Rugulactone derivatives act as inhibitors of NF-jB activation and modulates the transcription of NF-jB dependent genes in MDA-MB-231cells Debendra K. Mohapatra a,⇑ , D. Sai Reddy a , M. Janaki Ramaiah b , Sowjanya Ghosh b , Vikram Pothula a , Swetha Lunavath a , Shine Thomas b , S. N. C. V. L. Pushpa Valli b , Manika Pal Bhadra b,⇑ , Jhillu S. Yadav a,⇑ a Natural Products Chemistry Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, India b Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, India article info Article history: Received 15 September 2013 Revised 20 December 2013 Accepted 11 January 2014 Available online 20 January 2014 Keywords: Nuclear Factor-kappa B (NF-jB) Histone deacetylase (HDAC) Chromatin immunoprecipitation (ChIP) Luciferase (Luc) Polymerase chain reaction (PCR) Poly (ADP)-ribose polymerase (PARP) abstract Rugulactone and its analogues were synthesized following Horners–Wadsworth–Emmons and ring-clos- ing metathesis as the key reactions. A library of new rugulactone analogues were designed, synthesized and evaluated for their anticancer activity in breast cancer cells. All analogues have shown anti-prolifer- ative activity, while some of them exhibited significant cytotoxicity. In assays related to cell-cycle distri- bution, these conjugates induced G1 cell-cycle arrest in MDA-MB-231 cells. The cell cycle arrest nature was further confirmed by examining the effect on Cyclin E and Cdk2 proteins that acts at G1-S phase tran- sition. Immunocytochemistry assay revealed that these compounds inhibited nuclear translocation of NF- jB protein, thereby activation of NF-jB was inhibited. The expression of NF-jB target genes such as Cyclin D1 and Bcl-xL were severely affected. Apart from acting on NF-jB, these compounds also regulate class I Histone deacetylase proteins such as (HDAC-3 and 8) that have a crucial and regulatory role in cell- proliferation. Simultaneously, the apoptotic inducing nature of these compounds was confirmed by acti- vation of PARP protein, a protein that plays a key role in DNA damage and repair pathways. Among all compounds of this series 3g is the most potent compound and can be used for further studies. Ó 2014 Elsevier Ltd. All rights reserved. Breast cancer is the most frequent cancers in women in the Uni- ted States and Europe and is the leading cause of deaths world- wide. Chemoresistance is the main obstacle for the development of new drugs and strategies. The expression of estrogen receptor (ER) plays a prominent role in better prognosis and for anti-estro- gen therapy. In general ER-negative breast cancer cells are highly resistant to anti-estrogen therapy and more aggressive in nature. 1 Nuclear Factor-kappa B (NF-jB) is an eukaryotic transcription fac- tor present in virtually all animal cell types and has a vital role in cellular responses to various kinds of stimuli. 2 The transcription factor Nuclear Factor-kappa B (NF-jB) acts as a hub for a variety of intracellular signalling networks associated with carcinogenesis including cell survival, cell growth, inflammation, differentiation, immune response, apoptosis, invasion and metastasis. 3 Inappropri- ate regulation of NF-jB is associated with improper immune development, cancer, inflammatory and autoimmune diseases, septic shock, viral infection, synaptic plasticity and memory tech- nology. 4 Constitutive activation of NF-jB in tumor cells is one of the causal factors implicated in chemoresistance. 5 Tumors of breast, prostate and colon contain the activated form NF-jB and is the leading cause for the invasiveness particularly in breast can- cer. 6,7 The interesting aspects regarding the NF-jB activation is in controlling both the signals related to cell proliferation and inhib- iting apoptosis. 8 Thus targeting NF-jB is considered to be impor- tant for the development of anti-cancer drugs. 9 NF-jB exists in the cytoplasm as an inactive heterodimer complexed to inhibitor of kappa B (IjB). Activation of NF-jB signaling involves phosphor- ylation of IjB at the serine residues by inhibitor of kappa B kinase (IKK). Phosphorylated IjB subsequently undergoes proteasomal degradation allowing the translocation of the free heterodimer to the nucleus. Binding of the heterodimer (p65/p50) to jB enhancer elements in DNA results in transactivation of target genes that play a pivotal role in oncogenic progression. 9–11 Todate, five NF-jB subunits p50, p52, p65 (Rel A), cRel and Rel B are known which can form homo (or) heterodimeric complexes. These dimers can bind and are inhibited by three different cyto- plasmic inhibitory proteins IjBa,IjBb and IjBepsilon. NF-jB be- comes activated by stress, growth factors, proinflammatory http://dx.doi.org/10.1016/j.bmcl.2014.01.030 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved. ⇑ Corresponding authors. Tel.: +91 40 27193128; fax: +91 40 27160512 (D.K.M.). E-mail addresses: mohapatra@iict.res.in (D.K. Mohapatra), manika@iict.res.in (J.S. Yadav). Bioorganic & Medicinal Chemistry Letters 24 (2014) 1389–1396 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl