The Cell Death-Inducing Activity of the Peptide Containing Noxa Mitochondrial-Targeting Domain Is Associated with Calcium Release

Y.-W. Seo, H.-N. Woo, S. Piya, A. R. Moon, J.-W. Oh, C.-W. Yun, K.-K. Kim, J.-Y. Min, S.-Y. Jeong, S. Chung, P. I. Song, S.-Y. Jeong (+3 others)
2009 Cancer Research  
DNA damage stabilizes the p53 tumor suppressor protein that determines the cell fate by either cell cycle arrest or cell death induction. Noxa, the BH3-only Bcl-2 family protein, was shown to be a key player in p53-induced cell death through the mitochondrial dysfunction; however, the molecular mechanism by which Noxa induces the mitochondrial dysfunction to cause cell death in response to genotoxic agents is largely unknown. Here, we show that the mitochondrial-targeting domain (MTD) of Noxa
more » ... a prodeath domain. Peptide containing MTD causes massive necrosis in vitro through cytosolic calcium increase; it is released from the mitochondria by opening the mitochondrial permeability transition pore. MTD peptide-induced cell death can be inhibited by calcium chelator BAPTA-AM. Moreover, MTD peptide shows the potent tumor-killing activities in mice by joining with tumor-homing motifs. [Cancer Res 2009;69(21):8356-65] 8356 Noxa 5′-(1)-primer plus the L45A 3′-primer (TTTGGATATCGCATTCA-GAAGTTTCTGCCGG), respectively, and the PCR products were then digested with BglII and EcoRV and cloned into pEGFP-Noxa (1-54) digested with BglII and EcoRV. For L49A conversion, PCR was carried out using Noxa 5′-(1)-primer plus L49A 3′-primer (CGAATTCT-CAGGTTCCTGAGCAGAAGGCTTTGGATATCAGATTCAG), and then digested with BglII and EcoR1 followed by cloning it into pEGFP-c1 at BglII and EcoR1 sites. For 4Lmt and 5Lmt, wild-type (WT) Noxa and L29A constructs, respectively, were used as templates for PCRs done with the Noxa 5′ primer plus the 4mt 3′-primer (GTTTGGATATCGCATTCG-CAGCTTTCTGCCGGAAG). PCR products digested with BglII and EcoRV were cloned into the vector generated from pEGFP-Noxa L49A. All Noxa mutant constructs were confirmed by DNA sequence analysis. Peptide synthesis. Peptides were synthesized and purified by high performance liquid chromatography to obtain peptides of 98% purity (Peptron). Peptides were suspended in 50% DMSO at 0.5 mmol/L and stored at −20°C. Measurement of intracellular calcium. For Ca 2+ measurements in the cytosol, HeLa cells were cultured in an Lab-Tek Chamber glass slide and loaded with Fluo-4-AM at a final concentration of 3 μmol/L for 30 min, followed by washing with PBS at pH 7.4 and the addition of fresh Ca 2+ -free Krebs-ringer modified buffer [KRB: 125 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L Na 3 PO 4 , 1 mmol/L MgSO 4 , 5.5 mmol/L glucose, and 20 mmol/L HEPES (pH 7.4), at 37°C] containing the indicated peptides. Time-lapse images were obtained at 488-nm excitation with the Argon laser scanning confocal microscope (Leica TCS SP5 Microsystems) at 10-s intervals for 5 min to visualize Fluo-4-AM. Cobalt-quenched calcein assay. HeLa cells were loaded with 1 μmol/L Calcein-AM and 2 mmol/L cobalt in serum-free DMEM for 15 min, followed by adding 25 μmol/l MitoTracker for 2 min to stain the mitochondria. Then, HeLa cells were briefly washed with HBSS and were treated with MTD peptide in calcium-free media containing 10% FBS. Time-lapse images were obtained at 10-s intervals for 10 min. Cell death assay. The percentage of cell death was determined by counting enhanced green fluorescent protein (EGFP)-positive dead cells with morphology using the fluorescent microscope. Minimally, 300 cells in three separate fields were counted for each measurement. Syngeneic animal tumor model. We followed the university's institutional guidelines and regulations for animal experiments. Tumors were established in BALB/c mice by s.c. injection of CT-26 cells (1.5 × 10 5 cells) into the mouse as described (14) . Tumor volume was calculated as length × width 2 × 0.5. Tumor cells were grown for 7 to 8 d, and TU2:MTD peptide (385 μg/mouse), TU3:MTD peptide (230 μg/mouse), or PBS was i.v. injected through the tail vein every 2 d until the mice were sacrificed.
doi:10.1158/0008-5472.can-09-0349 pmid:19826054 fatcat:e26nypl3wndjffh2uojxiiyhzy