The effects of tocotrienols on murine liver cell viability and their apoptotic events were studied over a dose range of 0-32 microg mL(-1). Normal murine liver cells (BNL CL.2) and murine liver cancer cells (BNL 1ME A.7R.1) were treated with tocotrienols (T(3)), alpha tocopherol (alpha-T) and the chemo drug, Doxorubicin (Doxo, as a positive control). Cell viability assay showed that T(3) significantly (P < or = 0.05) lowered the percentage of BNL 1ME A.7R.1 cell viability in a dose-responsive manner (8-16 microg mL(-1)), whereas T did not show any significant (P>0.05) inhibition in cell viability with increasing treatment doses of 0-16 microg mL(-1). The IC(50) for tocotrienols were 9.8, 8.9, 8.1, 9.7, 8.1 and 9.3 microg mL(-1) at 12, 24, 36, 48, 60 and 72 hours respectively. Early apoptosis was detected 6 hours following T(3) treatment of BNL 1ME A.7R.1 liver cancer cells, using Annexin V-FITC fluorescence microscopy assay for apoptosis, but none were observed for the non-treated liver cancer cells at the average IC(50) of 8.98 microg mL(-1) tocotrienols for liver cancer cells. Several apoptotic bodies were detected in BNL 1ME A.7R.1 liver cancer cells at 6 hours post-treatment with tocotrienols (8.98 microg mL(-1)) using Acridine Orange/Propidium Iodide fluorescence assay. However, only a couple of apoptotic bodies were seen in the non-treated liver cancer cells and the BNL CL.2 normal liver cells. Some mitotic bodies were also observed in the T(3)-treated BNL 1ME A.7R.1 liver cancer cells but were not seen in the untreated BNL 1ME A.7R.1 cells and the BNL CL.2 liver cells. Following T(3)-treatment (8.98 microg mL(-1)) of the BNL 1ME A.7R.1 liver cancer cells, 24.62%, 25.53% and 44.90% of the cells showed elevated active caspase 3 activity at 9, 12 and 24 hours treatment period, respectively. DNA laddering studies indicated DNA fragmentation occurred in the T(3)-treated liver cancer cells, BNL 1ME A.7R.1 but not in non-treated liver cancer cells and the T(3)-treated and non-treated normal liver cells. These results suggest that tocotrienols were able to reduce the cell viability in the murine liver cancer cells at a dose of 8-32 microg mL(-1) and that this decrease in percentage cell viability may be due to apoptosis.
Photodynamic therapy is a treatment modality that involves site-directed generation of cytotoxic reactive oxygen species by light-activated photosensitisers.
An increasing large body of research on Parkinson's disease (PD) has focused on the understanding of the mechanisms behind the potential neuro protection offered by antioxidants and iron chelating agents. In this study, the protective effect of the bioflavonoid quercetin on 6-hydroxydopamine (6-OHDA)-induced model of PD was investigated. PD was induced by a single intracisternal injection of 6-hydroxydopamine (300μg) to male Sprague-Dawley rats. Quercetin treatment (30mg/kg body weight) over 14 consecutive days markedly increased the striatal dopamine and antioxidant enzyme levels compared with similar measurements in the group treated with 6-OHDA alone. There was a significant decrease in protein carbonyl content in the striatum compared with that of rats that did not receive quercetin. A significant increase in neuronal survivability was also found with quercetin treatment in rats administered 6-OHDA. In conclusion, treatment with quercetin defended against the oxidative stress in the striatum and reduced the dopaminergic neuronal loss in the rat model of PD.
Anti-inflammatory actions of the vitamin E fragment tocotrienol have not been described for microglia. Here, we screened palm α-, γ- and δ-tocotrienol isoforms and Tocomin® 50% (contains spectrum of tocotrienols and tocopherols) for their ability to limit nitric oxide (NO) production by BV2 microglia. Microglia were treated with varying doses of tocotrienols for 24h and stimulated with 1 μg/ml lipopolysaccharide (LPS). All tocotrienol isoforms reduced NO release by LPS-stimulated microglia, with 50 μM being the most potent tocotrienol dose. Of the isoforms tested, δ-tocotrienol lowered NO levels the most, reducing NO by approximately 50% at 48 h post-LPS treatment (p
Murraya koenigii is an edible herb widely used in folk medicine. Here we report that girinimbine, a carbazole alkaloid isolated from this plant, inhibited the growth and induced apoptosis in human hepatocellular carcinoma, HepG2 cells. The MTT and LDH assay results showed that girinimbine decreased cell viability and increased cytotoxicity in a dose-and time-dependent manner selectively. Girinimbine-treated HepG2 cells showed typical morphological features of apoptosis, as observed from normal inverted microscopy and Hoechst 33342 assay. Furthermore, girinimbine treatment resulted in DNA fragmentation and elevated levels of caspase-3 in HepG2 cells. Girinimbine treatment also displayed a time-dependent accumulation of the Sub-G(0)/G(1) peak (hypodiploid) and caused G(0)/G(1)-phase arrest. Together, these results demonstrated for the first time that girinimbine could effectively induce programmed cell death in HepG2 cells and suggests the importance of conducting further investigations in preclinical human hepatocellular carcinoma models, especially on in vivo efficacy, to promote girinimbine for use as an anticancer agent against hepatocellular carcinoma.
In our screening program for new photosensitizers from Malaysian biodiversity for photodynamic therapy (PDT) of cancer, MeOH extracts of ten terrestrial plants from Cameron Highlands in Pahang, Peninsular Malaysia, were tested. In a short-term 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, 20 μg/ml each of these extracts were incubated in a pro-myelocytic leukemia cell-line, HL60, with or without irradiation with 9.6 J/cm(2) of a broad spectrum light. Three samples, Labisia longistyla, Dichroa febrifuga, and Piper penangense, were photocytotoxic by having at least twofold lower cell viability when irradiated compared to the unirradiated assay. The extract of the leaves of Piper penangense, a shrub belonging to the family Piperaceae and widely distributed in the tropical and subtropical regions in the world, was subsequently subjected to bioassay-guided fractionation using standard chromatography methods. Eight derivatives of pheophorbide-a and -b were identified from the fractions that exhibited strong photocytotoxicity. By spectroscopic analysis, these compounds were identified as pheophorbide-a methyl ester (1), (R,S)-13(2) -hydroxypheophorbide-a methyl ester (2 and 3), pheophorbide-b methyl ester (4), 13(2) -hydroxypheophorbide-b methyl ester (5), 15(2) -hydroxylactone pheophorbide-a methyl ester (6), 15(2) -methoxylactone pheophorbide-a methyl ester (7), 15(2) -methoxylactone pheophorbide-b methyl ester (8).
Dendritic cells (DC) are efficient and potent antigen-presenting cells. Pilot clinical trials indicated that DC loaded with tumour antigen could induce tumour-specific immune responses in various cancers including B-cell lymphoma, melanoma and prostate cancer. Owing to extensively low number of DC in the blood circulation, a variety of sources have been used to generate DC including monocytes, CD34+ stem cells and even with leukaemic blast cells. We demonstrate here a simple method to generate DC from acute myeloid leukaemia (AML) cells and monocytes from healthy donor or remission samples. AML cells or monocytes were cultured in RPMI 1640 media supplemented with foetal bovine serum or autologous serum where possible and different combinations of cytokines GM-CSF, IL-4 and TNF-alpha. The generated DC were evaluated for their morphology by phase contrast microscopy and May Grunwald Giemsa staining. Viability of cells was determined by trypan blue dye exclusion. Percentage of yields and immunophenotypes were carried out by flow cytometry. We found that cultured AML cells and monocytes developed morphological and immuno-phenotypic characteristics of DC. Monocytes are better than AML blast in generating DC and serve as a ready source for dendritic cell vaccine development.
The SRB cytotoxicity assay was used to screen extracts and isolated constituents of some traditional medicinal plants from Malaysia and Thailand against two human cancer cell lines, COR L23 lung cancer cell line and MCF7 breast cancer cell line and the non-cancer MCF5 cell line. Five out of the seven species tested, i.e. Thai Alpinia galanga, Alpinia officinarum, Cayratia japonica, Physalis minima, Tabernaemontana divaricata, exhibited interesting cytotoxicity activity and this is the first report of cytotoxicity from any Cayratia species. Following bioassay-guided fractionation, 1'-acetoxychavicol acetate (48h exposure against COR L23 cells, IC(50) 7.8 microM against MCF7 cells, IC(50) 23.9 microM) was isolated as the major cytotoxic component of the Alpinia species, physalin F as the major cytotoxic component of Physalis minima (48 h exposure against COR L23 cells IC(50) 0.4 microM against MCF7 cells, IC(50) 0.59 microM). The Malaysian Alpinia galanga showed weak activity compared with the Thai sample and this was shown to be due to the relatively high amounts of 1'-acetoxychavicol acetate present in the Thai sample.
Skin is the largest organ in human system and plays a vital role as a barrier against environment and pathogens. Skin regeneration is important in tissue engineering especially in cases of chronic wounds. With the tissue engineering technology, these skins equivalent have been use clinically to repair burns and wounds. Consented redundant skin samples were obtained from patients aged 9 to 65 years old. Skin samples were digested with dispase, thus separating the epidermis and the dermis layer. The epidermis layer was trypsinized and cultured in DKSFM in 6-well plate at 37 degrees C and 5% CO2. Once confluent, the culture were trypsinized and the cells were pooled. Cells were counted using haemacytometer. Doubling time and viability were calculated and analysed. From the result, we conclude that doubling time and viability of in vitro keratinocytes cultured in DKSFM media is not age dependant.
The cytotoxicity of cell-free culture filtrates of 31 isolates of Vibrio cholerae O1 and O139, 5 reference strains and 26 clinical isolates, was tested on Madin Darby Bovine Kidney (MDBK) cells and Vero cells. The 3-[4,5-dimethylthiazol-2-y]-2, 5-diphenyltetrazolium bromide (MTT) test was used to detect the effect of the filtrates on the proliferation and viability of cultured cell populations. The filtrates were prepared from serial ten-fold dilutions of inoculated AKI and APW broth media with and without the addition of polymyxin B. The APW culture filtrates of both V. cholerae O1 and O139 with and without added polymyxin B showed greater toxicity to MDBK cells as compared to AKI filtrates. The cytotoxicity of AKI-grown V. cholerae O139 to MDBK cells was greater than that of V. cholerae O1 grown in the same medium. The cytotoxicity of APW filtrates on Vero cells was low and only noted when polymyxin was added to the medium.
Ficus deltoidea (Mas cotek) water extract has been widely used for woman health in Malaysia. Our investigation focused to identify anti-melanogenic efficacy of F. deltoidea since it has been known to have strong anti-oxidant activities. Anti-melanogenic effect of F. deltoidea extract was analyzed using cultured B16F1 melanoma cells. Cytotoxicity of the extract was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and determined the highest concentration of the extract that did not affect cell viability as 0.1% (w/v). α-MSH-induced melanin synthesis was significantly inhibited with dose-dependent manner by treatment of F. deltoidea leave extract, which was comparable to that of kojic acid. The extract directly inhibited mushroom tyrosinase activity and intracellular tyrosinase activity of B16F1 as well. The inhibition of intracellular tyrosinase activity was found to be exerted at the protein expression level when analyzed by immunoblot and tyrosinase zymography. The expression of microphthalmia-associated transcription factor (MITF) was also reduced by the F. deltoidea extract. In conclusion, F. deltoidea extract has strong anti-melanogenic activity that is exerted by direct inhibition of tyrosinase enzyme activity and by down-regulation of the expression of genes involved in the melanogenesis pathways. Collectively, data shown in this study strongly suggest that F. deltoidea extract has potential to be used as a novel depigmenting agent for cosmetics.
Malaysian locally processed raw food products are widely used as main ingredients in local cooking. Previous studies showed that these food products have a positive correlation with the incidence of cancer. The cytotoxicity effect was evaluated using MTT assay (3-(4,5-dimetil-2-thiazolil)-2,5-diphenyl-2H-tetrazolium bromide) against Chang liver cells at 2000 microg/ml following 72 h incubation. Findings showed all methanol extracts caused a tremendous drop in the percentage of cell viability at 2000 microg/ml (shrimp paste - 41.69+/-3.36%, salted fish - 37.2+/-1.06%, dried shrimp - 40.32+/-1.8%, p<0.05). To detect DNA damage in a single cell, alkaline Comet Assay was used. None of the extracts caused DNA damage to the Chang liver cells at 62.5 microg/ml following 24 h incubation, as compared to the positive control, hydrogen peroxide (tail moment - 9.50+/-1.50; tail intensity - 30.50+/-2.50). Proximate analysis which was used for the evaluation of macronutrients in food showed that shrimp paste did not comply with the protein requirement (<25%) as in Food Act 1983. Salt was found in every sample with the highest percentage being detected in shrimp paste which exceeded 20%. Following heavy metal analysis (arsenic, cadmium, lead and mercury), arsenic was found in every sample with dried shrimps showing the highest value as compared to the other samples (6.16 mg/kg). In conclusion, several food extracts showed cytotoxic effect but did not cause DNA damage against Chang liver cells. Salt was found as the main additive and arsenic was present in every sample, which could be the probable cause of the toxicity effects observed.
The present study was undertaken to determine whether Ligularia fischeri leaf extract (LF) is efficacious against collagen-induced arthritis (CIA) in mice. DBA/1J mice were immunized with bovine type II collagen and treated with LF (100 and 200 mg/kg) for 49 days. Mice were assessed regularly for signs of arthritis and the levels of rheumatoid factor, anti-type II collagen antibody, cytokines, AST, ALT, and creatinine in serum were also examined after the animals were killed. The arthritis score and paw edema were markedly suppressed in the groups treated with LF. Moreover, levels of rheumatoid factor, anti-type II collagen antibody, tumor necrosis factor-alpha, interleukin (IL)-1, and IL-6 in sera were reduced by LF administration. These data suggest that L. fischeri might be effective for the treatment of inflammatory arthritis like human rheumatoid arthritis.
Mesenchymal stromal cells (MSCs) have shown great promise for cell therapy of a wide range of diseases such as diabetes. However, insufficient viability of transplanted cells reaching to damaged tissues has limited their potential therapeutic effects. Expression of estrogen receptors on stem cells may suggest a role for 17β-estradiol (E2) in regulating some functions in these cells. There is evidence that E2 enhances homing of stem cells. Induction of hypoxia-inducible factor-1α (HIF-1α) by E2 and the profound effect of HIF-1α on migration of cells have previously been demonstrated. We investigated the effect of E2 on major mediators involved in trafficking and subsequent homing of MSCs both in vitro and in vivo in diabetic rats.
The present study was conducted to determine the effects of supplementing α-linolenic acid (ALA) into BioXcell(®) extender on post-cooling, post-thawed bovine spermatozoa and post thawed fatty acid composition. Twenty-four semen samples were collected from three bulls using an electro-ejaculator. Fresh semen samples were evaluated for general motility using computer assisted semen analyzer (CASA) whereas morphology and viability with eosin-nigrosin stain. Semen samples extended into BioXcell(®) were divided into five groups to which 0, 3, 5, 10 and 15 ng/ml of ALA were added, respectively. The treated samples were incubated at 37°C for 15 min for ALA uptake by sperm cells before being cooled for 2 h at 5°C. After evaluation, the cooled samples were packed into 0.25 ml straws and frozen in liquid nitrogen for 24 h before thawing and evaluation for semen quality. Evaluation of cooled and frozen-thawed semen showed that the percentages of all the sperm parameters improved with 5 ng/ml ALA supplement. ALA was higher in all treated groups than control groups than control group. In conclusion, 5 ng/ml ALA supplemented into BioXcell(®) extender improved the cooled and frozen-thawed quality of bull spermatozoa.
Among the array of nanomaterials, carbon nanotubes have shown great potential as drug carriers in the field of nanomedicine, owing to their attractive physicochemical structure, which facilitates functionalization of therapeutic molecules onto their external walls or being encapsulated inside the tubes. The aim of this preliminary study was to formulate betulinic acid (BA), a poorly water-soluble drug, in oxidized multiwalled carbon nanotubes (MWCNT-COOH) for enhanced delivery efficiency into cancer cells with reduced cytotoxicity. The synthesized MWCNT-BA nanocomposite was characterized using ultraviolet-visible, Fourier transform infrared, thermogravimetric analysis, powder X-ray diffraction, and field emission scanning electron microscopy techniques. The loading of BA in MWCNT-COOH nanocarrier was estimated to be about 14.5%-14.8% (w/w), as determined by ultraviolet-visible and thermogravimetric analysis. Fourier transform infrared study shows that the peaks of the resulting MWCNT-BA nanocomposite correlate to the characteristic functional groups of BA and MWCNT-COOH. The powder X-ray diffraction results confirmed that the tubular structures of MWCNT-COOH were not affected by the drug loading mechanism of BA. The release profiles demonstrated that approximately 98% of BA could be released within 22 hours by phosphate-buffered saline solution at pH 7.4 compared with about 22% within 24 hours at pH 4.8. The biocompatibility studies revealed that MWCNT-BA at concentrations <50μg/mL expressed no cytotoxicity effects for mouse embryo fibroblast cells after 72 hours of treatment. The anticancer activity of MWCNT-BA was observed to be more sensitive to human lung cancer cell line when compared with human liver cancer cell line, with half maximal inhibitory concentration values of 2.7 and 11.0μg/mL, respectively. Our findings form a fundamental platform for further investigation of the MWCNT-BA formulation against different types of cancer cells.
Tocotrienol rich fraction (TRF) is an extract of palm oil, which consists of 25% alpha tocopherol (α-TCP) and 75% tocotrienols. TRF has been shown to possess potent antioxidant, anti-inflammatory, anticancer, neuroprotection, and cholesterol lowering activities. Glutamate is the main excitatory amino acid neurotransmitter in the central nervous system of mammalian, which can be excitotoxic, and it has been suggested to play a key role in neurodegenerative disorders like Parkinson's and Alzheimer's diseases. In this present study, the effects of vitamin E (TRF and α-TCP) in protecting astrocytes against glutamate injury were elucidated. Astrocytes induced with 180 mM of glutamate lead to significant cell death. However, glutamate mediated cytotoxicity was diminished via pre and post supplementation of TRF and α-TCP. Hence, vitamin E acted as a potent antioxidant agent in recovering mitochondrial injury due to elevated oxidative stress, and enhanced better survivability upon glutamate toxicity.
The primary challenge in finding a treatment for tuberculosis (TB) is patient non-compliance to treatment due to long treatment duration, high dosing frequency, and adverse effects of anti-TB drugs. This study reports on the development of a nanodelivery system that intercalates the anti-TB drug isoniazid into Mg/Al layered double hydroxides (LDHs). Isoniazid was found to be released in a sustained manner from the novel nanodelivery system in humans in simulated phosphate buffer solutions at pH 4.8 and pH 7.4. The nanodelivery formulation was highly biocompatible compared to free isoniazid against human normal lung and 3T3 mouse fibroblast cells. The formulation was active against Mycobacterium tuberculosis and gram-positive bacteria and gram-negative bacteria. Thus results show significant promise for the further study of these nanocomposites for the treatment of TB.