Urease and human glioblastoma (U87) cells growth inhibitory iridoid glycoside acetates from Nyctanthes arbor-tristis Linn.

The present study was undertaken on the chemical constituents of ethanol extract of aerial parts of Nyctanthes arbor-tristis Linn., and their determination of growth inhibitory activity against glioblastoma multiforme cell line (U87) and urease inhibitory activity. Six constituents were isolated including two new arbortristoside F tetraacetate (1) and arbortristoside G heptaacetate (2) and four known arborside A tetraacetate (3), arborside C pentaacetate (4), 6,7-di-O-acetyl-6ß-hydroxyloganin hexaacetate (5) and nyctanthoside heptaacetate (6) iridoid glycoside acetates. Their structures were elucidated using spectroscopic methods, including 1D and 2D NMR and mass analyses. Compounds 3 and 6 showed significant growth inhibition of U87 cell line (76.41 and 87.62% inhibition) respectively while 2, 4 and 5 showed moderate inhibition. 3 and 6 showed notable urease inhibition (IC50 = 17.2 ± 0.4 and 17.2 ± 0.7 µM) respectively, and IC50 of 2, 4 and 5 were 23.8 - 56.3 µM. Compound 1 was inactive.

Modulation of Apoptotic and Akt/PI3K/mTOR pathways to target Glioblastoma Cells using synthetic compound PGEA-AN.

Objectives: To investigate the anticancer potential of a novel synthetic derivative of a naturally occurring diterpenoid against glioblastoma. METHODS: The in vitro study was conducted at the Ojha Campus of Dow University of Health Sciences, Karachi, from February to December 2021, and comprised U87 cells. The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine the growth inhibitory effect of 16(R and S) - phenylamino-cleroda3, 13(14) Zdiene- 15, 16 olide and standard drug temozolomide against glioblastoma cells, and half-maximal inhibitory concentration was calculated. Microscopy and immunocytochemistry were used to investigate apoptotic morphology and active caspase-3 and B-cell lymphoma 2 (Bcl-2) expression. Quantitative real time polymerase chain reaction was used to investigate the expression of proliferation markers. Data was analysed using SPSS 21. RESULTS: Both the synthetic derivative and the standard drug significantly inhibited growth of U87 cells (p<0.001) with half-maximal inhibitory concentration of 19µM and 185µM, respectively. Apoptotic morphology and upregulation of active caspase-3 protein expression was observed in cells treated with half-maximal inhibitory concentration doses of both the synthetic derivative (p<0.05) and the standard drug (p<0.001), and Bcl-2 was downregulated in both the synthetic derivative (p<0.01) and the standard drug (p=0.05). However, no significant difference was observed in the expression of proliferation markers (p>0.05). CONCLUSIONS: The synthetic diterpene derivative PGEA-AN showed growth inhibitory actiity against glioblastoma.

Neurogenin-2 induced neuronal differentiation in co-cultured bone marrow stromal and neonatal cortical cells.

Objective: To explore a novel and dynamic role for neurogenin-2 in promoting cortical neurogenesis in cells produced from co-culturing neonatal cortical neural progenitor cells with bone marrow stromal cells. METHODS: The experimental study was conducted from June 2016 to January 2019 at the neuropharmacology laboratory of the Hussein Ebrahim Jamal Research Institute of Chemistry, International Centre for Chemical and Biological Sciences, Karachi. The growth of cells at different stages in harvested cells was determined by 3-(4, 5- dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay. Immunocytochemistry was used to evaluate the protein expressions of neuronal markers and transcription factors. Data was analysed using SPSS 20. RESULTS: Data showed significant generation of neuronal cells and this was also verified by increased expression of nesting in cortical co-cultures with bone marrow stromal cells. Immunoreactive outcomes showed over expressions in co-cultured chlorotoxin cells. Subsequently, neurogenin-2 was found intermixed with induced expressions of transcriptional factor NeuroD1 and reduced glial fibrillary acidic protein-labelled cells. Conclusion: Better understanding of the mechanisms underlying transcriptional modulation of neurogenic events hold the key for emerging treatment approaches towards neurodegeneration.

Alpha terpineol directs bone marrow mesenchymal stem cells toward neuronal lineage through regulation of wnt signaling pathway.

Central nervous system anomalies give rise to neuropathological consequences with immense damage to the neuronal tissues. Cell based therapeutics have the potential to manage several neuropathologies whereby the differentiated cells are explored for neuronal regeneration. The current study analyzes the effect of a bioactive compound, alpha terpineol (AT) on the differentiation of rat bone marrow derived mesenchymal stem cells (BM-MSCs) toward neuronal lineage, and explores regulation of differentiation process through the study of Wnt pathway mediators. BM-MSCs were cultured and characterized based on their surface markers and tri-lineage differentiation. Safe dose of AT as optimized by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium bromide assay, was used for the treatment of MSCs. Treated cells were analyzed for the neuronal, astroglial and germ layer transition markers at the gene and protein levels, by quantitative polymerase chain reaction and immunocytochemistry, respectively. Temporal expression of Wnt pathway genes was assessed during the course of neuronal differentiation. AT treated group showed significant upregulation of neuron specific (NSE, MAP2, Tau, Nestin, and NefL) and astroglial (GFAP) genes with positive expression of late neuronal markers. Germ layer transition analysis showed the overexpression of ectodermal markers (NCAM, Nestin, and Pax6), whereas endodermal (AFP, MixL1, and Sox17), and mesodermal (Mesp1 and T Brachyury) markers were also found to be upregulated. Wnt signaling pathway was activated during the initial phase (30 min) of differentiation, which later was downregulated at 1, 3, and 5 h. AT efficiently induces neuronal differentiation of BM-MSCs by regulating Wnt signaling. Overexpression of both early and late neuronal markers indicate their neuro-progenitor state and thus can be utilized as a promising approach in cellular therapeutics to treat various neurodegenerative ailments. In addition, exploration of the molecular pathways may be helpful to understand the mechanism of cell-based neuronal regeneration.

Opuntiol Inhibits Growth and Induces Apoptosis in Human Glioblastoma Cells by Upregulating Active Caspase 3 Expression.

BACKGROUND: Glioblastoma Multiforme (GBM) is a deadly tumor with poor prognosis. Resistance to apoptosis considered as an important factor in treatment failure. Therefore, identification of new compounds that facilitates apoptosis is crucial. Natural Anti-inflammatory compounds have emerged as potential anti-cancer agents and should be explored for their apoptotic activity against GBM. Therefore, the present study aims to evaluate growth inhibitory and apoptotic activity of a natural anti-inflammatory compound "Opuntiol" against GBM cell line U87. METHODS: MTT assay was performed to determine the effect of Temozolomide and Opuntiol on growth inhibition of U87 cell. While, TUNEL assay was used to assess their apoptotic activity. To further assess apoptosis, nuclear condensation and nuclear area factor (NAF) was evaluated through DAPI staining. Whereas, active caspase-3 protein expression determined using immunocytochemistry. RESULTS: Significant growth inhibition was observed in U87 cells treated with Temozolomide (IC50 380 µM) and Opuntiol (IC50 357 µM). Temozolomide (p<0.001) and Opuntiol (p<0.001) significantly improved rate of apoptosis when compared to control group. A significant decrease in NAF was also observed in Temozolomide (p < 0.05) and Opuntiol (p < 0.05) treated cells. There was a significant increase in active caspase-3 expression when observed in Temozolomide (p<0.001) and Opuntiol (p<0.05) treated groups as compared to control. CONCLUSION: In conclusion our findings suggests, Opuntiol repress cell viability and possess strong apoptotic activity against GBM cell line U-87. However, further mechanistic studies will be required to confirm whether it can be develop as a potential drug against GBM.

Synthesis of lactobionic acid based bola-amphiphiles and its application as nano-carrier for curcumin delivery to cancer cell cultures in-vitro.

Curcumin is highly effective against various types of cancers; however, its low aqueous solubility, high metabolism and non-specificity hinder its efficacy. This study reports the synthesis of three lactobionic acid containing bola-amphiphiles and their investigation for curcumin nano-vesicular delivery into cancer cells. Synthesized bola-amphiphiles were capable of forming nano-vesicles and curcumin loading in a lipophilicity dependent manner. Bola-amphiphile with higher lipophilicity (C12) caused 89.55 ± 5.52% drug encapsulation in its spherical shape nano-vesicles (195.90 ± 0.83 nm). Bola-amphiphile resulting increased curcumin encapsulation with minimum vesicles size was further investigated for cellular uptake and in-vitro anticancer activity. Anticancer activity of curcumin significantly increased against the tested cancer cells upon loading in bola-amphiphile nano-vesicles. Furthermore, nano-vesicular drug delivery of curcumin enhanced its cellular uptake even at the lowest concentration of 1.25 µg/mL.It is concluded that the synthesized bola-amphiphile based nano-vesicles can efficiently deliver curcumin to the tested cancer cells and needs to be tested for established anticancer drugs against different cancer cell lines for effective treatment of cancer.

The effects of aspirin gel and mouthwash on levels of salivary biomarkers PGE2, TNF-α and nitric oxide in patients with periodontal diseases.

Inflammation and its mediators have an important role in gingivitis and periodontitis. Prostaglandin is one of the eicosanoid involved in many chronic inflammatory diseases, including periodontal diseases. Aspirin irreversibly acetylates cyclooxygenase and inactivate this enzyme responsible for the production of PGE2 that mediates pain and inflammation. The aim of the study was to prepare aspirin gel and mouthwash in 1% concentration and use it in patients with periodontal diseases during the non-surgical periodontal treatment and to assess its anti-inflammatory effects on salivary biomarkers PGE2, TNF-α, and nitric oxide. Thirty patients were divided into three treatment groups, standard treatment group, second received scaling and root planning with gel application of 1% aspirin, third received scaling and root planning followed by rinsing with 1% aspirin mouthwash. Results indicated that the levels of PGE2, TNF-α and nitric oxide in the groups of patients received gel treatment and mouthwash treatment was decreased to significant levels (p<0.001) as compared to the group of standard treatment. Aspirin gel was found to be more effective in reducing inflammatory biomarkers in contrast to aspirin mouthwash (p<0.001). We concluded from our study, that low concentration of aspirin oral preparations are highly active in reducing the inflammatory biomarkers associated with periodontal diseases.

N-(2-hydroxyphenyl) acetamide (NA-2) elicits potent antitumor effect against human breast cancer cell line (MCF-7).

Breast cancer is the most dominating malignancy in females worldwide. Treatment with conventional chemotherapeutics is associated with severe adverse effects. Thus need of new compounds, with better therapeutic potential and lesser side effects still exist. In this context the present study is planned to investigate therapeutic potential of anti-inflammatory compound N-(2- hydroxyphenyl) acetamide (NA-2) against breast cancer cells (MCF-7). The compound was selected on the basis of its reported anti-inflammatory, anti-arthritic and anti-glioblastoma activities in our previous studies. MTT, Annexin-V-FITC and wound healing assays were used to analyze the effect of compound on growth inhibition, apoptosis and metastasis. While flow cytometry, RT-PCR and immunocytochemistry techniques were used to assess the effect of NA-2 on cell cycle arrest, and expression of apoptotic markers (Bax and Bcl-2) at both mRNA and protein level respectively. Data analysis revealed that NA-2 significantly inhibits growth of MCF-7 cells after 48 h treatment (IC50 = 1.65 mM). NA-2 also delayed the wound healing process, arrested cell cycle at G0/G1 phase and induced apoptosis by enhancing Bax/Bcl-2 ratio. We concluded that NA-2 possesses strong anticancer activity against MCF-7 cells, which is mediated through different mechanisms, making it a useful molecule for the development of new antitumor drugs.

Pyrazinium thioacetate capped gold nanoparticles as Fe(III) sensor and Fe(III) marked anti-proliferating agent in human neuroblastoma cells.

Gold nanoparticles (AuNPs) stabilized by new cationic 1­(3­(acetylthio)propyl)pyrazin­1­ium ligand (PPTA) were synthesized. AuNPs stabilized by PPTA (PPTA-AuNPs) were found to be spherical and polydispersed with the average size of 60 nm. Human neuroblastoma (SHSY-5Y) cells permeability of PPTA-AuNPs was found to be a key feature to study the intracellular quenching of Fe(III) proliferative activity. In vitro MTT assay revealed non-cytotoxicity of PPTA and PPTA-AuNPs at 100 µM concentration, while treatment of 100 µM of Fe(III) with SHSY-5Y cells resulted into higher cells viability. Contrary, a mixture of 1:1 Fe(III) with PPTA-AuNPs showed no change in the viability of cells at same concentration which suggests the intracellular complexation and recognition of Fe(III) by PPTA-AuNPs. AFM morphological analysis of SHSY-5Y cells also supported the MTT assay results, and it is safe to conclude that PPTA-AuNPs can be used as Fe(III) probes in living cells. In addition, Fe(III) caused a significant decrease in the absorbance of surface plasmon resonance (SPR) band of PPTA-AuNPs in a wide range of concentration and pH, with limit of detection 4.3 µM. Moreover, the specific response of PPTA-AuNPs towards Fe(III) was unaffected by the interference of other metals and components of real samples of tap water.

N-(2-hydroxyphenyl)acetamide and its gold nanoparticle conjugation prevent glycerol-induced acute kidney injury by attenuating inflammation and oxidative injury in mice.

The protective activity of N-(2-hydroxyphenyl)acetamide (NA-2) and NA-2-coated gold nanoparticles (NA-2-AuNPs) in glycerol-treated model of acute kidney injury (AKI) in mice was investigated. NA-2 (50 mg/kg) and NA-2-AuNPs (30 mg/kg) were given to the animals for four days followed by 24-h water deprivation and injection of 50% glycerol (10 ml/kg im). The animals were sacrificed on the next day. Blood and kidneys were collected for biochemical investigations (urea and creatinine), histological studies (hematoxylin and eosin; and periodic acid-Schiff staining), immunohistochemistry (actin and cyclooxygenase-2, Cox-2), and real-time RT-PCR (inducible nitric oxide synthase, iNOS; nuclear factor-κB p50, NFκB; hemeoxygenase-1, HO-1; and kidney injury molecule-1, Kim-1). NA-2 protected renal tubular necrosis and inflammation, though the result of NA-2-AuNPs was better than compound alone and it also exhibited the activity at far less dose. The test compound and its gold nano-formulation decreased the levels of serum urea and creatinine level in the treated animals. Both NA-2 and NA-2-AuNPs also conserved actin cytoskeleton, and lowered COX-2 protein expression. Moreover, the mRNA expressions of iNOS and NFkB p50 were down-regulated, and HO-1 and Kim-1 genes were up-regulated. We conclude that NA-2 and NA-2-AuNPs ameliorates kidney inflammation and injury in glycerol-induced AKI animal model via anti-oxidant and anti-inflammatory mechanisms which make it a suitable candidate for further studies. We believe that these findings will contribute in the understanding of the mechanism of action of paracetamol-like drugs and can be considered for clinical research for the prevention of AKI.

Design and synthesis of mixed micellar system for enhanced anticancer efficacy of Paclitaxel through its co-delivery with Naringin.

Emergence of multidrug resistance (MDR) has limited the success of chemotherapeutic agents. Reversal of drugs efflux systems through combination therapy has got wider attention for increasing anticancer drugs efficacy. This study aims at co-encapsulation of Paclitaxel with Naringin in mixed polymeric micelles for enhanced anticancer activity of the drug. Drug-loaded micelles were prepared using two different amphiphilic block co-polymers and were characterized for morphology, size, zeta potential, drug encapsulation, in vitro release and stability using atomic force microscope (AFM), zetasizer, UV spectrophotometer, and FT-IR. MTT assay and fluorescence microscopy were used for in vitro cytotoxicity and cellular uptake studies. Nano-size micelles with spherical morphology and negative charge encapsulated 76.52 ± 0.94% and 32.87 0.61% Paclitaxel and Naringin, respectively. The micelles were thermally stable and retained 87.05 ± 0.69% and 92.88 ± 2.17% Paclitaxel and Naringin upon one-month storage. Maximum drug release was achieved at fourth hour of the study for both the loaded drugs. Paclitaxel co-encapsulation with Naringin synergistically improved its intracellular uptake and 65% in vitro cytotoxicity against breast cancer cells was achieved at its lower dose of 15 µg/mL. Results suggest that co-encapsulation of Paclitaxel with Naringin in mixed micelles is an effective strategy for achieving its higher anticancer activity.

N-(2-hydroxy phenyl) acetamide: a novel suppressor of Toll-like receptors (TLR-2 and TLR-4) in adjuvant-induced arthritic rats.

Toll-like receptors (TLRs) are key recognition structures of immune system and recently emerged as potential contributors to the inflammation observed in human and rodent models of arthritis. Present study aims to investigate the effect of N-(2-hydroxy phenyl)-acetamide (NA-2) on modulation of TLRs in the development of adjuvant-induced arthritis. Arthritis was induced by intradermal administration of heat-killed Mycobacterium tuberculosis H37Ra. The treatment of NA-2 (5 mg/kg) and indomethacin (5 mg/kg) was started in their respective group on the day of arthritis induction. Body weights, paw volume measurements, and nociception sensation (Plantar test) were done on alternate days to monitor the progression of the disease until arthritis score of four was observed in arthritic control group. Along with the clinical signs, histopathology of knee joints was also performed. The splenocytes cultures were prepared from each group; TLR-2 and TLR-4 mRNAs were analyzed in 48-h cultured splenocytes using RT-PCR; and the supernatants were used to determine IL-1ß and TNF-α by ELISA. A significant reversal of deficit seen in body weights of the arthritic control group was observed in NA-2-treated animals with a parallel decrease in paw edema and transmission of nociception. Remission of the clinical signs and nociception was associated with improved histology. Compared with arthritic control, NA-2 treatment significantly decreased the level of IL-1ß (p < 0.003) and TNF-α (p < 0.001) in the supernatants of cultured splenocytes. Likewise, NA-2 also reduced the expression of TLRs mRNA. Our findings suggest that NA-2 affects AIA in a pleiotropic manner, suppressing TLRs-mediated joint inflammation and related symptoms.

Modulation of c-Fos and BDNF protein expression in pentylenetetrazole-kindled mice following the treatment with novel antiepileptic compound HHL-6.

Brain-derived neurotrophic factor (BDNF) and c-Fos are shown to promote epileptogenesis and are taken as a marker of neuronal activity. The present study investigated the expression of BDNF and c-Fos in mice brain with pentylenetetrazol- (PTZ-) induced generalized seizure and evaluated the effect of novel tryptamine derivative HHL-6 on the expression of these two markers. The subconvulsive dose of PTZ (50 mg/kg) was administered on alternate days in the experimental groups until the seizure scores 4-5 developed in the PTZ-control group. At the end of each experiment, animals were sacrificed, brain samples were collected and cryosectioned, and immunohistochemical analysis of BDNF and c-Fos protein was performed. Data obtained from two sections per mouse (n = 12 animals/group) is presented as means ± S.E.M. The test compound HHL-6 demonstrated a potent anticonvulsant activity in the PTZ-induced seizure in mice. Significant reduction in the BDNF (P < 0.003) and c-Fos (P < 0.01) protein expression was observed in the HHL-6 treated group. Based on these results we suggest that one of the possible mechanisms of HHL-6 to inhibit epileptogenesis might be due to its controlling effect on the cellular and molecular expression of the factors that contribute to the development of epileptogenic plasticity in the CNS.

The anti-arthritic and anti-oxidative effect of NBD (6-nitro-1,3-benzodioxane) in adjuvant-induced arthritis (AIA) in rats.

OBJECTIVES: The present study evaluated the anti-arthritic and anti-oxidative effects of 6-nitro-1,3-benzodioxane in the adjuvant-induced arthritis model in rats. METHODS: Arthritis was induced in female rats by intradermal injection of MT37Ra. Arthritis was evaluated by arthritic score, body weight loss, paw volume measurement, and histological changes. The plantar test was used to evaluate the effect of NBD on hyperalgesia. RESULTS: The hyperalgesia (p < 0.0001) and hind paw inflammation (p < 0.034) was significantly decreased with parallel increase in the body weight of the NBD-treated (25 mg/kg) group compared to arthritic control rats. The antioxidant activity analysis demonstrated that the treatment of NBD significantly suppressed the levels of nitric oxide (p < 0.001) and peroxide (p < 0.002) with a significant increase in the glutathione (p < 0.021) compared to the arthritic control group. Since the IL-1ß and TNF-α are key pro-inflammatory cytokines in arthritis, we therefore measured their levels in the serum samples. In comparison to the arthritic control group, the NBD treatment significantly reduced the levels of IL-1ß (p < 0.003) and TNF-α (p < 0.026). CONCLUSION: Our results suggests that NBD is an anti-arthritic agent that not only reduces the severity of the disease process but also affects contributing factors of arthritic inflammation including free radicals and inflammatory cytokines production.

Suppression of c-Fos protein and mRNA expression in pentylenetetrazole-induced kindled mouse brain by isoxylitones.

An early immediate gene c-fos has been proposed as the gene responsible for turning on molecular events that might underlie the long-term neural changes occurring during kindling. We have evaluated the effects of novel anticonvulsant isomeric compounds isoxylitones [(E/Z)-2-propanone-1,3,5,5-trimethyl-2-cyclohexen-1-ylidine] on the c-Fos protein and mRNA expression in the brain samples of kindled mice and compared it with the normal and untreated kindled groups. Kindling was induced in male NMRI mice by repeated administration of sub-convulsive dose (50 mg/kg) of pentylenetetrazole (PTZ) until a seizure score of 4-5 was achieved. The c-Fos expression was quantified by combination of immunohistochemistry and RT-PCR protocols. Both the immunohistochemical and RT-PCR analysis revealed a marked increase in the expression of c-fos mRNA and protein in the brain regions tested in case of PTZ-kindled control group compared to normal control. In contrast, the isoxylitone (30 mg/kg)-treated group demonstrated significant reduction of c-Fos expression compared to PTZ-kindled control animals. However, low expression of c-fos mRNA was only detected in the thalamus of the isoxylitone-treated brain samples. Based on these observations, we suggest that isoxylitones may have the capacity to control the seizure pattern by mechanism such as the suppression of c-Fos protein and mRNA levels in different regions of the brain. Further investigations to explore the mechanism of action of these compounds are under process.

N-(2-hydroxy phenyl) acetamide inhibits inflammation-related cytokines and ROS in adjuvant-induced arthritic (AIA) rats.

The present study was carried out to study the anti-arthritic and anti-inflammatory activity of N-(2-hydroxy phenyl) acetamide in adjuvant-induced arthritis in adult female Sprague Dawley rats. During experimental period, body weight and paw oedema volume were observed. At the end of each experiment, plasma and serum samples were collected and used for estimation of pro-inflammatory cytokines IL-1 beta and TNF-alpha and oxidative stress markers i.e., nitric oxide, peroxide and GSH. Our results suggested that, the reduction in body weight and increase in paw oedema volume were significantly retarded in the AIA rats receiving 5mg/kg and 10mg/kg doses of N-(2-hydroxy phenyl) acetamide as compared to diseased control animals. The serum levels of IL-1 beta and TNF-alpha were reduced as compared to those in the diseased control group. Treatment with N-(2-hydroxy phenyl) acetamide also altered oxidative stress markers in relation to its anti-inflammatory activity. Based on our results, it can be concluded that N-(2-hydroxy phenyl) acetamide possesses promising anti-arthritic property.