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TGF-beta Pathway

TGF-beta Inhibitors

SB 431542 is a potent and specific transforming growth factor-β superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7 inhibitor with an IC50 of 94 nM for inhibition of Smad3 phosphorylation. Transforming growth factor β (TGF-β) is a member of a large superfamily of pleiotropic cytokines that are involved in many cellular activities, including growth, differentiation, migration, cell survival and adhesion. Transforming growth factor β1 (TGF-β1) is responsible for the production of extracellular matrix, acting through the TGF-β type I and type II receptors and activating intracellular mediators such as Smad proteins, p38 MAPK (mitogen-activated protein kinase) and the extracellular signal-regulated kinase (ERK) pathway. SB 431542 prevented TGF-beta1-induced nuclear Smad3 localization. SB 431542 prevented TGF-beta1-induced collagen Ialpha1 (col Ialpha1). SB 431542 has no effect on components of the ERK, JNK, or p38 MAP kinase pathways or on components of the signaling. [1] SB 431542 and SB203580 (a p38 kinase inhibitor) attenuated TGF-β1-induced phosphorylation of Smad2/3 protein. SB 431542 and gefitinib attenuated TGF-β1-induced phosphorylation of ERK1/2 and p38 kinase. SB 431542 and gefitinib also attenuated TGF-β1-induced cyclin D1 protein expression. [2] SB 431542 inhibited the TGF-beta-induced proliferation of osteosarcoma cells in humans.

Protocol:
Biochemical assay: Macrophages and ECs were serum starved overnight and then pre-incubated or not with the TGF-β receptor kinase inhibitor, SB 431542 (SB431542, SB-431542), at a final concentration of 10 µM, 1 h before M. avium infection. After that period cells were washed twice with PBS and then submitted or not to infection with M. avium in completed medium. Culture supernatants and glass coverslips were harvested at each time point during the kinetics of infection either for measurement of TGF-β secretion or for mycobacterial growth, as described above. For detection of ERK1/2 phosphorylation in ECs treated with SB431542, ECs were serum starved overnight and then pre-treated or not with 10 µM SB431542 for 1 h. After that period cells were washed twice with PBS and then submitted or not to infection with M. avium in medium without serum. Four hours post-infection cells were harvested and total cell extract were produced for evaluation of ERK1/2 phosphorylation by immunoblot. [3]

Cell assay: A498 cells were seeded at 5,000 to 10,000 cells/well in 96-well plates. The cells were serum-deprived for 24 h and then treated with SB 431542 (SB431542, SB-431542) for 48 h to assess the cellular toxicity. Cell viability is determined by incubating cells for 4 h with XTT labeling and electron coupling reagent according to the manufacturer’s directions. Live cells with active mitochondria produce an orange-colored product, formazan, which is detected using a plate reader at between A450 nm andA500 nm with a reference wavelength greater than 600 nm. The absorbance values correlate with the number of viable cells. [1]

[1] Laping NJ et al. Mol Pharmacol. 2002 Jul;62(1):58-64.
[2] Chen SC et al. Transl Res. 2011 Oct;158(4):214-24.
[3] L’Abbate C et al. PLoS One. 2011;6(6):e21465.

LY2157299 is a novel selective small molecule transforming growth factor beta receptor (TGF-βR) kinase inhibitor with IC50 of 86 nM and 2 nM for TβR1 and 2 nM, respectively. LY2157299 was well tolerated at both doses of 40 and 80 mg and no drug-related CTCAE Grade 3 or 4 toxicities were observed. Absorption of LY2157299 was rapid as determined by plasma concentrations at the first sampling time (0.5 hours). Daily oral administration of LY2157299 was safe and well tolerated at the two dose levels and the pharmacokinetic profile was consistent with the prediction derived from preclinical PK/PD model. As would be predicted for an inhibitor of TGF-β signaling, LY2157299 blocked the phsophorylation of Smad2 but had no effect on fibroblast growth factor (FGF) or platelet-derived growth factor (PDGF) signaling. However, another study reported that LY2157299 induced angiogenesis and enhanced VEGF- and basic-fibroblast-growth-factor- induced angiogenesis in a Matrigel-plug assay, whereas adding an alpha5-integrin -neutralizing antibody to the Matrigel selectively inhibited this enhanced response. [1][2][3][4]

References on LY2157299:
[1] Bueno L et al. Eur J Cancer. 2008 Jan;44(1):142-50.
[2] Workman P. Future Oncol. 2005 Jun;1(3):315-8.
[3] http://aacrmeetingabstracts.org/cgi/content/abstract/2005/1/1463-a
[4] Liu Z et al. J Cell Sci. 2009 Sep 15;122(Pt 18):3294-302. 

IM-412 is 3-(2-chlorobenzyl)-1,7-dimethyl-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione, which significantly decreased TGF-β stimulated reporter activity in a dose-dependent manner. In addition, IM-412 inhibited TGF-β-induced expression of the fibrotic markers α-smooth muscle actin (α-SMA) and fibronectin, and collagen accumulation in CCD-18Lu human normal lung fibroblasts without cell cytotoxicity. IM-412 decreased Smad2 and -3 phosphorylation as well as JNK and ERK activity. Moreover, expression levels of TGF-β receptor I (TβRI) and receptor II (TβRII) were down-regulated by IM-412 in a dose-dependent manner. The small molecule IM-412 could attenuate TGF-β-mediated fibroblast differentiation through inhibition of the overall TGF-β response and may be a promising novel agent for the treatment of pathological fibrotic conditions.

References on IM-412:
Park S, Ahn JY, Lim MJ, Kim MH, Lee SL, Yun YS, Jeong G, Song JY. IM-412 inhibits transforming growth factor beta-induced fibroblast differentiation in human lung fibroblast cells. Biochem Biophys Res Commun. 2010 Aug 20;399(2):268-73.

ARP 101 as one of the most potent inducer of autophagy, is a selective matrix metalloproteinase-2 (MMP-2) inhibitor . ARP101 treatment was highly effective in inducing the formation of autophagosome and conversion of LC3I into LC3II. Moreover, ARP101-induced autophagy was completely blocked in mouse embryo fibroblasts that lacked autophagy related gene 5 (ATG5(-/-) MEF). Interestingly, cell death induced by ARP101 was not inhibited by zVAD, a pan caspase inhibitor, whereas, it was efficiently suppressed by addition of 3-methyladenine, an autophagy inhibitor. These results suggest that the selective MMP-2 inhibitor, ARP101, induces autophagy and autophagy-associated cell death.

References on ARP101:
Jo YK, Park SJ, Shin JH, Kim Y, Hwang JJ, Cho DH, Kim JC. ARP101, a selective MMP-2 inhibitor, induces autophagy-associated cell death in cancer cells. Biochem Biophys Res Commun. 2011 Jan 28;404(4):1039-43. Epub 2010 Dec 25.