Neem oil limonoids induces p53-independent apoptosis and autophagy.
Azadirachta indica, commonly known as neem, has a wide range of medicinal properties. Neem extracts and its purified products have been examined for induction of apoptosis in multiple cancer cell types; however, its underlying mechanisms remain undefined. We show that neem oil (i.e., neem), which contains majority of neem limonoids including azadirachtin, induced apoptotic and autophagic cell death. Gene silencing demonstrated that caspase cascade was initiated by the activation of caspase-9, whereas caspase-8 was also activated late during neem-induced apoptosis. Pretreatment of cancer cells with pan caspase inhibitor, z-VAD inhibited activities of both initiator caspases (e.g., caspase-8 and -9) and executioner caspase-3. Neem induced the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria, suggesting the involvement of both caspase-dependent and AIF-mediated apoptosis. p21 deficiency caused an increase in caspase activities at lower doses of neem, whereas p53 deficiency did not modulate neem-induced caspase activation. Additionally, neem treatment resulted in the accumulation of LC3-II in cancer cells, suggesting the involvement of autophagy in neem-induced cancer cell death. Low doses of autophagy inhibitors (i.e., 3-methyladenine and LY294002) did not prevent accumulation of neem-induced LC3-II in cancer cells. Silencing of ATG5 or Beclin-1 further enhanced neem-induced cell death. Phosphoinositide 3-kinase (PI3K) or autophagy inhibitors increased neem-induced caspase-3 activation and inhibition of caspases enhanced neem-induced autophagy. Together, for the first time, we demonstrate that neem induces caspase-dependent and AIF-mediated apoptosis, and autophagy in cancer cells.
Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
Pragya
P
Srivastava
Neelu
N
Yadav
Ravi
R
Lella
Andrea
A
Schneider
Anthony
A
Jones
Timothy
T
Marlowe
Gabrielle
G
Lovett
Kieran
K
O'Loughlin
Hans
H
Minderman
Raghu
R
Gogada
Dhyan
D
Chandra
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D051033:Apoptosis Inducing Factor / Q000378:metabolism; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects*; D000071186:Beclin-1; D015153:Blotting, Western; D020169:Caspases / Q000737:chemistry / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D045304:Cytochromes c / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005989:Glycerides / Q000737:chemistry*; D006801:Humans; D007306:Insecticides / Q000494:pharmacology; D036701:Limonins / Q000494:pharmacology*; D008565:Membrane Proteins / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D013729:Terpenes / Q000737:chemistry*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D051033:Apoptosis Inducing Factor; D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; D005989:Glycerides; D007306:Insecticides; D036701:Limonins; D008565:Membrane Proteins; C495901:TP53 protein, human; D013729:Terpenes; D016159:Tumor Suppressor Protein p53; C002443:neem oil; C025946:3-methyladenine; D045304:Cytochromes c; D020169:Caspases; D000225:Adenine; C010329:azadirachtin