Subhashis Pal

Thioaryl naphthylmethanone oxime ether analogs as novel anticancer agents

Employing a rational design of thioaryl naphthylmethanone oxime ether analogs containing functional properties of various anticancer drugs, a series of compounds were identified that displayed potent cytotoxicity toward various cancer cells, out of which 4-(methylthio)phenyl)(naphthalen-1-yl)methanone O-2-(diethylamino)ethyl oxime (MND) exhibited the best safety profile. MND induced apoptosis, inhibited migration and invasion, strongly inhibited cancer stem cell population on a par with salinomycin, and demonstrated orally potent tumor regression in mouse MCF-7 xenografts. Mechanistic studies revealed that MND strongly abrogated EGF-induced proliferation, migration, and tyrosine kinase (TK) signaling in breast cancer cells. However, MND failed to directly inhibit EGFR or other related receptor TKs in a cell-free system. Systematic investigation of a putative target upstream of EGFR revealed that the biological effects of MND could be abrogated by pertussis toxin. Together, MND represents a new nonquinazoline potential drug candidate having promising antiproliferative activity with good safety index.

Prunetin signals via G-protein-coupled receptor, GPR30(GPER1): Stimulation of adenylyl cyclase and cAMP-mediated activation of MAPK signaling induces Runx2 expression in osteoblasts to promote bone regeneration ☆ ☆☆

Prunetin is found in red clover and fruit of Prunus avium (red cherry). The effect of prunetin on osteoblast function, its mode of action and bone regeneration in vivo were investigated. Cultures of primary osteoblasts, osteoblastic cell line and HEK293T cells were used for various in vitro studies. Adult female rats received drill-hole injury at the femur diaphysis to assess the bone regenerative effect of prunetin. Prunetin at 10nM significantly (a) increased proliferation and differentiation of primary cultures of osteoblasts harvested from rats and (b) promoted formation of mineralized nodules by bone marrow stromal/osteoprogenitor cells. At this concentration, prunetin did not activate any of the two nuclear estrogen receptors (α and β). However, prunetin triggered signaling via a G-protein-coupled receptor, GPR30/GPER1, and enhanced cAMP levels in osteoblasts. G15, a selective GPR30 antagonist, abolished prunetin-induced increases in osteoblast proliferation, differentiation and intracellular cAMP. In osteoblasts, prunetin up-regulated runt-related transcription factor 2 (Runx2) protein through cAMP-dependent Erk/MAP kinase activation that ultimately resulted in the up-regulation of GPR30. Administration of prunetin at 0.25mg/kg given to rats stimulated bone regeneration at the site of drill hole and up-regulated Runx2 expression in the fractured callus and the effect was comparable to human parathyroid hormone, the only clinically used osteogenic therapy. We conclude that prunetin promotes osteoinduction in vivo and the mechanism is defined by signaling through GPR30 resulting in the up-regulation of the key osteogenic gene Runx2 that in turn up-regulates GPR30.

Theophylline, a methylxanthine drug induces osteopenia and alters calciotropic hormones, and prophylactic vitamin D treatment protects against these changes in rats

The drug, theophylline is frequently used as an additive to medications for people suffering from chronic obstructive pulmonary diseases (COPD). We studied the effect of theophylline in bone cells, skeleton and parameters related to systemic calcium homeostasis. Theophylline induced osteoblast apoptosis by increasing reactive oxygen species production that was caused by increased cAMP production. Bone marrow levels of theophylline were higher than its serum levels, indicating skeletal accumulation of this drug. When adult Sprague-Dawley rats were treated with theophylline, bone regeneration at fracture site was diminished compared with control. Theophylline treatment resulted in a time-dependent (at 4- and 8 weeks) bone loss. At 8 weeks, a significant loss of bone mass and deterioration of microarchitecture occurred and the severity was comparable to methylprednisone. Theophylline caused formation of hypomineralized osteoid and increased osteoclast number and surface. Serum bone resorption and formation marker were respectively higher and lower in the theophylline group compared with control. Bone strength was reduced by theophylline treatment. After 8 weeks, serum 25-D3 and liver 25-hydroxylases were decreased in theophylline group than control. Further, theophylline treatment reduced serum 1, 25-(OH)2 vitamin D3 (1,25-D3), and increased parathyroid hormone and fibroblast growth factor-23. Theophylline treated rats had normal serum calcium and phosphate but displayed calciuria and phosphaturia. Co-administration of 25-D3 with theophylline completely abrogated theophylline-induced osteopenia and alterations in calcium homeostasis. In addition, 1,25-D3 protected osteoblasts from theophylline-induced apoptosis and the attendant oxidative stress. We conclude that theophylline has detrimental effects in bone and prophylactic vitamin D supplementation to subjects taking theophylline could be osteoprotective.

IL-3 Decreases Cartilage Degeneration by Downregulating Matrix Metalloproteinases and Reduces Joint Destruction in Osteoarthritic Mice

Osteoarthritis (OA) is a chronic disease of articular joints that leads to degeneration of both cartilage and subchondral bone. These degenerative changes are further aggravated by proinflammatory cytokines including IL-1β and TNF-α. Previously, we have reported that IL-3, a cytokine secreted by activated T cells, protects cartilage and bone damage in murine models of inflammatory and rheumatoid arthritis. However, how IL-3 protects cartilage degeneration is not yet known. In this study, we investigated the role of IL-3 on cartilage degeneration under both in vitro and in vivo conditions. We found that both mouse and human chondrocytes show strong expression of IL-3R at gene and protein levels. IL-3 increases the expression of mouse chondrocyte-specific genes, Sox9 and collagen type IIa, which were downregulated by IL-1β. Moreover, IL-3 downregulated IL-1β– and TNF-α–induced expression of matrix metalloproteinases in both mouse and human chondrocytes. Interestingly, IL-3 reduces the degeneration of articular cartilage and subchondral bone microarchitecture in a mouse model of human OA. Moreover, IL-3 showed the preventive and therapeutic effects on cartilage degeneration induced by IL-1β in micromass pellet cultures of human mesenchymal stem cells. Thus, to our knowledge, we provide the first evidence that IL-3 has therapeutic potential in amelioration of degeneration of articular cartilage and subchondral bone microarchitecture associated with OA.

The Thiocarbamate Disulphide Drug, Disulfiram Induces Osteopenia in Rats by Inhibition of Osteoblast Function Due to Suppression of Acetaldehyde Dehydrogenase Activity

Dithiocarbamates (DTC), a sulfhydryl group containing compounds, are extensively used by humans that include metam and thiram due to their pesticide properties, and disulfiram (DSF) as an alcohol deterrent. We screened these DTC in an osteoblast viability assay. DSF exhibited the highest cytotoxicity (IC50 488nM). Loss in osteoblast viability and proliferation was due to induction of apoptosis via G1 arrest. DSF treatment to osteoblasts reduced glutathione (GSH) levels and exogenous addition of GSH prevented DSF-induced reactive oxygen species generation and osteoblast apoptosis. DSF also inhibited osteoblast differentiation in vitro and in vivo, and the effect was associated with inhibition of aldehyde dehydrogenase (ALDH) activity. Out of various ALDH isozymes, osteoblasts expressed only ALDH2 and DSF downregulated its transcript as well as activity. Alda-1, a specific activator of ALDH2, stimulated osteoblast differentiation. Subcutaneous injection of DSF over the calvarium of new born rats reduced the differentiation phenotype of calvarial osteoblasts but increased the mRNA levels of Runx-2 and osteocalcin. DSF treatment at a human-equivalent dose of 30 mg/kg p.o. to adult Sprague Dawley rats caused trabecular osteopenia and suppressed the formation of mineralized nodule by bone marrow stromal cells. Moreover, DSF diminished bone regeneration at the fracture site. In growing rats, DSF diminished growth plate height, primary and secondary spongiosa, mineralized osteoid and trabecular strength. Substantial decreased bone formation was also observed in the cortical site of these rats. We conclude that DSF has a strong osteopenia inducing effect by impairing osteoblast survival and differentiation due to the inhibition of ALDH2 function.

Globular adiponectin reverses osteo-sarcopenia and altered body composition in ovariectomized rats

Adiponectin regulates various metabolic processes including glucose flux, lipid breakdown and insulin response. We recently reported that adiponectin receptor1 (adipoR1) activation by a small molecule reverses osteopenia in leptin receptor deficient db/db (diabetic) mice. However, the role of adiponectin in bone metabolism under the setting of post-menopausal (estrogen-deficiency) osteopenia and associated metabolic derangements has not been studied. Here, we studied the therapeutic effect of the globular form of adiponectin (gAd), which is predominantly an adipoR1 agonist, in aged ovariectomized (OVX) rats and compared it with standard-of-care anti-osteoporosis drugs. In OVX rats with established osteopenia, gAd completely restored BMD and load bearing capacity and improved bone quality. Skeletal effects of gAd were comparable to PTH (osteoanabolic) but better than alendronate (anti-catabolic). Both osteoanabolic and anti-catabolic mechanisms led to the anti-osteoporosis effect of gAd. In cultured osteoblasts and bones, gAd increased a) adipoR1 and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) expression to promote mitochondrial respiration, which likely fueled osteoblast differentiation, b) suppressed sclerostin (a wnt antagonist) in a sirtuin1-dependent manner and c) decreased receptor-activator of nuclear factor κB ligand (RANKL) to achieve its anti-catabolic effect. The OVX-induced sarcopenia and insulin resistance were also improved by gAd. We conclude that gAd has therapeutic efficacy in estrogen deficiency-induced osteoporosis, sarcopenia and insulin resistance and hold metabolic disease modifying potential in postmenopausal women.

M2 polarization of macrophages by Oncostatin M in hypoxic tumor microenvironment is mediated by mTORC2 and promotes tumor growth and metastasis

Oncostatin M (OSM), an inflammatory cytokine belonging to the interleukin-6 (IL-6) superfamily, plays a vital role in multitude of physiological and pathological processes. Its role in breast tumor progression and metastasis to distant organs is well documented. Recent reports implicate OSM in macrophage M2 polarization, a key pro-tumoral phenomenon. M2 polarization of macrophages is believed to promote tumor progression by potentiating metastasis and angiogenesis. In the current study, we delineated the mechanism underlying OSM induced macrophage M2 polarization. The findings revealed that OSM skews macrophages towards an M2 polarized phenotype via mTOR signaling complex 2 (mTORC2). mTORC2 relays signals through two effector kinases i.e. PKC-α and Akt. Our results indicated that mTORC2 mediated M2 polarization of macrophages is not dependent on PKC-α and is primarily affected via Akt, particularly Akt1. In vivo studies conducted on 4T1/BALB/c mouse orthotropic model of breast cancer further corroborated these observations wherein i.v. reintroduction of mTORC2 abrogated monocytes into orthotropic mouse model resulted in diminished acquisition of M2 specific attributes by tumor associated macrophages. Metastasis to distant organs like lung, liver and bone was reduced as evident by decrease in formation of focal metastatic lesions in mTORC2 abrogated monocytes mice. Our study pinpoints key role of mTORC2-Akt1 axis in OSM induced macrophage polarization and suggests for possible usage of Oncostatin-M blockade and/or selective mTORC2 inhibition as a potential anti-cancer strategy particularly with reference to metastasis of breast cancer to distant organs such as lung, liver and bone.

Guava fruit extract and its triterpene constituents have osteoanabolic effect: Stimulation of osteoblast differentiation by activation of mitochondrial respiration via the Wnt/β-catenin signaling☆☆☆

The aim of this study was to evaluate the skeletal effect of guava triterpene-enriched extract (GE) in rats and identify osteogenic compounds thereof, and determine their modes of action. In growing female rats, GE at 250 mg/kg dose increased parameters of peak bone mass including femur length, bone mineral density (BMD) and biomechanical strength, suggesting that GE promoted modeling-directed bone growth. GE also stimulated bone regeneration at the site of bone injury. In adult osteopenic rats (osteopenia induced by ovariectomy, OVX) GE completely restored the lost bones at both axial and appendicular sites, suggesting a strong osteoanabolic effect. Serum metabolomics studies showed changes in several metabolites (some of which are related to bone metabolism) in OVX compared with ovary-intact control and GE treatment to OVX rats reversed those. Out of six abundantly present triterpenes in GE, ursolic acid (UA) and 2α-hydroxy ursolic acid (2α-UA) induced osteogenic differentiation in vitro as did GE by activating Wnt/β-catenin pathway assessed by phosphorylation of GSK-3β. Over-expressing of constitutively active GSK-3β (caGSK-3β) in osteoblasts abolished the differentiation-promoting effect of GE, UA and 2α-UA. All three increased both glycolysis and mitochondrial respiration but only rotenone (inhibitor of mitochondrial electron transfer) and not 2-deoxyglucose (to block glycolysis) inhibited osteoblast differentiation. In addition, caGSK-3β over-expression attenuated the enhanced mitochondrial respiration caused by GE, UA and 2α-UA. We conclude that GE has osteoanabolic effect which is contributed by UA and 2α-UA, and involve activation of canonical Wnt signaling which in turn modulates cellular energy metabolism leading to osteoblast differentiation.

Bacillus clausii inhibits bone loss by skewing Treg-Th17 cell equilibrium in postmenopausal osteoporotic mice model

OBJECTIVES Postmenopausal osteoporosis is one of most commonly occurring skeletal diseases leading to bone loss and fragility. Probiotics have been associated with various immunomodulatory properties and thus can be exploited to enhance bone health. In the present study, we report, to our knowledge for the first time, that oral administration of Bacillus clausii (BC) in postmenopausal osteoporotic (OVX) mice model enhances bone health. METHODS BC was selected as probiotic of choice due to its established immunomodulatory properties. BC skews the Treg-Th17 cell balance in vivo by inhibiting osteoclastogenic Th17 cells and promoting antiosteoclastogenic Treg cell development in postmenopausal osteoporotic mice. Mice were divided into three groups (sham, OVX, and OVX + BC), and BC was administered orally in drinking water for 6 wk post-ovariectomy. At the end of experiment, mice were sacrificed and bones were analyzed for various parameters, along with lymphoid tissues for Treg-Th17 cells and serum cytokines. RESULTS We observed that BC administration enhanced bone health. This effect of BC administration was found due to skewing of Treg-Th17 cell balance (enhanced Treg and decreased Th17 cells) in vivo. BC administration reduced levels of proinflammatory cytokines (interleukin [IL]-6, IL-17, IFN-γ and tumor necrosis factor-α) and increased levels of anti-inflammatory cytokine (IL-10). CONCLUSIONS The present study strongly supports and establishes the osteoprotective potential of BC leading to enhanced bone health in postmenopausal osteoporotic mice model.

Pharmacological activation of aldehyde dehydrogenase 2 promotes osteoblast differentiation via bone morphogenetic protein-2 and induces bone anabolic effect

Abstract Aldehyde dehydrogenases (ALDHs) are a family of enzymes involved in detoxifying aldehydes. Previously, we reported that an ALDH inhibitor, disulfiram caused bone loss in rats and among ALDHs, osteoblast expressed only ALDH2. Loss‐of‐function mutation in ALDH2 gene is reported to cause bone loss in humans which suggested its importance in skeletal homeostasis. We thus studied whether activating ALDH2 by N‐(1, 3‐benzodioxol‐5‐ylmethyl)‐2, 6‐dichlorobenzamide (alda‐1) had osteogenic effect. We found that alda‐1 increased and acetaldehyde decreased the differentiation of rat primary osteoblasts and expressions of ALDH2 and bone morphogenetic protein‐2 (BMP‐2). Silencing ALDH2 in osteoblasts abolished the alda‐1 effects. Further, alda‐1 attenuated the acetaldehyde‐induced lipid‐peroxidation and oxidative stress. BMP‐2 is essential for bone regeneration and alda‐1 increased its expression in osteoblasts. We then showed that alda‐1 (40 mg/kg dose) augmented bone regeneration at the fracture site with concomitant increase in BMP‐2 protein compared with control. The osteogenic dose (40 mg/kg) of alda‐1 attained a bone marrow concentration that was stimulatory for osteoblast differentiation, suggesting that the tissue concentration of alda‐1 matched its pharmacologic effect. In addition, alda‐1 promoted modeling‐directed bone growth and peak bone mass achievement, and increased bone mass in adult rats which reiterated its osteogenic effect. In osteopenic ovariectomized (OVX) rats, alda‐1 reversed trabecular osteopenia with attendant increase in serum osteogenic marker (procollagen type I N‐terminal peptide) and decrease in oxidative stress. Alda‐1 has no effect on liver and kidney function. We conclude that activating ALDH2 by alda‐1 had an osteoanabolic effect involving increased osteoblastic BMP‐2 production and decreased OVX‐induced oxidative stress. HighlightsAlda‐1 induced osteoblast differentiation that involved upregulation of ALDH2 and BMP‐2Alda‐1 attenuated acetaldehyde‐induced inhibition of osteoblast differentiationAlda‐1 enhanced bone regeneration at the fracture site and peak bone mass achievementAlda‐1 reversed trabecular osteopenia in OVX rats via an osteoanabolic mechanism