Synthesis and cytotoxic effects of (E)-3-(2,3-dimethoxyphenyl)-1-(5-methylfuran-2-yl) prop-2-en-1-one in MDA-MB231 and MCF-7 breast cancer cell lines

A chalcone derivative, (E)-3-(2,3-dimethoxyphenyl)-1-(5-methylfuran-2-yl)-prop-2-en-1-one (DMMF) was synthesized and evaluated against various cancerous cell lines including colon adenocarcinoma (HT-29), myloplasticleukemia (HL60), breast cancer (MCF-7 and MDA-MB231), normal hepatic cell (WRL-68) and normal breast cell (MCF-10A). The structure of DMMF was determined by EI-MS, 1H NMR and single X-ray crystallographic techniques. The DMMF possessed the highest cytotoxic effect against MCF-7 breast cancer cell (2.01 ± 1.53 μg/mL) and lowest against normal hepatic WRL-68 and breast cell lines after 24 h of treatment. Induction of apoptosis and regulation of cell cycle progression results indicates the significant increase in early apoptosis and G2/M arrest after 48 h of treatment in MCF-7 cells. Meanwhile, in MDA-MB231 cells, there was an increase in Sub G0/G1 cells population and early/late apoptotic cells upon treatment with DMMF. Additionally, DMMF effectively induced G2/M cell cycle arrest in MCF-7 cells and apoptosis in both MCF-7 and MDA-MB231 cells

Synthesis and Cytotoxic Effects of (E)-3-(2,3-dimethoxyphenyl)-1-(5-methylfuran-2-yl) prop-2-en-1-one in MDA-MB231 and MCF-7 Breast Cancer Cell Lines

A chalcone derivative, (E)-3-(2,3-dimethoxyphenyl)-1-(5-methylfuran-2-yl)-prop-2-en-1-one (DMMF) was synthesized and evaluated against various cancerous cell lines including colon adenocarcinoma (HT-29), myloplasticleukemia (HL60), breast cancer (MCF-7 and MDA-MB231), normal hepatic cell (WRL-68) and normal breast cell (MCF-10A). The structure of DMMF was determined by EI-MS, 1H NMR and single X-ray crystallographic techniques. The DMMF possessed the highest cytotoxic effect against MCF-7 breast cancer cell (2.01 ± 1.53 μg/mL) and lowest against normal hepatic WRL-68 and breast cell lines after 24 h of treatment. Induction of apoptosis and regulation of cell cycle progression results indicates the significant increase in early apoptosis and G2/M arrest after 48 h of treatment in MCF-7 cells. Meanwhile, in MDA-MB231 cells, there was an increase in Sub G0/G1 cells population and early/late apoptotic cells upon treatment with DMMF. Additionally, DMMF effectively induced G2/M cell cycle arrest in MCF-7 cells and apoptosis in both MCF-7 and MDA-MB231 cells

alpha-Mangostin from Cratoxylum arborescens demonstrates apoptogenesis in MCF-7 with regulation of NF-kappa B 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 cance

Beta-mangostin demonstrates apoptogenesis in murine leukaemia (WEHI-3) cells in vitro and in vivo

Abstract Background Beta-mangostin (BM) is a xanthone-type of natural compound isolated from Cratoxylum arborescens. This study aimed to examine the apoptosis mechanisms induced by BM in a murine monomyelocytic cell line (WEHI-3) in vitro and in vivo. 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

Histopathology of the spleen tissues.

<p>(A) Control where BALB/c mice were not injected with WEHI-3 cells; the white pulp, red pulp and lymphocyte cells were clearly shown. (B) BALB/c mice injected with WEHI-3 cells and not treated. The leukemia sectioning showed widened white pulp, but the red pulp became tiny. The yellow arrows indicated neoplastic cells. (C) BALB/c mice injected with WEHI-3 cells and treated with 100 mg/kg thymoquinone. The white pulp decreased in size, and the red pulp exhibited a little increase in size. The treatment with 100 mg/kg thymoquinone showed similarity to the control.</p

Effects of thymoquinone on the weights of liver and spleen tissues from BALB/c mice.

<p>The animals were injected with WEHI-3 cells (1×10⁶) for 3-week time period and treated with or without thymoquinone (TQ) for 3 weeks. (A) Livers were individually collected, photographed, and (B) weighed. (C) Spleen was individually collected, photographed and (D) weighed. Thymoquinone affected the weight of liver and spleen tissues from BALB/c mice. ‘*’ indicates statistically significant at p<0.05. (n = 5).</p

Fluorescent microscope analysis of nuclei fragmentation by Hoechst 33342 and AO/PI Double-Staining.

<p>(A) Staining with Hoechst 33342. Cells in the control were untreated WEHI-3 cells. Apoptotic cells appeared (white arrows) after 24 and 48 h. In 72 h, all of the cells were apoptotic. (B) AO/PI staining. After 72 h, the untreated cells showed normal structure without prominent apoptosis and necrosis. Early apoptosis features were seen after 24 h representing intercalated AO (bright green) amongst the fragmented DNA; blebbing, orange color representing late apoptosis were noticed after 48 h treatment; bright red colored secondary necrosis were visible after 72 h. White arrows: viable cells; red arrows: membrane blebbing; pink arrows: early apoptosis; blue arrows: late apoptosis; yellow arrows: secondary necrosis. Images are representative of one of three similar experiments. (C) Early and late apoptosis increased significantly (*p<0.05) compared to control, in a time-dependent manner. However, no significant difference was observed in necrosis cells. VI: viable cell, EA: early apoptosis, LA: late apoptosis, SN: secondary necrosis</p

Effect of thymoquinone on early apoptosis, cell cycle analysis and protein expression.

<p>(A) Staining with FITC-conjugated Annexin V and PI; cells were analyzed by flow cytometry. Control cells (no drug treatment), 24, 48 and 72 h were in a time-dependent manner. The early apoptotic events (Annexin+/PI-) are shown in lower right quadrant (Q4) of each panel. Quadrant (Q2) represents Annexin+/PI+ late stage of apoptosis/dead cells. (n = 2). (B) Histograms for cell cycle analysis. (C) Cell cycle graph; Induction of S phase arrest in the cell cycle progression. “*” Indicates statistically significant at p<0.05, where the arrest at 24, 48 and 72 h was individually compared to control. (D) Effect of thymoquinone on the levels of apoptosis regulatory proteins at 3, 6, 12 and 24 h with β- actin as a loading control, ‘*’ indicates statistically significant at p<0.05. Shapiro-Wilk test: Hsb70, P = 0.339; Bcl2, P = 0.57; Bax, P = 0.192, where P value are greater than α level of 0.05, showing that the data have normally distributed population.</p

Detection of apoptosis by TUNEL assay in (A) the spleen and (B) the liver tissues of BALB/c mice.

<p>Normal sections show less apoptotic cells (white arrows). TUNEL staining in positive control section of BALB/c mice (induced with leukemia and without treatment) shows aggressive cell proliferation without apoptotic cells. Tissues of BALB/c mice induced with leukemia and treated with 100 mg/kg thymoquinone (TQ) show the evidence of apoptosis by the represented apoptotic cells (white arrows).</p

Effects of thymoquinone on animal body weight.

<p>(A) Representative images of normal BALB/c mice; BALB/c mice injected with WEHI-3 cells (1×10⁶) and BALB/c mice injected with WEHI-3 cells (1×10⁶) and treated with thymoquinone 100 mg/kg for 3 weeks. The animals were then sacrificed, and photographed. The arrows are pointing at the size of the liver (blue arrow) and spleen (red arrow). (B) Body weight changes of BALB/c mice treated with or without thymoquinone (TQ) for 3 weeks. Values are average of five mice.</p