Comparisson of Molecular Mechanisms Altered by Treatment with Different Drugs (Doxorubicin, Melphalan, Bortezomib, Aplidin and Arsenic Trioxide) in Multiple Myeloma Patients Using Oligonucleotide Microarrays.

The development of novel targeted therapies in Multiple Myeloma (MM) has opened promising expectations for the treatment of this incurable hematological malignancy. However, the molecular mechanisms of both novel biologically based therapies and conventional treatments are still unclear. The purpose of the present study was to evaluate the changes in the gene expression profile of the human multiple myeloma cell line (MM.1S) following exposure to Doxorubicin, Melphalan, Bortezomib, Aplidin and Arsenic Trioxide. We have focused on the analysis of the early steps of activation of molecular mechanisms that lead to MM cell death. For this purpose we investigated with a time-course the onset of the apoptosis for every drug, according to the flow cytometric analysis with Annexin V- FITC Apoptosis Detection Reagent Kit. Based on these results the optimal concentration and time of exposure for each of the drugs were as follows Melphalan 50 μM, 9hours; Doxorubicin 1 μM, 17hours; Bortezomib 10 nM, 6 hours; Aplidin 50 nM, 4 hours and Arsenic Trioxide 5 mM, 3 hours. Affymetrix HG-U133A array containing around 15,000 full-length genes was used for mRNA expression profiling. All the experiments were performed in duplicate. The DNA- Chip Analyzer (DChip) was used to normalize and compare samples. Genes with expression changes greater than twofold in either direction were considered significant. A total of 269, 74, 74, 808 and 525 genes showed a significantly altered expression pattern, in response to Melphalan, Doxorubicin, Bortezomib, Aplidin and Arsenic Trioxide, respectively. Our results demonstrate that treatment with Melphalan inhibits DNA replication and transcription (underexpression of POLA, RFC1) as well as proliferation and survival (underexpression of IGF-1); it blocks the cell cycle (overexpression of CDKN1A and PA26) and induces apoptosis (overexpression of GADD45B). Doxorubicin deregulates many genes involved in cell cycle arrest (high expression of CDKN1A, PA26, GADD45A and low expression of CCND2 y CDC20) as well as up-regulation of several members of the TNF family (CD95, TRAILR2 and CD27). Bortezomib increases the expression of many “heat shock proteins” (HSP 110, HSP 70B, HSP 70B′) and decreases the level expression of IGF-1. Aplidin triggers early induction of many genes involved in apoptosis (overexpression of MAP4K3 and EGR2) and down-regulation of genes that play and important role in G2/M phase transition (NEK2, CENPF, BUB1). Arsenic Trioxide induces underexpression of essential genes for G1/S phase transition and cell cycle progression (CDC7L1, CDC25A) as well as underexpression of genes that mediate spindle formation and cromosome segregation (STK6, PRC1, HCAP-G, ZWINT, KIF4A, BUB1). Moreover, Arsenic Trioxide alike Bortezomib treatment up-regulates several HSP (HSP 40, HSP70B, HSP27). TNFSF9 which inhibits proliferation was the only gene up-regulated by all the drugs. Microarray technology demonstrates that treatment with novel targeted therapies (Bortezomib, Aplidin and Arsenic Trioxide) induces deregulation of molecular mechanisms which are not involved in anti-myeloma activity of conventional treatments (Melphalan, Doxorubicin). In addition it is an efficient tool to understand the differences in mechanisms of action of novel drugs.