Kalle M. Arunasree

Alteration of mitochondrial membrane potential by Spirulina platensis C‐phycocyanin induces apoptosis in the doxorubicinresistant human hepatocellular‐carcinoma cell line HepG2

C‐PC (C‐phycocyanin) is a water‐soluble biliprotein from the filamentous cyanobacterium Spirulina platensis with potent antioxidant, anti‐inflammatory and anticancerous properties. In the present study, the effect of C‐PC was tested on the proliferation of doxorubicin‐sensitive (S‐HepG2) and ‐resistant (R‐HepG2) HCC (hepatocellular carcinoma) cell lines. These studies indicate a 50% decrease in the proliferation of S‐ and R‐HepG2 cells treated with 40 and 50 μM C‐PC for 24 h respectively. C‐PC also enhanced the sensitivity of R‐HepG2 cells to doxorubicin. R‐HepG2 cells treated with C‐PC showed typical apoptotic features such as membrane blebbing and DNA fragmentation. Flow‐cytometric analysis of R‐HepG2 cells treated with 10, 25 and 50 μM C‐PC for 24 h showed 18.8, 39.72 and 65.64% cells in sub‐G0/G1‐phase respectively. Cytochrome c release, decrease in membrane potential, caspase 3 activation and PARP [poly(ADP‐ribose) polymerase] cleavage were observed in C‐PC‐treated R‐HepG2 cells. These studies also showed down‐regulation of the anti‐apoptotic protein Bcl‐2 and up‐regulation of the pro‐apoptotic Bax (Bcl2‐associated X‐protein) protein in the R‐HepG2 cells treated with C‐PC. The present study thus demonstrates that C‐PC induces apoptosis in R‐HepG2 cells and its potential as an anti‐HCC agent.

Anti-leukemic effects of gallic acid on human leukemia K562 cells: downregulation of COX-2, inhibition of BCR/ABL kinase and NF-κB inactivation.

Gallic acid (GA) induces apoptosis in various cancer cell lines. In this study, we investigated the apoptotic activity induced by GA on chronic myeloid leukemia (CML) cell line-K562 and the underlying mechanism. GA reduced the viability of K562 cells in a dose and time dependent manner. GA led to G0/G1 phase arrest in K562 cells by promoting p21 and p27 and inhibiting the levels of cyclin D and cyclin E. Further studies indicated apoptosis with impaired mitochondrial function as a result of deranged Bcl-2/Bax ratio, leakage of cytochrome c and PARP cleavage along with DNA fragmentation and by up-regulating the expression of caspase-3. GA also activated the protein expressions of fatty acid synthase ligand and caspase-8. GA is more effective in imatinib resistant-K562 (IR-K562) cells (IC50 4 μM) than on K562 cells (IC50 33 μM). GA inhibited cyclooxygenase-2 (COX-2) in K562 as well as IR-K562 cells appears to be COX-2 involved in the suppression of growth. Interestingly, GA also inhibited BCR/ABL tyrosine kinase and NF-κB. In conclusion, GA induced apoptosis in K562 cells involves death receptor and mitochondrial-mediated pathways by inhibiting BCR/ABL kinase, NF-κB activity and COX-2.

Synthesis of new secretory phospholipase A2-inhibitory indole containing isoxazole derivatives as anti-inflammatory and anticancer agents

Secretory phospholipase A2 (sPLA2) is an important enzyme that plays a key role in various inflammatory diseases including cancer and its inhibitors have been developed as preventive or therapeutic agents. In the present study, a series of new indole containing isoxazole derivatives (10a-10o) is synthesized and evaluated for their sPLA2 inhibitory activities. All compounds (10a-10o) showed significant sPLA2 inhibition activities both in vitro and in vivo studies which is substantiated in in silico studies. Among all the tested compounds, 10o showed potent sPLA2 inhibition activity, that is comparable or more to ursolic acid (positive control). Further studies demonstrated that 10o showed in vitro antiproliferative activity when tested against MCF-7 breast and DU145 prostate cancer cells. Furthermore, compounds 10a-10o obeyed lipinskys rule of 5 and suggesting druggable properties. The in vitro, in vivo and in silico results are encouraging and warrant pre-clinical studies to develop sPLA2-inhibitory compound 10o as novel therapeutic agent for various inflammatory disorders and several malignancies.

Ethylenediamine diacetate (EDDA) mediated synthesis of aurones under ultrasound: Their evaluation as inhibitors of SIRT1

An improved synthesis of functionalized aurones has been accomplished via the reaction of benzofuran-3(2H)-one with a range of benzaldehydes in the presence of a mild base EDDA under ultrasound. A number of aurones were synthesized (within 5-30min) and the molecular structure of a representative compound determined by single crystal X-ray diffraction study confirmed Z-geometry of the C-C double bond present within the molecule. Some of the compounds synthesized have shown SIRT1 inhibiting as well as anti proliferative properties against two cancer cell lines in vitro. Compound 3a [(Z)-2-(5-bromo-2-hydroxybenzylidene) benzofuran-3(2H)-one] was identified as a potent inhibitor of SIRT1 (IC(50)=1μM) which showed a dose dependent increase in the acetylation of p53 resulting in induction of apoptosis.

Effects of (15S)‐hydroperoxyeicosatetraenoic acid and (15S)‐hydroxyeicosatetraenoic acid on the acute‐ lymphoblastic‐leukaemia cell line Jurkat: activation of the Fas‐mediated death pathway

The antiproliferative effects of 15‐LOX (15‐lipoxygenase) metabolites of arachidonic acid {(15S)‐HPETE [(15S)‐hydroperoxyeicosatetraenoic acid] and (15S)‐HETE [(15S)‐hydroxyeicosatetraenoic acid]} and the mechanism(s) involved were studied in the human T‐cell leukaemia cell line Jurkat. (15S)‐HPETE, the hydroperoxy metabolite of 15‐LOX, inhibited the growth of Jurkat cells 3 h after exposure and with an IC50 value of 10 μM. The hydroxy metabolite of 15‐LOX, (15S)‐HETE, on the other hand, inhibited the growth of Jurkat cells after 6 h of exposure and with an IC50 value of 40 μM. The cells exposed to 10 μM (15S)‐HPETE for 3 h or to 40 μM (15S)‐HETE for 6 h showed increased expression of Fas ligand and FADD (Fas‐associated death domain), caspase 8 activation, Bid (BH3‐interacting domain death agonist) cleavage, decrease in mitochondrial membrane potential, cytochrome c release, caspase 3 activation, PARP‐1 [poly(ADP‐ribose) polymerase‐1] cleavage and DNA fragmentation, suggesting the involvement of both extrinsic and intrinsic death pathways. Further studies on ROS (reactive oxygen species) generation revealed the involvement of NADPH oxidase. In conclusion, the present study indicates that NADPH oxidase‐induced ROS generation activates the Fas‐mediated death pathway.

Design, Synthesis and Screening Studies of Potent Thiazol-2-Amine Derivatives as Fibroblast Growth Factor Receptor 1 Inhibitors

Fibroblast growth factor receptor 1 (FGFR1) a tyrosine kinase receptor, plays important roles in angiogenesis, embryonic development, cell proliferation, cell differentiation, and wound healing. The FGFR isoforms and their receptors (FGFRs) considered as a potential targets and under intense research to design potential anticancer agents. Fibroblast growth factors (FGFs) and its growth factor receptors (FGFR) plays vital role in one of the critical pathway in monitoring angiogenesis. In the current study, quantitative pharmacophore models were generated and validated using known FGFR1 inhibitors. The pharmacophore models were generated using a set of 28 compounds (training). The top pharmacophore model was selected and validated using a set of 126 compounds (test set) and also using external validation. The validated pharmacophore was considered as a virtual screening query to screen a database of 400,000 virtual molecules and pharmacophore model retrieved 2800 hits. The retrieved hits were subsequently filtered based on the fit value. The selected hits were subjected for docking studies to observe the binding modes of the retrieved hits and also to reduce the false positives. One of the potential hits (thiazole-2-amine derivative) was selected based the pharmacophore fit value, dock score, and synthetic feasibility. A few analogues of the thiazole-2-amine derivative were synthesized. These compounds were screened for FGFR1 activity and anti-proliferative studies. The top active compound showed 56.87% inhibition of FGFR1 activity at 50 µM and also showed good cellular activity. Further optimization of thiazole-2-amine derivatives is in progress.

Anti-inflammatory profile of Aegle marmelos (L) Correa (Bilva) with special reference to young roots grown in different parts of India

Background Aegle marmelos (Bilva) is being used in Ayurveda for the treatment of several inflammatory disorders. The plant is a member of a fixed dose combination of Dashamoola in Ayurveda. However, the usage of roots/root bark or stems is associated with sustainability concerns. Objectives The present study is aimed to compare the anti-inflammatory properties of different extracts of young roots (year wise) and mature parts of Bilva plants collected from different geographical locations in India, so as to identify a sustainable source for Ayurvedic formulation. Materials and methods A total of 191 extracts (petroleum ether, ethyl acetate, ethanol and aqueous) of roots, stems and leaves of A. marmelos (collected from Gujarat, Maharashtra, Odisha, Chhattisgarh, Karnataka and Andhra Pradesh region) were tested for anti-inflammatory effects in vitro on isolated target enzymes cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX), lymphocyte proliferation assay (LPA), cytokine profiling in LPS induced mouse macrophage (RAW 264.7) cell line and in vivo carrageenan induced paw edema in mice. Results Of 191 extracts, 44 extracts showed COX-2 inhibition and 38 extracts showed COX-1 inhibition, while none showed 5-LOX inhibition. Cytokine analysis of the 44 extracts showing inhibition of COX-2 suggested that only 17 extracts modulated the cytokines by increasing the anti-inflammatory cytokine IL-2 and reducing the pro-inflammatory cytokines like IL-1β, MIP1-α and IL-6. The young (2 and 3 years) roots of Bilva plants from Gujarat and young (1 yr) roots from Odisha showed the most potent anti-inflammatory activity by suppressing the pro-inflammatory cytokines and inducing anti-inflammatory cytokines. These three extracts have also shown in vivo anti-inflammatory activity comparable to that in adult stem and root barks. Conclusion The present study reveals that young roots of Bilva plants from Gujarat and Odisha region could form a sustainable source for use in Ayurvedic formulations with anti-inflammatory activities. The present study also indicates that the region in which the plants are grown and the age of the plants play an important role in exhibiting the anti-inflammatory effect.