Discovery of a New Molecular Marker, Prohibitin, and Development of Candidates for New Chemotherapeutic Agents Targeting Prohibitin in Primary AML Cells.

In light of the recent studies that showed significant causal relationship between mitochondrial genetic mutation and apoptosis in pathology of various tumors and degenerative diseases, mitochondrial proteins have been interesting targets for the study of apoptosis in leukemia and other malignancies. This observation prompted us to analyze mitochondrial proteins and develop new anti-proliferative agents targeting them. Mitochondria were isolated from AML cells by density-gradient ultracentrifugation using swelling buffer and sucrose buffer. We identified 48 spots corresponding to 38 proteins in primary AML cells using 2-DE and mass spectrometry (MALDI-TOF/TOF technology), the expression of which were altered significantly compared to that of normal hematopoietic cells. Out of these deregulated proteins, 12 and 20 proteins were observed in up- or down-regulated spots, respectively. Interestingly, prohibitin (PHB) (gi4505773) was highly expressed in primary AML cells, which was confirmed by Western blot, immunohistochemistry and immunofluorecenct study in the primary AML bone marrow cells and sections. To assess the functional significance of aberrant prohibitin expression, we applied siRNA delivery for silencing of prohibitin. Transduction with a siRNA 11867 construct resulted in 75% decrease of AML cells as compared to the nonsilencing control construct after two days. Potent chemical substances that can alkylate PHB in AML cells, cyclohexylphenyl-chloroethyl urea (CCEU) and iodophenyl-chloroethyl urea (ICEU), were synthesized in our laboratory. Time and dose dependent manner of proliferation suppression when treated with CCEU and ICEU was observed in leukemic cell lines including THP-1, K-562 and Kasumi-1. Moreover, notable morphological transformation of leukemic cells was observed when treated with 50 – 200 umol of CCEU and ICEU for 24 hours. Cell cycle analysis of CCEU-and-ICEU-treated- THP-1 and Kasumi-1 cell lines showed a remarkable increase of the sub-G1 phase. Immunoblotting experiment revealed the change of cytoplasmic and nucleoplasmic PHB in K-562 cell line. Expression of cleaved caspase3 and poly ADP-ribose polymerases were also observed to have increased in primary AML cells and cell lines. By analyzing AML cell mitochondrial protein we discovered a new molecular marker, PHB, characteristically overexpressed in AML cells and developed new anti-cancer agents such as CCEU and ICEU that target against PHB in AML cells.