Influence of organic supplements on accumulation of β-boswellic acid, acetyl-β-boswellic acid, 11-keto-β-boswellic acid and acetyl-11-keto-β-boswellic acid in callus culture of Boswellia serrata Roxb.

Boswellia serrata Roxb. is a source of several bioactive triterpenoids. Boswellic acid, obtained from oleo-gum resin of the tree, is a major potentially bioactive and medicinal compound. Unrestricted exploitation of its natural resource has led to its listing among the threatened and endangered species. Accumulation of the compound through tissue culture seems a promising option. The present work was conducted to study the effect of sodium pyruvate, l-phenylalanine, glycine, ferulic acid and sucrose on the growth of callus and accumulation of four principal isomers of boswellic acids, viz. β-boswellic acid (BBA), acetyl-β-boswellic acid (ABBA), 11-keto-β-boswellic acid (KBBA) and acetyl-11-keto-β-boswellic acid (AKBBA). Callus cultures obtained from embryo explants of Boswellia serrata on Murashige and Skoog medium containing 2.5 μM 6-benzyladenine, 15 μM indole acetic acid and 200 mg l^?1 polyvinyl pyrrolidone was supplemented with varying concentrations of the supplements. Sodium pyruvate was most beneficial for the production of AKBBA (77 folds), BBA (27 folds) and ABBA (27 folds) at 10 mg l^?1 and for KBBA (47 folds) at 5 mg l^?1 when compared with control. It was closely followed by sucrose (50 g l^?1) resulting in KBBA (22-fold), AKBBA (25-fold), BBA (17-fold) and ABBA (10-fold). Glycine, l-phenylalanine and ferulic acid were relatively less effective. It can be concluded that callus cultures manipulated with different concentrations of organic supplements, sodium pyruvate or sucrose, in particular, could be considered as an alternate strategy for direct production of boswellic acid and help in the conservation of the species.     

A propionyloxy derivative of 11-keto-β-boswellic acid induces apoptosis in HL-60 cells mediated through topoisomerase I & II inhibition

Boswellic acids have invariably been reported for their antiproliferative potential in various cell systems. In the present study the growth inhibitory effect of propionyloxy derivative of 11-keto-β-boswellic acid (PKBA; a semisynthetic analogue of 11-keto-β-boswellic acid) on HL-60 promyelocytic leukemia cells is being reported for the first time. In the preliminary studies, in vitro cytotoxicity of PKBA was investigated against eight human cancer cell lines viz., IMR-32, SF-295 (both neuroblastoma), PC-3 (prostate), Colo-205 (colon), MCF-7 (breast), OVCAR-5 (ovary), HL-60, Molt-4 (both leukemia) and their respective IC(50) values were found to be 5.95, 7.11, 15.2, 14.5, 15, 15.9, 8.7 & 9.5μg/ml, respectively. For determining the mechanism of cell death in HL-60 cells, PKBA was subjected to different mechanistic studies. DNA relaxation assay of PKBA revealed inhibition of both topoisomerases I & II. The fragmentation analysis of DNA revealed typical ladders indicating the cytotoxic effect to be mediated by induction of apoptosis. The morphologic studies of PKBA showed the presence of true apoptotic bodies. Apoptosis was confirmed further by flow-cytometric detection of sub-G(1) peaks and enhanced annexin-V-FITC binding of the cells. The activation of apoptotic cascade by PKBA in HL-60 cells was found to be associated with the loss of mitochondrial membrane potential, release of cytochrome c, activation of initiator and executioner caspases and cleavage of poly ADP ribose polymerase (PARP). In vivo studies of PKBA revealed anti-tumoral activity against both ascitic and solid murine tumor models. These studies thus demonstrate PKBA to induce apoptosis in HL-60 cells due to the inhibition of topoisomerases I and II.     

Acetyl-11-keto-β-boswellic acid modulates membrane dynamics in benzo(a)pyrene-induced lung carcinogenesis

Molecular and Cellular Biochemistry - Membrane fluidity is the most important physiochemical property of cell membranes and governs its functional attributes. The current investigations were...     

Microbial transformation of acetyl-11-keto-β-boswellic acid and their inhibitory activity on LPS-induced NO production

The capabilities of 20 strains of fungi to transform acetyl-11-keto-β-boswellic (AKBA) were screened. And biotransformation of AKBA by Cunninghamella blakesleana AS 3.970 afforded five metabolites (1–5), while two metabolites (6, 7) were isolated from biotransformation of Cunninghamella elegans AS 3.1207. The chemical structures of these metabolites were identified by spectral methods including 2D NMR and their structures were elucidated as 7β-hydroxy-3-acety-11-keto-β-boswellic acid (1), 21β-dihydroxy-3-acety-11-keto-β-boswellic acid (2), 7β,22α-dihydroxy-3-acety-11-keto-β-boswellic acid (3), 7β,16α-dihydroxy-3-acety-11-keto-β-boswellic acid (4), 7β,15α-dihydroxy-3-acety-11-keto-β-boswellic acid (5); 7β,15α,21β-trihydroxy-3-acety-11-keto-β-boswellic acid (6) and 15α,21β-dihydroxy-3-acety-11-keto-β-boswellic acid (7). All these products are previously unknown. Their primary structure–activity relationships (SAR) of inhibition activity on LPS-induced NO production in RAW 264.7 macrophage cells were evaluated.     

GSK-3β inhibition protects human nucleus pulposus cell against oxidative stress-inducing apoptosis through mitochondrial pathway

Molecular Biology Reports - Oxidative stress in the intervertebral disc leads to nucleus pulposus (NP) degeneration by inducing cell apoptosis. However, the molecular mechanisms underlying this...     

A new cytochrome P450 system from Bacillus megaterium DSM319 for the hydroxylation of 11-keto-β-boswellic acid (KBA)

In the genome of Bacillus megaterium DSM319, a strain who has recently been sequenced to fully exploit its potential for biotechnological purposes, we identified a gene encoding the cytochrome P450 CYP106A1 as well as genes encoding potential redox partners of CYP106A1. We cloned, expressed, and purified CYP106A1 and five potential autologous redox partners, one flavodoxin and four ferredoxins. The flavodoxin and three ferredoxins were able to support the activity of CYP106A1 displaying the first cloned natural redox partners of a cytochrome P450 from B. megaterium. The CYP106A1 system was able to convert the pentacyclic triterpene 11-keto-β-boswellic acid (KBA) belonging to the main bioactive constituents of Boswellia serrata gum resin extracts, which are used to treat inflammatory disorders and arthritic diseases. In order to provide sufficient amounts of the KBA products to characterize them structurally by NMR spectroscopy, recombinant whole-cell biocatalysts were constructed based on B. megaterium MS941. The main product has been identified as 7β-hydroxy-KBA, while the side product (～20 %) was shown to be a mixture of 7β,15α-dihydroxy-KBA and 15α-hydroxy-KBA. Without further optimization 560.7 mg l^?1 day^?1 of the main product, 7β-hydroxy-KBA, could be obtained thus providing a suitable starting point for the efficient production of modified KBA by chemical tailoring to produce novel KBA derivatives with increased bioavailability and this way more efficient drugs.     

Melphalan induced germ cell toxicity and dose-dependent effects of β-aminoisobutyric acid in experimental rat model: Role of oxidative stress,inflammation and apoptosis

The success of chemotherapy regimens has led to an increase in cancer survival rate over the last decades. Melphalan has been widely used for the treatment of several types of cancers despite its gonadotoxic effects. Due to its ability to cause mutations in the spermatogonial stem cells and spermatids, melphalan can exert a negative impact on male reproductive health in young cancer survivors. β-aminoisobutyric acid (BAIBA), a myokine released by skeletal muscles, has been reported to have beneficial effects in diabetic nephropathy, cardiomyopathy and hepatic toxicity. However, the exact role of BAIBA in chemotherapy-induced germ cell toxicity is still unexplored. The present study aims to determine the dose-dependent (25, 50, and 100 mg/kg) effects of BAIBA on melphalan-induced (1.5 mg/kg) germ cell toxicity in sprague−dawley (SD) rats. The evaluation parameters included quantification of oxidative stress biomarkers, sperm count, sperm motility and head morphology, sperm and testicular DNA damage, sperm mitochondrial membrane potential, ultrastructural changes in sperms, histological and protein expression studies in testes. Melphalan treatment significantly altered all the above-mentioned parameters and the high dose (100 mg/kg) of BAIBA restored melphalan-induced toxicity in a significant manner by exerting antioxidant, anti-inflammatory and antiapoptotic effects. However, the medium dose (50 mg/kg) of BAIBA decreased the toxicity of melphalan and the low dose (25 mg/kg) of BAIBA failed to counteract the melphalan-induced male germ cell toxicity as well as the peripheral blood micronucleus induction. The antioxidant, anti-inflammatory and antiapoptotic role of BAIBA in melphalan-induced gonadal damage is a novel finding in an experimental rat model.     

17β-Estradiol regulates oxidative stress in prostate cancer cell lines according to ERalpha/ERbeta ratio

Estrogen action is mediated by the two receptor isoforms: estrogen receptor alpha and beta. Both receptors are expressed in human prostate tissue and have different action profiles. ERalpha is positively correlated with the malignancy of prostate cancer, while ERbeta may protect against abnormal prostate cell growth. 17β-Estradiol (E2), at least in part, induces cancerous transformations by causing deleterious mutations through the formation of reactive oxygen species (ROS). The aim was to study the effect of E2 on oxidative stress and the expression of uncoupling proteins (UCPs) and antioxidant enzymes in several prostate cancer cell lines with different ERalpha/ERbeta ratios. The cell prostate lines with a lower ERalpha/ERbeta ratio had lower oxidative stress, which could be partially explained by the increased expression of antioxidant enzymes and UCPs. Moreover, the action of E2 on the expression of antioxidant enzymes and UCPs was dual and dependent on the ERalpha/ERbeta ratio. Treatments with 0.1 nM E2 in cell lines with high ERalpha/ERbeta ratio produced a decrease in antioxidant enzymes and UCPs levels, with an increase in ROS production. These effects disappeared when the treatment was done in the presence of an ERalpha antagonist (MPP). In the cell lines with greatest levels of ERbeta and the lowest ERalpha/ERbeta ratio, E2 treatment caused the up-regulation of antioxidant enzymes and UCPs with a look-up decrease in ROS production. These effects were reversed when the cells were treated with E2 in the presence of an ERbeta antagonist (R,R-THC). On the whole, our results suggest a dual E2 effect; increasing or decreasing oxidative stress in part by modulation of UCPs and antioxidant enzymes according to the abundance ERbeta and ERalpha/ERbeta ratio in prostate cancer cell lines.     

Modulation of YY1 and p53 expression by transforming growth factor-β3 in prostate cell lines

Transforming growth factor-β (TGF-β) is the prototype of a family of secreted polypeptide growth factors. These cytokines play very important roles during development, as well as in normal physiological and disease processes, by regulating a wide array of cellular processes, such as cell growth, differentiation, migration, apoptosis, and extracellular matrix production. TGF-β utilizes a multitude of intracellular signalling pathways in addition to Smads with actions that are dependent on circumstances, including dose, target cell type, and context. The aims of this research were (i) to verify the effects of dose-dependent TGF-β3 treatment on YY1 and p53 expression, in BPH-1 cell line, human benign prostate hyperplasia, and two prostate cancer cell lines, LNCaP, which is androgen-sensitive, and DU-145, which is androgen-non responsive, (ii) establish a correlation between p53 and YY1 and (iii) determine the expression of a number of important intracellular signalling pathways in TGF-β3-treated prostate cell lines. The expression of YY1, p53, PI3K, AKT, pAKT, PTEN, Bcl-2, Bax, and iNOS was evaluated through Western blot analysis on BPH-1, LNCaP, and DU-145 cultures treated with 10 and 50 ng/ml of TGF-β3 for 24 h. The production of nitric oxide (NO) was determined by Griess reagent and cell viability through MTT assay. The results of this research demonstrated profound differences in the responses of the BPH-1, LNCaP, and DU-145 cell lines to TGF-β3 stimulation. We believe that the findings could be important because of the clinical relevance that they may assume and the therapeutic implications for TGF-β treatment of prostate cancer.     

Chronic Akt activation attenuated lipopolysaccharide-induced cardiac dysfunction via Akt/GSK3β-dependent inhibition of apoptosis and ER stress

Sepsis is characterized by systematic inflammation and contributes to cardiac dysfunction. This study was designed to examine the effect of protein kinase B (Akt) activation on lipopolysaccharide-induced cardiac anomalies and underlying mechanism(s) involved. Mechanical and intracellular Ca^2 + properties were examined in myocardium from wild-type and transgenic mice with cardiac-specific chronic Akt overexpression following LPS (4 mg/kg, i.p.) challenge. Akt signaling cascade (Akt, phosphatase and tensin homologue deleted on chromosome ten, glycogen synthase kinase 3 beta), stress signal (extracellular-signal-regulated kinases, c-Jun N-terminal kinases, p38), apoptotic markers (Bcl-2 associated X protein, caspase-3/-9), endoplasmic reticulum (ER) stress markers (glucose-regulated protein 78, growth arrest and DNA damage induced gene-153, eukaryotic initiation factor 2α), inflammatory markers (tumor necrosis factor α, interleukin-1β, interleukin-6) and autophagic markers (Beclin-1, light chain 3B, autophagy-related gene 7 and sequestosome 1) were evaluated. Our results revealed that LPS induced marked decrease in ejection fraction, fractional shortening, cardiomyocyte contractile capacity with dampened intracellular Ca^2 + release and clearance, elevated reactive oxygen species (ROS) generation and decreased glutathione and glutathione disulfide (GSH/GSSG) ratio, increased ERK, JNK, p38, GRP78, Gadd153, eIF2α, BAX, caspase-3 and -9, downregulated B cell lymphoma 2 (Bcl-2), the effects of which were significantly attenuated or obliterated by Akt activation. Akt activation itself did not affect cardiac contractile and intracellular Ca^2 + properties, ROS production, oxidative stress, apoptosis and ER stress. In addition, LPS upregulated levels of Beclin-1, LC3B and Atg7, while suppressing p62 accumulation. Akt activation did not affect Beclin-1, LC3B, Atg7 and p62 in the presence or absence of LPS. Akt overexpression promoted phosphorylation of Akt and GSK3β. In vitro study using the GSK3β inhibitor SB216763 mimicked the response elicited by chronic Akt activation. Taken together, these data showed that Akt activation ameliorated LPS-induced cardiac contractile and intracellular Ca^2 + anomalies through inhibition of apoptosis and ER stress, possibly involving an Akt/GSK3β-dependent mechanism.     

Growth inhibition induced by transforming growth factor-β1 in human oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is a world-wide health problem and its incidence accounts for 1.9–3.5% of all malignant tumors. Transforming growth factor beta/Smads (TGF-β/Smads) signaling pathway plays an important role in oncogenesis, but its function and molecular mechanisms in OSCC remain unclear. Expression of transforming growth factor-β receptor type II (TβRII) and Smad4 was studied by immunohistochemistry in 108 OSCC patients and 10 normal controls. Function and molecular mechanisms of TGF-β/Smads signaling pathway was then investigated in two human tongue squamous carcinoma cell lines with high and low metastasis (Tb and Tca8113) by RT-PCR, Western Blot, immunofluorescence, cell growth curve and flow cytometry (FCM), respectively. TβRII and Smad4 were significantly down-regulated in tumor tissues (with or without lymph node metastasis) compared to normal oral epithelium tissues (P < 0.05). TGF-β1 induced arrest of the cell cycle rather than cell death in Tca8113 and Tb cells, and this influence was mediated by the increasing the expression and changing the location of its downstream components of TGF-β1/Smads signaling pathway. TGF-β1 rapidly increased the expression of p15 and p21 in both Tca8113 and Tb cells. TGF-β1 did not increase p27 expression in Tca8113 cells, but p27 expression was increased in Tb cells. These indicated that TGF-β1 induced G₁ arrest of cell cycle through a different regulating pathway in Tb cells compared with Tca8113 cells. Thus, we conclude that TGF-β/Smads signaling pathway play a important role on cell growth and metastasis potential in OSCC. Xiumei Wang, Wenjing Sun, and Jing Bai contributed equally to this paper.     

Antistaphylococcal and biofilm inhibitory activities of acetyl-11-keto-β-boswellic acid from <Emphasis Type="Italic">Boswellia serrata</Emphasis>

Background  

Boswellic acids are pentacyclic triterpenes, which are produced in plants belonging to the genus Boswellia. Boswellic acids appear in the resin exudates of the plant and it makes up 25-35% of the resin. β-boswellic acid, 11-keto-β-boswellic acid and acetyl-11-keto-β-boswellic acid have been implicated in apoptosis of cancer cells, particularly that of brain tumors and cells affected by leukemia or colon cancer. These molecules are also associated with potent antimicrobial activities. The present study describes the antimicrobial activities of boswellic acid molecules against 112 pathogenic bacterial isolates including ATCC strains. Acetyl-11-keto-β-boswellic acid (AKBA), which exhibited the most potent antibacterial activity, was further evaluated in time kill studies, postantibiotic effect (PAE) and biofilm susceptibility assay. The mechanism of action of AKBA was investigated by propidium iodide uptake, leakage of 260 and 280 nm absorbing material assays.     

Asymmetric dimethylarginine exacerbates Aβ-induced toxicity and oxidative stress in human cell and Caenorhabditis elegans models of Alzheimer disease

Growing evidence suggests a strong association between cardiovascular risk factors and incidence of Alzheimer disease (AD). Asymmetric dimethylarginine (ADMA), the endogenous nitric oxide synthase inhibitor, has been identified as an independent cardiovascular risk factor and is also increased in plasma of patients with AD. However, whether ADMA is involved in the pathogenesis of AD is unknown. In this study, we found that ADMA content was increased in a transgenic Caenorhabditis elegans β-amyloid (Aβ) overexpression model, strain CL2006, and in human SH-SY5Y cells overexpressing the Swedish mutant form of human Aβ precursor protein (APPsw). Moreover, ADMA treatment exacerbated Aβ-induced paralysis and oxidative stress in CL2006 worms and further elevated oxidative stress and Aβ secretion in APPsw cells. Knockdown of type 1 protein arginine N-methyltransferase to reduce ADMA production failed to show a protective effect against Aβ toxicity, but resulted in more paralysis in CL2006 worms as well as increased oxidative stress and Aβ secretion in APPsw cells. However, overexpression of dimethylarginine dimethylaminohydrolase 1 (DDAH1) to promote ADMA degradation significantly attenuated oxidative stress and Aβ secretion in APPsw cells. Collectively, our data support the hypothesis that elevated ADMA contributes to the pathogenesis of AD. Our findings suggest that strategies to increase DDAH1 activity in neuronal cells may be a novel approach to attenuating AD development.     

The role of glycogen synthase kinase-3β in glioma cell apoptosis induced by remifentanil

The aim of malignant glioma treatment is to inhibit tumor cell proliferation and induce tumor cell apoptosis. Remifentanil is a clinical anesthetic drug that can activate the N-methyl-D-aspartate (NMDA) receptor. NMDA receptor signaling activates glycogen synthase kinase-3β (GSK-3β). Discovered some 32 years ago, GSK-3β was only recently considered as a therapeutic target in cancer treatment. The purpose of this study was to assess whether remifentanil can induce the apoptosis of C6 cells through GSK-3β activation. 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was used to detect cell viability. Hoechst 33342 staining and flow cytometry were used to detect cell apoptosis. The effect of GSK-3β activation was detected using a GSK-3β activation assay kit and 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), a potent and selective small molecule inhibitor of GSK-3β. The MTT assay indicated that remifentanil induced C6 cell death in a concentration- and time-dependent manner. Hoechst 33342 staining and flow cytometry showed that remifentanil significantly induced C6 cell apoptosis. The measurement of GSK-3β activation showed that remifentanil increased the cellular level of GSK-3β. All of these toxic effects can be attenuated by treatment with TDZD-8. These results suggest that remifentanil is able to induce C6 cell apoptosis through GSK-3β activation, which provides a basis for its potential use in the treatment of malignant gliomas.     

The protective role of DL-α-lipoic acid in the oxidative vulnerability triggered by Aβ-amyloid vaccination in mice

Recent reports indicate that -amyloid peptide (A) vaccine based therapy for Alzheimers disease (AD) may be on the horizon. There are however, concerns about the safety of this approach. Immunization with A has several disadvantages, because it crosses the blood brain barrier and cause inflammation and neurotoxicity. The present work is aimed to study the protective effective of -lipoic acid (LA) in the oxidative vulnerability of -amyloid in plasma, liver, spleen and brain, when A fibrils are given intraperitoneally in inflammation induced mice. Result shows that reactive oxygen species (ROS) in the astrocytes of inflammation induced mice along with A (IA) has shown 2.5-fold increase when compared with LA treated mice. The increased level of lipid peroxidase (LPO) (p < 0.05) and decreased antioxidant status (p < 0.05) were observed in the plasma, liver, spleen and brain of IA induced mice when compared with LA treated mice.Data shows that there were no significant changes observed between the control and LA treated mice. Our biochemical and histological results highlight that significant oxidative vulnerability was observed in IA treated mice, which was prevented by LA therapy. Our findings suggest that the antioxidant effect of LA when induced with A may serve as a potent therapeutic tool for inflammatory AD models. (Mol Cell Biochem 270: 29–37, 2005)