Suberoylanilide hydroxamic acid as a potential therapeutic agent for human breast cancer treatment.
BACKGROUND
Suberoylanilide hydroxamic acid (SAHA) is a prototype of the newly developed, second-generation, hybrid polar compounds. It is a novel histone deacetylase inhibitor with high potency for inducing cell differentiation of cultured murine erythroleukemia cells. Studies with SAHA have primarily been performed with hematopoietic tumor cells. Here we extent these studies with SAHA to human breast cancer cell lines in an attempt to find better therapeutic agents for breast cancer treatment.


METHODS
Human breast cancer cell lines, MCF7, MDA-MB-231, and MDA-MB-435, as well as normal cells, including the normal breast epithelial cell line MCF-10A, and fibroblasts, were treated with SAHA. Cells assayed for cell survival by using trypan blue exclusion assay, colony formation assay, and cell cycle and apoptosis analysis. The effects of SAHA on cell cycle and apoptosis regulatory proteins were examined by Western blots analysis. The identification of additional target genes was carried out by differential display (DD) and reverse transcription-polymerase chain reaction (RT-PCR).


RESULTS
SAHA inhibited clonogenic growth of MCF7, MDA-MB-231, and MDA-MB-435 breast cancer cells. These cells were more sensitive to SAHA-mediated cytotoxic effects than normal breast epithelial cells and fibroblasts. The cytotoxic effects of SAHA on breast cancer cells were manifested by G1 and G2/M cell cycle arrest and eventual apoptosis. The pan-caspase inhibitor, Z-VAD.fmk, blocked SAHA-induced cell death, DNA laddering, and cleavage of poly(ADP-ribose) polymerase, indicating the involvement of caspases in SAHA-mediated apoptosis. In addition, SAHA modulated cell cycle and apoptosis regulatory proteins. For example, cyclin-dependent kinase (CDK) inhibitors p21WAF1/Cip1 and p27Kip1 were induced, and retinoblastoma protein pRb was hypophosphorylated. Moreover, SAHA induced several genes associated with differentiation and/ or growth inhibition. These genes encode gelsolin, isopentenyl-diphosphate delta isomerase (IDI1), and 1,25-dihydroxyvitamin D-3 up-regulated protein 1 (VDUP1), the last two of which were identified by DD. Induction of these genes may contribute to SAHA-mediated pro-differentiating and antiproliferative effects.


CONCLUSIONS
SAHA induced growth inhibition, cell cycle arrest, and eventual apoptosis in human breast cancer cells, possibly by modulating cell cycle and apoptosis regulatory proteins, such as CDK inhibitors p21 and p27, pRb, and other differentiation and/or growth inhibition-associated genes, including gelsolin, IDI1 and VDUP1. This, together with the low toxicity in normal cells, suggests that SAHA might have therapeutic potential for the treatment of human breast cancers.
Huang
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Dana-Farber Cancer Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.
Pardee
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D000970:Antineoplastic Agents / Q000494:pharmacology / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D001483:Base Sequence; D001943:Breast Neoplasms / Q000188:drug therapy*; D061945:Caspase Inhibitors; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D017931:DNA Primers; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology / Q000627:therapeutic use*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D015854:Up-Regulation / Q000187:drug effects; D000077337:Vorinostat
D000970:Antineoplastic Agents; D061945:Caspase Inhibitors; D017931:DNA Primers; D004791:Enzyme Inhibitors; D006877:Hydroxamic Acids; D000077337:Vorinostat