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.
Clausena excavata Burm. f. has been used in folk medicines in eastern Thailand for the treatment of cancer.
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.
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.
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.
Arbab IA
，Abdul AB
，Sukari MA
，Abdullah R
，Syam S
，Kamalidehghan B
，Ibrahim MY
，Taha MM
，Abdelwahab SI
，Ali HM
，Mohan S
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Chloroform Fraction of Methanolic Extract of Seeds of Annona muricata Induce S Phase Arrest and ROS Dependent Caspase Activated Mitochondria-Mediated Apoptosis in Triple-Negative Breast Cancer.
Triple Negative Breast Cancers (TNBCs) have high morbidity and shorter survival rate in the population. These types of cancers have high aggressiveness, lymphatic invasion, and absence of receptors. The treatment options for these types of cancers are also scarce. Several studies have been conducted to investigate the effectiveness of seeds of Annona muricata for its anti-cancer activities in various cancer cell lines, such as lung A549, breast MCF7, colon HT-29, oral KB, and human hepatoma cell lines. But works related to its anti-cancer effect and mechanism of action in TNBCs have not been elucidated.
The present study was undertaken to evaluate the in vitro, in vivo, and in silico anti-cancer potential of chloroform fraction of methanolic extract of seeds of Annona muricata (CMAM) against TNBC along with elucidation of its mechanistic pathway.
In vitro cytotoxicity- and antiproliferative- studies in three triple-negative breast cancer cell lines were conducted using the MTT and SRB assays, respectively. The mechanism through which CMAM exerts its pharmacological effect was elucidated in vitro employing cell morphological assessment studies using Acridine Orange/Ethidium Bromide (AO/EB), intracellular reactive oxygen species assay, DNA fragmentation assay, agarose gel electrophoresis, terminal deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) assay, cell cycle analysis, annexin binding assay, and caspase-activated mitochondria-mediated apoptotic assays using western blot. In vivo evaluation in 4T1 induced murine mammary tumor model was also conducted. Phytoconstituents in CMAM were analyzed using liquid chromatography mass spectroscopy. In silico binding studies with various annonaceous acetogenins against BCL-2 and cyclin E were performed.
Cytotoxicity studies in MDA-MD-231, 4TI, and BT-549 revealed the IC50 value of CMAM to be 2.5±0.14, 4.8±0.3, and 4.5±0.16μg/mL, respectively. Anti-proliferative studies in 4T1, MDA-MB-231, and BT- 549 revealed the GI50 values to be 0.128+0.03, 18.03+0.20, 0.95+0.04μg/mL, respectively. CMAM exhibited its cytotoxicity through the lysis of cell membrane, ROS dependent caspase-activated mitochondria-mediated apoptosis, and arresting the S phase of the cell cycle. In vivo evaluation also supported the tumoricidal property of CMAM, as evidenced by a reduction in tumor volume and serum biomarkers. Histopathologically, there was a marked reduction in cellularity, nuclear chromatin condensation, and a few normal cells in the group treated with CMAM at a dose of 31mg/Kg. Phytoconstituent evaluation has revealed the presence of annonaceous acetogenins in CMAM. Among the various annonaceous acetogenins, muricatacin alone showed lipophilicity and binding affinity towards BCL-2 and cyclin E1.
The current study shows the effectiveness of CMAM against TNBC both in vitro and in vivo. This anticancerous effect of CMAM could be by virtue of its ROS dependent caspase-activated mitochondriamediated apoptosis and the S-phase arrest of the cell cycle in the TNBCs. Our results indicate that the presence of annonaceous acetogenins, especially muricatacin, could be contributing to this anticancerous effect of CMAM. Thus, muricatacin could be a potential candidate for the targeted therapy of TNBCs.
Kariyil BJ
，Ayyappan UPT
，Gopalakrishnan A
，George AJ
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