Hesperetin suppresses RANKL-induced osteoclastogenesis and ameliorates lipopolysaccharide-induced bone loss

Destructive bone diseases caused by osteolysis are increasing in incidence. They are characterized by an excessive imbalance of osteoclast formation and activation. During osteolysis, the activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways are triggered by receptor activator of NF-κB ligand (RANKL), inflammatory factors, and oxidative stress. Previous studies have indicated that the common flavanone glycoside compound hesperetin exhibits anti-inflammatory and antioxidant activity by inhibition of NF-κB and MAPK signaling pathways. However, the direct relationship between hesperetin and osteolysis remain unclear. In the present study, we investigated the effects of hesperetin on lipopolysaccharide (LPS)-induced osteoporosis and elucidated the related mechanisms. Hesperetin effectively suppressed RANKL-induced osteoclastogenesis, osteoclastic bone resorption, and F-actin ring formation in a dose-dependent manner. It also significantly suppressed the expression of osteoclast-specific markers including tartrate-resistant acid phosphatase, matrix metalloproteinase-9, cathepsin K, c-Fos, and nuclear factor of activated T-cells cytoplasmic 1. Furthermore, it inhibited osteoclastogenesis by inhibiting activation of NF-κB and MAPK signaling, scavenging reactive oxygen species, and activating the nuclear factor E2 p45-related factor 2/heme oxygenase 1 (Nrf2/HO-1) signaling pathway. Consistent with in vitro results, hesperetin effectively ameliorated LPS-induced bone loss, reduced osteoclast numbers, and decreased the RANKL/OPG ratio in vivo. As such, our results suggest that hesperetin may be a great candidate for developing a novel drug for destructive bone diseases such as periodontal disease, tumor bone metastasis, rheumatoid arthritis, and osteoporosis.     

Effect of heat shock preconditioning on NF-kappaB/I-kappaB pathway during I/R injury of the rat liver

Hepatic ischemia-reperfusion (I/R) injury continues to be a fatal complication after liver surgery. Heat shock (HS) preconditioning is an effective strategy for protecting the liver from I/R injury, but its exact mechanism is still unclear. Because the activation of nuclear factor-kappaB (NF-kappaB) is an important event in the hepatic I/R-induced inflammatory response, the effect of HS preconditioning on the pathway for NF-kappaB activation was investigated. In the control group, NF-kappaB was activated 60 min after reperfusion, but this activation was suppressed in the HS group. Messenger RNA expressions of proinflammatory mediators during reperfusion were also reduced with HS preconditioning. Concomitant with NF-kappaB activation, NF-kappaB inhibitor I-kappaB proteins were degraded in the control group, but this degradation was suppressed in the HS group. This study shows that HS preconditioning protected the liver from I/R injury by suppressing the activation of NF-kappaB and the subsequent expression of proinflammatory mediators through the stabilization of I-kappaB proteins.     

Identification of an autoregulatory feedback pathway involving interleukin-1alpha in induction of constitutive NF-kappaB activation in pancreatic cancer cells

We previously reported that NF-kappaB is constitutively activated in most human pancreatic cancer tissues and cell lines but not in normal pancreatic tissues and immortalized pancreatic ductal epithelial cells. IkappaBalphaM-mediated inhibition of constitutive NF-kappaB activity in human pancreatic cancer cells suppressed tumorigenesis and liver metastasis in an orthotopic nude mouse model, suggesting that constitutive NF-kappaB activation plays an important role in pancreatic tumor progression and metastasis. However, the underlying mechanism by which NF-kappaB is activated in pancreatic cancer remains to be elucidated. In this study, we found that an autocrine mechanism accounts for the constitutive activation of NF-kappaB in metastatic human pancreatic cancer cell lines. Further investigation showed that interleukin-1alpha was the primary cytokine secreted by these cells that activates NF-kappaB. Neutralization of interleukin-1alpha activity suppressed the constitutive activation of NF-kappaB and the expression of its downstream target gene, urokinase-type plasminogen activator, in metastatic pancreatic cancer cell lines. Our results demonstrate that regulation of interleukin-1alpha expression is primarily dependent on AP-1 activity, which is in part induced by signaling pathways that are epidermal growth factor receptor-dependent and -independent. In conclusion, our findings suggest a possible mechanism for the constitutive activation of NF-kappaB in metastatic human pancreatic cancer cells and a possible missing mechanistic link between inflammation and cancer.     

Indirubin enhances tumor necrosis factor-induced apoptosis through modulation of nuclear factor-kappa B signaling pathway

Although indirubin is known to exhibit anti-cancer and anti-inflammatory activities, very little is known about its mechanism of action. In this study, we investigated whether indirubin mediates its effects through interference with the NF-kappaB pathway. As examined by the DNA binding of NF-kappaB, we found that indirubin suppressed tumor necrosis factor (TNF)-induced NF-kappaB activation in a dose- and time-dependent manner. Indirubin also suppressed the NF-kappaB activation induced by various inflammatory agents and carcinogens. Further studies showed that indirubin blocked the phosphorylation and degradation of IkappaB alpha through the inhibition of activation of IkappaB alpha kinase and phosphorylation and nuclear translocation of p65. NF-kappaB reporter activity induced by TNFR1, TNF receptor-associated death domain, TRAF2, TAK1, NF-kappaB-inducing kinase, and IKKbeta was inhibited by indirubin but not that induced by p65 transfection. We also found that indirubin inhibited the expression of NF-kappaB-regulated gene products involved in antiapoptosis (IAP1, IAP2, Bcl-2, Bcl-xL, and TRAF1), proliferation (cyclin D1 and c-Myc), and invasion (COX-2 and MMP-9). This correlated with enhancement of the apoptosis induced by TNF and the chemotherapeutic agent taxol in human leukemic KBM-5 cells. Indirubin also suppressed cytokine-induced cellular invasion. Overall, our results indicate that anti-cancer and anti-inflammatory activities previously assigned to indirubin may be mediated in part through the suppression of the NF-kappaB activation pathway.     

Synthesis and biological evaluation of hesperetin derivatives as agents inducing apoptosis

A flavanone, hesperetin, has been known to exert antitumor activity by inducing apoptosis. To find hesperetin derivatives showing better antitumor activity, 12 derivatives were designed and synthesized. Their antitumor activities were measured using a long-term survival clonogenic assay. Among the compounds, K-5b, hesperetin-7-butyrate, showed the half-maximal cell growth inhibitory concentration three times as low as that of hesperetin. To compare the cytotoxicity of hesperetin and K-5b, the HCT116 human colon cancer cell line was treated with various concentrations of each compound. K-5b decreased the cell viability to a larger extent than hesperetin and triggered apoptosis more efficiently than hesperetin in an apoptosis detection assay using fluorescein isothiocyanate-labeled annexin V. Immunoblotting analysis showed that K-5b promoted caspase-mediated apoptosis more efficiently than hesperetin. Because hesperetin has been reported to induce apoptosis through the activation of the c-Jun N-terminal kinase (JNK) pathway, we tested whether K-5b activates JNKs. K-5b stimulated JNK1 and JNK2 more efficiently than hesperetin as shown by western blot analysis. In conclusion, hesperetin derivatives exerting better antitumor activity than hesperetin by inducing apoptosis were found.     

NF-kappaB inhibitor dehydroxymethylepoxyquinomicin suppresses osteoclastogenesis and expression of NFATc1 in mouse arthritis without affecting expression of RANKL, osteoprotegerin or macrophage colony-stimulating factor

Inhibition of NF-kappaB is known to be effective in reducing both inflammation and bone destruction in animal models of arthritis. Our previous study demonstrated that a small cell-permeable NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), suppresses expression of proinflammatory cytokines and ameliorates mouse arthritis. It remained unclear, however, whether DHMEQ directly affects osteoclast precursor cells to suppress their differentiation to mature osteoclasts in vivo. The effect of DHMEQ on human osteoclastogenesis also remained elusive. In the present study, we therefore examined the effect of DHMEQ on osteoclastogenesis using a mouse collagen-induced arthritis model, and using culture systems of fibroblast-like synovial cells obtained from patients with rheumatoid arthritis, and of osteoclast precursor cells from peripheral blood of healthy volunteers. DHMEQ significantly suppressed formation of osteoclasts in arthritic joints, and also suppressed expression of NFATc1 along the inner surfaces of bone lacunae and the eroded bone surface, while serum levels of soluble receptor activator of NF-kappaB ligand (RANKL), osteoprotegerin and macrophage colony-stimulating factor were not affected by the treatment. DHMEQ also did not suppress spontaneous expression of RANKL nor of macrophage colony-stimulating factor in culture of fibroblast-like synovial cells obtained from patients with rheumatoid arthritis. These results suggest that DHMEQ suppresses osteoclastogenesis in vivo, through downregulation of NFATc1 expression, without significantly affecting expression of upstream molecules of the RANKL/receptor activator of NF-kappaB/osteoprotegerin cascade, at least in our experimental condition. Furthermore, in the presence of RANKL and macrophage colony-stimulating factor, differentiation and activation of human osteoclasts were also suppressed by DHMEQ, suggesting the possibility of future application of NF-kappaB inhibitors to rheumatoid arthritis therapy.     

Modulatory efficacy of hesperetin (citrus flavanone) on xenobiotic-metabolizing enzymes during 1,2-dimethylhydrazine-induced colon carcinogenesis

Colorectal cancer is the second leading cause of cancer death worldwide with diet playing a prominent role in disease initiation and progression. Diet and nutrition play an important role during this multistep colon carcinogenic process. We have investigated the modulatory efficacy of hesperetin on aberrant crypt foci (ACF) and xenobiotic-metabolizing enzymes on 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis. Male albino Wistar rats were randomly divided into six groups. Group 1 served as control, received modified pellet diet and group 2 rats received 20 mg/kg body weight of hesperetin p.o. every day. Groups 3-6 rats were given subcutaneous injections of 1,2-dimethylhydrazine (20 mg/kg body weight) once a week for 15 weeks to induce ACF in the colon. In addition, rats in group 4 received hesperetin as in group 2 orally for the first 15 weeks (initiation), group 5 rats received hesperetin as in group 2 after the last injection of DMH and continued till the end of the experimental period (post-initiation). Group 6 received hesperetin as in group 2 throughout the entire period of 32 weeks. DMH exposure showed high incidence (90%) of ACF (280 ± 24.5 aberrant crypt/colon) and dysplastic ACF, elevated activities of phase I enzymes and reduced the activities of phase II enzymes in the liver and colonic mucosa of colon cancer bearing rats. Hesperetin supplementation significantly reversed these effects, the effect being more pronounced in group 6 rats (hesperetin supplemented throughout the study period).These findings suggest that hesperetin can significantly reduce the formation of preneoplastic lesions and effectively modulate the xenobiotic-metabolizing enzymes in rats.     

Effect of hesperetin on tyrosinase: inhibition kinetics integrated computational simulation study

Tyrosinase inhibitors have potential applications in medicine, cosmetics and agriculture to prevent hyperpigmentation or browning effects. Some of the flavonoids mostly found in herbal plants and fruits are revealed as tyrosinase inhibitors. We studied the inhibitory effects of one such flavonoid, hesperetin, on mushroom tyrosinase using inhibition kinetics and computational simulation. Hesperetin reversibly inhibited tyrosinase in a competitive manner with K_i = 4.03 ± 0.26 mM. Measurements of ANS-binding fluorescence showed that hesperetin induced the hydrophobic disruption of tyrosinase. For further insight, we used the docking algorithms to simulate binding between tyrosinase and hesperetin. Simulation was successful (binding energies for Dock6.3: −34.41 kcal/mol and for AutoDock4.2: −5.67 kcal/mol) and showed that a copper ion coordinating with 3 histidine residues (HIS61, HIS85, and HIS259) within the active site pocket was chelated via hesperetin binding. Our study provides insight into the inhibition of tyrosinase in response to flavonoids. A combination of inhibition kinetics and computational prediction may facilitate the identification of potential natural tyrosinase inhibitors such as flavonoids and the prediction of their inhibitory mechanisms.     

Hesperetin-etoposide combinations induce cytotoxicity in U2OS cells: Implications on therapeutic developments for osteosarcoma

Osteosarcoma chemotherapy has improved survival rates, however, chemoresistance and drug toxicity still limit therapy. Drug combinations may overcome these limitations by allowing fewer chemoresistant cells to survive. The aim of this study was to evaluate the cytotoxic potential of hesperetin to osteosarcoma and to analyze the cell cycle effects of combinations of hesperetin with chemotherapeutic agents. For this, the U2OS human osteosarcoma cell line was exposed to hesperetin or hesperetin combined with etoposide or doxorubicin in defined proportions. Hesperetin was less cytotoxic compared to chemotherapeutic agents, as shown by cell growth, viability and clonogenic assays. Notwithstanding, hesperetin combined with etoposide showed additive effects on the inhibition of cell growth. Furthermore, hesperetin induced G2-phase arrest, associated with decreased gene expression of cyclins B1 and E1 and cyclin-dependent kinases 1 and 2. The combination with higher additive effect resulted in higher percentage of cells in G2-phase, showing that G2-phase arrest is associated with cytotoxicity. Moreover, hesperetin induced cytostatic effects. In conclusion, our results suggest that G2-phase arrest is an important step for hesperetin-induced cytotoxicity in U2OS cells. Hesperetin shows potential cytotoxicity when combined with etoposide, which may have implications on therapeutic developments for osteosarcoma.     

Exploring the Effect of Hesperetin–HSPC Complex—A Novel Drug Delivery System on the In Vitro Release, Therapeutic Efficacy and Pharmacokinetics

Hesperetin is known to exhibit a variety of pharmacological activities in mammalian cell systems. Although it shows appreciable bioavailability when administered orally, its faster elimination from body creates the need of frequent administration to maintain effective plasma concentration. To overcome this limitation, a phospholipid complex of hesperetin was prepared and evaluated for antioxidant activity and pharmacokinetic profile. The hesperetin content of the complex was determined by a spectrophotometer and the surface characteristics of the complex were studied by means of microscope. The antioxidant activity was evaluated in carbon-tetrachloride-intoxicated rats at a dose level of 100 mg/kg body weight, p.o. The complex was studied for in vitro drug release characteristics and effect of complexation on serum concentration of hesperetin in rats was also studied along with main pharmacokinetic parameters. The results showed that the complex has a sustained release property and enhanced antioxidant activity (P < 0.05 and <0.01) as compared to free hesperetin at the same dose level. Pharmacokinetic study depicted that the complex has higher relative bioavailability and acted for a longer period of time. The study therefore suggests that phospholipid complex of hesperetin produced better antioxidant activity than free drug at the same dose level and the effect persisted for a longer period of time, which may be helpful in solving the problems of faster elimination of the molecule.     

Oxidative modulation of NF-kappaB signaling by oxidized low-density lipoprotein

Oxidized low-density lipoprotein (oxLDL) modifies macrophage inflammatory responses in the pathogenesis of atherosclerosis. In the present study, we focused on gamma-glutamylcysteine synthetase (gamma-GCS), a rate limiting enzyme of glutathione synthesis, and examined whether inflammatory stimulation of gamma-GCS gene in macrophages by lipopolysaccharide (LPS) is modified when the cells were exposed to oxLDL. We found that the nuclear factor-kappaB (NF-kappaB)-mediated induction of gamma-GCS by LPS (100 ng/ml) was suppressed by a 48-h pre-treatment with oxLDL (50 micro/ml), and this was due to a decrease in the DNA-binding activity of NF-kappaB. Furthermore, pre-treatment with oxLDL caused a carbonylation of NF-kappaB subunit p65. With alpha-tocopherol, the oxLDL-induced carbonylation of proteins decreased with a restoration of DNA-binding activity of NF-kappaB. Together, these indicate that oxidative modification of NF-kappaB suppresses LPS-induced expression of gamma-GCS gene in ox-LDL-treated cells, suggesting an implication of oxLDL-induced modulation of NF-kappaB signaling with atherosclerosis.     

The citrus flavonone hesperetin prevents letrozole-induced bone loss in a mouse model of breast cancer

Aromatase is a key enzyme in estrogen synthesis, and aromatase inhibitors (AIs) have been developed for treating estrogen-responsive breast cancer. Because of its nondiscriminatory inhibition of estrogen synthesis, patients treated with AIs also contract diseases typically associated with estrogen deficiency, such as bone deterioration. Our laboratory found that the citrus flavonone hesperetin could inhibit aromatase, and the selective estrogen receptor modulator nature of flavonoid might counteract the undesirable effect of AIs. In the present study, we employed an established postmenopausal model for breast carcinogenesis to examine the drug interaction between hesperetin and letrozole, one of the AIs. Athymic mice were ovariectomized and transplanted with aromatase-overexpressing MCF-7 cells (MCF-7aro). Hesperetin was administered in the diet at 5000 ppm, and letrozole was injected sc at different doses. Results showed that either hesperetin or letrozole could reduce plasma estrogen level and inhibit tumor growth. Most importantly, the letrozole-induced bone loss measured as bone volume fraction was reversed by hesperetin without compromising on the deterrence of MCF-7aro tumor growth. Taken together, the present study suggested that hesperetin could be a potential cotherapeutic agent to AI.     

Hesperetin impairs glucose uptake and inhibits proliferation of breast cancer cells

The flavanone hesperetin is known to decrease basal glucose uptake, although the inhibitory mechanism is largely unknown. Here, we used MDA‐MB‐231 breast cancer cells to investigate the molecular pathways affected by hesperetin. The results indicate that the suppression of glucose uptake is caused by the down‐regulation of glucose transporter 1 (GLUT1). Hesperetin was also found to inhibit insulin‐induced glucose uptake through impaired cell membrane translocation of glucose transporter 4 (GLUT4). In addition, the phosphorylation of the insulin receptor‐beta subunit (IR‐beta) and Akt was suppressed. Hesperetin also decreased cellular proliferation, which is likely due to the inhibition of glucose uptake. Cancer cells are highly dependent on glucose and hesperetin may, therefore, have potential application as an anticancer agent. Copyright © 2012 John Wiley & Sons, Ltd.     

7,7'-Dihydroxy bursehernin inhibits the expression of inducible nitric oxide synthase through NF-kappaB DNA binding suppression.

This study isolated a lignan, 7,7'-dihydroxy bursehernin, from Geranium thunbergii and investigated whether or not the lignan affects the induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in RAW264.7 macrophages stimulated with lipopolysaccharide (LPS). The gel shift analysis and luciferase reporter gene assays using the iNOS promoter and nuclear factor-kappaB (NF-kappaB) minimal promoter showed that a treatment with 7,7'-dihydroxy bursehernin reduced the reporter activities and binding of NF-kappaB to the NF-kappaB consensus sequence, while it had no effect on the nuclear translocation of p65 and the phosphorylation/degradation of I-kappaBalpha. It was reported that a few natural compounds directly suppressed the binding activity of the NF-kappaB components to DNA. The NF-kappaB binding activity was not reversed by the in vitro exposure of the nuclear extracts to 7,7'-dihydroxy bursehernin, which suggest that a metabolite(s) of 7,7'-dihydroxy bursehernin might target the binding of the NF-kappaB complex to the DNA binding domain region in the promoter region of the iNOS gene. After incubation of RAW264.7 cells with 7,7-dihydroxy bursehernin for 18h, the levels of parent compound were negligible; while a main metabolite, 4-[4-(n-hydroxy-phenyl)-2,3-dimethyl-buta-1,3-dienyl]-benzene-1,2-diol was detected in cell lysates and culture medium.     

Sensitization of TNF-induced cytotoxicity in lung cancer cells by concurrent suppression of the NF-kappaB and Akt pathways

Blockage of either nuclear factor-kappaB (NF-kappaB) or Akt sensitizes cancer cells to TNF-induced apoptosis. In this study, we investigated the undetermined effect of concurrent blockage of these two survival pathways on TNF-induced cytotoxicity in lung cancer cells. The results show that Akt contributes to TNF-induced NF-kappaB activation in lung cancer cells through regulating phosphorylation of the p65/RelA subunit of NF-kappaB. Although individually blocking IKK or Akt partially suppressed TNF-induced NF-kappaB activation, concurrent suppression of these pathways completely inhibited TNF-induced NF-kappaB activation and downstream anti-apoptotic gene expression, and synergistically potentiated TNF-induced cytotoxicity. Moreover, suppression of Akt inhibited the Akt-mediated anti-apoptotic pathway through dephosphorylation of BAD. These results indicate that concurrent suppression of NF-kappaB and Akt synergistically sensitizes TNF-induced cytotoxicity through blockage of distinct survival pathways downstream of NF-kappaB and Akt, which may be applied in lung cancer therapy.     

Antioxidative effects of hesperetin against 7,12-dimethylbenz(a)anthracene-induced oxidative stress in mice

We investigated the effects of the chronic administration of hesperetin on the activation of the antioxidant defence system in mice in which oxidative stress had been induced by 7,12-dimethylbenz(a)anthracene (DMBA). Mice were divided randomly into three treatment groups. Hesperetin was administered orally to two of the three groups at 10 and 50 mg/kg body weight for 5 weeks. Subsequently, each group was subdivided randomly into DMBA-treated and untreated groups. The DMBA-treated groups were intragastrically administered a dose of 34 mg/kg BW in corn oil vehicle twice a week for 2 weeks. The TBARS value showed a tendency to decrease following hesperetin treatment; these decreases were significantly greater in the DMBA-treated than the untreated groups. Hesperetin significantly decreased the carbonyl content at the high dose in both DMBA-treated and untreated mice. Catalase and SOD activity were increased by hesperetin; this increase was more pronounced in DMBA-treated than untreated mice. Catalase, Mn-SOD, and CuZn-SOD expression analyses supported these results. Although the GSH-px and GR activity were little affected, hesperetin treatment significantly increased the GSH/GSSG ratio in the DMBA-treated group in a dose-dependent manner. These results suggest that hesperetin shows antioxidant activity and plays a protective role against DMBA-induced oxidative stress.     

Pyrrolidine dithiocarbamate inhibits TNF-alpha-dependent activation of NF-kappaB by increasing intracellular copper level in human aortic smooth muscle cells

Pyrrolidine dithiocarbamate (PDTC) is a metal-chelating compound that acts as antioxidant or pro-oxidant and is widely used to study redox regulation of cell function. In the present study, we investigated effects of PDTC and another antioxidant, N-acetyl-l-cysteine (NAC), on TNF-alpha-dependent activation of NF-kappaB in human aortic smooth muscle cells (HASMC). Treatment of the cells with TNF-alpha induced the activation of p65/p50 heterodimer NF-kappaB and increased the mRNA levels of monocyte chemoattractant protein (MCP)-1. Pretreatment with PDTC markedly suppressed the NF-kappaB activation and expression of MCP-1 by inhibiting IkappaB-alpha degradation. In contrast, NAC had no effect. PDTC concomitantly increased the intracellular levels of copper, and bathocuproinedisulfonic acid, a non-cell-permeable chelator of Cu(1+), inhibited the PDTC-induced increase in intracellular copper level and reversed the PDTC effects on IkappaB-alpha, NF-kappaB, and MCP-1. These results indicate that TNF-alpha-dependent expression of MCP-1 in HASMC is tightly regulated by NF-kappaB and that intracellular copper level is crucial for the TNF-alpha-dependent activation of NF-kappaB in HASMC.     

Hesperetin: a potent antioxidant against peroxynitrite

Peroxynitrite (ONOO_-) is a reactive oxidant formed from superoxide (•O₂^-) and nitric oxide (•NO), that can oxidize several cellular components, including essential protein, non-protein thiols, DNA, low-density lipoproteins (LDL), and membrane phospholipids. ONOO_- has contributed to the pathogenesis of diseases such as stroke, heart disease, Alzheimer's disease, and atherosclerosis. Because of the lack of endogenous enzymes to thwart ONOO_- activation, developing a specific ONOO_- scavenger is remarkably important. In this study, the ability of hesperetin (3',5,7-trihydroxy-4-methoxyflavanone) to scavenge ONOO_- and to protect cells against ONOO_- and ROS was investigated. The data gained show that hesperetin can efficiently scavenge authentic ONOO_-. In spectrophotometric analysis, the data revealed that hesperetin led to declined ONOO_--mediated nitration of tyrosine through electron donation. Hesperetin exhibited significant inhibition on the nitration of bovine serum albumin (BSA) by ONOO_- in a dose-dependent manner. Hesperetin also manifested cytoprotection from cell damage induced by ONOO_- and ROS. The present study suggests that hesperetin is a powerful ONOO_- scavenger and promotes cellular defense activity in the protection against ONOO_- involved diseases.