Tribbles 2 pseudokinase confers enzalutamide resistance in prostate cancer by promoting lineage plasticity.

Enzalutamide, a second-generation antiandrogen, is commonly prescribed for the therapy of advanced prostate cancer, but enzalutamide-resistant, lethal, or incurable disease invariably develops. To understand the molecular mechanism(s) behind enzalutamide resistance, here, we comprehensively analyzed a range of prostate tumors and clinically relevant models by gene expression array, immunohistochemistry, and Western blot, which revealed that enzalutamide-resistant prostate cancer cells and tumors overexpress the pseudokinase, Tribbles 2 (TRIB2). Inhibition of TRIB2 decreases the viability of enzalutamide-resistant prostate cancer cells, suggesting a critical role of TRIB2 in these cells. Moreover, the overexpression of TRIB2 confers resistance in prostate cancer cells to clinically relevant doses of enzalutamide, and this resistance is lost upon inhibition of TRIB2. Interestingly, we found that TRIB2 downregulates the luminal markers androgen receptor and cytokeratin 8 in prostate cancer cells but upregulates the neuronal transcription factor BRN2 (Brain-2) and the stemness factor SOX2 (SRY-box 2) to induce neuroendocrine characteristics. Finally, we show that inhibition of either TRIB2 or its downstream targets, BRN2 or SOX2, resensitizes resistant prostate cancer cells to enzalutamide. Thus, TRIB2 emerges as a potential new regulator of transdifferentiation that confers enzalutamide resistance in prostate cancer cells via a mechanism involving increased cellular plasticity and lineage switching.

MK591 (Quiflapon), a 5-lipoxygenase inhibitor, kills pancreatic cancer cells via downregulation of protein kinase C-epsilon.

Introduction: Pancreatic tumors and cell lines derived from them exhibit elevated expression of 5-lipoxygenase (5-Lox), whereas non-tumor glands or normal cells do not exhibit this overexpression. Arachidonic acid stimulates pancreatic cancer cell growth via metabolic conversion through the 5-Lox pathway, and inhibition of 5-Lox activity decreases the viability of pancreatic cancer cells. However, the downstream signaling mechanisms through which 5-Lox exerts its effects on the survival of pancreatic cancer cells remain to be elucidated. Methods: The effects of 5-Lox inhibition on cell proliferation, apoptosis, and invasive potential were investigated in pancreatic cancer cells. The protein expression was analyzed by Western blot. Apoptosis was analyzed by Annexin-V binding assay and by detecting the degradation of chromatin-DNA to nucleosomal fragments. The protein kinase C-epsilon (PKCε) activity was measured by an immunoprecipitation-kinase assay. The in vivo effects of MK591 were evaluated in pancreatic tumor xenograft model. Results: MK591, a specific inhibitor of 5-Lox activity, killed pancreatic cancer cells via induction of apoptosis, involving externalization of phosphatidylserine, cleavage of PARP (poly-ADP ribose polymerase) and degradation of chromatin DNA to nucleosomes. MK591 effectively blocked in vitro invasion and soft-agar colony formation by pancreatic cancer cells and decreased pancreatic tumor growth in nude mice xenografts. Furthermore, inhibition of 5-Lox downregulated K-Ras and inhibited phosphorylation of c-Raf and ERKs. Interestingly, 5-Lox inhibition induced apoptosis in pancreatic cancer cells without the inhibition of Akt but the protein level of PKCε was dramatically downregulated. Furthermore, inhibition of 5-Lox decreased the phosphorylation of Stat3 at Serine-727. Pre-treatment of pancreatic cancer cells with peptide activators of PKCε prevented apoptosis induced by 5-Lox inhibition, suggesting that the mechanism by which 5-Lox inhibition causes cell death in pancreatic cancer involves downregulation of PKCε. The combination of low doses of MK591 and gemcitabine synergistically reduced the oncogenic phenotype and killed pancreatic cancer cells by inducing apoptosis. Discussion: These findings indicate that inhibition of 5-Lox interrupts an Akt-independent, PKCε-dependent survival mechanism in pancreatic cancer cells and suggest that metabolism of arachidonic acid through the 5-Lox pathway plays an integral part in the survival of pancreatic cancer cells via signaling through PKCε, an oncogenic, pro-survival serine/threonine kinase.

Daclatasvir, an Antiviral Drug, Downregulates Tribbles 2 Pseudokinase and Resensitizes Enzalutamide-Resistant Prostate Cancer Cells.

FDA-approved enzalutamide is commonly prescribed to reduce the growth of advanced prostate cancer by blocking androgen receptor function. However, enzalutamide-resistant prostate cancer (ERPC) invariably develops and progresses to metastatic, lethal disease. Management of ERPC poses a special problem not only because available therapeutic regimens cannot effectively kill ERPC cells but also due to their propensity to invade large bones. Moreover, molecular mechanism(s) behind enzalutamide resistance is not properly understood, which is delaying development of newer agents. We found that the pseudokinase, Tribbles 2 (TRIB2), is overexpressed in ERPC cells and plays a critical role in their survival. Forced overexpression of TRIB2 enhances prostate cancer cell growth and confers resistance to physiologic doses of enzalutamide, suggesting that TRIB2 plays an important role in the development and progression of ERPC. Though TRIB2 has emerged as an excellent molecular target for ERPC, suitable inhibitors are not commercially available for effective targeting. By designing a luciferase-tagged TRIB2 fusion protein-based assay system, we screened a library of about 1,600 compounds and found that daclatasvir (DCV), an antiviral drug, effectively inhibits TRIB2-luciferase. We also found that DCV degrades TRIB2 proteins by direct binding and resensitizes ERPC cells to enzalutamide treatment. Moreover, DCV at lower, sublethal doses synergizes with enzalutamide to decrease the viability and induce apoptosis in prostate cancer cells. Because DCV is already approved by the FDA and well tolerated in humans, based on our findings, it appears that DCV is a promising new agent for development of an effective therapy for advanced, enzalutamide-resistant, lethal prostate cancer.

(+)-Cyanidan-3-ol inhibits epidermoid squamous cell carcinoma growth via inhibiting AKT/mTOR signaling through modulating CIP2A-PP2A axis.

BACKGROUND: Despite recent advances in the treatment of squamous cell skin cancer (SCSC), the disease persists, and treatment resistance develops. Thus, identifying new targets and developing new therapeutic approaches showing low vulnerability to drug resistance is highly needed. PURPOSE: This study aimed to reveal a novel targeted phytotherapeutic strategy for SCSC treatment alone or in combination with standard targeted anticancer molecules. STUDY DESIGN: A library of natural products was utilized to identify molecules that inhibit the growth of skin cancer cells. The anticancer potential of the selected compound was evaluated in human skin squamous carcinoma models, in vitro and in vivo. A comprehensive ingenuity pathway analysis (IPA) strategy and molecular biology technology was adopted to investigate the therapeutic mechanisms in human SCSC. METHODS: The Matrigel invasion chamber, foci formation and soft agar colony formation assays were employed to study the cells invasion and migration potential in vitro. In vivo antitumor effects were evaluated in DMBA/TPA-induced skin papilloma and A431 human skin squamous carcinoma xenograft tumor models. An integrative IPA was employed to identify mechanisms and protein targets in human SCSC.Compounds synergies were determined by the bliss model and evaluated using human SCSC cell lines and xenograft tumors. Histological staining, immunofluorescence imaging, real-time PCR, Western blots, and flow cytometric analyses were employed to analyze apoptosis and cell signaling mechanisms. RESULTS: We identified (+)-cyanidan-3-ol (CD-3) as a selective compound for inhibiting the growth of SCSC cell lines. CD-3 inhibited tumor growth and burden without apparent toxicity and prolonged the survival of tumor-bearing mice. CD-3 inhibitory effects on SCSC growth are mediated via cell cycle arrest and caspase-dependent apoptosis induction. Mechanistic studies showed that CD-3 activates PP2A via inhibiting CIP2A and produces tumor growth inhibitory effects via promoting dephosphorylation of oncogenic AKT/mTOR signaling proteins in SCSC cells and xenograft tumors in a PP2A dependent manner. Furthermore, the combination of CD-3 and mTOR inhibitors (mTORi) synergistically reduced oncogenic phenotypes. CONCLUSIONS: Our study suggests that PP2A activation is an effective strategy for SCSC treatment and the CD-3 and mTORi combination may serve as a promising treatment for SCSC.

Cerebroprotective effect of Ocimum gratissimum against focal ischemia and reperfusion-induced cerebral injury.

CONTEXT: Oxidative stress is believed to increase delayed neuronal death in the brain following ischemia. As a consequence, many attempts to reduce the damage resulting from cerebral ischemia under more highly oxidized conditions have focused on treatments aimed at maintaining the redox equilibrium of the local environment. Many antioxidants were shown to be neuroprotective in experimental models of cerebral ischemia and reperfusion. OBJECTIVE: The present study was designed to investigate the potential protective effects of ethanol extract of Ocimum gratissimum Linn. (Lamiaceae) (EEOg) against focal ischemia and reperfusion (I/R) insult in rat brain. MATERIALS AND METHODS: The animal model of focal I/R was established by occluding the middle cerebral artery (MCA) of male Wistar rats for 2 h, followed by 24 h reperfusion. The thiobarbituric acid reactive substances concentration, superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity were determined by colorimetric assays. The characterization and quantitative analysis of phenolic content was determined using HPLC. RESULTS: MCA occlusion led to significant rise in cerebral infarct volume and lipid peroxidation, and depletion in SOD and GPx in brain. The neurological deficits were also significantly elevated by MCA occlusion. All the brain oxidative stress, damage and neurological deficits were significantly attenuated by pre-treatment with EEOg (150 or 300 mg/kg, p.o.). CONCLUSION: The overall finding suggests the neuroprotective potential of O. gratissimum in cerebral ischemia, and is mediated through its antioxidant activity. Therefore, O. gratissimum should be investigated further as a possible strategy against cerebral stroke.

Antimelanoma and radioprotective activity of alcoholic aqueous extract of different species of Ocimum in C(57)BL mice.

CONTEXT: Various Ocimum species (Labiateae) are commonly used for the treatment of inflammation, stress, diarrhea, and as an antioxidant drug in the Indian ethnic system of medicine. OBJECTIVE: The present study was carried out to investigate the antimelanoma and radioprotective activity of different species of Ocimum in C(57)BL and Swiss albino mice. MATERIALS AND METHODS: The antimelanoma activity of 50% alcoholic aqueous leaf extract of five species of Ocimum [Ocimum sanctum (SE), Ocimum gratissimum (GE), Ocimum basilicum (BE), Ocimum canum (CE), and Ocimum kilimandscharicum (KE)] alone or in combination with radiotherapy was determined on the basis of tumor volume, body weight, and survival rate of animals. The radioprotective potential of different species of Ocimum was determined by chromosomal aberration assay. The effect of the alcoholic aqueous extract of different species of Ocimum was also evaluated for the estimation of glutathione level and glutathione S-transferase activity in Swiss albino mice. RESULTS: The 50% alcoholic aqueous extract of different species of Ocimum administered orally (200 mg/kg, p.o.) resulted in significant reduction in tumor volume, increase in average body weight, and survival rate of mice. The various extracts showed modulatory influence against lethal irradiation doses of gamma radiation in terms of radiation-induced chromosomal damage, while at the same time induced an increase in reduced glutathione level and GST activity. DISCUSSION AND CONCLUSION: These findings demonstrate that Ocimum species have antimelanoma and radioprotective activity against B(16)F(10) metastatic melanoma cell line-induced metastasis and could be exploited as one of the potential sources for plant-based pharmaceutical products.

Isolation optimisation, synthesis, molecular docking and in silico ADMET studies of lantadene a and its derivatives.

A simple and economical method was developed for the extraction and isolation of pentacyclic triterpenoid lantadene A from the leaves of Lantana camara. The lantadene A displays significant anti-inflammatory and anticancer properties via the inhibition of IKK-mediated NF-κB protein. Therefore, the derivatives of lantadene A were synthesised to further optimise the pharmacophore for anti-inflammatory and anticancer activities. The synthesised compounds were docked into the active site of IKK to find the most potent inhibitor of IKK. Molecular docking studies revealed that 3ß,22ß-diisobutyl substituted lantadene derivative (10) binds to the IKK protein with the highest affinity. Furthermore, in the in silico ADMET studies, the lead IKK inhibitor (10) was found to be Ames non-toxic, non-carcinogen, and a weak inhibitor of hERG.[Figure: see text].

Bioinformatic Analyses of Canonical Pathways of TSPOAP1 and its Roles in Human Diseases.

TSPO-associated protein 1 (TSPOAP1) is a cytoplasmic protein and is closely associated with its mitochondrial transmembrane protein partner translocator protein (TSPO). To decipher the canonical signalling pathways of TSPOAP1, its role in human diseases and disorders, and relationship with TSPO; expression analyses of TSPOAP1- and TSPO-associated human genes were performed by Qiagen Ingenuity Pathway Analysis (IPA). In the expression analysis, necroptosis and sirtuin signalling pathways, mitochondrial dysfunction, and inflammasome were the top canonical pathways for both TSPOAP1 and TSPO, confirming the close relationship between these two proteins. A distribution analysis of common proteins in all the canonical pathways predicted for TSPOAP1 revealed that tumor necrosis factor receptor 1 (TNFR1), vascular cell adhesion molecule 1 (VCAM1), cyclic AMP response element-binding protein 1 (CREB1), T-cell receptor (TCR), nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 (NLRP3), DNA-dependent protein kinase (DNA-PK or PRKDC), and mitochondrial permeability transition pore (mPTP) were the major interaction partners of TSPOAP1, highlighting the role of TSPOAP1 in inflammation, particularly neuroinflammation. An analysis of the overlap between TSPO and TSPOAP1 Homo sapiens genes and top-ranked canonical pathways indicated that TSPO and TSPOAP1 interact via voltage-dependent anion-selective channels (VDAC1/2/3). A heat map analysis indicated that TSPOAP1 has critical roles in inflammatory, neuroinflammatory, psychiatric, and metabolic diseases and disorders, and cancer. Taken together, this information improves our understanding of the mechanism of action and biological functions of TSPOAP1 as well as its relationship with TSPO; furthermore, these results could provide new directions for in-depth functional studies of TSPOAP1 aimed at unmasking its detailed functions.

Pharmacological and genetic targeting of 5-lipoxygenase interrupts c-Myc oncogenic signaling and kills enzalutamide-resistant prostate cancer cells via apoptosis.

Much of the morbidity and mortality due to prostate cancer happen because of castration-resistant prostate cancer (CRPC) which invariably develops after anti-androgenic therapy. FDA-approved enzalutamide is commonly prescribed for CRPC which works by blocking androgen receptor function. However, even after initial good response, enzalutamide-resistant prostate cancer (ERPC) develops which eventually leads to widespread metastasis. Management of ERPC is extremely difficult because available therapeutic regimen cannot effectively kill and eliminate ERPC cells. Though the mechanism behind enzalutamide-resistance is not properly understood, over-activation of c-Myc has been found to be a common event which plays an important role in the maintenance and progression of ERPC phenotype. However, direct-targeting of c-Myc poses special problem because of its non-enzymatic nature and certain amount of c-Myc activity is needed by non-cancer cells as well. Thus, c-Myc has emerged as an elusive target which needs to be managed by novel agents and strategies in a cancer-specific way. We investigated the effects of pharmacological and genetic inhibition of 5-lipoxygenase (5-Lox) on cell proliferation, apoptosis and invasive potential of enzalutamide-resistant prostate cancer cells. Transcriptional activity of c-Myc was analyzed by DNA-binding, luciferase-assays, and expression of c-Myc-target genes. We found that 5-Lox regulates c-Myc signaling in enzalutamide-resistant prostate cancer cells and inhibition of 5-Lox by Quiflapon/MK591 or shRNA interrupts oncogenic c-Myc signaling and kills ERPC cells by triggering caspase-mediated apoptosis. Interestingly, MK591 does not affect normal, non-cancer cells in the same experimental conditions. Our findings indicate that inhibition of 5-Lox may emerge as a promising new approach to effectively kill ERPC cells sparing normal cells and suggest that development of a long-term curative therapy of prostate cancer may be possible by killing and eliminating ERPC cells with suitable 5-Lox-inhibitors.

HDAC1 Governs Iron Homeostasis Independent of Histone Deacetylation in Iron-Overload Murine Models.

AIMS: Iron-overload disorders are common and could lead to significant morbidity and mortality worldwide. Due to limited treatment options, there is a great need to develop novel strategies to remove the excess body iron. To discover potential epigenetic modulator in hepcidin upregulation and subsequently decreasing iron burden, we performed an epigenetic screen. The in vivo effects of the identified compounds were further tested in iron-overload mouse models, including Hfe-/-, Hjv-/-, and hepatocyte-specific Smad4 knockout (Smad4fl/fl;Alb-Cre+) mice. RESULTS: Entinostat (MS-275), the clinical used histone deacetylase 1 (HDAC1) inhibitor, was identified the most potent hepcidin agonist. Consistently, Hdac1-deficient mice also presented higher hepcidin levels than wild-type controls. Notably, the long-term treatment with entinostat in Hfe-/- mice significantly alleviated iron overload through upregulating hepcidin transcription. In contrast, entinostat showed no effect on hepcidin expression and iron levels in Smad4fl/fl;Alb-Cre+ mice. Further mechanistic studies revealed that HDAC1 suppressed expression of hepcidin through interacting with SMAD4 rather than deacetylation of SMAD4 or histone-H3 on the hepcidin promoter. INNOVATION: The findings uncovered HDAC1 as a novel hepcidin suppressor through complexing with SMAD4 but not deacetylation of either histone 3 or SMAD4. In addition, our study suggested a novel implication of entinostat in treating iron-overload disorders. CONCLUSIONS: Based on our results, we conclude that entinostat strongly activated hepcidin in vivo and in vitro. HDAC1 could serve as a novel hepcidin suppressor by binding to SMAD4, effect of which is independent of BMP/SMAD1/5/8 signaling. Antioxid. Redox Signal. 28, 1224-1237.

In Vitro and In Vivo Evaluation of Small Cationic Abiotic Lipopeptides as Novel Antifungal Agents.

We investigated the antifungal potential of short lipopeptides against clinical fungal isolates with an objective to evaluate their clinical feasibility. All tested lipopeptides exhibit good antifungal activity with negligible difference between the MICs against susceptible and drug-resistant clinical fungal isolates. The MTT assay results revealed the lower cytotoxicity of lipopeptides toward mammalian cells (NRK-52E). In particular, LP24 displayed highest potency against most of the tested fungal isolates with MICs in the range of 1.5-4.5 µg/mL. Calcein dye leakage experiments with model membrane suggested the membrane-active mode of action for LP24. Extending our work from model membranes to intact Aspergillus fumigatus in scanning electron micrographs, we could visualize surface perturbation caused by LP24. LP24 (5 mg/kg) significantly reduces the A. fumigatus burden among the various organs of infected animals, and 70% of the infected mice survived when observed for 28 days. This study underscores the potential of small cationic abiotic lipopeptides to develop into the next-generation antimicrobial therapy.

Topical (+)-catechin emulsified gel prevents DMBA/TPA-induced squamous cell carcinoma of the skin by modulating antioxidants and inflammatory biomarkers in BALB/c mice.

An emulsified gel of (+)-catechin was developed and evaluated topically against 7,12-dimethylbenz(a)anthracene-induced and 12-O-tetradecanoylphorbol-13-acetate-promoted (DMBA-induced and TPA-promoted) squamous cell carcinoma of the skin in BALB/c mice. The biological evaluation outcome indicated that the (+)-catechin emulsified gel increased the activity of oxidative stress biomarkers glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), glutathione reductase (GR), and glutathione peroxidase (GPx), whereas it decreased the level of malondialdehyde (MDA). The mechanistic study showed that genes implicated in the inflammation and cancer, such as cyclooxygenase-2 (COX-2), nuclear factor-kappa B (NF-κB), and inducible nitric-oxide synthase (iNOS), were down-regulated by (+)-catechin emulsified gel while inhibiting an inflammatory mediator prostaglandin E2 (PGE2). The (+)-catechin emulsified gel further suppressed the activity of pro-inflammatory cytokines, viz. tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), and interleukin-6 (IL-6). The in vitro permeation study revealed that release of (+)-catechin from an emulsified gel base reached a steady state after 6 h, while pH of the entire emulsified gel was found to be between 6.2 and 6.5 that falls well within the normal pH range of the skin.

In vitro and in vivo antibacterial evaluation and mechanistic study of ornithine based small cationic lipopeptides against antibiotic resistant clinical isolates.

We investigated the in vitro activities of short lipopeptides against a large panel of clinical isolates of antibiotic resistant bacteria. In the animal model, LP16 (5 mg/kg) significantly decreased the burden of viable colony forming unit (CFU) of bacteria. MTT assay results revealed the high selectivity of lipopeptides toward microbial cells. Calcein dye leakage experiments and flow cytometric analysis suggests the membranolytic effect of lipopeptides, which was further confirmed by visualizing bacterial damage via electron microscopy tool (SEM & TEM). Moreover, stability in human blood plasma and no sign of resistance development against clinical isolates of Escherichia coli and Staphylococcus aureus were observed for lead lipopeptides. These results demonstrate the potential of short lipopeptides as a novel class of anti-infectives.

Human breast adenocarcinoma cytotoxicity and modulation of 7,12-dimethylbenz[a]anthracene-induced mammary carcinoma in Balb/c mice by Acacia catechu (L.f.) Wild heartwood.

OBJECTIVE: The chemopreventive potential of (+)-catechin-rich extract of Acacia catechu (L.f.) Willd. heartwood (AQCE) was evaluated against human breast adenocarcinoma cell line (MCF-7) and 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinoma in Balb/c mice. METHODS: Cell cytotoxicity was investigated using different colorimetric assays. Apoptosis was observed using diphenylamine assay and fluorescent microscopy. AQCE was further evaluated for antitumor activity against DMBA-induced mammary carcinoma. The levels of tumor markers and oxidative stress were measured. Furthermore, level of transcription factors was measured by enzyme-linked immunosorbent assay. RESULTS: The results showed that administration of AQCE showed a dose-dependent growth inhibition response and DNA fragmentation in MCF-7 cells. Tumor multiplicity was significantly decreased to 42.91% with AQCE when compared with DMBA-treated animals. The levels of tumor markers such as total sialic acid and lipid-associated sialic acid were substantially increased after DMBA treatment. However, AQCE treatment restored tumor markers level. AQCE also significantly reduced elevated levels of nitrite and malondialdehyde in DMBA-treated animals. Additionally, AQCE also increased the activities of antioxidant enzymes, viz., catalase, superoxide dismutase, total thiol, reduced glutathione, protein thiol, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase in the mammary tissue and liver mitochondria of DMBA-administered animals. Significant increase in the protein levels of p53, c-jun, and p65 were observed in DMBA-treated mice, whereas less expression was observed in AQCE-treated animals. Eventually, AQCE also significantly improved body weight and maintained the mammary tissue architecture in normal range. CONCLUSIONS: The present data strongly suggest that anticancer potentiality of (+)-catechin-rich AQCE may be attributable to its ability to positively modulate tumor markers as well as the antioxidant system that could decompose the peroxides and, thereby, offer a protection against lipid peroxidation and linked to the expression of transcription factors during DMBA-induced mammary carcinoma.

Cytotoxicity and apoptosis induction in human breast adenocarcinoma MCF-7 cells by (+)-cyanidan-3-ol.

The objective of this study was to evaluate the cytotoxicity and possible signalling pathway implicated in (+)-cyanidan-3-ol (CD-3) induced apoptosis in the human breast adenocarcinoma cell line (MCF-7). The effects of CD-3 on cell proliferation of MCF-7 cells were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), sulforhodamine B (SRB) and lactate dehydrogenase (LDH) assays. Cell apoptosis was detected by Hoechst 33258 (HO) and acridine orange/ethylene dibromide (AO/EB) staining and DNA fragmentation analysis. The expressions of apoptosis-related genes were assessed by RT-PCR and ELISA. Our data revealed that CD-3 induced MCF-7 cell death in a dose-dependent manner. Marked changes in apoptotic morphology was clearly observed after CD-3 treatment. CD-3 induced cell death was considered to be apoptotic by observing the typical apoptotic morphological change under fluorescent microscopy and DNA fragmentation assays. The induction of apoptosis is correlated with the increased mRNA expressions of p53, Bax, and caspase-3, -7, -8 and -9 and decreased mRNA expressions of bcl-2. Subsequently, CD-3 decreased the mRNA expressions of mdm2, p65, c-jun, c-fos in MCF-7 cells. The protein levels of p53, Bax, and caspase-3 were increased, whereas, that of p65, c-jun and Bcl-2 were decreased in MCF-7 cells on CD-3 treatment. These results clearly demonstrated that CD-3 effectively induced growth inhibition and apoptosis in MCF-7 cells.

Growth inhibition and apoptosis induction by (+)-Cyanidan-3-ol in hepatocellular carcinoma.

The objective of this study was to evaluate the cytotoxicity of (+)-cyanidan-3-ol (CD-3) in human hepatocellular carcinoma cell line (HepG2) and chemopreventive potential against hepatocellular carcinoma (HCC) in Balb/c mice. The HepG2 cell line was treated with CD-3 at various concentrations and the proliferation of the HepG2 cells was measure by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), sulforhodamine B (SRB) and lactate dehydrogenase (LDH) assays. Cell apoptosis was detected by Hoechst 33258 (HO), Acridine orange/ethylene dibromide (AO/EB) staining, DNA fragmentation analysis and the apoptosis rate was detected by flow cytometry. The HCC tumor model was established in mice by injecting N-nitrosodiethylamine/carbon tetrachloride (NDEA/CCl4) and the effect of CD-3 on tumor growth in-vivo was studied. The levels of liver injury markers, tumor markers, and oxidative stress were measured. The expression levels of apoptosis-related genes in in-vitro and in vivo models were determined by RT-PCR and ELISA. The CD-3 induced cell death was considered to be apoptotic by observing the typical apoptotic morphological changes under fluorescent microscopy and DNA fragmentation analysis. Annexin V/PI assay demonstrated that apoptosis increased with increase in the concentration of CD-3. The expression levels of apoptosis-related genes that belong to bcl-2 and caspase family were increased and AP-1 and NF-κB activities were significantly suppressed by CD-3. Immunohistochemistry data revealed less localization of p53, p65 and c-jun in CD-3 treated tumors as compared to localization in NDEA/CCl4 treated tumors. Taken together, our data demonstrated that CD-3 could significantly inhibit the proliferation of HepG2 cells in-vitro and suppress HCC tumor growth in-vivo by apoptosis induction.

Chemopreventive efficacy of (+)-catechin-rich aqueous extract of Acacia catechu Willd. Heartwood against 7,12-dimethylbenz[a]anthracene-induced hepatocarcinoma in Balb/c mice.

The objective of this study was to investigate the chemopreventive potential of (+)-catechin-rich extract of Acacia catechu heartwood (AQCE) against 7,12-dimethylbenz[a]anthracene (DMBA)-induced hepatocellular carcinoma in Balb/c mice. The levels of liver injury markers, tumor markers, and oxidative stress were measured in serum and liver tissues. Furthermore, the levels of transcription factors were measured by ELISA. Tumor incidence was found to be 100% in DMBA-treated animals (group 2), whereas, in AQCE-treated animals (group 3), it was 37.5%. AQCE treatment reduced liver injury and restored tumor-marker levels. AQCE also significantly reduced elevated levels of nitrite and hepatic malondialdehyde (MDA) in DMBA-treated animals. Additionally, AQCE modulated the activity of different antioxidant enzymes in liver tissues. Eventually, AQCE also significantly improved body weight, prevented the increase of relative liver weight, and maintained the liver cellular architecture within the normal range of the control. A significant increase in the protein levels of p53, c-jun, and NF-κB (p65) were observed in DMBA-treated mice, whereas low levels of these markers were observed in DMBA+AQCE-treated animals. These findings strongly suggest (1) that (+)-catechin-rich AQCE exerts a chemopreventive effect by modulating the levels of lipid peroxidation and by promoting the enzymatic and non-enzymatic antioxidant defense system and (2) that this effect is linked to the expression of transcription factors during hepatocarcinogenesis.

Human epithelial carcinoma cytotoxicity and inhibition of DMBA/TPA induced squamous cell carcinoma in Balb/c mice by Acacia catechu heartwood.

OBJECTIVES: Acacia catechu heartwood contains significant amounts of polyphenolic compounds that exhibit powerful antioxidant activity. The purpose of this study was to evaluate the cytotoxicity of A. catechu heartwood extracts in a human epithelial carcinoma cell line (A431) and antitumour activity against DMBA/TPA induced squamous cell carcinoma in Balb/c mice. METHODS: Various extracts, including aqueous, ethyl acetate, chloroform and n-hexane, were tested for cytotoxic properties on a human epithelial carcinoma cell line (A431) by using MTT, sulforhodamine B and lactate dehydrogenase leakage assays. The standardized A. catechu heartwood aqueous extract (AQCE) was further evaluated for antitumour activity against 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) induced skin carcinoma in Balb/c mice. KEY FINDINGS: The results showed that administration of AQCE showed a dose-dependent growth inhibition response, with an IC50 value of 78.56 µg/ml. Tumour incidence was significantly decreased (P < 0.001) to 30% with AQCE compared with 100% in the DMBA/TPA group. The AQCE was also found to significantly upregulate different antioxidant enzymes in skin and liver tissue. CONCLUSIONS: The results suggest that AQCE may exert its chemopreventive activity by acting as an antioxidant.

Calcified thoracic intervertebral disc at two levels as a cause of mid-back pain in a child: a case report.

An uncommon case of calcified disc at two levels in the thoracic spine is presented. A 12-year-old girl presented with intervertebral thoracic disc calcification from the levels D7-D8 and D11-D12. Level D7-D8 remained asymptomatic, whereas at level D11-D12 she had spontaneous pain for 10 months. Her symptoms were self-limiting and disappeared with bed rest, analgesics, and the use of a brace. The radiological changes were the same at 4-year follow-up, but the patient remained asymptomatic.