Cytotoxicity and apoptotic activities of alpha-, gamma- and delta-tocotrienol isomers on human cancer cells

Background: Tocotrienols, especially the gamma isomer was discovered to possess cytotoxic effects associated with the induction of apoptosis in numerous cancers. Individual tocotrienol isomers are believed to induce dissimilar apoptotic mechanisms in different cancer types. This study was aimed to compare the cytotoxic potency of alpha-, gamma- and delta-tocotrienols, and to explore their resultant apoptotic mechanisms in human lung adenocarcinoma A549 and glioblastoma U87MG cells which are scarcely researched. Methods: The cytotoxic effects of alpha-, gamma- and delta-tocotrienols in both A549 and U87MG cancer cells were first determined at the cell viability and morphological aspects. DNA damage types were then identified by comet assay and flow cytometric study was carried out to support the incidence of apoptosis. The involvements of caspase-8, Bid, Bax and mitochondrial membrane permeability (MMP) in the execution of apoptosis were further expounded. Results: All tocotrienols inhibited the growth of A549 and U87MG cancer cells in a concentration- and time-dependent manner. These treated cancer cells demonstrated some hallmarks of apoptotic morphologies, apoptosis was further confirmed by cell accumulation at the pre-G1 stage. All tocotrienols induced only double strand DNA breaks (DSBs) and no single strand DNA breaks (SSBs) in both treated cancer cells. Activation of caspase-8 leading to increased levels of Bid and Bax as well as cytochrome c release attributed by the disruption of mitochondrial membrane permeability in both A549 and U87MG cells were evident. Conclusions: This study has shown that delta-tocotrienol, in all experimental approaches, possessed a higher efficacy (shorter induction period) and effectiveness (higher induction rate) in the execution of apoptosis in both A549 and U87MG cancer cells as compared to alpha- and gamma-tocotrienols. Tocotrienols in particular the delta isomer can be an alternative chemotherapeutic agent for treating lung and brain cancers

Acalypha wilkesiana ethyl acetate extract enhances the in vitro cytotoxic effects of α-tocopherol in human brain and lung cancer cells

Multi-combinatorial approachesare considered nowadays to enhance the effectiveness of cancer treatment. In this study, α-tocopherol was tested in combination with the ethyl acetate extract from Acalypha wilkesiana for cytotoxicity activity against U87MG and A549 cell lines. The GI50 values for α-tocopherol against U87MG and A549 cells were 0.923±0.411μg/ml and 5.290±1.952μg/ml respectively in cell viability tests; when A. wilkesiana extract was added in adjunct with the treatment of α-tocopherol in minimum inhibitory concentration (MIC), the GI50 values of α-tocopherol improved significantly (p<0.05) to <0.43μg/ml (1μM) for both cell lines tested. Histological staining signified that both α-tocopherol and A. wilkesiana extract treated cancer cell lines exhibited apoptotic morphological characteristics. Single cell gel electrophoresis (SCGE) comet assays revealed that α-tocopherol caused only double strand DNA breaks; whereas A. wilkesiana extract caused both single strand and double strand DNA breaks in U87MG and A549 cells. It is proposed that α-tocopherol and A. wilkesiana extract might trigger apoptosis in both U87MG and A549 cells through different apoptotic pathways that might complement each other to enhance their antiproliferative efficacy against the cancer cells

Quantitative approach in screening the antiviral properties of Kandis Hutan in animal cell culture

This main aim of this study was to determine the concentration of Kandis Hutan leaf extracts that can inhibit the infectivity of pseudorabies virus (PrV) in Vero cells. The leaf extracts were crude and extracted with 3 organic solvents namely hexane, ethyl acetate and ethanol. The cytotoxic effect of extracts on Vero cells was assessed by both MTT assay and cell cytotoxicity scoring method. Two-fold serial dilutions of each extracts were prepared from the highest concentration of 1000µg/ml in 0.1% DMSO. For MTT assay, the highest cytotoxicity was found in the hexane extract (CC50 <1.25 µg/mL), followed by ethyl acetate extract (CC50 = 237.5 µg/mL), whilst minimal cell cytotoxicity was observed in ethanol extracts (CC50 = 555.0 µg/mL). There was a significant correlation between cell scoring system and MTT assay in term of cell cytotoxicity whereby the least toxic was ethanol extracts, followed by ethyl acetate extract and the most toxic hexane extract. Four antiviral assays were conducted for each extracts, namely plaque reduction assay, cytopathic effect (CPE) reduction assay, inhibition assay and virucidal assay. The most promising result was obtained from the inhibition assay, in which ethyl acetate extracts produced 75% viral inhibition at 125 µg/mL concentration. In virucidal assay, both ethyl acetate and ethanol extracts produced 100% viral inhibition at 250 µg/mL. For plaque reduction assay, there was a significant dose dependent inhibition for ethyl acetate extract but not for ethanol extract and hexane extract. In comparison to CPE reduction assay, findings from plaque reduction assay showed better viral inhibition by ethyl acetate extract (47%) at concentration of 300 µg/mL. The estimated selectivity index (ESI) calculated from the inhibition assay showed the highest antiviral response by ethyl acetate extract (2.7) in comparison with ethanol extract (1.8) and hexane extract (0.1). Therefore, the most promising antiviral activity was produced by ethyl acetate extract which showed consistent viral inhibition in all tested antiviral assays. In contrary, hexane extracts showed the least antiviral efficacy among the tested extracts

Data on the Lignosus rhinocerotis water soluble sclerotial extract affecting intracellular calcium level in rat dorsal root ganglion cells

The data in this article contain supporting evidence for the research manuscript entitled “Bronchodilator effects of Lignosus rhinocerotis extract on rat isolated airways is linked to the blockage of calcium entry” by Lee et al. (2018) [1]. The data were obtained by calcium imaging technique with fluorescent calcium indicator dyes, Fura 2-AM, to visualize calcium ion movement in the rat dorsal ganglion (DRG) cells. The effects of L. rhinocerotis cold water extract (CWE1) on intracellular calcium levels in the DRG cells were presented. Keywords: Lignosus rhinocerotis, Medicinal mushroom, Bronchodilators, Calcium dynamic

Antiviral properties of Berembang Bukit and Kandis Hutan against psedorabies virus in animal cell culture

Tropical rainforest in Malaysia represents and untapped potential source of antiviral compounds. Bioactive compounds in plant species from the same genus as Kandis Hutan such as xanthones, benzophenones, biflavonoids and lupeol had been studied. Eugeniin is an anti-herpesvirus compound which had also been found n Berembang Bukit. This preliminary study was carried out to discover the presence of antiviral properties in Berembang Bukit and Kandis Hutan using different antiviral assays. In this study, MTT cell viability assay was used in addition to microscopic evaluation of pseudorabies virus (PrV)- induced cytopathic effects (CPE) on Vero cells. The cellular toxicity of DMSO was also evaluated. DMSO was less than 10% cytotoxic at concentration of 0.1% to Vero cells and its effect can be negligible. Both plants had demonstrated antiviral properties in thyl acetate and ethanol extracts. From our findings from all three antiviral assays, the ethanol-extracted Kandis Hutan possessed the most promising antiviral properties. Nevertheless, antiviral potential of ethyl acetate and ethanol-extracted Berembang Bukit and ethyl acetate-extracted Kandis Hutan also merit further investigatio

Quantitative in vitro assays for screening antiviral characteristics of Berembang Bukit leaf extracts

Berembang Bukit or Megawasih, is a native of our tropical rainforest and other ASEAN countries. In a preliminary review, a qualitative screening on the antiviral activities of its leaf extracts revealed various degrees of antiviral potential. Therefore this study was done to evaluate its antiviral characteristics in a quantitative approach. The leaf extracts were prepared using hexane, ethyl acetate and ethanol extractions and dissolved in DMSO. DMSO cytotoxicity was initially evaluated to ensure a safe working concentration. A negligible cytotoxicity was observed at a concentration of ≤ 0.1% DMSO. The cytotoxic effect of extracts on Vero cells was assessed by both MTT assay and cell cytotoxicity scoring method. Two-fold serial dilutions of each extracts were prepared from the highest concentration of 1000µg/mL in 0.1% DMSO. For MTT assay, the highest cytotoxicity was found in the ethyl acetate extract (CC50 = 218µg/mL), whilst minimal cell cytotoxicity was observed in both hexane (CC50 = 833µg/ml) and ethanol (CC50 = >1000µg/mL) extracts. However, there were no correlation between MTT and cell scoring for cytotoxicity in this study. A series of experiments including CPE reduction assay, plaque reduction assay, inhibition assay and virucidal assay were done to evaluate the total antiviral potential of the leaf extracts. The leaf extracts produced a dose-dependent antiviral response. Both ethyl acetate and ethanol extracts showed 100% plaque formation inhibition in plaque reduction assay, inhibition assay and virucidal assay. Hexane extracts showed absence of plaque inhibition in all the tested antiviral assays. In inhibition assay, the estimated selective index (ESI) for ethanol and ethyl acetate extracts were 8.3 and 1.9, respectively. Whilst in CPE reduction assay, the ESI for the respective extracts were 6.7 and 2.9. In conclusion, the ethanol extracts exhibited the highest antiviral efficacy among the tested extracts

Acalypha wilkesiana extracts induce apoptosis by causing single strand and double strand DNA breaks

Ethnopharmacological relevance: The seeds of Acalypha wilkesiana have been used empirically by traditional healers in Southwest Nigeria together with other plants as a powder mixture to treat patients with breast tumours and inflammation. Aim of the study: There is an increasing interest among researchers in searching for new anticancer drugs from natural resources, particularly plants. This study aimed to investigate the anticancer properties of Acalypha wilkesiana extracts and the characteristics of DNA damage against brain and lung cancer cells. Materials and methods: The antiproliferative activity of Acalypha wilkesiana extracts (ethyl acetate, hexane, and ethanol) was examined on human glioma (U87MG), human lung carcinoma (A549), and human lung fibroblast (MRC5) cells. Results: Cell viability MTT assay revealed that ethyl acetate extract of the plant possessed significant antiproliferative effects against both U87MG (GI50 = 28.03 ± 6.44 μg/ml) and A549 (GI50 = 89.63 ± 2.12 μg/ml) cells (p value 300 μg/ml). The ethanol extract showed no antiproliferative effects on any cell line examined. Haematoxylin & Eosin (H & E) staining and single cell gel electrophoresis (SCGE) comet assay confirmed that plant extract-treated cells underwent apoptosis and not necrosis. SCGE comet assays confirmed that plant extracts caused both single strand (SSB) and double strand (DSB) DNA breaks that led to the execution of apoptosis. Conclusion: The extracts (especially ethyl acetate and hexane) of Acalypha wilkesiana possess valuable cytotoxic effects that trigger apoptosis in U87MG and A549 cancer cells through induction of DNA SSBs and DSBs

Antiproliferation and induction of caspase-8-dependent mitochondria-mediated apoptosis by β-tocotrienol in human lung and brain cancer cell lines

The pure vitamin isomer, β-tocotrienol has the least abundance among the other vitamin E isomers that are present in numerous plants. Hence, it is very scarcely studied for its bioactivity. In this study, the antiproliferative effects and primary apoptotic mechanisms of β-tocotrienol on human lung adenocarcinoma A549 and glioblastoma U87MG cells were investigated. It was evidenced that β-tocotrienol had inhibited the growth of both A549 (GI50=1.38±0.334μM) and U87MG (GI50=2.53±0.604μM) cells at rather low concentrations. Cancer cells incubated with β-tocotrienol were also found to exhibit hallmarks of apoptotic morphologies including membrane blebbing, chromatin condensation and formation of apoptotic bodies. The apoptotic properties of β-tocotrienol in both A549 and U87MG cells were the results of its capability to induce significant (P<0.05) double-strand DNA breaks (DSBs) without involving single-strand DNA breaks (SSBs). β-Tocotrienol is said to induce activation of caspase-8 in both A549 and U87MG cells guided by no activation when caspase-8 inhibitor, z-IETD-fmk was added. Besides, disruption on the mitochondrial membrane permeability of the cells in a concentration- and time-dependent manner had occurred. The induction of apoptosis by β-tocotrienol in A549 and U87MG cells was confirmed to involve both the death-receptor mediated and mitochondria-dependent apoptotic pathways. These findings could potentiate the palm oil derived β-tocotrienol to serve as a new anticancer agent for treating human lung and brain cancers

Reduction of MTT to purple formazan by vitamin E isomers in the absence of cells

The yellow tetrazolium salt 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) is widely used to determine cell viability in cell proliferation and cytotoxic assays. MTT is reduced by metabolically active cells to form an insoluble purple formazan product that is quantifiable by spectrophotometry. It is the most common and direct assay for cell viability. However, in this present study, we demonstrated that the vitamin E isomers α-β-γ-δ-tocotrienols and α-tocopherol were able to reduce MTT into a formazan product, despite the absence of living cells. For comparison, a second method for determining cell viability, which is the neutral red uptake assay, was used in parallel with the MTT assay. The results showed that neutral red did not interact with the vitamin E isomers. Our findings suggest that the MTT assay is not suitable for studying the proliferative effects of vitamin E isomers on cell growth

Electrical conduction by interface states in semiconductor heterojunctions

peer reviewedaudience: researcher, professionalElectrical conduction in semiconductor heterojunctions containing defect states in the interface region is studied. As the classical drift-diffusion mechanism cannot in any case explain electrical conduction in semiconductor heterojunctions, tunnelling involving interface states is often considered as a possible conduction path. A theoretical treatment is made where defect states in the interface region with a continuous energy distribution are included. Electrical conduction through this defect band then allows the transit of electrons from the conduction band of one semiconductor to the valence band of the second component. The analysis is initiated by electrical measurements on n-CdS/p-CdTe heterojunctions obtained by chemical vapour deposition of CdS on (111) oriented CdTe single crystals, for which current--voltage and capacitance--frequency results are shown. The theoretical analysis is based on the numerical resolution of Poisson's equation and the continuity equations of electrons, holes and defect states, where a current component corresponding to the defect band conduction is explicitly included. Comparison with the experimental curves shows that this formalism yields an efficient tool to model the conduction process through the interface region. It also allows us to determine critical values of the physical parameters when a particular step in the conduction mechanism becomes dominant