Neem oil limonoids induces p53-independent apoptosis and autophagy

Abstract

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.

Immunomodulatory action of SGI-110, a hypomethylating agent, in acute myeloid leukemia cells and xenografts

“…Whole protein was extracted from cell lines and/or tissue samples (38) and quantitated. 30–50 μg of protein was loaded onto a NuPAGE® Novex® 4–12% Bis-Tris gel (Invitrogen) and transferred to a nitrocellulose membrane (Invitrogen).…”

Neem components as potential agents for cancer prevention and treatment

“…Extracts from seeds and leaves of neem induce apoptosis in different types of cancer cells such as leukemia [16, 30], prostate [18,31,32], cervical [23], colon [32], stomach [33], and breast [20,32] as well as choriocarcinoma [26], and hepatocarcinoma cells [26]. These findings suggest a proapoptotic effect of neem extracts on a broad spectrum of cancer cell types.…”

“…Neem limonoids induce apoptosis through the intrinsic pathway in prostate cancer and cervical cancer cells, accompanied by increased release of cytochrome c from mitochondria [23,32]. The mitochondrial release of cytochrome c is one of the initiating events during apoptosis via intrinsic apoptotic pathway [37,38].…”

Ayurveda as an Adjuvant Medication for Combating Cancer: A Review

“…Different studies indicate its use against buccal carcinogenesis, skin carcinogenesis, prostate cancer, Ehrlich carcinoma and B16 melanoma [7]. Neem extracts have been shown to possess potent anti-cancer properties against oral squamous cell carcinoma, [8,9] and induces apoptosis in 4T1 breast cancer BALB/c Mice [10]. Nimbolide, a limonoid present in leaves and flowers of the neem tree, have apoptosisinducing property [11,12] thus beneficial in human breast cancer [13].…”