Mohamed Yousif Ibrahim

Zerumbone induces apoptosis in T-acute lymphoblastic leukemia cells

Zerumbone (ZER) is a potential anticancer natural compound, isolated from Zingiber zerumbet Smith. In this investigation, the anticancer properties of ZER were evaluated on cancer cells of T-acute lymphoblastic leukemia, CEM-ss. The results showed that ZER has cytotoxic effect against CEM-ss cells with an IC(50) of 8.4 ± 1.9 μg/ml (coefficient of variation < 30%). Comparatively, 5-fluorouracil (positive control), imposed an inhibitory effect on CEM-ss cells with an IC(50) of 1.94 ± 0.06 μg/ml. Scanning electron microscopy (SEM) results revealed abnormalities such as membrane blebbing, holes and cytoplasmic extrusions, all of which are characteristics of apoptosis. In addition, ZER has increased the number of TUNEL-positive stain and the cellular level of caspase-3, the hallmarks of apoptosis, on treated CEM-ss cells. It could be concluded that, ZER was able to produce apoptosis on T-acute lymphoblastic leukemia, CEM-ss. The current findings suggest that ZER might be helpful for improving the usefulness of anticancer agents in the therapy of leukemia.

Thymoquinone Induces Mitochondria-Mediated Apoptosis in Acute Lymphoblastic Leukaemia in Vitro

There has been a growing interest in naturally occurring compounds from traditional medicine with anti-cancer potential. Nigella sativa (black seed) is one of the most widely studied plants. This annual herb grows in countries bordering the Mediterranean Sea and India. Thymoquinone (TQ) is an active ingredient isolated from Nigella sativa. The anti-cancer effect of TQ, via the induction of apoptosis resulting from mitochondrial dysfunction, was assessed in an acute lymphocyte leukemic cell line (CEMss) with an IC50 of 1.5 µg/mL. A significant increase in chromatin condensation in the cell nucleus was observed using fluorescence analysis. The apoptosis was then confirmed by Annexin V and an increased number of cellular DNA breaks in treated cells were observed as a DNA ladder. Treatment of CEMss cells with TQ encouraged apoptosis with cell death-transducing signals by a down-regulation of Bcl-2 and up-regulation of Bax. Moreover, the significant generation of cellular ROS, HSP70 and activation of caspases 3 and 8 were also observed in the treated cells. The mitochondrial apoptosis was clearly associated with the S phase cell cycle arrest. In conclusion, the results from the current study indicated that TQ could be a promising agent for the treatment of leukemia.

Dentatin isolated from Clausena excavata induces apoptosis in MCF-7 cells through the intrinsic pathway with involvement of NF-κB signalling and G0/G1 cell cycle arrest: a bioassay-guided approach.

ETHNOPHARMACOLOGICAL RELEVANCE Clausena excavata Burm. f. has been used in folk medicines in eastern Thailand for the treatment of cancer. MATERIALS AND METHODS To investigate the apoptosis mechanism, we isolated dentatin (DTN) from this plant using a bioassay-guided approach. DTN-induced cytotoxicity was observed with the MTT assay. Acridine orange/propidium iodide staining was used to detect cells in early apoptosis and high content screening (HCS) to observe nuclear condensation, cell permeability, mitochondrial membrane potential (MMP) and cytochrome c release. Apoptosis was confirmed with a clonogenic assay, DNA laddering and caspase 3/7 and 9 assays. Reactive oxygen species (ROS) formation, Bcl-2 and Bax expression, and cell cycle arrest were also investigated. The involvement of nuclear factor-kappa B (NF-κB) was analysed with the HCS assay. RESULTS A significant increase in chromatin condensation in the cell nucleus was observed by fluorescence analysis. Apoptosis was confirmed by the reduced number of colonies in the clonogenic assay and the increased number of cellular DNA breaks in treated cells observed as a DNA ladder. Treatment of MCF-7 cells with DTN encouraged apoptosis with cell death-transducing signals that reduced MMP by down-regulation of Bcl-2 and up-regulation of Bax, triggering cytochrome c release from the mitochondria to the cytosol. The released cytochrome c triggered the activation of caspase 9 followed by the executioner caspase 3/7. DTN treatment significantly arrested MCF-7 cells at the G0/G1 phase (p<0.05) and ROS was significantly elevated. Moreover, DTN significantly blocked the induced translocation of NF-κB from cytoplasm to nucleus. CONCLUSION Together, the results demonstrated that the DTN isolated from Clausena excavata inhibited the proliferation of MCF-7 cells, leading to cell cycle arrest and programmed cell death, which was confirmed to occur through the mitochondrial pathway with involvement of the NF-κB signalling pathway.

Involvement of NF-ΚB and HSP70 signaling pathways in the apoptosis of MDA-MB-231 cells induced by a prenylated xanthone compound, α-mangostin, from Cratoxylum arborescens

Background Cratoxylum arborescens has been used traditionally in Malaysia for the treatment of various ailments. Methods α-Mangostin (AM) was isolated from C. arborescens and its cell death mechanism was investigated. AM-induced cytotoxicity was observed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Acridine orange/propidium iodide staining and annexin V were used to detect cells in early phases of apoptosis. High-content screening was used to observe the nuclear condensation, cell permeability, mitochondrial membrane potential, and cytochrome c release. The role of caspases-3/7, -8, and -9, reactive oxygen species, Bcl-2 and Bax expression, and cell cycle arrest were also investigated. To determine the role of the central apoptosis-related proteins, a protein array followed by immunoblot analysis was conducted. Moreover, the involvement of nuclear factor-kappa B (NF-κB) was also analyzed. Results Apoptosis was confirmed by the apoptotic cells stained with annexin V and increase in chromatin condensation in nucleus. Treatment of cells with AM promoted cell death-transducing signals that reduced MMP by downregulation of Bcl-2 and upregulation of Bax, triggering cytochrome c release from the mitochondria to the cytosol. The released cytochrome c triggered the activation of caspase-9 followed by the executioner caspase-3/7 and then cleaved the PARP protein. Increase of caspase-8 showed the involvement of extrinsic pathway. AM treatment significantly arrested the cells at the S phase (P<0.05) concomitant with an increase in reactive oxygen species. The protein array and Western blotting demonstrated the expression of HSP70. Moreover, AM significantly blocked the induced translocation of NF-κB from cytoplasm to nucleus. Conclusion Together, the results demonstrate that the AM isolated from C. arborescens inhibited the proliferation of MDA-MB-231 cells, leading to cell cycle arrest and programmed cell death, which was suggested to occur through both the extrinsic and intrinsic apoptosis pathways with involvement of the NF-κB and HSP70 signaling pathways.

Thymoquinone inhibits murine leukemia WEHI-3 cells in vivo and in vitro.

Background Thymoquinone is an active ingredient isolated from Nigella sativa (Black Seed). This study aimed to evaluate the in vitro and in vivo anti-leukemic effects of thymoquinone on WEHI-3 cells. 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.

α-Mangostin from Cratoxylum arborescens demonstrates apoptogenesis in MCF-7 with regulation of NF-κB and Hsp70 protein modulation in vitro, and tumor reduction in vivo.

Cratoxylum arborescens is an equatorial plant belonging to the family Guttiferae. In the current study, α-Mangostin (AM) was isolated and its cell death mechanism was studied. HCS was undertaken to detect the nuclear condensation, mitochondrial membrane potential, cell permeability, and the release of cytochrome c. An investigation for reactive oxygen species formation was conducted using fluorescent analysis. To determine the mechanism of cell death, human apoptosis proteome profiler assay was conducted. In addition, using immunofluorescence and immunoblotting, the levels of Bcl-2-associated X protein (Bax) and B-cell lymphoma (Bcl)-2 proteins were also tested. Caspaces such as 3/7, 8, and 9 were assessed during treatment. Using HCS and Western blot, the contribution of nuclear factor kappa-B (NF-κB) was investigated. AM had showed a selective cytotoxicity toward the cancer cells with no toxicity toward the normal cells even at 30 μg/mL, thereby indicating that AM has the attributes to induce cell death in tumor cells. The treatment of MCF-7 cells with AM prompted apoptosis with cell death-transducing signals. This regulated the mitochondrial membrane potential by down-regulation of Bcl-2 and up-regulation of Bax, thereby causing the release of cytochrome c from the mitochondria into the cytosol. The liberation of cytochrome c activated caspace-9, which, in turn, activated the downstream executioner caspace-3/7 with the cleaved poly (ADP-ribose) polymerase protein, thereby leading to apoptotic alterations. Increase of caspace 8 had showed the involvement of an extrinsic pathway. This type of apoptosis was suggested to occur through both extrinsic and intrinsic pathways and prevention of translocation of NF-κB from the cytoplasm to the nucleus. Our results revealed AM prompt apoptosis of MCF-7 cells through NF-κB, Bax/Bcl-2 and heat shock protein 70 modulation with the contribution of caspaces. Moreover, ingestion of AM at (30 and 60 mg/kg) significantly reduced tumor size in an animal model of breast cancer. Our results suggest that AM is a potentially useful agent for the treatment of breast cancer.

In vivo Assessment of Antioxidant and Wound Healing Improvement of a New Schiff Base Derived Co (II) Complex in Rats

Co (II) complex (CMLA) was investigated to evaluate the rate of wound healing in rats. Animals were placed into four groups: gum acacia, Intrasite gel, 10 and 20 mg/ml of CMLA. Wounds were made on the dorsal neck area, then treated with Intrasite gel or CMLA; both of these treatments led to faster healing than with gum acacia. Histology of the wounds dressed with CMLA or Intrasite gel displayed a smaller scar width, required less time to heal and showed more collagen staining and fewer inflammatory cells in comparison to wounds dressed with the vehicle. Immunohistochemistry for Hsp70 and TGF-β showed greater staining intensity in the treated groups compared to the vehicle group. Bax staining was less intense in treated groups compared to the vehicle group, suggesting that CMLA and Intrasite gel provoked apoptosis, responsible for the development of granulation tissue into a scar. CD31 protein analysis showed that the treated groups enhanced angiogenesis and increased vascularization compared to the control group. Furthermore, a significant increase in the levels of GPx and SOD and a decrease in MDA were also observed in the treated groups. This results suggest that CMLA is a potentially promising agent for the wounds treatment.

In vitro assessment of anti-proliferative effect induced by α-mangostin from Cratoxylum arborescens on HeLa cells

Natural medicinal products possess diverse chemical structures and have been an essential source for drug discovery. Therefore, in this study, α-mangostin (AM) is a plant-derived compound was investigated for the apoptotic effect on human cervical cancer cells (HeLa). The cytotoxic effects of AM on the viability of HeLa and human normal ovarian cell line (SV40) were evaluated by using MTT assay. Results showed that AM inhibited HeLa cells viability at concentration- and time-dependent manner with IC50 value of 24.53 ± 1.48 µM at 24 h. The apoptogenic effects of AM on HeLa were assessed using fluorescence microscopy analysis. The effect of AM on cell proliferation was also studied through clonogenic assay. ROS production evaluation, flow cytometry (cell cycle) analysis, caspases 3/7, 8, and 9 assessment and multiple cytotoxicity assays were conducted to determine the mechanism of cell apoptosis. This was associated with G2/M phase cell cycle arrest and elevation in ROS production. AM induced mitochondrial apoptosis which was confirmed based on the significant increase in the levels of caspases 3/7 and 9 in a dose-dependent manner. Furthermore, the MMP disruption and increased cell permeability, concurrent with cytochrome c release from the mitochondria to the cytosol provided evidence that AM can induce apoptosis via mitochondrial-dependent pathway. AM exerted a remarkable antitumor effect and induced characteristic apoptogenic morphological changes on HeLa cells, which indicates the occurrence of cell death. This study reveals that AM could be a potential antitumor compound on cervical cancer in vitro and can be considered for further cervical cancer preclinical and in vivo testing.

Acute Toxicity and Gastroprotection Studies of a New Schiff Base Derived Manganese (II) Complex against HCl/Ethanol-Induced Gastric Ulcerations in Rats.

Manganese is a crucial element for health. In this study, the gastroprotective efficacy of Mn (II) complex (MDLA) against acidified ethanol (HCl/Ethanol)-induced gastric ulceration in rats was evaluated. The animals were distributed into 5 groups. Groups 1 and 2 received carboxymethylcellulose (CMC), group 3 was pretreated with omeprazole, and groups 4 and 5 were given 10 and 20 mg/kg of MDLA, respectively. After one hour, CMC and HCl/Ethanol were given to groups 2–5 whilst the animals in group 1 were ingested with CMC. After sacrifice, gastric lesions were evaluated by wall mucus, gross appearance, histology, antioxidant enzymes and immunohistochemistry. Group 2 displayed severe gastric damage with a significant reduction in wall mucus. Conversely, gastric lesions were reduced in groups 3–5 by 85.72%, 56.51% and 65.93%, respectively. The rats in groups 3–5 showed up-regulation of heat shock protein 70 (Hsp70) with down-regulation of Bcl-2-associated protein x (Bax). Pretreatment with omeprazole or MDLA led to an increase in the uptake of Periodic Acid Schiff (PAS) stain in the glandular part of the gastric tissue, raised levels of prostaglandin E2 (PGE2) and superoxide dismutase (SOD), and a reduction in malondialdehyde (MDA) concentrations. These results suggested the gastroprotective action of Mn (II) complex.

Beta-mangostin from Cratoxylum arborescens activates the intrinsic apoptosis pathway through reactive oxygen species with downregulation of the HSP70 gene in the HL60 cells associated with a G0/G1 cell-cycle arrest

Xanthones are phytochemical compounds found in a number of fruits and vegetables. Characteristically, they are noted to be made of diverse properties based on their biological, biochemical, and pharmacological actions. Accordingly, the apoptosis mechanisms induced by beta-mangostin, a xanthone compound isolated from Cratoxylum arborescens in the human promyelocytic leukemia cell line (HL60) in vitro, were examined in this study. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was done to estimate the cytotoxicity effect of β-mangostin on the HL60 cell line. Acridine orange/propidium iodide and Hoechst 33342 dyes and Annexin V tests were conducted to detect the apoptosis features. Caspase-3 and caspase-9 activities; reactive oxygen species; real-time polymerase chain reaction for Bcl-2, Bax, caspase-3, and caspase-9 Hsp70 genes; and western blot for p53, cytochrome c, and pro- and cleavage-caspase-3 and caspase-9 were assessed to examine the apoptosis mechanism. Cell-cycle analysis conducted revealed that β-mangostin inhibited the growth of HL60 at 58 µM in 24 h. The administration of β-mangostin with HL60 caused cell morphological changes related to apoptosis which increased the number of early and late apoptotic cells. The β-mangostin-catalyzed apoptosis action through caspase-3, caspase-7, and caspase-9 activation overproduced reactive oxygen species which downregulated the expression of antiapoptotic genes Bcl-2 and HSP70. Conversely, the expression of the apoptotic genes Bax, caspase-3, and caspase-9 were upregulated. Meanwhile, at the protein level, β-mangostin activated the formation of cleaved caspase-3 and caspase-9 and also upregulated the p53. β-mangostin arrested the cell cycle at the G0/G1 phase. Overall, the results for β-mangostin showed an antiproliferative effect in HL60 via stopping the cell cycle at the G0/G1 phase and prompted the intrinsic apoptosis pathway.