteoglycan loss. Furthermore, the expressions of MMPs of MMP-2, MMP-3, MMP-9, and MMP-13 and p53 were increased. The addition of 30 µM S-Equol could lessen those caused by SNP. Moreover, S-Equol activates the PI3K/Akt pathway, which is an upstream regulation of p53 and NO production and is associated with apoptosis and matrix degradation. As a pretreatment of phosphoinositide 3-kinases (PI3K) inhibitor, all S-Equol protective functions against SNP decrease or disappear. In conclusion, through PI3K/Akt activation, S-Equol can protect chondrocytes against SNP-induced matrix degradation and apoptosis, which are commonly found in OA, suggesting S-Equol is a potential for OA prevention.

Zinc protects chondrocytes from monosodium iodoacetate-induced damage by enhancing ATP and mitophagy.

Osteoarthritis (OA) is characterized with articular cartilage degradation, and monosodium iodoacetate (MIA)-treated chondrocyte is the most commonly used model for mimicking OA progression. Zinc protects chondrocytes from MIA-induced damage. Here, we explored the protective effects of 25 µM zinc on 5 µM MIA-treated SW1353 cells (human chondrosarcoma cell line) through the analysis of energy metabolism- and autophagy-related parameters. We found that the exposure of SW1353 cells to MIA decreased ATP levels, expression of glycolysis-related proteins, including glucose transporter 1, hexokinase 2, and pyruvate dehydrogenase E1 component subunit alpha, and the levels of mitochondrial complex I, II, IV, and V subunits of the oxidative phosphorylation pathway. MIA treatment also decreased the expression of autophagy-related proteins, including autophagic elongation protein 5 (ATG5), ATG7, and microtubule-associated protein 1A/1B light chain 3B (LC3-II) and mitophagy-related proteins, including phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1), ubiquitin, and p62. These results indicate that MIA interferes with energy metabolism and the autophagic clearance of dysfunctional mitochondria (so called mitophagy). Interestingly, zinc exposure could almost completely reverse the effects of MIA, suggesting its potential protective role against OA progression.

Zinc Protects Articular Chondrocytes through Changes in Nrf2-Mediated Antioxidants, Cytokines and Matrix Metalloproteinases.

Osteoarthritis (OA) is an age-related degenerative joint disease characterized by high oxidative stress, chondrocyte death and cartilage damage. Zinc has been implicated in the antioxidant capacity of the cell, and its deficiency might inhibit chondrocyte proliferation. The present study examined the potential of zinc as a preventive supplement against OA using the in vitro chondrosarcoma cell line SW1353 and an in vivo Wistar rat model to mimic OA progress induced by monosodium iodoacetate (MIA). The results demonstrated that, in SW1353 cells, 5 µM MIA exposure increased oxidative stress and decreased the expression of GPx1 and Mn-SOD but still increased GSH levels and HO-1 expression and enhanced the expression of interleukin (IL)-10, IL-1ß, and matrix metalloproteinase (MMP)-13. Zinc addition could block these changes. Besides, the expression of Nrf2 and phosphorylated (p)-Akt was dramatically increased, implicating the p-Akt/Nrf2 pathway in the effects of zinc on MIA-treated cells. A rat model achieved similar results as those of cell culture, and 1.6 mg/kg/day of zinc supplementation is sufficient to prevent OA progress, while 8.0 mg/kg/day of zinc supplementation does not have a better effect. These findings indicate that zinc supplementation exerts a preventive effect with respect to MIA-induced OA progress.

Epirubicin induces apoptosis in osteoblasts through death-receptor and mitochondrial pathways.

Epirubicin is an anthracycline and is widely used in tumor treatment, but has toxic and undesirable side effects on wide range of cells and hematopoietic stem cells (HSC). Osteoblasts play important roles in bone development and in supporting HSC differentiation and maturation. It remains unknown whether epirubicin-induced bone loss and hematological toxicity are associated with its effect on osteoblasts. In primary osteoblast cell cultures, epirubicin inhibited cell growth and decreased mineralization. Moreover, epirubicin arrested osteoblasts in the G2/M phase, and this arrest was followed by apoptosis in which both the extrinsic (death receptor-mediated) and intrinsic (mitochondrial-mediated) apoptotic pathways were evoked. The factors involved in the extrinsic apoptotic pathway were increased FasL and FADD as well as activated caspase-8. Those involved in the intrinsic apoptotic pathway were decreased Bcl-2; increased reactive oxygen species, Bax, cytochrome c; and activated caspase-9 and caspase-3. These results demonstrate that epirubicin induced osteoblast apoptosis through the extrinsic and intrinsic apoptotic pathways, leading to the destruction of osteoblasts and consequent lessening of their functions in maintaining bone density and supporting hematopoietic stem cell differentiation and maturation.

Arecoline Increases Glycolysis and Modulates pH Regulator Expression in HA22T/VGH Hepatoma Cells, Leading to Increase of Intracellular Ca2+, Reactive Oxygen Species, and Anoikis.

Background: Cancer cells proliferate rapidly and are resistant to cell death, relying on aggravated glycolysis to satisfy their increased demand for energy and biosynthetic precursors. However, this process may create unfavorable microenvironments, such as increased acidity, leading to cytotoxicity. Our previous study demonstrated that arecoline induces anoikis of HA22T/VGH hepatoma cells. The present study aimed to examine if arecoline induced anoikis is related to the glycolytic pathway and explore the underlying mechanisms. Methods: HA22T/VGH cells were treated with arecoline and changes in the glycolytic end products lactate and ATP, glycolytic-related gene expression, intracellular and extracellular pH, pH-regulating gene expression, reactive oxygen species (ROS) levels, intracellular Ca2+ concentration ([Ca2+]i) and mitochondrial membrane potential were examined, relative to untreated cells. Cell viability and morphology were also assessed. Results: Arecoline increased lactate and ATP production through induction of glycolytic genes, including glucose transporter 3 (Glut3), hexokinase 1 (HK1), hexokinase 2 (HK2), and pyruvate kinase (PK). The intracellular pH was not changed, despite increased lactate levels, implying that intracellular H+ was exported out of the cells. mRNA expression of pH regulators including monocarboxylate transporter 1 and 4 (MCT 1 and 4), sodium bicarbonate cotransporter 1 (NBC1), carbonic anhydrases (CA) IX and XII and vacuolar ATPase (V-ATPase) were down-regulated. Na+/H+ exchanger 1 (NHE1) mRNA levels remained unchanged while Na+/Ca2+ exchanger (NCX) was up-regulated and eventually [Ca2+]i was increased. ROS generation was increased and mitochondrial membrane potential was decreased followed by cell detachment and death. Addition of 2-deoxy-d-glucose, a glucose competitor that interferes with glycolysis, attenuated arecoline induction of lactate [Ca2+]i, ROS and cell detachment. Similarly, ROS scavengers could block the effects of arecoline. Conclusions: This study demonstrated that arecoline induced glycolysis and modulated the mRNA expression of pH-regulator genes in HA22T/VGH cells. This phenomenon led to the elevation of [Ca2+]i, ROS generation, and subsequent cell detachment.

Vitamin C Protects Chondrocytes against Monosodium Iodoacetate-Induced Osteoarthritis by Multiple Pathways.

Osteoarthritis (OA) is the most prevalent joint disease. Dietary intake of vitamin C relates to a reduction in cartilage loss and OA. This study examined the efficacy of vitamin C to prevent OA with the in vitro chondrosarcoma cell line (SW1353) and the in vivo monosodium iodoacetate (MIA)-induced OA rat. Results demonstrated that, in SW1353 cells, treatment with 5 µM MIA inhibited cell growth and increased oxidative stress, apoptosis, and proteoglycan loss. In addition, the expression levels of the pro-inflammatory cytokines IL-6, IL-17A, and TNF-α and matrix metalloproteinases (MMPs) MMP-1, MMP-3, and MMP-13 were increased. All of these MIA-induced changes could be prevented with treatment of 100 µM vitamin C. In an animal model, intra-articular injection of MIA-induced cartilage degradation resembled the pathological changes of OA, and treatment of vitamin C could lessen these changes. Unexpectedly, vitamin C's effects did not strengthen with the increasing dosage, while the 100 mg/kg dosage was more efficient than the 200 or 300 mg/kg dosages. Vitamin C possessed multiple capacities for prevention of OA progress, including a decrease in apoptosis and in the expression of pro-inflammatory cytokines and MMPs in addition to the well-known antioxidation.

Osteoblasts activate the Nrf2 signalling pathway in response to arsenic trioxide treatment.

Arsenic trioxide is used to treat a variety of leukaemia types and causes tumour cell death. However, it is not well known whether arsenic trioxide is toxic to bone osteoblast cells, the precursor cells from which leukaemia cells originate. The aim of this study was to examine the response of osteosarcoma cell line MG63 and primary cultured osteoblasts to arsenic trioxide treatment. After 24h of treatment, arsenic trioxide was more effective at inhibiting cell growth and increasing oxidative stress and DNA damage in MG63 cells than in osteoblasts. In addition, arsenic trioxide arrested cell cycle progression in the G2/M phase, and induced apoptosis in MG63 cells, but not in primary cultured osteoblasts. The results further showed that the expression of transcription factor Nrf2 and its downstream antioxidant effectors, including hemeoxygenase-1, glutathione, and superoxide dismutase, was increased in primary cultured osteoblasts. Additionally, expression of heat shock proteins was also increased. Experiments using inhibitors of antioxidant enzymes in the presence of arsenic trioxide-treated osteoblasts demonstrated that glutathione and superoxide dismutase were responsible for reducing oxidative stress, caspase-3 activity, and apoptosis and that heat shock proteins helped reduce caspase-3 activity. Unexpectedly, there was no apparent effect of the markedly increased hemeoxygenase-1, suggesting that other functions might exist for hemeoxygenase-1. These findings demonstrate that osteosarcoma cells are more sensitive to arsenic trioxide treatment than primary cultured osteoblasts and that primary cultured osteoblasts activate the Nrf2 signalling pathway in response to arsenic trioxide exposure to escape from oxidative damage and apoptosis.

Progesterone increases apoptosis and inversely decreases autophagy in human hepatoma HA22T/VGH cells treated with epirubicin.

Hepatocellular carcinoma (HCC) is the leading cause of cancer-related deaths worldwide. Epirubicin can induce intracellular reactive oxygen species and is widely used to treat unresectable HCC. Progesterone has been found to inhibit the proliferation of hepatoma cells. This study was designed to test the combined effects of epirubicin and progesterone on human hepatoma cell line, HA22T/VGH. These cells were treated with different concentrations of epirubicin with or without the coaddition of 30 µM progesterone and then analyzed for apoptosis, autophagy, and expressions of apoptotic-related proteins and multidrug-resistant gene. Epirubicin treatment dose-dependently inhibited the growth of HA22T/VGH cells. Addition of 30 µM progesterone, which was inactive alone, augmented the effect of epirubicin on the inhibition of growth of HA22T/VGH cells. Cotreatment with progesterone enhanced epirubicin-induced apoptosis, as evidenced by greater increase in caspase-3 activity and in the ratio of the apoptosis-regulating protein, Bax/Bcl-X(L). The combination also caused a decrease in autophagy and in the expression of multidrug resistance-related protein 1 mRNA compared to epirubicin alone. This study shows the epirubicin/progesterone combination was more effective in increasing apoptosis and inversely decreasing autophagy on HA22T/VGH cells treated with epirubicin alone, suggesting that this combination can potentially be used to treat HCC.

Progesterone augments epirubicin-induced apoptosis in HA22T/VGH cells by increasing oxidative stress and upregulating Fas/FasL.

BACKGROUND: Although epirubicin, an anthracycline drug, is widely used to treat hepatocellular carcinoma, its therapeutic efficacy is disappointing. Thus, the efficacy of epirubicin may be improved when combined with other drugs. This study investigated the therapeutic potential of combination of progesterone and epirubicin in the treatment of the human hepatoma cell line HA22T/VGH and the possible mechanisms through which this combination might induce apoptosis. MATERIALS AND METHODS: HA22T/VGH cells were treated without or with 25 µM progesterone and/or 0.5 µM epirubicin and analyzed for oxidative stress, redox status, Fas/FasL expression, caspase activity, and apoptosis. RESULTS: HA22T/VGH cells treated with epirubicin increased the production of reactive oxygen species and nitric oxide, the expression of Fas, FasL, and Fas-associated death domain, and the activities of caspase-8 and caspase-3. Epirubicin treatment also decreased glutathione resulting in the induction of apoptosis. Treatment with progesterone alone increased nitric oxide production, but it did not affect the other parameters. However, when HA22T/VGH cells were treated with progesterone and epirubicin, the effects of epirubicin were enhanced. CONCLUSIONS: Our observations suggest that progesterone enhances the efficacy of epirubicin. The increased efficacy is potentially attributed to progesterone's enhancement of epirubicin-induced oxidative stress, thereby reducing redox status. In addition, progesterone sequentially upregulates Fas/FasL to induce the caspase-8 and caspase-3 pathways, thereby resulting in increased apoptosis. The combination had a greater effect on the induction of HA22T/VGH cell apoptosis and could potentially serve as a more effective treatment for hepatocellular carcinoma than epirubicin alone.

Osteoblasts survive the arsenic trioxide treatment by activation of ATM-mediated pathway.

Arsenic trioxide (ATO) is widely used in tumor treatment, but excessive arsenic exposure can have adverse effects. We recently found that, in primary osteoblasts, ATO produces oxidative stress and causes DNA tailing, but does not induce apoptosis. We further examined the signaling pathway by which osteoblasts survive ATO treatment, and found that they were arrested at G2/M phase of the cell cycle at 30h and overrode the G2/M boundary at 48h. After treatment for 30h, there was increased Cdc2 phosphorylation and expression of Wee1, a Cdc2 kinase, and expression of the cell cycle inhibitor, p21(waf1/cip1), which interacts with Cdc2. Furthermore, levels of the phosphatase Cdc25C, which activates Cdc2, were decreased, while the ratio of its phosphorylated/inactivated form to the total amount was increased. Moreover, phosphorylation/activation of the checkpoint kinases Chk1, Chk2 and p53 levels were increased, as were levels of activated ATM and γ-H2AX. The cell viability was decreased as an ATM inhibitor was added. Additionally, these effects of ATO on γ-H2AX, Chk1, Chk2, p53, and p21(waf1/cip1) were reduced by an ATM inhibitor. These findings suggest that G2/M phase arrest of osteoblasts is mediated by Chk1/Chk2 activation via an ATM-dependent pathway by which osteoblasts survive.

Combined effect of soy isoflavones and vitamin D3 on bone loss in ovariectomized rats.

OBJECTIVE: Several studies have shown that soy isoflavones have estrogen-like activities and might constitute an alternative to hormone replacement treatment. The present study investigated the effects of soy isoflavones alone and combined with vitamin D3 on prevention of bone loss. METHODS: Sprague-Dawley rats were sham-operated (n = 8) or ovariectomized (OVX; n = 40), and then the OVX rats were randomly assigned to five groups that were untreated or treated for 14 wk with vitamin D3, 17ß-estradiol, soy isoflavone extract (SIE), or vitamin D3 plus SIE. The effects of the isoflavones and 1α,25(OH)(2)D(3) on cultured osteoblasts and osteoclasts also were investigated. RESULTS: In OVX rats, the bone mineral density and trabecular bone volume loss were improved by 17ß-estradiol, SIE, or SIE plus vitamin D3 treatment. SIE treatment was more effective than vitamin D3 or 17ß-estradiol in inhibiting increases in serum tumor necrosis factor-α levels and osteoblast osteoprotegerin expression. SIE plus vitamin D3 was more effective in increasing osterix expression than each alone. Bone cell cultures showed that the isoflavones induced preosteoblasts to differentiate into osteoblasts and increased osteoblast mineralization. Isoflavones inhibited preosteoclasts and osteoclast proliferation and decreased osteoclast resorption. The combination of isoflavones plus 1α,25(OH)(2)D(3) showed additive effects on the increase in cell proliferation of cultured preosteoblasts. CONCLUSION: Treatment with soy isoflavones might be an alternative to hormone replacement therapy in decreasing bone loss from postmenopausal estrogen deficiency. In addition, there are further effects on increasing transcription factor osterix expression and preosteoblast proliferation when these were combined with vitamin D3.

Arsenic trioxide affects bone remodeling by effects on osteoblast differentiation and function.

Arsenic trioxide (ATO) is widely used in tumor treatment, but excessive arsenic exposure can have adverse health effects. This study was to examine the association between ATO treatment and bone remodeling. The effects of ATO on osteoblast function were investigated in primary cell cultures and in an in vivo study in rats. Sprague-Dawley rats (n=30) were randomly assigned to 3 groups which were injected intraperitoneally with saline or 5 or 10 mg/kg of ATO for 4 weeks. In cell culture, ATO decreased osteoblast mineralization by decreasing alkaline phosphatase (ALP) expression and this effect was prevented by co-addition of inorganic phosphate (Pi). Moreover, levels of mRNAs for the transcription factors runt-related transcription factor 2 (Runx2) and osterix, the osteoblast osteogenic gene osteocalcin, and the adherence molecule vascular cell adhesion molecule-1 (VCAM-1) were decreased by ATO. Levels of mRNAs for the cytokine IL-6 were also decreased, whereas GM-CSF mRNA levels were increased. Similar effects of ATO on osteoblasts were seen in in vivo experiments in the rat. Moreover, decreases of bone turnover markers of osteocalcin, Procollagen type I N-terminal propeptide (PINP), and C-terminal cross-linked telopeptide (CTX) as well as bone mineral density (BMD) and trabecular bone volume of femur were observed in ATO-treated rats. These results suggest that ATO interferes with bone remodeling mostly through changes in osteoblast differentiation and function.

Arecoline decreases interleukin-6 production and induces apoptosis and cell cycle arrest in human basal cell carcinoma cells.

Arecoline, the most abundant areca alkaloid, has been reported to decrease interleukin-6 (IL-6) levels in epithelial cancer cells. Since IL-6 overexpression contributes to the tumorigenic potency of basal cell carcinoma (BCC), this study was designed to investigate whether arecoline altered IL-6 expression and its downstream regulation of apoptosis and the cell cycle in cultured BCC-1/KMC cells. BCC-1/KMC cells and a human keratinocyte cell line, HaCaT, were treated with arecoline at concentrations ranging from 10 to 100µg/ml, then IL-6 production and expression of apoptosis- and cell cycle progress-related factors were examined. After 24h exposure, arecoline inhibited BCC-1/KMC cell growth and decreased IL-6 production in terms of mRNA expression and protein secretion, but had no effect on HaCaT cells. Analysis of DNA fragmentation and chromatin condensation showed that arecoline induced apoptosis of BCC-1/KMC cells in a dose-dependent manner, activated caspase-3, and decreased expression of the anti-apoptotic protein Bcl-2. In addition, arecoline induced progressive and sustained accumulation of BCC-1/KMC cells in G2/M phase as a result of reducing checkpoint Cdc2 activity by decreasing Cdc25C phosphatase levels and increasing p53 levels. Furthermore, subcutaneous injection of arecoline led to decreased BCC-1/KMC tumor growth in BALB/c mice by inducing apoptosis. This study demonstrates that arecoline has potential for preventing BCC tumorigenesis by reducing levels of the tumor cell survival factor IL-6, increasing levels of the tumor suppressor factor p53, and eliciting cell cycle arrest, followed by apoptosis.

Hemeoxygenase-1 expression in response to arecoline-induced oxidative stress in human umbilical vein endothelial cells.

BACKGROUND: Arecoline, the most abundant areca alkaloid, has been reported to stimulate reactive oxygen species (ROS) production in several cell types. Overproduction of ROS has been implicated in atherogenesis. Hemeoxygenase-1 (HO-1) has cytoprotective activities in vascular tissues. This study investigated the effect of arecoline on adhesion molecule expression and explored the role of HO-1 in this process. METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with arecoline, then ROS levels and the expression of adhesion molecules and HO-1 were analyzed and potential signaling pathways investigated. RESULTS: After 2h of arecoline treatment, ROS production was stimulated and reached a maximum at 12h. Expression of the adhesion molecules ICAM and VCAM was also induced. Glutathione pretreatment completely blocked arecoline-stimulated ROS production and VCAM expression, but not ICAM expression. Arecoline also induced HO-1 expression and this effect was partly due by ROS stimulation. Inhibition of c-jun N-terminal kinase (JNK) by SP600125, p38 by SB 203580, or tyrosine kinase by genistein reduced arecoline-induced HO-1 expression. In contrast, inhibition of ERK (extracellular signal-related MAP kinase) by PD98059 had no effect. Transfection of HUVECs with the GFP/HO-1 gene, which resulted in a 5-fold increase in HO-1 activity, markedly, but not completely, inhibited the decrease in cell viability caused by arecoline. CONCLUSIONS: This study demonstrates that, in HUVECs, arecoline stimulates ROS production and ICAM and VCAM expression. HO-1 expression is also upregulated through the ROS, tyrosine kinase, and MAPK (JNK and p38) signaling pathways.

Combined arginine and ascorbic acid treatment induces apoptosis in the hepatoma cell line HA22T/VGH and changes in redox status involving the pentose phosphate pathway and reactive oxygen and nitrogen species.

Arginine is a physiological substrate for nitric oxide synthase to generate nitric oxide (NO), which can influence tumor cell survival, while ascorbic acid is selectively toxic for cancer cells. This study explored the effect of an arginine/ascorbic acid combination on human cancer cell lines. The hepatoma cell line HA22T/VGH was the most sensitive of the tested cells to combination treatment. A combination of 5.74 mM of arginine and 0.57 mM of ascorbic acid induced HA22T/VGH cell death through apoptosis and an increase in levels of reactive oxygen species and NO, as well as its stable products NO(2)(-) and NO(3)(-). The combination also reduced the activity of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and transaldolase in the pentose phosphate pathway, a major mechanism for producing NADPH, resulting in a marked decrease in intracellular NADPH levels. A dramatic decrease in intracellular glutathione (GSH) levels, a decrease in the mitochondrial membrane potential, ATP depletion and release of cytochrome c were also seen. Caspase-9 and caspase-3 were activated, apoptotic protein Bax expression increased and the expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL decreased. These results suggest that this combination induced HA22T/VGH cell death by interfering with redox state regulation by a reduction in pentose phosphate pathway activity and increasing oxidative and nitrosative stress.

Arecoline induces HA22T/VGH hepatoma cells to undergo anoikis - involvement of STAT3 and RhoA activation.

BACKGROUND: Our previous study showed that, in basal cell carcinoma cells, arecoline reduces levels of the tumor cell survival factor interleukin-6 (IL-6), increases levels of tumor suppressor factor p53, and elicits cell cycle arrest, followed by apoptosis. In preliminarily studies, we observed that arecoline induces detachment of the human-derived hepatoma cell line HA22T/VGH from the extracellular matrix. In the present study, we explored the fate of the detached HA22T/VGH cells and investigated the underlying mechanism. METHODS: HA22T/VGH cells or primary cultured rat hepatocytes were treated with arecoline, then changes in morphology, viability, apoptosis, and the expression of surface beta1-integrin, apoptosis-related proteins, and IL-6 were examined. Furthermore, activation of the signal transducer and activator of transcription 3 (STAT3) pathway and the RhoA/Rock signaling pathway, including p190RhoGAP and Src homology-2 domain-containing phosphatase SHP2, was examined. RESULTS: A low concentration of arecoline (

Combined effects of terazosin and genistein on a metastatic, hormone-independent human prostate cancer cell line.

Metastatic prostate cancer progresses from androgen-dependent to androgen-independent. Terazosin, a long-acting selective alpha1-adrenoreceptor antagonist, induces apoptosis of prostate cancer cells in an alpha1-adrenoreceptor-independent manner, while genistein, a major soy isoflavone, inhibits the growth of several types of cancer cells. The present study was designed to test the therapeutic potential of a combination of terazosin and genistein using a metastatic, hormone-independent prostatic cancer cell line, DU-145. Terazosin or genistein treatment inhibited the growth of DU-145 cells in a dose-dependent manner, whereas had no effect on normal prostate epithelial cells. Addition of 1 microg/ml of terazosin, which was inactive alone, augmented the growth inhibitory effect of 5 microg/ml of genistein. Co-treatment with terazosin resulted in the genistein-induced arrest of DU-145 cells in G2/M phase being overridden and an increase in apoptotic cells, as evidenced by procaspase-3 activation and PARP cleavage. The combination also caused a greater decrease in the levels of the apoptosis-regulating protein, Bcl-XL, and of VEGF165 and VEGF121 than genistein alone. In conclusion, the terazosin/genistein combination was more effective in inhibiting cell growth and VEGF expression as well as inducing apoptosis of the metastatic, androgen-independent prostate cancer cell line, DU-145, than either alone. The doses used in this study are in lower and nontoxic anticancer dosage range, suggesting this combination has potential for therapeutic use.

Zinc at pharmacologic concentrations affects cytokine expression and induces apoptosis of human peripheral blood mononuclear cells.

OBJECTIVE: The present study examined the effect of zinc at concentrations of the apoptotic signaling pathway and immune function of peripheral blood mononuclear cells (PBMCs). METHODS: PBMCs from healthy subjects were treated in vitro with various zinc concentrations to imitate different serum statuses of physiologic (2 to 15 microM) and pharmacologic (15 to 100 microM) concentrations to higher than 100 microM and analyzed their expressions of cytokines and apoptotically related factors. RESULTS: Although a normal physiologic concentration of zinc had no effect on immunologic function or apoptosis of PBMCs, a pharmacologic concentration (100 microM) or higher affected both functions. Zinc decreased cell proliferation at concentrations higher than 100 microM and stimulated cytokine expression at concentrations of at least 100 microM. Further, at concentrations of at least 100 microM, apoptosis was induced, and expressions of caspase-3 and proapoptotic genes, including Fas (FasL) and c-fos, which trigger apoptosis through receptor-mediated extrinsic and mitochondrion-mediated apoptotic pathways, respectively, were increased. At concentrations at least 300 microM, expressions of antiapoptotic factors nuclear factor-kappaB, Bcl-2, and Bcl-X(L) were markedly decreased. CONCLUSIONS: Zinc stimulates cytokine expression and induces apoptosis of PBMCs from healthy subjects only at concentrations equal to or greater than the serum pharmacologic range. Receptor-mediated extrinsic and mitochondrial-mediated intrinsic pathways are involved in this zinc-induced apoptosis.