Cheppail Ramachandran*, Gilda Portalatin, Karl-W Quirin, Enrique Escalon, Ziad Khatib and Steven J. Melnick Inhibition of AKT signaling by supercritical CO 2 extract of mango ginger (Curcuma amada Roxb.) in human glioblastoma cells DOI 10.1515/jcim-2015-0005 Received February 1, 2015; accepted July 15, 2015; previously published online October 6, 2015 Abstract Background: Mango ginger (Curcuma amada Roxb.) is a less-investigated herb for anticancer properties than other related Curcuma species. AKT (a serine/threonine protein kinase B, originally identified as an oncogene in the trans- forming retrovirus AKT8) plays a central role in the devel- opment and promotion of cancer. In this investigation, we have analyzed the effect of supercritical CO2 extract of mango ginger (CA) on the genetic pathways associated with AKT signaling in human glioblastoma cells. Methods: The inhibitory effect of supercritical CO 2 extract of mango ginger (Curcuma amada) on AKT signaling was investigated in U-87MG glioblastoma cells. Results: CA was highly cytotoxic to glioblastoma cell line (IC 50 = 4.92 ± 0.81 μg/mL) compared to mHypoE-N1 normal mouse hypothalamus cell line (IC 50 = 40.57 ± 0.06 μg/mL). CA inhibits AKT (protein Kinase B) and adenosine mono- phophate -activated protein kinase α (AMPKα) phosphory- lation significantly in a dose-dependent manner. The cell migration which is necessary for invasion and metastasis was also inhibited by CA treatment, with about 43% reduc- tion at 20 μg/mL concentration. Analysis of mRNA and protein expression of genes associated with apoptosis, cell proliferation and angiogenesis showed that CA modulates expression of genes associated with apoptosis (Bax, Bcl-2, Bcl-X, BNIP3, caspase-3, mutant p53 and p21), cell prolifera- tion (Ki67) and angiogenesis vascular endothelial growth factor (VEGF). Additionally, heat shock protein 90 (HSP90) and AMPKα genes interacting with the AKT signaling path- way were also downregulated by CA treatment. Conclusions: These results indicate the molecular targets and mechanisms underlying the anticancer effect of CA in human glioblastoma cells. Keywords: AKT signaling, cell migration, gene expres- sion, glioblastoma, mango ginger Introduction AKT (serine/threonine protein kinase B) has emerged as central in diverse signaling cascades that regulate cell proliferation, cell size, response to nutrient availability, glucose metabolism, cell invasiveness, genome instability and angiogenesis [1]. Deregulation of these processes is considered as the hallmark of cancer, and a large body of literature suggests a role of frequent hyper-activation of AKT signaling in many human cancers [2]. AKT also sits at the crux of multiple oncogenic and tumor suppressor net- works and almost all known oncogenic growth factors, angiogenic factors and cytokines activate AKT by binding to cognate receptors on cell surface [3]. Several recent studies have also shown that overexpression and/or acti- vation of AKT renders tumor cells resistant to chemother- apeutic drugs and signal molecule inhibitors such as Gleevec, Iressa, Herceptin and retinoid acid [4–8]. AKT is activated by phosphorylation at multiple sites. Initially the Thr 308 residue is phosphorylated, causing a charge-induced binding site conformational change. Although phosphorylation at Thr 308 partially activates AKT, full activation of AKT requires phosphorylation on a second site, the Ser 473 residue, which greatly amplifies the rate of catalysis and downstream effects of AKT activation [9, 10]. Activated AKT is a well-established survival factor, exerting anti-apoptotic activity in part by preventing the release of cytochrome c from the mitochondria [11]. AKT also phosphorylates and inactivates the pro-apoptotic fac- tors BAD and procaspase-9 [12]. Moreover, AKT phosphor- ylates and inactivates the FOXO transcription factors, which mediate the expression of genes critical for apoptosis, such as the Fas ligand gene. In another anti-apoptotic mechan- ism, AKT promotes the phosphorylation and translocation *Corresponding author: Cheppail Ramachandran, Miami Children’s Hospital, Miami, FL, USA; Dharma Biomedical LLC, Miami, FL, USA, E-mail: cheppail.ramachandran@mch.com Gilda Portalatin, Dharma Biomedical LLC, Miami, FL, USA Karl-W Quirin, Flavex Naturextrakte, GmbH, Rehlingen, Germany Enrique Escalon, Ziad Khatib, Miami Children’s Hospital, Miami, FL, USA Steven J. Melnick, Miami Children’s Hospital, Miami, FL, USA; Dharma Biomedical LLC, Miami, FL, USA J Complement Integr Med. 2015; 12(4): 307–315 Brought to you by | University of Saskatchewan Authenticated Download Date | 2/19/16 12:45 AM