MOLECULAR CARCINOGENESIS 55:964–976 (2016) PI3K Target Based Novel Cyano Derivative of Betulinic Acid Induces Its Signalling Inhibition by Down-Regulation of pGSK3b and Cyclin D1 and Potentially Checks Cancer Cell Proliferation Rabiya Majeed, 1,2 Aashiq Hussain, 1 Payare L. Sangwan, 2,3 Praveen K. Chinthakindi, 2 Imran Khan, 2 Parduman R. Sharma, 1 Surrinder Koul, 2 Ajit K. Saxena, 1 and Abid Hamid 1,3 * 1 Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road Jammu, India 2 Bioorganic Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road Jammu, India 3 CSIR-Academy of Scientific & Innovative Research, Govt. of India, New Delhi, India In spite of the Betulinic acid (BA) being recognized as anticancerous source; its further use in clinical development is greatly hampered because of its poor pharmacokinetic properties. To circumvent these limitations, we synthesized a PI3K target based library of 18 triazole based derivatives and we identified a C-3 cyano analog of betulinic acid (CBA) with significant cell death effects with 5–7 fold higher potency than BA in various cancers. Importantly, no such report is available demonstrating the involvement of BA or its structural analogs in the modulation of PI3K pathway. Using, human leukemia HL-60 cells as a model, we for the first time report that CBA decreased expression of PI3K p110a, p85a, and pAKT in HL-60. Furthermore, we could find significant depletion of pGSK3b, cyclin D1 and increased expression of p21/ cip, p27/Kip proteins. CBA induced G0/G1 cell cycle arrest, increased sub-G0 DNA fraction and annexin V binding of the cells besides imparting the typical surface features of cell death. Also, this target specific inhibition was associated with mitochondrial apoptosis as was reflected by expression studies of various proteins together with reactive oxygen species generation and decline in mitochondrial trans membrane potential. The apoptotic effectors i.e., caspase 8 and caspase 9 were found to get upregulated besides PI3K associated DNA repair enzyme i.e., PARP cleavage was observed. Thus, our results elucidated that CBA or other BA based small molecules inhibit PI3K/AKT pathway with induction of subsequent cancer cell death which may be useful therapeutic strategy against leukemias and possibly other cancers. © 2015 Wiley Periodicals, Inc. Key words: leukemia; phosphotidylinositol-3 kinase; GSK3b; apoptosis; betulinic acid; metabolism INTRODUCTION Leukemia is currently the sixth leading cause of cancer related deaths worldwide [1]. In the treatment perspectives, one of the strong considerations is the use of natural products as a platform for the drug development with target-based approach to address the limitations associated with current therapies. Also, in current scientific promise of chemopreven- tion, there is an overwhelming need to develop the new chemopreventive agents that are both effective and safe. Approximately, half of the drugs currently used in the clinic are derived from natural products and in the case of cancer this proportion surpasses 60% [2]. Natural compounds of diverse structures and functional groups have been considered prototypes, leads or heads of series. Importantly, betulinic acid (BA) is a naturally occurring, widely distributed pentacyclic triterpene that exhibits potent antitumor effects besides stem cell modulatory effects [1,3]. The mode of the action of BA in cancer cells is linked to the activation of mitochondrial intrinsic apoptotic path- way and to perturbations in the cell cycle progres- sion [4]. Treatment with BA results in loss of the mitochondrial membrane potential (MMP), followed by release of cytochrome c and apoptotis-inducing factor (AIF), leading to activation of caspases and formation of the apoptosome. Also, ROS generation was linked to activation of pro apoptotic p38 and SAP/ JNK kinase. However, BA induces apoptosis in a p53- and CD95-independent manner [3]. Betulinic acid has been reported to modulate activity of the transcription factor nuclear factor-kB (NF-kB), and can also inhibit aminopeptidase N, an enzyme that is involved in the regulation of angiogenesis and over- expressed in several cancers [5]. There are reports which suggested that triterpenoids induce cell cycle arrest and apoptosis in cancer cells via the modulation Conflict of interests: none. *Correspondence to: Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road Jammu 180001, J & K, India. CSIR-Academy of Scientific & Innovative Research, Govt. of India. Received 17 January 2015; Revised 23 April 2015; Accepted 1 May 2015 DOI 10.1002/mc.22339 Published online 27 May 2015 in Wiley Online Library (wileyonlinelibrary.com). ß 2015 WILEY PERIODICALS, INC.