Reversal of Chemoresistance in Human Lung Cancer Cell Line A549/Taxol by Synthetic Second Mitochondria-Derived Activator of Caspase Peptide.

BACKGROUND: Chemoresistance is a leading cause of treatment failure in advanced lung cancer, including that with the extensively prescribed taxol. Recently, a series of structurally unique second mitochondria-derived activators of caspase (Smac) that act as antagonists of inhibitor of apoptosis proteins (IAPs) have been discovered, exhibiting the ability of inducing enhanced apoptosis of various cancer cell types when combined with chemotherapy. In the present study, we synthesized the second mitochondria-derived activator of caspase peptide (Smac-N7 for short) and explored its capacity in combination with taxol in vitro. METHODS: The sensitivity assay and reversal ability of Smac-N7 were tested by MTT. Flow cytometry was used to analyze apoptosis of cells with Annexin V/PI double staining technique. Cell cloning ability was performed to reflect its biological behavior in each group. RESULTS: Concentrations with inhibitory rates < 10% were selected as the reversal value of Smac-N7 peptide using MTT. The reversal folds were 2.52, 3.26, 3.67, and 5.4 in taxol + Smac-N7 (0.0390625, 0.078125, 0.15625, 0.3125 µg/mL, respectively), and concentrations of Smac-N7 and reversal folds appeared in an obvious positive correlation (r(s) = 1, p = 0.000). Apoptosis analyzed at 48 hours by flow cytometry showed the apoptotic rates in taxol and 0.0390625, 0.078125, 0.15625, and 0.3125 µg/mL Smac-N7 + taxol groups were 15.4 ± 1.09%, 20.8% ± 2.18%, 28.4% ± 4.17%, 37.64% ± 6.41%, and 46.6% ± 7.76%, respectively. Concentrations of Smac-N7 appeared to have negative correlations with PE and SF (r(s) = -1, p < 0.05), which showed that the cells' cloning ability in 0.3125 µg/mL Smac-N7 + taxol group was worse than that of other groups. CONCLUSIONS: When combined with taxol, 0.3125 µg/mL Smac-N7 peptide may significantly increase taxol-induced apoptosis in chemoresistant A549/taxol lung cells at 48 hours, and is potentially useful as a reversal agent in lung cancer therapy.

Smac mimetic SM-164 potentiates APO2L/TRAIL- and doxorubicin-mediated anticancer activity in human hepatocellular carcinoma cells.

BACKGROUND: The members of inhibitor of apoptosis proteins (IAPs) family are key negative regulators of apoptosis. Overexpression of IAPs are found in hepatocellular carcinoma (HCC), and can contribute to chemotherapy resistance and recurrence of HCC. Small-molecule Second mitochondria-derived activator of caspases (Smac) mimetics have recently emerged as novel anticancer drugs through targeting IAPs. The specific aims of this study were to 1) examine the anticancer activity of Smac mimetics as a single agent and in combination with chemotherapy in HCC cells, and 2) investigate the mechanism of anticancer action of Smac mimetics. METHODS: Four HCC cell lines, including SMMC-7721, BEL-7402, HepG2 and Hep3B, and 12 primary HCC cells were used in this study. Smac mimetic SM-164 was used to treat HCC cells. Cell viability, cell death induction and clonal formation assays were used to evaluate the anticancer activity. Western blotting analysis and a pancaspase inhibitor were used to investigate the mechanisms. RESULTS: Although SM-164 induced complete cIAP-1 degradation, it displayed weak inhibitory effects on the viability of HCC cells. Nevertheless, SM-164 considerably potentiated Apo2 ligand or TNF-related apoptosis-inducing ligand (APO2L/TRAIL)- and Doxorubicin-mediated anticancer activity in HCC cells. Mechanistic studies demonstrated that SM-164 in combination with chemotherapeutic agents resulted in enhanced activation of caspases-9, -3 and cleavage of poly ADP-ribose polymerase (PARP), and also led to decreased AKT activation. CONCLUSIONS: Smac mimetics can enhance chemotherapeutic-mediated anticancer activity by enhancing apoptosis signaling and suppressing survival signaling in HCC cells. This study suggests Smac mimetics are potential therapeutic agents for HCC.

Design, synthesis, and evaluation of potent, nonpeptidic mimetics of second mitochondria-derived activator of caspases.

A series of new Smac mimetics have been designed, synthesized, and evaluated. The most potent compound 10 binds to XIAP, cIAP-1, and cIAP-2 BIR3 proteins with K(i) of 3.9, 0.37, and 0.25 nM, respectively. Compound 10 antagonizes XIAP in a cell-free functional assay and induces rapid cIAP-1 degradation in cancer cells. Compound 10 inhibits cell growth in the MDA-MB-231 cancer cell line with an IC(50) of 8.9 nM.

A novel polyarginine containing Smac peptide conjugate that mediates cell death in tumor and healthy cells.

The seven N-terminal amino acids AVPIAQK (SmacN7) of the mitochondrial protein Smac (second mitochondria-derived activator of caspase) promote caspase activation by binding specifically to inhibitor of apoptosis proteins (IAPs) and blocking their inhibitory activity. SmacN7 cannot pass through the cell membrane, but to be of therapeutic use it would be essential for it to enter the cell. To achieve transmembrane transport of SmacN7 we coupled it to a novel fluorescein isothiocyanate (FITC)-labelled transmembrane transport peptide RRRRK(FITC)RRRR via ss-alanine to produce the conjugate AVPIAQKssA RRRRK(FITC)RRRR. Because IAPs are much more strongly expressed in the cytoplasm of tumor cells, we expected this conjugate to produce staining of the cytoplasm, and for this to be stronger in tumor cells than in healthy cells. Surprisingly, we found strong nuclear uptake of the Smac conjugate and of the transport peptide alone without subsequent release in both tumor cells and healthy cells from the bladder, prostate, and brain. This was accompanied by cell death. In contrast to expectations, it appears that the apoptotic effects observed do not result from the SmacN7 cargo alone.

Design and characterization of bivalent Smac-based peptides as antagonists of XIAP and development and validation of a fluorescence polarization assay for XIAP containing both BIR2 and BIR3 domains.

XIAP (X-chromosome-linked inhibitor of apoptosis protein) is an inhibitor of apoptosis by binding to and inhibition of caspase-3 and caspase-7 through its BIR2 domain and caspase-9 through its BIR3 domain. Smac (second mitochondria-derived activator of caspases) protein is an endogenous antagonist of XIAP. Smac forms a dimer and concurrently binds both the BIR2 and BIR3 domains in XIAP, functioning as a highly efficient and potent cellular inhibitor of XIAP. In this article, we have designed and synthesized a bivalent Smac-based ligand (Smac-1) and its fluorescent labeled analogue (Smac-1F) and characterized their interaction with different constructs of XIAP. Our study demonstrates that bivalent Smac-based ligands bind concurrently to both the BIR2 and BIR3 domains of XIAP and are more than 500 times more potent than the corresponding monovalent Smac-based ligands. Bivalent Smac-based ligands also function as much more potent antagonists of XIAP than do the corresponding monovalent Smac-based ligands in cell-free functional assays. Using Smac-1F and XIAP containing both BIR2 and BIR3 domains, we also developed and validated a new fluorescence polarization-based assay. Hence, our designed bivalent Smac-based peptides mimic the mode of dimeric Smac protein in their interaction with XIAP containing both BIR2 and BIR3 domains and achieve extremely high potency in binding and functional assays. Our study provides new insights into the mode of action of bivalent Smac ligands targeting XIAP and a basis for the design and development of cell-permeable, bivalent Smac mimetics.

Synthetic Smac peptide enhances the effect of etoposide-induced apoptosis in human glioblastoma cell lines.

Smac/DIABLO is a mitochondrial protein released into cytosol during the progression of apoptosis. Smac/DIABLO promotes apoptosis by neutralizing the inhibitory effect of the inhibitor of apoptosis proteins (IAPs) on the processing and activity of the effecter of caspase. Here, we generated synthetic Smac peptide which possesses an IAP-binding domain and Drosophila antennapaedia penetration sequence, and examined whether it enhances the effect of the chemotherapeutic agent etoposide in the human glioblastoma cell line. Cellular uptake of Smac peptide in several glioma cell lines was most prominent at 6-12 h after addition. Caspase activity assay showed that our peptide successfully increased the activity of caspase-3 and caspase-9 in etoposide-induced apoptosis. In addition, Smac peptide increased the amount of cleaved PARP (poly ADP-ribose polymerase), but control peptides did not. Moreover, the addition of z-VAD-fmk, a caspase inhibitor, counterbalanced the effect of Smac peptide. Finally, we demonstrated that Smac peptide could enhance the growth inhibition effect of etoposide compared with control peptides. These results suggest that synthetic Smac peptide may be a new molecular targeting anti-tumor therapy for human glioblastoma.

Total synthesis of human and rat coupling factor-6 amide and pressor effects in the rat.

Mitochondrial coupling factor-6 (CF-6) is a component of the ATP synthase complex essential for energy transduction. CF-6, which is localized to the surface of endothelial cells (ECs) and released by shear stress, has been implicated as an endogenous vasoconstrictor. Previous methods of obtaining CF-6 through purification and recombinant methods were laborious and inefficient. Here, we describe the chemical synthesis of human CF-6, (33-108)-NH(2), its C-terminal fragment (55-108)-NH(2), which is termed pCF-6; the rat CF-6, (33-108)-NH(2), its C-terminal fragment pCF-6, (55-108)-NH(2); and two N-terminal fragments of the rat pro-coupling factor-6, (24-52)-NH(2) and (33-52)-NH(2). Biological activities of each peptide were initially screened with bioassays and verified by in vivo studies. Accordingly, intravenous administration of CF-6, pCF-6, rat CF-6, and rat pCF-6 produced a modest but statistically significant increase in blood pressure and heart rate in urethane anesthetized rats, whereas the N-terminal rat pro-coupling factor-6, (24-52)-NH(2) and (33-52)-NH(2) caused no significant pressor response. Thus, the biologically active site probably resides at the C-terminal portion of CF-6 peptides.

Predominant suppression of apoptosome by inhibitor of apoptosis protein in non-small cell lung cancer H460 cells: therapeutic effect of a novel polyarginine-conjugated Smac peptide.

The inhibitor of apoptosis proteins (IAPs) plays a central role in repressing caspase-mediated cell death. However, little is known about the actual role of endogenously expressed IAPs in cancer cells. We found that the cytochrome c/apoptotic protease-activating factor-1 (apoptosome)-dependent caspase activation is deficient in human non-small cell lung cancer (NSCLC) NCI-H460 cells. This dysfunctional apoptosome activity was not correlated with any decrease of apoptosome component factors, but it was linked to an increased X-linked inhibitor of apoptosis protein (XIAP). In H460 cells, the overexpressed XIAP, but not c-IAP1, bound to the processed form of caspase-9 and suppressed the activation of downstream effector caspases. Moreover, the defect in apoptosome activity in H460 cells was dramatically restored by the IAP-targeting SmacN7 peptide, which disrupted XIAP-caspase-9 binding, indicating an essential role of the IAP in the apoptosome inhibition. However, the SmacN7 did not show any striking effect on the apoptosome activity of normal lung fibroblast cells, although these cells also expressed modest amounts of IAP. To explore the therapeutic approach, we additionally developed SmacN7(R)8, a newly designed cell permeable peptide. The SmacN7(R)8 selectively reversed the apoptosis resistance of H460 cells, and when in combination with chemotherapy, regressed the tumor growth in vivo with little toxicity to the mice. Our results indicate that IAP-dependent suppression of apoptosome predominantly occurs in IAP-overexpressing tumor, and the IAP-targeting Smac peptide is an effective molecule to increase tumor cell death induced by chemotherapy in vitro and in vivo.