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.
METHODS: Sprague Dawley rats clustered into five groups, normal control (A) and cancer controls were subcutaneously injected with normal saline and 15 mg/kg azoxymethane, respectively, and nourished on 10% tween 20 and fed on 10% tween 20; reference control (C), injected with 15 mg/kg azoxymethane and injected (intraperitoneal) with 35 mg/kg 5-fluorouracil (5-FU); D and E rat groups received a subcutaneous injection of 15 mg/kg azoxymethane and nourished on 30 and 60 mg/kg of PN, respectively.
RESULTS: The acute toxicity trial showed a lack of any abnormal signs or mortality in rats ingested with 250 and 500 mg/kg of PN. The gross morphology of colon tissues revealed significantly lower total colonic aberrant crypt foci incidence in PN-treated rats than that of cancer controls. Histological examination of colon tissues showed increased aberrant crypt foci availability with bizarrely elongated nuclei, stratified cells and higher depletion of the submucosal glands in cancer controls. PN treatment caused positive modulation of apoptotic (Bax and Bcl-2) proteins and inflammatory cytokines (TNF-α, IL-6 and IL-10). Moreover, rats fed on PN had significantly higher antioxidants (superoxide dismutase) and lower malondialdehyde concentrations in their colon tissue homogenates.
CONCLUSION: The chemoprotective efficiency of PN against azoxymethane-induced aberrant crypt foci is shown by lower aberrant crypt foci values and higher aberrant crypt foci inhibition percentage, possibly through augmentation of genes responsible for apoptotic cascade and inflammations originating from azoxymethane oxidative stress insults.
METHODS AND RESULTS: Five groups of rats: normal control, cancer control, TPHE low dose, TPHE high dose and positive control (tamoxifen) were used for the in vivo study. Histopathological examination showed that TPHE significantly suppressed the carcinogenic effect of LA7 tumour cells. The tumour sections from TPHE-treated rats demonstrated significantly reduced expression of Ki67 and PCNA compared to the cancer control group. Using a bioassay-guided approach, the cytotoxic compound of TPHE was identified as a tricyclic sesquiterpene lactone, namely, 8β- hydroxyl- 4β, 15- dihydrozaluzanin C (HDZC). Signs of early and late apoptosis were observed in MCF7 cells treated with HDZC and were attributed to the mitochondrial intrinsic pathway based on the up-regulation of Bax and the down-regulation of Bcl-2. HDZC induced cell cycle arrest in MCF7 cells and increased the expression of p21 and p27 at the mRNA and protein levels.
CONCLUSION: This results of this study substantiate the anticancer effect of TPHE and highlight the involvement of HDZC as one of the contributing compounds that act by initiating mitochondrial-mediated apoptosis.
METHODS: A WEHI-3 cell line was used to evaluate the cytotoxicity of BM by MTT. AO/PI and Hoechst 33342 dyes, Annexin V, multiparametric cytotoxicity 3 by high content screening (HCS); cell cycle tests were used to estimate the features of apoptosis and BM effects. Caspase 3 and 9 activities, ROS, western blot for Bcl2, and Bax were detected to study the mechanism of apoptosis. BALB/c mice injected with WEHI-3 cells were used to assess the apoptotic effect of BM in vivo.
RESULTS: BM suppressed the growth of WEHI-3 cells at an IC50value of 14 ± 3 μg/mL in 24 h. The ROS production was increased inside the cells in the treated doses. Both caspases (9 and 3) were activated in treating WEHI-3 cells at 24, 48 and 72 h. Different signs of apoptosis were detected, such as cell membrane blebbing, DNA segmentation and changes in the asymmetry of the cell membrane. Another action by which BM could inhibit WEHI-3 cells is to restrain the cell cycle at the G1/G0 phase. In the in vivo study, BM reduced the destructive effects of leukaemia on the spleen and liver by inducing apoptosis in leukaemic cells.
CONCLUSION: BM exerts anti-leukaemic properties in vitro and in vivo.
METHODOLOGY: The experimental set included different animal groups. Specifically, four groups with gastric mucosal lesions were receiving either a) Ulcer control group treated with absolute ethanol (5 ml/kg), b) 20 mg/kg of omeprazole as reference group, c) 25 of biochanin A, d) 50 mg/kg of biochanin A. Histopathological sectioning followed by immunohistochemistry staining were undertaken to evaluate the influence of the different treatments on gastric wall mucosal layer. The gastric secretions were collected in the form of homogenate and exposed to superoxide dismutase (SOD) and nitric oxide enzyme (NO) and the level of malondialdehyde (MDA) and protein content were measured. Ulceration and patchy haemorrhage were clearly observed by light microscopy. The morphology of the gastric wall as confirmed by immunohistochemistry and fluorescent microscopic observations, exhibited sever deformity with notable thickness, oedematous and complete loss of the mucosal coverage however the biochanin-pretreated animals, similar to the omeprazole-pretreated animals, showed less damage compared to the ulcer control group. Moreover, up-regulation of Hsp70 protein and down-regulation of Bax protein were detected in the biochanin A pre-treated groups and the gastric glandular mucosa was positively stained with Periodic Acid Schiff (PAS) staining and the Leucocytes infiltration was commonly seen. Biochanin A displayed a great increase in SOD and NO levels and decreased the release of MDA.
CONCLUSIONS: This gastroprotective effect of biochanin A could be attributed to the enhancement of cellular metabolic cycles perceived as an increase in the SOD, NO activity, and decrease in the level of MDA, and also decrease in level of Bax expression and increase the Hsp70 expression level.