Macrobrachium rosenbergii nodavirus (MrNv) causes white tail disease (WTD) in giant freshwater prawns, which leads to devastating economic losses in the aquaculture industry. Despite extensive research on MrNv, there is still no antiviral agent to treat WTD. Thus, the main aim of this study was to identify potential anti-MrNv molecules. A 12-mer phage-displayed peptide library was biopanned against the MrNv virus-like particle (VLP). After four rounds of biopanning, two dominant phages harbouring the amino acid sequences HTKQIPRHIYSA and VSRHQSWHPHDL were selected. An equilibrium binding assay in solution was performed to determine the relative dissociation constant (KDrel) of the interaction between the MrNv VLP and the selected fusion phages. Phage-HTKQIPRHIYSA has a KDrel value of 92.4±22.8 nM, and phage-VSRHQSWHPHDL has a KDrel value of 12.7±3.8 nM. An in-cell elisa was used to determine the inhibitory effect of the synthetic peptides towards the entry of MrNv VLP into Spodoptera frugiperda (Sf9) cells. Peptides HTKQIPRHIYSA and VSRHQSWHPHDL inhibited the entry of the MrNv VLP into Sf9 cells with IC50 values of 30.4±3.6 and 26.5±8.8 µM, respectively. Combination of both peptides showed a significantly higher inhibitory effect with an IC50 of 4.9±0.4 µM. An MTT assay revealed that the viability of MrNv-infected cells increased to about 97 % in the presence of both peptides. A real-time RT-PCR assay showed that simultaneous application of both peptides significantly reduced the number of MrNv per infected cell, from 97±9 to 11±4. These peptides are lead compounds which can be further developed into potent anti-MrNv agents.
The aims of this study were to compare the effectiveness of different drying methods and to investigate the effects of adding a series of individual protectant such as skim milk, sucrose, maltodextrin, and corn starch for preserving Lactobacillus acidophilus FTDC 3081 cells during spray and freeze-drying and storage at different temperatures. Results showed a remarkable high survival rate of 70-80% immediately after spray- and freeze-drying in which the cell viability retained at the range of 109 to 1010 CFU/mL. After a month of storage, maltodextrin showed higher protective ability on both spray- and freeze-dried cells as compared to other protective agents at 4°C, 25°C, and 40°C. A complete loss in viability of spray-dried L. acidophilus FTDC 3081 was observed after a month at 40°C in the absence of protective agent.
Compelling evidences posited that high level of saturated fatty acid gives rise to mitochondrial dysfunction and inflammation in the development of insulin resistance in skeletal muscle. Celastrol is a pentacyclic triterpenoid derived from the root extracts of Tripterygium wilfordii that possesses potent anti-inflammatory properties in a number of animal models with metabolic diseases. However, the cellular mechanistic action of celastrol in alleviating obesity-induced insulin resistance in skeletal muscle remains largely unknown. Therefore, the present investigation evaluated the attributive properties of celastrol at different concentrations (10, 20, 30 and 40nM) on insulin resistance in C2C12 myotubes evoked by palmitate. We demonstrated that celastrol improved mitochondrial functions through significant enhancement of intracellular ATP content, mitochondrial membrane potential, citrate synthase activity and decrease of mitochondrial superoxide productions. Meanwhile, augmented mitochondrial DNA (mtDNA) content with suppressed DNA oxidative damage were observed following celastrol treatment. Celastrol significantly enhanced fatty acid oxidation rate and increased the level of tricarboxylic acid (TCA) cycle intermediates in palmitate-treated cells. Further analysis revealed that the improvement of glucose uptake activity in palmitate-loaded myotubes was partly mediated by celastrol via activation of PI3K-Akt insulin signaling pathway. Collectively, these findings provided evidence for the first time that the protection from palmitate-mediated insulin resistance in C2C12 myotubes by celastrol is likely associated with the improvement of mitochondrial functions-related metabolic activities.
Mesenchymal stem cells (MSCs) are known to secrete cardioprotective paracrine factors that can potentially activate endogenous cardiac c-kit cells (CCs). This study aims to optimise MSC growth conditions and medium formulation for generating the conditioned medium (CdM) to facilitate CC growth and expansion in vitro. The quality of MSC-CdM after optimisation of seeding density during MSC stabilisation and medium formulation used during MSC stimulation including glucose, ascorbic acid, serum and oxygen levels and the effects of treatment concentration and repeated CdM harvesting were assessed based on CC viability in vitro under growth factor- and serum-deprived condition. Our data showed that functional CdM can be produced from MSCs with a density of 20,000 cells/cm2, which were stimulated using high glucose (25 mM), ascorbic acid supplemented, serum-free medium under normoxic condition. The generated CdM, when applied to growth factor- and serum-deprived medium at 1:1 ratio, improved CC viability, migration and proliferation in vitro. Such an effect could further be augmented by generating CdM concentrates without compromising CC gene and protein expressions, while retaining its capability to undergo differentiation to form endothelial, smooth muscle and cardiomyocytes. Nevertheless, CdM could not be repeatedly harvested from the same MSC culture, as the protein content and its effect on CC viability deteriorated after the first harvest. In conclusion, this study provides a proof-of-concept strategy to standardise the production of CdM from MSCs based on rapid, stepwise assessment of CC viability, thus enabling production of CdM favourable to CC growth for in vitro or clinical applications.
Alzheimer's disease (AD) is a neurodegenerative disease that leads to progressive loss of neurons which often results in deterioration of memory and cognitive function. The development of AD is highly associated with the formation of senile plaques and neurofibrillary tangles. Amyloid β (Aβ) induces neurotoxicity and contributes to the development of AD. Recent evidences also highlighted the importance of neuroglobin (Ngb) in ameliorating AD. This study assessed the ability of fucosterol, a phytosterol found in brown alga, in protecting SH-SY5Y cells against Aβ-induced neurotoxicity. Its effects on the mRNA levels of APP and Ngb as well as the intracellular Aβ levels were also determined in Aβ-induced SH-SY5Y cells. SH-SY5Y cells were exposed to fucosterol prior to Aβ treatment. The effect on apoptosis was determined using Annexin V FITC staining and mRNA expression was studied using RT-PCR. Flow cytometry confirmed the protective effects of fucosterol on SH-SY5Y cells against Aβ-induced apoptosis. Pretreatment with fucosterol increased the Ngb mRNA levels but reduced the levels of APP mRNA and intracellular Aβ in Aβ-induced SH-SY5Y cells. These observations demonstrated the protective properties of fucosterol against Aβ-induced neurotoxicity in neuronal cells.
Recent in vitro and in vivo studies have demonstrated that zerumbone (ZER) possesses anticancer properties. The main objective of this study was to examine the effectiveness of the combination of ZER and cisplatin (CIS) to treat cervical intraepithelial neoplasia (CIN) in vivo. Microculture tetrazolium assay and immunohistochemistry of proliferating cellular nuclear antigen were used to study the antitumor effect of ZER. Prenatally exposed female BALB/c mice were used as a model. The progenies with CIN were injected peritoneally with isotonic sodium chloride solution (positive control), CIS, ZER, and a combination of both compounds. All treated and untreated mice were humanely killed, and serum and cervix were obtained for interleukin 6 analysis and histopathologic studies using hematoxylin and eosin staining, respectively. Zerumbone has revealed an antitumor effect on human cervical cancer cells and downregulates immunoexpression of proliferating cellular nuclear antigen (P < 0.05). In vivo study indicates that ZER at 16 mg/kg and CIS at 10 mg/kg have a regressing effect on CIN. The combination of ZER and CIS also showed similar effectiveness in regressing CIN. Our results indicate that the combination of ZER and CIS has modulated the serum level of interleukin 6 when compared with that in mice treated with isotonic sodium chloride solution (P < 0.05). The effectiveness of combining ZER and CIS could be further explored as a new therapeutic intervention of early precancerous stages of carcinogenesis before the invasive stage begins.
Peperomia pellucida leaf extract was characterized for its anticancer, antimicrobial, antioxidant activities, and chemical compositions. Anticancer activity of P. pellucida leaf extract was determined through Colorimetric MTT (tetrazolium) assay against human breast adenocarcinoma (MCF-7) cell line and the antimicrobial property of the plant extract was revealed by using two-fold broth micro-dilution method against 10 bacterial isolates. Antioxidant activity of the plant extract was then characterized using α, α-diphenyl-β-picrylhydrazyl (DPPH) radical scavenging method and the chemical compositions were screened and identified using gas chromatography-mass spectrometry (GC-MS). The results of present study indicated that P. pellucida leaf extract possessed anticancer activities with half maximal inhibitory concentration (IC(50)) of 10.4 ± 0.06 µg/ml. The minimum inhibitory concentration (MIC) values were ranged from 31.25 to 125 mg/l in which the plant extract was found to inhibit the growth of Edwardsiella tarda, Escherichia coli, Flavobacterium sp., Pseudomonas aeruginosa and Vibrio cholerae at 31.25 mg/l; Klebsiella sp., Aeromonas hydrophila and Vibrio alginolyticus at 62.5 mg/l; and it was able to control the growth of Salmonella sp. and Vibrio parahaemolyticus at 125 mg/l. At the concentration of 0.625 ppt, the plant extract was found to inhibit 30% of DPPH, free radical. Phytol (37.88%) was the major compound in the plant extract followed by 2-Naphthalenol, decahydro- (26.20%), Hexadecanoic acid, methyl ester (18.31%) and 9,12-Octadecadienoic acid (Z,Z)-, methyl ester (17.61%). Findings from this study indicated that methanol extract of P. pellucida leaf possessed vast potential as medicinal drug especially in breast cancer treatment.
Methanol extracts of seven Malaysian medicinal plants were screened for antioxidant and nitric oxide inhibitory activities. Antioxidant activity was measured by using FTC, TBA and DPPH free radical scavenging methods and Griess assay was used for the measurement of nitric oxide inhibition in lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma)-treated RAW 264.7 cells. All the extracts showed strong antioxidant activity comparable to or higher than that of alpha-tocopherol, BHT and quercetin in FTC and TBA methods. The extracts from Leea indica and Spermacoce articularis showed strong DPPH free radical scavenging activity comparable with quercetin, BHT and Vit C. Spermacoce exilis showed only moderate activity but other species were weak as compared to the standards. In the Griess assay Lasianthus oblongus, Chasalia chartacea, Hedyotis verticillata, Spermacoce articularis and Leea indica showed strong inhibitory activity on nitric oxide production in LPS and IFN-gamma-induced RAW 264.7 cells. Extracts from Psychotria rostrata and Spermacoce exilis also inhibited NO production but this was due to their cytotoxic effects upon cells during culture.
Gemcitabine (GEM) is used to treat various cancers such as breast, pancreatic, non-small lung, ovarian, bladder, and cervical cancers. GEM, however, has the problem of non-selectivity. Water-soluble, fluorescent, and mono-dispersed carbon dots (CDs) were fabricated by ultrasonication of sucrose. The CDs were further conjugated with GEM through amide linkage. The physical and morphological properties of these carbon dot-gemcitabine (CD-GEM) conjugates were determined using different analytical techniques. In vitro cytotoxicity and apoptosis studies of CD-GEM conjugates were evaluated by various bioactivity assays on human cell lines, MCF-7 (human breast adenocarcinoma), and HeLa (cervical cancer) cell lines. The results of kinetic studies have shown a maximum drug loading efficacy of 17.0 mg of GEM per 50.0 mg of CDs. The CDs were found biocompatible, and the CD-GEM conjugates exhibited excellent bioactivity and exerted potent cytotoxicity against tumor cells with an IC50 value of 19.50 μg ml-1 in HeLa cells, which is lower than the IC50 value of pure GEM (∼20.10 μg ml-1). In vitro studies on CD-GEM conjugates demonstrated the potential to replace the conventional administration of GEM. CD-GEM conjugates are more stable, have a higher aqueous solubility, and are more cytotoxic as compared to GEM alone. The CD-GEM conjugates show reduced side effects in the normal cells along with excellent cellular uptake. Hence, CD-GEM conjugates are more selective toward cancerous cell lines as compared to non-cancerous cells. Also, the CD-GEM conjugates successfully induced early and late apoptosis in cancer cell lines and might be effective and safe to use for in vivo applications.
Metastases account for more than 90% of all cancer deaths and respond poorly to most therapies. There remains an urgent need for new therapeutic modalities for the treatment of advanced metastatic cancers. The benzimidazole methylcarbamate drugs, commonly used as anti-helmitics, have been suggested to have anticancer activity, but progress has been stalled by their poor water solubility and poor suitability for systemic delivery to disseminated cancers. We synthesized and characterized the anticancer activity of novel benzimidazoles containing an oxetane or an amine group to enhance solubility. Among them, the novel oxetanyl substituted compound 18 demonstrated significant cytotoxicity toward a variety of cancer cell types including prostate, lung, and ovarian cancers with strong activity toward highly aggressive cancer lines (IC50: 0.9-3.8 μM). Compound 18 achieved aqueous solubility of 361 μM. In a mouse xenograft model of a highly metastatic human prostate cancer, compound 18 (30 mg/kg) significantly inhibited the growth of established tumors (T/C: 0.36) without noticeable toxicity.
Physalin F (a secosteroid derivative), is well recognized as a potent anticancer compound from Physalis minima L., a plant that is traditionally used to treat cancer. However, the exact molecular anticancer mechanism remains to be elucidated.
Phytochemical investigation on the stem bark of Shorea maxwelliana yielded five oligostilbenoids identified as α-viniferin (1), maximol A (2), vaticanol A (3), suffruticosol A (4) and vaticanol G (5). Chemotaxonomy of isolated compounds was discussed briefly. Major compounds were tested for neurotoxic and cytotoxic activities. Neurotoxicity for all tested compounds did not pose any toxic effect against cultured cell (cell viability range ±100-94%). Compounds 2-5 possessed active cyctotoxic activity against HL60 cell line with IC50 values range of 2.7-78 µg mL(-1).
Regrowth of the cryopreserved protocorm-like bodies (PLBs) of Dendrobium Bobby Messina was assessed based on the plant vitrification solution 2 (PVS2) optimisation conditions. The optimized protocol obtained based on TTC spectrophotometrical analysis and growth recovery were 3-4 mm of PLBs size precultured in 0.2 M sucrose for 1 day, treated with a mixture of 2 M glycerol and 0.4 M sucrose supplemented with half-strength liquid MS media at 25 °C for 20 min and subsequently dehydrated with PVS2 at 0 °C for 20 min prior to storage in liquid nitrogen. Following rapid warming in a water bath at 40 °C for 90 s, PLBs were treated with unloading solution containing half-strength liquid MS media supplemented with 1.2 M sucrose. Subsequently, the PLBs were cultured on half-strength semi-solid MS media supplemented with 2 % (w/v) sucrose without any growth regulators and resulted in 40 % growth recovery. In addition, ascorbic acid treatment was used to evaluate the regeneration process of cryopreserved PLBs. However, growth recovery rates of non-cryopreserved and cryopreserved PLBs were 30 and 10 % when 0.6 mM ascorbic acid was added. Scanning electron microscopy analysis indicates that there are not much damages observed on both cryopreserved and non-cryopreserved PLBs in comparison to PLBs stock culture.
Early measurements of tissue viability after myocardial infarction (MI) are essential for accurate diagnosis and treatment planning but are challenging to obtain. Here, manganese, a calcium analogue and clinically approved magnetic resonance imaging (MRI) contrast agent, is used as an imaging biomarker of myocardial viability in the first hours after experimental MI. Safe Mn2+ dosing is confirmed by measuring in vitro beating rates, calcium transients, and action potentials in cardiomyocytes, and in vivo heart rates and cardiac contractility in mice. Quantitative T1 mapping-manganese-enhanced MRI (MEMRI) reveals elevated and increasing Mn2+ uptake in viable myocardium remote from the infarct, suggesting MEMRI offers a quantitative biomarker of cardiac inotropy. MEMRI evaluation of infarct size at 1 h, 1 and 14 days after MI quantifies myocardial viability earlier than the current gold-standard technique, late-gadolinium-enhanced MRI. These data, coupled with the re-emergence of clinical Mn2+ -based contrast agents open the possibility of using MEMRI for direct evaluation of myocardial viability early after ischemic onset in patients.
The delivery of macromolecular platinum drugs into cancerous cells is enhanced by conjugating the polymer to albumin. The monomers N-(2-hydroxypropyl)methacrylamide (HPMA) and Boc protected 1,3-diaminopropan-2-yl acrylate (Ac-DAP-Boc) are copolymerized in the presence of a furan protected maleimide functionalized reversible addition-fragmentation chain transfer (RAFT) agent. The resulting polymer with a composition of P(HPMA14 -co-(Ac-DAP-Boc)9 ) and a molecular weight of Mn = 7600 g mol(-1) (Đ = 1.24) is used as a macromolecular ligand for the conjugation to the platinum drug. Thermogravimetric analysis reveals full conjugation. After deprotection of the maleimide functionality of the polymer, the reactive polymer is conjugated to albumin using the Cys34 functionality. The conjugation is monitored using size exclusion chromatography, MALDI-TOF (matrix assisted laser desorption ionization time-of-flight), and SDS Page (sodium dodecyl sulphate polyacrylamide gel electrophoresis). The polymer-albumin conjugates self-assemble in water into nanoparticles of sizes of around 80 nm thanks to the hydrophobic nature of the platinum drugs. The albumin coated nanoparticles are readily taken up by ovarian cancer cell lines and they show superior toxicity compared to a control sample without protein coating.
Andrographolide has been reported with anticancer and anti-inflammatory properties through the inhibition of the activity of signaling molecules such as v-Src, nuclear factor-κB (NF-κB), STAT3, and PI3K. NF-κB has been proven to promote cancer cell survival, and targeting this pathway will halt the growth of cancer cells. Efforts have been made to produce semisynthetic derivatives of andrographolide with improved anticancer potency and selectivity. Subsequently, the effect of a selected derivative, 3,14,19-tripropionylandrographolide (SRS06), was tested for its action against NF-κB.
Andrographolide (AGP) is the main bioactive constituent isolated from the traditional medicinal, Andrographis paniculata which contributes towards its various biological activities, including anticancer property. In this study, a series of new AGP derivatives were semi-synthesised and screened against the NCI in vitro 60 cell lines. From the screening results, we had identified SRS07 as the most potent AGP derivative, against breast and colon cancer cell lines. Subsequently, SRS07 was tested for its capability to induce cell cycle arrest and apoptosis in MCF-7 and HCT116 cancer cells. SRS07 effectively induced G1 cell cycle arrest in both cell lines and ultimately apoptosis by inducing DNA fragmentation in HCT116 cells. The apoptotic cell death induced by SRS07 was confirmed via FITC Annexin-V double staining. Western blot analysis of SRS07-treated HCT116 cells revealed that the compound induced apoptosis be activating caspase 8 which in turn cleaved Bid to t-Bid to initiate cell death cascade. Prediction of the possible mode of action of SRS07 by utilising NCI COMPARE analysis failed to reveal a distinct mechanism category. Hence, it is speculated that SRS07 possesses novel mechanism of action. In conclusion, SRS07 demonstrated superior in vitro anticancer profiles and emerged as a potential lead anticancer candidate.
Limited tumor penetrability of anti-cancer drugs is recognized as one of the major factors that lead to poor anti-tumor activity. SRJ09 (3,19-(2-bromobenzylidene) andrographolide) has been identified as a lead anti-cancer agent for colon cancer. Recently, this compound was shown by us to be a mutant K-Ras binder. In this present study, the penetrability of SRJ09 through the DLD-1 colon cancer multicell layer (MCL) was evaluated. The amount of SRJ09 that penetrated through the MCL was quantitated by utilizing high performance liquid chromatography (HPLC). Histopathological staining was used to visualize the morphology of MCL. A chemosensitivity assay was performed to assess the anti-cancer activity of SRJ09 in DLD-1 cells. SRJ09 was able to penetrate through DLD-1 MCL and is inversely proportional with the MCL thickness. The flow rates for SRJ09 through MCL were 0.90 ± 0.20 μM/min/cm(2) and 0.56 ± 0.06 μM/min/cm(2) for days 1 and 5, respectively, which are better than doxorubicin. Histopathological examination revealed that the integrity of the DLD-1 MCL was retained and no visible damage was inflicted on the cell membrane, confirming the penetration of SRJ09 was by diffusion. Short term exposure (1 h) in DLD-1 cells demonstrated SRJ09 had IC50 of 41 μM which was approximately 4-folds lower than andrographolide, the parent compound of SRJ09. In conclusion, SRJ09 successfully penetrated through DLD-1 MCL by diffusion and emerged as a potential candidate to be developed as a clinically viable anti-colon cancer drug.
The plant Andrographis paniculata found throughout Southeast Asia contains Andrographolide 1, a diterpenoid lactone, which has antitumour activities against in vitro and in vivo breast cancer models. In the present study, we report on the synthesis of andrographolide derivatives, 3,19-isopropylideneandrographolide (2), 14-acetyl-3,19-isopropylideneandrographolide (3) and 14-acetylandrographolide (4), and their in vitro antitumour activities against a 2-cell line panel consisting of MCF-7 (breast cancer cell line) and HCT-116 (colon cancer cell line). Compounds 2 and 4 were also screened at the US National Cancer Institute (NCI) for their activities against a panel of 60 human cancer cell lines derived from nine cancer types. Compound 2 was found to be selective towards leukaemia and colon cancer cells, and compound 4 was selective towards leukaemia, ovarian and renal cancer cells at all the dose-response parameters. Compounds 2 and 4 showed non-specific phase of the cell cycle arrest in MCF-7 cells treated at different intervals with different concentrations. NCI's COMPARE and SOM mechanistic analyses indicated that the anticancer activities of these new class of compounds were not similar to that of standard anticancer agents, suggesting novel mechanism(s) of action.
The growth inhibiting potential of andrographolide was evaluated in three acute promyelocytic leukaemia cell line models (HL-60, NB4 and all-trans retinoic acid (ATRA)-resistant NB4-R2).