Evaluation of the Moonlighting Histone H3 Specific Protease (H3ase) Activity and the Dehydrogenase Activity of Glutamate Dehydrogenase (GDH).

The N-terminus of Histone H3 is proteolytically processed in aged chicken liver. A histone H3 N-terminus specific endopeptidase (named H3ase) has been purified from the nuclear extract of aged chicken liver. By sequencing and a series of biochemical methods including the demonstration of H3ase activity in bacterially expressed GDH, it was established that the H3ase activity was a moonlighting protease activity of glutamate dehydrogenase (GDH). However, the active site for the H3ase in the GDH remains elusive. Here, using cross-linking studies of the homogenously purified H3ase, we show that the GDH and the H3ase remain in the same native state. Further, the H3ase and GDH activities could be uncoupled by partial denaturation of GDH, suggesting strong evidence for the involvement of different active sites for GDH and H3ase activities. Through densitometry of the H3ase clipped H3 products, the H3ase activity was quantified and it was compared with the GDH activity of the chicken liver nuclear GDH. Furthermore, the H3ase mostly remained distributed in the perinuclear area as demonstrated by MNase digestion and immuno-localization of H3ase in chicken liver nuclei, as well as cultured mouse hepatocyte cells, suggesting that H3ase demonstrated regulated access to the chromatin. The present study thus broadly compares the H3ase and GDH activities of the chicken liver GDH.

SWI/SNF Chromatin Remodelers: Structural, Functional and Mechanistic Implications.

The nuclear events of a eukaryotic cell, such as replication, transcription, recombination and repair etc. require the transition of the compactly arranged chromatin into an uncompacted state and vice-versa. This is mediated by post-translational modification of the histones, exchange of histone variants and ATP-dependent chromatin remodeling. The SWI/SNF chromatin remodeling complexes are one of the most well characterized families of chromatin remodelers. In addition to their role in modulating chromatin, they have also been assigned roles in cancer and health-related anomalies such as developmental, neurocognitive, and intellectual disabilities. Owing to their vital cellular and medical connotations, developing an understanding of the structural and functional aspects of the complex becomes imperative. However, due to the intricate nature of higher-order chromatin as well as compositional heterogeneity of the SWI/SNF complex, intra-species isoforms and inter-species homologs, this often becomes challenging. To this end, the present review attempts to present an amalgamated perspective on the discovery, structure, function, and regulation of the SWI/SNF complex.

Extra-nuclear histones: origin, significance and perspectives.

Histones are classically known to organize the eukaryotic DNA into chromatin. They are one of the key players in regulating transcriptionally permissive and non-permissive states of the chromatin. Nevertheless, their context-dependent appearance within the cytoplasm and systemic circulation has also been observed. The past decade has also witnessed few scientific communications on the existence of vesicle-associated histones. Diverse groups have attempted to determine the significance of these extra-nuclear histones so far, with many of those studies still underway. Of note amongst these are interactions of extra-nuclear or free histones with cellular membranes, mediated by mutual cationic and anionic natures, respectively. It is here aimed to consolidate the mechanism of formation of extra-nuclear histones; implications of histone-induced membrane destabilization and explore the mechanisms of their association/release with extracellular vesicles, along with the functional aspects of these extra-nuclear histones in cell and systemic physiology.

Differential Expression of SWI/SNF Chromatin Remodeler Subunits Brahma and Brahma-Related Gene During Drug-Induced Liver Injury and Regeneration in Mouse Model.

The chromatin remodeling activity of mammalian SWI/SNF complex is carried out by either Brahma (BRM) or Brahma-related gene (BRG-1). The BRG-1 regulates genes involved in cell proliferation, whereas BRM is associated with cell differentiation, and arrest of cell growth. Global modifications of histones and expression of genes of chromatin-remodeling subunits have not been studied in in vivo model systems. In the present study, we investigate epigenetic modifications of histones and the expression of genes in thioacetamide (TAA)-induced liver injury and regeneration in a mouse model. In the present study, we report that hepatocyte proliferation and H3S10 phosphorylation occur during 60 to 72 h post TAA treatment in mice. Furthermore, there was change in the H3K9 acetylation and H3K9 trimethylation pattern with respect to liver injury and regeneration phase. Looking into the expression pattern of Brg-1 and Brm, it is evident that they contribute substantially to the process of liver regeneration. The SWI/SNF remodeler might contain BRG-1 as its ATPase subunit during injury phase. Whereas, BRM-associated SWI/SNF remodeler might probably be predominant during decline of injury phase and initiation of regeneration phase. Furthermore, during the regeneration phase, BRG-1-containing remodeler again predominates. Considering all these observations, the present study depicts an interplay between chromatin interacting machineries in different phases of thioacetamide-induced liver injury and regeneration.

Chicken liver glutamate dehydrogenase (GDH) demonstrates a histone H3 specific protease (H3ase) activity in vitro.

Site-specific proteolysis of the N or C-terminus of histone tails has emerged as a novel form of irreversible post-translational modifications assigned to histones. Though there are many reports describing histone specific proteolysis, there are very few studies on purification of a histone specific protease. Here, we demonstrate a histone H3 specific protease (H3ase) activity in chicken liver nuclear extract. H3ase was purified to homogeneity and identified as glutamate dehydrogenase (GDH) by sequencing. A series of biochemical experiments further confirmed that the H3ase activity was due to GDH. The H3ase clipped histone H3 products were sequenced by N-terminal sequencing and the precise clipping sites of H3ase were mapped. H3ase activity was only specific to chicken liver as it was not demonstrated in other tissues like heart, muscle and brain of chicken. We assign a novel serine like protease activity to GDH which is specific to histone H3.

Purification and characterization of a novel histone H2A specific protease (H2Asp) from chicken liver nuclear extract.

The proteolysis of the N- or the C-terminal tails of histones have recently emerged as a novel form of irreversible posttranslational modifications of histones. However, there are very few reports describing purification of a histone specific protease. Here, we report a histone H2A specific protease (H2Asp) activity in the chicken liver nuclear extract. The H2Asp was purified to homogeneity and was found to be a ~10.5kDa protein. It demonstrated high specificity to histone H2A and was an aspartic acid like protease as shown by protease inhibition assay. The H2Asp, in the in vitro cleavage assay generated a single clipped H2A product which comigrated along with histone H4 in the SDS-PAGE and migrated as a single band when single H2A was used as substrates. The expression of H2Asp was independent of age and was tissue specific, which was demonstrated only in the nuclear extracts of chicken liver and not from the same of other tissues like brain, muscles and erythrocytes. It was also seen that H2Asp activity also exists in other classes of vertebrates from Pisces to Mammals. This report forms the first such report describing purification of a histone H2A specific protease.

Triptolide, histone acetyltransferase inhibitor, suppresses growth and chemosensitizes leukemic cells through inhibition of gene expression regulated by TNF-TNFR1-TRADD-TRAF2-NIK-TAK1-IKK pathway.

Triptolide, a diterpene triepoxide, from the Chinese herb Tripterygium wilfordii Hook.f, exerts its anti-inflammatory and immunosuppressive activities by inhibiting the transcription factor nuclear factor-κB (NF-κB) pathway, through a mechanism not yet fully understood. We found that triptolide, in nanomolar concentrations, suppressed both constitutive and inducible NF-κB activation, but did not directly inhibit binding of p65 to the DNA. The diterpene did block TNF-induced ubiquitination, phosphorylation, and degradation of IκBα, the inhibitor of NF-κB and inhibited acetylation of p65 through suppression of binding of p65 to CBP/p300. Triptolide also inhibited the IκBα kinase (IKK) that activates NF-κB and phosphorylation of p65 at serine 276, 536. Furthermore, the NF-κB reporter activity induced by TNF-TNFR1-TRADD-TRAF2-NIK-TAK1-IKKß was abolished by the triepoxide. Triptolide also abrogated TNF-induced expression of cell survival proteins (XIAP, Bcl-x(L), Bcl-2, survivin, cIAP-1 and cIAP-2), cell proliferative proteins (cyclin D1, c-myc and cyclooxygenase-2), and metastasis proteins (ICAM-1 and MMP-9). This led to enhancement of apoptosis induced by TNF, taxol, and thalidomide by the diterpene and to suppression of tumor invasion. Overall, our results demonstrate that triptolide can block the inflammatory pathway activated by TNF-TNFR1-TRADD-TRAF2-NIK-TAK1-IKK, sensitizes cells to apoptosis, and inhibits invasion of tumor cells.

Identification of novel anti-inflammatory agents from Ayurvedic medicine for prevention of chronic diseases: "reverse pharmacology" and "bedside to bench" approach.

Inflammation, although first characterized by Cornelius Celsus, a physician in first Century Rome, it was Rudolf Virchow, a German physician in nineteenth century who suggested a link between inflammation and cancer, cardiovascular diseases, diabetes, pulmonary diseases, neurological diseases and other chronic diseases. Extensive research within last three decades has confirmed these observations and identified the molecular basis for most chronic diseases and for the associated inflammation. The transcription factor, Nuclear Factor-kappaB (NF-kappaB) that controls over 500 different gene products, has emerged as major mediator of inflammation. Thus agents that can inhibit NF-kappaB and diminish chronic inflammation have potential to prevent or delay the onset of the chronic diseases and further even treat them. In an attempt to identify novel anti-inflammatory agents which are safe and effective, in contrast to high throughput screen, we have turned to "reverse pharmacology" or "bed to benchside" approach. We found that Ayurveda, a science of long life, almost 6,000 years old, can serve as a "goldmine" for novel anti-inflammatory agents used for centuries to treat chronic diseases. The current review is an attempt to provide description of various Ayurvedic plants currently used for treatment, their active chemical components, and the inflammatory pathways that they inhibit.

Thiocolchicoside exhibits anticancer effects through downregulation of NF-κB pathway and its regulated gene products linked to inflammation and cancer.

The discovery of new uses for older, clinically approved drugs is one way to expedite drug development for cancer. Thiocolchicoside, a semisynthetic colchicoside from the plant Gloriosa superba, is a muscle relaxant and used to treat rheumatologic and orthopedic disorders because of its analgesic and anti-inflammatory mechanisms. Given that activation of the transcription factor NF-κB plays a major role in inflammation and tumorigenesis, we postulated that thiocolchicoside would inhibit NF-κB and exhibit anticancer effects through the modulation of NF-κB-regulated proteins. We show that thiocolchicoside inhibited proliferation of leukemia, myeloma, squamous cell carcinoma, breast, colon, and kidney cancer cells. Formation of tumor colonies was also suppressed by thiocolchicoside. The colchicoside induced apoptosis, as indicated by caspase-3 and poly(ADP-ribose) polymerase cleavage, and suppressed the expression of cell survival [e.g., Bcl-2, X-linked inhibitor of apoptosis (XIAP), MCL-1, bcl-xL, cIAP-1, cIAP-2, and cFLIP] proteins. Cell proliferation biomarkers such as c-MYC and phosphorylation of phosphoinositide 3-kinase and glycogen synthase kinase 3ß were also blocked by thiocolchicoside. Because most cell survival and proliferation gene products are regulated by NF-κB, we studied the effect of thiocolchicoside on this transcription factor and found that thiocolchicoside inhibited NF-κB activation, degradation of inhibitory κBα (IκBα), IκBα ubiquitination, and phosphorylation, abolished the activation of IκBα kinase, and suppressed p65 nuclear translocation. This effect of thiocolchicoside on the NF-κB pathway led to inhibition of NF-κB reporter activity and cyclooxygenase-2 promoter activity. Our results indicate that thiocolchicoside exhibits anticancer activity through inhibition of NF-κB and NF-κB-regulated gene products, which provides novel insight into a half-century old drug.

Modification of cysteine 179 of IkappaBalpha kinase by nimbolide leads to down-regulation of NF-kappaB-regulated cell survival and proliferative proteins and sensitization of tumor cells to chemotherapeutic agents.

Reverse pharmacology, also called the "bedside to bench" approach, that deals with new uses for a well known molecular entity has been used extensively in cancer drug development to identify novel compounds and delineate their mechanisms of action. Here, we show that nimbolide, a triterpenoid isolated from Azadirachta indica, enhanced the apoptosis induced by inflammatory cytokines and chemotherapeutic agents in tumor cells. This limonoid abrogated the expression of proteins associated with cell survival (Bcl-2, Bcl-xL, IAP-1, and IAP-2), proliferation (cyclin D1), invasion (MMP-9), and angiogenesis (VEGF), all regulated by nuclear factor (NF)-κB. Nimbolide inhibited the activation of NF-κB induced by carcinogens and inflammatory stimuli. Constitutively active NF-κB found in most tumor cells was also inhibited. We found that suppression of NF-κB activation by nimbolide was caused by inhibition of IκB kinase (IKK), which led to suppression of IκBα phosphorylation and degradation, nuclear translocation, DNA binding, and gene transcription. Reducing agent reversed the action of the limonoid, suggesting the involvement of a cysteine residue. Replacement of Cys(179) of IKK-ß with alanine abolished the effect of nimbolide, suggesting that Cys(179) plays a critical role in inhibiting the NF-κB activation. Overall, our results indicate that nimbolide can sensitize tumor cells to chemotherapeutic agents through interaction with IKK, leading to inhibition of NF-κB-regulated proteins.

Oxidative stress, inflammation, and cancer: how are they linked?

Extensive research during the past 2 decades has revealed the mechanism by which continued oxidative stress can lead to chronic inflammation, which in turn could mediate most chronic diseases including cancer, diabetes, and cardiovascular, neurological, and pulmonary diseases. Oxidative stress can activate a variety of transcription factors including NF-κB, AP-1, p53, HIF-1α, PPAR-γ, ß-catenin/Wnt, and Nrf2. Activation of these transcription factors can lead to the expression of over 500 different genes, including those for growth factors, inflammatory cytokines, chemokines, cell cycle regulatory molecules, and anti-inflammatory molecules. How oxidative stress activates inflammatory pathways leading to transformation of a normal cell to tumor cell, tumor cell survival, proliferation, chemoresistance, radioresistance, invasion, angiogenesis, and stem cell survival is the focus of this review. Overall, observations to date suggest that oxidative stress, chronic inflammation, and cancer are closely linked.

Celastrol suppresses invasion of colon and pancreatic cancer cells through the downregulation of expression of CXCR4 chemokine receptor.

Although metastasis accounts for >90% of cancer-related deaths, no therapeutic that targets this process has yet been approved. Because the chemokine receptor CXCR4 is one of the targets closely linked with tumor metastasis, inhibitors of this receptor have the potential to abrogate metastasis. In the current report, we demonstrate that celastrol can downregulate the CXCR4 expression on breast cancer MCF-7 cells stably transfected with HER2, an oncogene known to induce the chemokine receptor. Downregulation of CXCR4 by the triterpenoid was not cell type-specific as downregulation occurred in colon cancer, squamous cell carcinoma, and pancreatic cancer cells. Decrease in CXCR4 expression was not due to proteolysis as neither proteasome inhibitors nor lysosomal stabilization had any effect. Quantitative reverse transcription polymerase chain reaction analysis revealed that downregulation of CXCR4 messenger RNA (mRNA) by celastrol occurred at the translational level. Chromatin immunoprecipitation analysis revealed regulation at the transcriptional level as well. Abrogation of the chemokine receptor by celastrol or by gene-silencing was accompanied by suppression of invasiveness of colon cancer cells induced by CXCL12, the ligand for CXCR4. This effect was not cell type-specific as celastrol also abolished invasiveness of pancreatic tumor cells, and this effect again correlated with the disappearance of both the CXCR4 mRNA and CXCR4 protein. Other triterpenes, such as withaferin A and gedunin, which are known to inhibit Hsp90, did not downregulate CXCR4 expression, indicating that the effects were specific to celastrol. Overall, these results show that celastrol has potential in suppressing invasion and metastasis of cancer cells by down-modulation of CXCR4 expression.

Gamma-tocotrienol promotes TRAIL-induced apoptosis through reactive oxygen species/extracellular signal-regulated kinase/p53-mediated upregulation of death receptors.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor superfamily, is in clinical trials for cancer therapy, but its anticancer potential is limited by the development of resistance. We investigated the ability of tocotrienol (T3), an unsaturated vitamin E present in palm oil, rice bran, barley, oats, and wheat germ, to sensitize tumor cells to TRAIL. Results from esterase staining, colony formation, caspase activation, and sub-G(1) cell cycle arrest revealed that gamma-T3 can sensitize human colon cancer cells to TRAIL. When examined for the mechanism, we found that gamma-T3 significantly downregulated the expression of antiapoptotic proteins (c-IAP2 and Bcl-xL). We also found that gamma-T3, but not tocopherol, induced the expression of the TRAIL receptors death receptor (DR)-4 and DR5. This induction was not cell type specific, as upregulation was also found in pancreatic, kidney, and leukemic cells. Upregulation of DRs by gamma-T3 required the production of reactive oxygen species (ROS), and sequestering of ROS abolished both upregulation of the receptors and potentiation of TRAIL-induced apoptosis. Induction of DRs by gamma-T3 also required activation of extracellular signal-regulated kinase 1 (ERK1), as silencing of ERK1 by specific siRNA abrogated the upregulation of TRAIL receptors. Further, induction of DRs by gamma-T3 required the expression of p53 and Bax, as no induction of the receptors was found in colon cancer cells with deletion of these genes. Overall, our results show that gamma-T3 sensitizes tumor cells to TRAIL by upregulating DRs through the ROS/ERK/p53 pathway and by downregulating cell survival proteins.

Delivery of antiinflammatory nutraceuticals by nanoparticles for the prevention and treatment of cancer.

Extensive research within the last two decades has revealed that most chronic illnesses, including cancer, diabetes, and cardiovascular and pulmonary diseases, are mediated through chronic inflammation. Thus, suppressing chronic inflammation has the potential to delay, prevent, and even treat various chronic diseases, including cancer. Various nutraceuticals from fruits, vegetables, vitamins, spices, legumes, and traditional Chinese and Ayurvedic medicine have been shown to safely suppress proinflammatory pathways; however, their low bioavailability in vivo limits their use in preventing and treating cancer. We describe here the potential of nanotechnology to fill this gap. Several nutraceuticals, including curcumin, green tea polyphenols, coenzyme Q, quercetin, thymoquinone and others, have been packaged as nanoparticles and proven to be useful in "nanochemoprevention" and "nano-chemotherapy".

Cyclodextrin-complexed curcumin exhibits anti-inflammatory and antiproliferative activities superior to those of curcumin through higher cellular uptake.

Curcumin, a yellow pigment present in the spice turmeric (Curcuma longa), has been linked with multiple beneficial activities, but its optimum potential is limited by poor bioavailability, in part due to the lack of solubility in aqueous solvents. To overcome the solubility problem, we have recently developed a novel cyclodextrin complex of curcumin (CDC) and examined here this compound for anti-inflammatory and antiproliferative effects. Using the electrophoretic mobility shift assay, we found that CDC was more active than free curcumin in inhibiting TNF-induced activation of the inflammatory transcription factor NF-kappaB and in suppressing gene products regulated by NF-kappaB, including those involved in cell proliferation (cyclin D1), invasion (MMP-9), and angiogenesis (VEGF). CDC was also more active than free curcumin in inducing the death receptors DR4 and DR5. Annexin V staining, cleavage of caspase-3 and PARP, and DNA fragmentation showed that CDC was more potent than free curcumin in inducing apoptosis of leukemic cells. Antiproliferative assays also demonstrated that CDC was more active than free curcumin in suppressing proliferation of various cancer cell lines. The cyclodextrin vehicle had no effect in these assays. Compared with free curcumin, CDC had a greater cellular uptake and longer half-life in the cells. Overall we demonstrated that CDC had superior attributes compared with free curcumin for cellular uptake and for antiproliferative and anti-inflammatory activities.

Targeting inflammatory pathways by flavonoids for prevention and treatment of cancer.

Observational studies have suggested that lifestyle risk factors such as tobacco, alcohol, high-fat diet, radiation, and infections can cause cancer and that a diet consisting of fruits and vegetables can prevent cancer. Evidence from our laboratory and others suggests that agents either causing or preventing cancer are linked through the regulation of inflammatory pathways. Genes regulated by the transcription factor NF- kappaB have been shown to mediate inflammation, cellular transformation, tumor cell survival, proliferation, invasion, angiogenesis, and metastasis. Whereas various lifestyle risk factors have been found to activate NF- kappaB and NF- kappaB-regulated gene products, flavonoids derived from fruits and vegetables have been found to suppress this pathway. The present review describes various flavones, flavanones, flavonols, isoflavones, anthocyanins, and chalcones derived from fruits, vegetables, legumes, spices, and nuts that can suppress the proinflammatory cell signaling pathways and thus can prevent and even treat the cancer.

Crotepoxide chemosensitizes tumor cells through inhibition of expression of proliferation, invasion, and angiogenic proteins linked to proinflammatory pathway.

Crotepoxide (a substituted cyclohexane diepoxide), isolated from Kaempferia pulchra (peacock ginger), although linked to antitumor and anti-inflammatory activities, the mechanism by which it exhibits these activities, is not yet understood. Because nuclear factor kappaB (NF-kappaB) plays a critical role in these signaling pathways, we investigated the effects of crotepoxide on NF-kappaB-mediated cellular responses in human cancer cells. We found that crotepoxide potentiated tumor necrosis factor (TNF), and chemotherapeutic agents induced apoptosis and inhibited the expression of NF-kappaB-regulated gene products involved in anti-apoptosis (Bcl-2, Bcl-xL, IAP1,(2) MCl-1, survivin, and TRAF1), apoptosis (Bax, Bid), inflammation (COX-2), proliferation (cyclin D1 and c-myc), invasion (ICAM-1 and MMP-9), and angiogenesis (VEGF). We also found that crotepoxide inhibited both inducible and constitutive NF-kappaB activation. Crotepoxide inhibition of NF-kappaB was not inducer-specific; it inhibited NF-kappaB activation induced by TNF, phorbol 12-myristate 13-acetate, lipopolysaccharide, and cigarette smoke. Crotepoxide suppression of NF-kappaB was not cell type-specific because NF-kappaB activation was inhibited in myeloid, leukemia, and epithelial cells. Furthermore, we found that crotepoxide inhibited TAK1 activation, which led to suppression of IkappaBalpha kinase, abrogation of IkappaBalpha phosphorylation and degradation, nuclear translocation of p65, and suppression of NF-kappaB-dependent reporter gene expression. Overall, our results indicate that crotepoxide sensitizes tumor cells to cytokines and chemotherapeutic agents through inhibition of NF-kappaB and NF-kappaB-regulated gene products, and this may provide the molecular basis for crotepoxide ability to suppress inflammation and carcinogenesis.

Lipopolysaccharide alters the vaginal electrical resistance in cycling and pregnant mice.

PROBLEM: Lipopolysaccharide (LPS) has been postulated to exert harmful biologic effects during pregnancy. The objective of present investigation is to measure the vaginal electrical resistance (VER) in LPS-treated normal cycling and pregnant female mice. METHOD OF STUDY: Minimum dose (MD) of LPS (250 microg/kg body weight) was injected in pregnant female mice through i.p. route on day 0.5 of pregnancy. VER was measured during different phases of reproductive cycle in female mice, which were pre-exposed to LPS and in untreated cycling female mice. VER was also measured in control pregnant female mice (saline-treated mice) through whole pregnancy and LPS-treated female mice in early stages of pregnancy. RESULTS: Vaginal electrical resistance was significantly higher during proestrous or early estrous stage as compared with any other stages of reproductive cycle in mouse. One peak of VER was observed during peri-implantation period of pregnancy in control female mice. The significant differences in the pattern of VER were found between LPS-treated and control female mice during peri-implantation period of pregnancy, and between cycling female mice, which were pre-exposed to LPS and untreated cycling female mice during proestrus. CONCLUSION: The presented results demonstrate, for the first time, that LPS exposure during pregnancy may be determined by measuring VER in mothers without any adverse effect on ongoing pregnancy and may help in refining the assisted reproduction techniques.

Simvastatin, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, suppresses osteoclastogenesis induced by receptor activator of nuclear factor-kappaB ligand through modulation of NF-kappaB pathway.

Simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, is a cholesterol-lowering drug that may play a role in bone metabolism through a mechanism that is not fully understood. Recently, receptor activator of NF-kappaB ligand (RANKL), a member of the TNF superfamily, has emerged as a major mediator of bone loss via activation of osteoclastogenesis. The latter is also associated with certain cancers such as multiple myeloma and breast cancer. Whether simvastatin can modulate RANKL-induced or cancer induced osteoclastogenesis was investigated. The effect of simvastatin on RANKL signaling and consequent osteoclastogenesis was investigated. RANKL induced NF-kappaB activation, whereas pretreatment with simvastatin completely suppressed such activation and correlated with suppression of RANKL-induced activation of IkappaBalpha kinase, IkappaBalpha phosphorylation and IkappaBalpha degradation. Similarly, RANKL induced the differentiation of monocytic cells to osteoclasts, whereas simvastatin suppressed it. The inhibition was maximal when cells were exposed to both simvastatin and RANKL simultaneously and minimal when simvastatin was added 1 day after RANKL treatment. Simvastatin also inhibited the osteoclastogenesis induced by human breast cancer and by multiple myeloma cells. Together, our results indicate that simvastatin inhibits the RANKL-induced NF-kappaB activation pathway that leads to suppression of osteoclastogenesis induced by RANKL and by tumor cells, thereby suggesting its therapeutic potential in osteoporosis and in cancer-related bone loss.