Analogue 3 also showed potent antiproliferative activity against the highly invasive non-small-cell lung cancer cell line CL1-5 with an ED50 value of 0.11 μM. To determine which genes were differentially expressed upon CL1-5 treatment with analogue 3, the genome-wide mRNA expression profiles of 3-treated cells and control cells were determined using Affymetrix human genome U133 plus 2.0 GeneChip according to the Manufacturer's protocols (Santa Clara, CA, http://www.affymetrix.com) by the Microarray Core Facility of National Research Program for Genomic Medicine of National Science Council in Taiwan as previously described. This Affymetrix GeneChip contains 54,675 probe sets to analyze the expression levels of 47,400 transcripts and variants, including 38,500 well-characterized human genes. GeneChips from the hybridization experiments were read by the Affymetrix GeneChip scanner 3000 7G, and raw data were processed using GC-RMA algorithm. The raw data were then analyzed by GeneSpring GX software version 11.01. 2.5 × 105 CL1-5 cells were treated for 24 h with 3 at a concentration of 0.05 μg/mL, and then total RNA was extracted by TRI zol (Life Technologies, Gaithersburg, MD) RNA from non-treated CL1-5 cells was used as a control. A total of 2,838 genes showed at least two-fold changes in expression levels between the CL1-5 treated with 3 and CL1-5 DMSO control. Analogue 3 up-regulated the expression of 1,112 genes and down-regulate 1,726 genes. The differentially expressed genes were analyzed for GeneGo canonical pathway maps by using MetaCore Analytical Suite (GeneGo Inc., St Joseph, MI). The top ten pathways involved in analog 3 affected genes were shown in . Seven pathways are cell cycle-related pathways and three are DNA damage-related. For example, in the spindle assembly checkpoint (SAC) or chromosome segregation pathway, the genes altered by the treatment with 3 encoded mitotic kinases (e.g., CDK1-cyclin B, Aurora A, Aurora B, and NEK2A), SAC proteins (e.g., MAD1, MAD2, securin, and separase), and motor proteins (e.g., dynein1, dynein activator complex dynactin, and KNSL1) (). The cyclin dependent kinase, Aurora kinases, and NEK2A kinases are critical for mitotic progression, through phosphorylation of their numerous substrates. NEK2A or Aurora kinases are required for spindle formation at the onset of mitosis or chromosome segregation and cytokinesis, respectively. The SAC proteins, such as MAD2, activate spindle checkpoint and inhibit securin degradation, until all chromosomes are aligned at the metaphase plate. When chromosomes are aligned correctly, the E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) inhibitor MAD2 is dissociated from the APC/C and removed from the attached kinetochore by dynein. Subsequently, APC/C is activated by CDC20 or CDH1. APC/C-CDC20 or -CDH1 recognizes substrates such as cyclins, NEK2A, and securin or Aurora kinases and cyclins, respectively. At the onset of anaphase, the separase inhibitor securin is poly-ubiquitinated by activated APC/C followed by digestion by the proteasome. Subsequently, activated separase cleaves thecohesin complex, resulting in separation of the sister chromatids. All of these proteins are expressed in a cell cycle-dependent manner. In our oligonucleotide microarray studies, genes encoding these proteins were up-regulated by the treatment with 3. The up-regulation of MAD2L1 transcript was confirmed by semi-quantitative RT-PCR (). Therefore, we assume that 3 may modulate SAC and chromosome separation, and conclude that 3 induces cell cycle arrest mainly in the G2/M-phase. Because oligonucleotide microarray data are quite complicated and can be contradictory, we will need to conduct additional experiments, such as real-time qPCR, to verify our results.
