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.
Cratoxylum arborescens has been used traditionally in Malaysia for the treatment of various ailments.



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.



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.



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.
Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
Mohamed Yousif
MY
Ibrahim
Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
Najihah
N
Mohd Hashim
Medical Research Centre, Jazan University, Jazan, Saudi Arabia.
Syam
S
Mohan
Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
Mahmood Ameen
MA
Abdulla
Medical Research Centre, Jazan University, Jazan, Saudi Arabia.
Siddig Ibrahim
SI
Abdelwahab
Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
Behnam
B
Kamalidehghan
Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia ; Epigenetics Lab, HIR Building, Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia.
Mostafa
M
Ghaderian
Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia ; Department of Physiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
Firouzeh
F
Dehghan
Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
Landa Zeenelabdin
LZ
Ali
Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
Hamed
H
Karimian
Department of Bioproduct Research and Innovation, Institute of Bioproduct Development (IBD), Universiti Teknologi Malaysia, UTM Johor Bahru, Johor, Malaysia.
Maizatulakmal
M
Yahayu
Department of Chemistry, Faculty of Science, Universiti Putra Malaysia (UPM), Serdang, Selangor, Malaysia.
Gwendoline Cheng Lian
GC
Ee
Institute of Tropical Agriculture, Universiti Putra Malaysia (UPM), Serdang, Selangor, Malaysia.
Abdoreza Soleimani
AS
Farjam
Department of Chemistry, University of Malaya, Kuala Lumpur, Malaysia.
Hapipah
H
Mohd Ali
D000972:Antineoplastic Agents, Phytogenic / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D028084:Clusiaceae / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004354:Drug Screening Assays, Antitumor; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D015394:Molecular Structure; D016328:NF-kappa B / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D013329:Structure-Activity Relationship; D044004:Xanthones / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*
D000972:Antineoplastic Agents, Phytogenic; D018840:HSP70 Heat-Shock Proteins; D016328:NF-kappa B; D044004:Xanthones; C021053:mangostin
caspase-3/7; mitochondria; protein array