METHODOLOGY/PRINCIPAL FINDINGS: The cytotoxic effect of thymoquinone was assessed using an MTT assay, while the inhibitory effect of thymoquinone on murine WEHI-3 cell growth was due to the induction of apoptosis, as evidenced by chromatin condensation dye, Hoechst 33342 and acridine orange/propidium iodide fluorescent staining. In addition, Annexin V staining for early apoptosis was performed using flowcytometric analysis. Apoptosis was found to be associated with the cell cycle arrest at the S phase. Expression of Bax, Bcl2 and HSP 70 proteins were observed by western blotting. The effects of thymoquinone on BALB/c mice injected with WEHI-3 cells were indicated by the decrease in the body, spleen and liver weights of the animal, as compared to the control.
CONCLUSION: Thymoquinone promoted natural killer cell activities. This compound showed high toxicity against WEHI-3 cell line which was confirmed by an increase of the early apoptosis, followed by up-regulation of the anti-apoptotic protein, Bcl2, and down-regulation of the apoptotic protein, Bax. On the other hand, high reduction of the spleen and liver weight, and significant histopathology study of spleen and liver confirmed that thymoquinone inhibited WEHI-3 growth in the BALB/c mice. Results from this study highlight the potential of thymoquinone to be developed as an anti-leukemic agent.
METHODS: MTT and trypan blue exclusion tests were conducted to determine the 50% inhibitory concentration (IC50) and cell proliferation. FITC Annexin and Guava® reagent were used to study the cell apoptosis and examine the cell cycle phases, respectively. The expression of JAK/STAT-negative regulator genes, SOCS-1, SOCS-3, and SHP-1, was investigated using reverse transcriptase- quantitative PCR (RT-qPCR).
RESULTS: TQ demonstrated a potential inhibition of HL60 cell proliferation and a significant increase in apoptotic cells in dose and time-dependent manner. TQ significantly induced cycle arrest at G0-G1 phase (P < 0.001) and enhanced the re-expression of JAK/STAT-negative regulator genes.
CONCLUSION: TQ potentially inhibited HL60 cell proliferation and significantly increased apoptosis with re-expression of JAK/STAT-negative regulator genes suggesting that TQ could be a new therapeutic candidate for leukemia therapy.
.
METHODS: BCR-ABL positive K562 CML cells were treated with TQ. Cytotoxicity was determined by Trypan blue exclusion assay. Apoptosis assay was performed by annexin V-FITC/PI staining assay and analyzed by flow cytometry. Transcription levels of BCR ABL, JAK2, STAT3, STAT5A and STAT5B genes were evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Protein levels of JAK2 and STAT5 were determined by Jess Assay analysis.
RESULTS: TQ markedly decreased the cell proliferation and induced apoptosis in K562 cells (P < 0.001) in a concentration dependent manner. TQ caused a significant decrease in the transcriptional levels of BCR ABL, JAK2, STAT3, STAT5A and STAT5B genes (P < 0.001). TQ induced a significant decrease in JAK2 and STAT5 protein levels (P < 0.001).
CONCLUSION: our results indicated that TQ inhibited cell growth of K562 cells via downregulation of BCR ABL/ JAK2/STAT3 and STAT5 signaling and reducing JAK2 and STAT5 protein levels.
METHOD: TQ-nanoparticles were prepared and optimized by using two different formulations with different drugs to PLGA-PEG ratio (1:20 and 1:7) and different PLGA-PEG to Pluronic F68 ratio (10:1 and 2:1). The morphology and size were determined using TEM and DLS. Characterization of particles was done using UV-VIS, ATR-IR, entrapment efficiency, and drug release. The effects of drug, polymer, and surfactants were compared between the two formulations. Cytotoxicity assay was performed using MTS assay.
RESULTS: TEM finding showed 96% of particles produced with 1:7 drug to PLGA-PEG were less than 90 nm in size and spherical in shape. This was confirmed with DLS which showed smaller particle size than those formed with 1:20 drug to PLGA-PEG ratio. Further analysis showed zeta potential was negatively charged which could facilitate cellular uptake as reported previously. In addition, PDI value was less than 0.1 in both formulations indicating monodispersed and less broad in size distribution. The absorption peak of PLGA-PEG-TQ-Nps was at 255 nm. The 1:7 drug to polymer formulation was selected for further analysis where the entrapment efficiency was 79.9% and in vitro drug release showed a maximum release of TQ of 50%. Cytotoxicity result showed IC50 of TQ-nanoparticle at 20.05 μM and free TQ was 8.25 μM.
CONCLUSION: This study showed that nanoparticle synthesized with 1:7 drug to PLGA-PEG ratio and 2:1 PLGA-PEG to Pluronic F68 formed nanoparticles with less than 100 nm and had spherical shape as confirmed with DLS. This could facilitate its transportation and absorption to reach its target. There was conserved TQ stability as exhibited slow release of this volatile oil. The TQ-nanoparticles showed selective cytotoxic effect toward UACC 732 cells compared to MCF-7 breast cancer cells.
METHODS AND RESULTS: TQRF was extracted from N. sativa seeds using supercritical fluid extraction. The regulatory effects of TQRF at 80 microg/ml and TQ at 2 microg/ml on LDLR and HMGCR gene expression were investigated in HepG2 cells using quantitative real-time PCR. The TQ content in TQRF was 2.77% (w/w) and was obtained at a temperature of 40 degrees C and a pressure of 600 bar. Treatment of cells with TQRF and TQ resulted in a 7- and 2-fold upregulation of LDLR mRNA level, respectively, compared with untreated cells. The mRNA level of HMGCR was downregulated by 71 and 12%, respectively, compared with untreated cells.
CONCLUSION: TQRF and TQ regulated genes involved in cholesterol metabolism by two mechanisms, the uptake of low-density lipoprotein cholesterol via the upregulation of the LDLR gene and inhibition of cholesterol synthesis via the suppression of the HMGCR gene.