Aug
12
Mon
2013
Invited Talk: Screening flavonoids for NF-kB inhibitory effect as potential breast cancer therapy @ Sathyam Hall
Aug 12 @ 11:00 am – 11:20 am

ayyappanAyyappan Nair, Ph.D.
Head, Business Development (Technologies, Discovery Biology), Anthem Biosciences & DavosPharma, New Jersey, USA


Inhibition of NF-κB regulated gene expression by chrysoeriol suppresses tumorigenesis in breast cancer cells

Amrutha K1, Pandurangan Nanjan1, Sanu K Shaji1, Damu Sunilkumar1, Subhalakshmi K1, Rashmi U Nair1, Lakshmi Rajakrishna2, Asoke Banerji1, Ayyappan Ramesh Nair1*,2

  1. School of Biotechnology, Amrita Vishwa Vidyapeetham, Amritapuri Campus, Clappana P.O., Kollam – 690 525, Kerala, India
  2. Anthem Biosciences, No 49, Canara Bank Road, Bommasandra Industrial Area, Phase 1,  Hosur Road, Bangalore – 560 099, Karnataka, India

Abstract:  A large number of effective cancer-preventing compounds inhibit the activation of nuclear factor-κ B (NF-κB).  It has been previously demonstrated that some flavonoids that are a vital component of our diet inhibits this pathway. As a consequence, many flavonoids inhibit genes involved in various aspects of tumorigenesis and have thus emerged as potential chemopreventive candidates for cancer treatment. We studied the effect of 17 different flavonoids, including the highly evaluated quercetin on the NF-κB pathway, and on the expression of MMP-9 and COX-2 (two NF-κB regulated genes involved in metastasis) in the highly invasive human breast cancer cell line MDA-MB-231.  The findings suggest that not all the quercetin like flavone backbone compounds inhibit the NF-κB pathway, and that the highly hydoxylated flavonols quercetagetin and gossypetin did not inhibit this pathway, nor did it inhibit the expression of MMP-9 and COX-2.  This indicates a correlation between inhibition of NF-κB and subsequent suppression of these NF-κB regulated genes. Here, we also report the novel observation that the not so well characterized methoxylated flavone chrysoeriol inhibited the NF-κB pathway, and was most potent in reducing the expression of MMP-9 and COX-2.  Based on these observations, the cellular effects of chrysoeriol were evaluated in MDA-MB-231.  Chrysoeriol caused cell cycle arrest at G2/M, inhibited migration and invasion, and caused cell death of macrophages that contributed to migration of these cancer cells.  These effects of chrysoeriol make it a potential therapeutic candidate for breast cancer metastasis.

Ayyappan

 

Delegate Talk: Protoplast fusion and transformation: A tool for activation of latent gene clusters @ Sathyam Hall
Aug 12 @ 3:15 pm – 3:35 pm
Delegate Talk: Protoplast fusion and transformation: A tool for activation of latent gene clusters @ Sathyam Hall | Vallikavu | Kerala | India

Abhijeet Kate, Arpana G Panicker, Diana Writer, Giridharan P, Keshav K V Ramamoorthy, Saji George, Shailendra K Sonawane


Protoplast fusion and transformation: A tool for activation of latent gene clusters

In the quest to discover new bioactive leads for unmet medical needs, actinomycetes present a treasure trove of undiscovered molecules. The ability of actinomycetes to produce antibiotics and other bioactive secondary metabolites has been underestimated due to sparse studies of cryptic gene clusters. These gene clusters can be tapped to explore scaffolds hidden in them. The up-regulation of the dormant genes is one of the most important areas of interest in the bioactive compounds discovery from microbial resources. Genome shuffling is a powerful tool for the activation of such gene clusters. Lei Yu, et al.1, reported enhancement of the lactic acid production in Lactobacillus rhamnosus through genome shuffling brought about by protoplast fusion. D. A. Hopwood et al.2 suggested that an interspecific recombination between strains producing different secondary metabolites, generate producers of ‘hybrid’ antibiotics. They also mentioned that an intraspecific fusion of actinomycetes protoplast bring about random and high frequency recombination. Protoplasts can also be used as recipients for isolated DNA, again in the presence of polyethylene glycol (PEG). In our study we had undertaken random genome shuffling by protoplast fusion of two, rather poorly expressed actinomycetes strains A (Figure 1) & B (Figure 2), mediated by PEG; and also by naked DNA transformation of Strain A protoplast with the DNA of Strain B. We generated eight protoplast fusants and seven transformants from parents considering their morphological difference from the two parent strains. These 15 recombinants were checked for their same colony morphologies for five generations to ensure phenotypic stability. Antibiotic resistance pattern was established by using antibiotic octodisc to generate a marker profile of the recombinants and the parent strains. Eight fusants (AP-18, AP-25, AP-2, AP-11, AP-14, AP-19, AP-11 and AP-27) and four transformants (TAP-30, TAP-31, TAP-32 and TAP-33) (Table 1) have shown a different antibiotic sensitivity pattern as compared to the parent strains. We envisage that these recombinants harbor shuffled gene clusters. To support array of conditions to express such shuffled/cryptic genes the recombinants were fermented in 11 different nutrient stress variants. The extracts generated were subjected to metabolite profiling by HPLC-ELSD, bioactivity screening for cytotoxicity and anti-infective capabilities. Two fusants AP-11 (Figure 3) and AP-25; one transformant TAP-32 (in growth media MBA-5 and MBA-7) displayed antifungal activity unlike parent strains (Table 2) Fusant AP-11 (Table 5) exhibited significant cell growth inhibition of five different cancer cell lines. The parents Strain A and Strain B did not exhibit any cell growth inhibition of these cell lines (Table 5). The metabolite profiling of fusant AP-11 and transformant TAP-32 was done by HPLC-ELSD. AP-11 showed the presence of five additional peaks (Figure 5 & Figure 6); TAP-32 extract from medium MBA-5 (Figure 7 & Figure 8) showed the presence of four additional peaks and TAP-32 extract from MBA-7 (Figure 9 & Figure 10) showed 14 additional peaks as compared to parent strains in similar medium and media controls. The study indicated that protoplast fusion and transformation have not only caused morphological changes but also shuffled genes responsible for synthesis of bioactive molecules. Further characterization of these new peaks is warranted.