Abstract
Resistance to chemotherapy is one of the major obstacles to successful cancer treatment. Many cancer patients develop metastatic cancers after responding to initial treatment, which are often refractory to chemotherapy, and remain incurable. Inhibition of apoptotic pathways due to up-regulation of anti-apoptotic elements, such as Bcl-2 and IAPs (inhibitors of apoptosis proteins) has been reported to account for the resistance to chemotherapeutic agents. Thus, it is critical to restore the inhibited apoptosis to re-sensitize the cancer to chemotherapy. In this study, we investigated whether a pro-apoptotic peptide and a proteasome inhibitor peptide can synergize to induce apoptosis in doxorubicin-resistant cancer cells in vitro. The pro-apoptotic peptide is a cationic amphipathic peptide which depolarizes mitochondrial membranes to release apoptosis initiators such as, cytochrom c and Smac/DIABLO to cell cytosols, which activate caspases, apoptosis executors by inhibiting IAPs (inhibitors of apoptosis proteins). MG132 (Z-Leu-Leu-Leu-CHO) has been reported to inhibit anti-apoptotic proteins including NF-[[Unable to Display Character: ĸ]]B and Bcl-2 family proteins to stimulate apoptosis. We hypothesized that when concurrently delivered to the same cells, KLA and MG132 would synergize to induce apoptosis in resistant cancer cells by acting on different stages of apoptotic pathways. To test this, we conjugated KLA and MG132 by forming acid-labile Schiff bases between ε-amino groups of lysines in KLA and the aldehyde group of MG132. We expected that the formed KLA-MG132 conjugates would be internalized into cells by pinocytosis then, dissociated in the acidic environment (pH 4 – 5) of lysosomes to release intact KLA and MG132. The conjugation efficiency was found to be ∼ 100%, as measured indirectly by quantifying amine groups of un-conjugated lysine residues by TNBS assay. The conjugation was confirmed further by determining the molecular masses of the formed conjugates by mass spectrometry after reducing the conjugates by NaBH4 to stabilize Schiff bases. The conjugates induced upto 50 % of cell death in doxorubicin-resistant human cancer cells at the concentration of 0.6 μM as determined by MTT assay meanwhile; un-conjugated MG132 and KLA were inactive at the same concentration. And, the levels of cytosolic cytochrome c were 2-fold greater in the cells treated with KLA-MG132 conjugates than in cells treated with MG132 alone. The levels of active caspase-3 were higher by up to 70 % in cells treated with KLA-MG132 conjugate than in cells treated with MG132 alone. In conclusion, we have observed that KLA and MG132 conjugated via Schiff base induced potent apoptosis in doxorubicin-resistant human cancer cell lines at sub-micromolar concentrations.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2655.
- ©2010 American Association for Cancer Research
Volume 70, Issue 8 Supplement
