Microarray analysis and signaling pathway profiling
The cell cycle arrest in CCRF-CEM cells was early detected after 24 h and the down regulation of important genes such as CHK1 and WEE1, as reported above might plays a considerable role. This allegation is strengthened by the fact that one of the key players in cell cycle regulation such as GADD45 is also up-regulated following GA treatment (Additional file 1: Figure S1).
HSPA6 (heat shock 70 kDa protein 6) and HIST1H2BD (histone cluster 1, H2bd) genes were found to be most up-regulated upon GA treatment. In cooperation with other chaperones, Hsp70s stabilize preexistent proteins against aggregation and mediate the folding of newly translated polypeptides in the cytosol as well as within organelles. They bind extended peptide segments with a net hydrophobic character exposed by polypeptides during translation and membrane translocation, or following stress-induced damage (http://www.uniprot.org). Interestingly, 70 kDa heat shock protein protects cells from ischemia and its expression is increased in consequence to hypoglycemia [17, 18]. In addition, closely connected genes to HSPA6, HSPA1A and DNAJB2, were found to be up-regulated in our anaylses. Whereas HSPA1A is coding for heat shock 70 kDa protein 1A/1B, DNAJB2 has as protein product cochaperon Hsp40 (DNAJ in bacteria) which is essential for Hsp70 function . Interesting genes which are up-regulated included FOSB and JUN. A combined up-regulation of these genes with Hsp70 mRNA was earlier observed in as stress response to high acceleration . This finding also confirmed the assumption that GA might cause hypoxic stress. Through high acceleration an induction of ischemia can be observed . Furthmore, dimerization of protein products of FOSB (a FOS family member) and JUN constitute the transcription factor AP-1 which is activated by oxidative stress . Surprisingly, a cofactor of JUN-activated transciption, DDX21, belonged to one of the most down-regulated genes in our microarray analysis . This findings give reason to speculate that GA might mimic under-supply of oxygen or glucose and, therefore, leads to apoptosis (see also Figure 2). Very interesting in the contest of the latter assumption was that GA inhibited angiogenesis (Figure 1). It would be worthwhile to investigate, whether the vascular endothelial growth factor (VEGF), its secretion or even the VEGF receptor are inhibited by GA leading to interrupted VEGF signaling, as normally VEGF is activated under hypoxic conditions . Thereby, the fact that we did not observed any significant change in VEGF mRNA expression (See Additional file 1: Table S3) does not mean that VEGF signaling was not targeted by GA. VEGF protein expression can independently vary from corresponding mRNA amounts in the cell .
Certain histone mRNAs were also up-regulated after treatment with GA HIST1H2BD, HIST2H2AC, HIST2H2AA4 (Table 3)]. This observation is surprising, as levels of histone mRNA usually increase during S-phase, but decrease back to baseline level between the S-phase and mitosis . This finding was also confirmed by our FACS analyses showing that cells treated with 1 × IC50 of GA for 72 h were more often arrested in the S- or G2/M phases than untreated control cells (see also Figure 2.C2 and D2).
CD52 is a membrane protein which is discussed as target molecule for leukemia therapy . Alemtuzumab is an antibody directly targeting CD52 and already approved for clinical use . In our experiments, the mRNA of this protein was strongly up-regulated. A combined treatment of GA with Ametuzumab may possibly boost the apoptotic effect of both drugs, as GA might sensitize cells to Alemtuzumab by inducing CD52 expression and activating CD52 downstream mechanisms. This speculations merits further experimentation in the future.
ACTB and ACTBL3 belonged to the most down-regulated genes. Beta-actin mRNA levels are known to be disturbed after ischemia , which is in line with our assumption that GA may mimic hypoxia. Another gene fitting to our hypothesis is PGAM1, which codes for phosphoglycerate mutase in glycolysis. Another gene coding for a protein similar to phosphoglycerate mutase processed protein was also down-regulated by GA, LOC728188. Down-regulation of glycolysis key molecules accompanied by hypoxic stress may destroy the entire energy production apperture ultimately leading to cell death. The mis-regulation in glyco-related mechanisms was also indicated by down-regulation of DHRS2, whose encoded protein preferentially binds to glucose and related sugars .
KPNA2 codes for importin alpha. This protein is a key player in the nuclear transport of macromolecules . Moreover, HNRNPA1P2 is rarely investigated yet, but seems to be involved to mRNA transport from the nucleus to cytosol (according to UniProt database, http://www.uniprot.org). THOC4 encoding a more investigated mRNA transporter molecule was also significantly down-regulated. The THOC4 protein is part of the TREX complex, which specifically associates with spliced mRNA . THOC4 is especially involved in nuclear export of Hsp70 transcripts .
Interestingly, RAB37 and TRAPPC6A encode also two proteins which are also involved to transport mechanisms (http://www.uniprot.org). They were also mis-regulated in their transcriptional activity after GA treatment. In summary, it seems that transport mechanisms are de-regulated as consequence of treatment with this compound.
Recapitulating, GA seems to cause hypoxia and hypoglycemia as several genes of these functions were affected. Furthermore, a considerable de-regulation of several protein- and mRNA-transporter genes was observed. The latter findings was also confirmed by Ingenuity Pathway Analyses of the microarray data pointing to “Molecular Transport” and “Protein Trafficking” with highest score (Table 3; Figure 3). In contrast, no molecules belonging to the other hits of Table 3 were found to be extremely up- or down-regulated.
In the signaling networks of Figures 3 and 4, more key players become apparent which do not belong to most mis-regulated genes in our investigation: HNRNPK LYAR and YBX1. According to UniProt database, HNRNPK is involved in RNA processing and splicing. However, it is interesting from our point of view that it seems to be important for enhanced proliferation, as increased levels of HNRNPK mRNA correlates with increased proliferative activity . LYAR is a gene involved in development and cell growth regulation and especially for leukemia cells, as mRNA levels are increased, but LYAR mRNA is not or only less expressed in cells of different healthy tissues, e.g. thymus, bone marrow, liver, heart, brain, kidney or spleen . YBX1 encodes for a protein, YB-1, which is involved in many mechanisms: proliferation, mRNA processing, DNA repair, transcription, splicing and drug resistence . The relation of YB-1 to drug resistance is due to its activation of the multidrug resistance gene, MDR1
. In addition, YBX1 expression is also important for deregulation of the oncogene, MYCN
 (Figure 3B).
MYCN is a prominent transcription factor important for tumorigenesis and chemotherapy. MYCN mRNA was found to be over-expressed in human tumor biopsies . Directly correlated to this finding is the down-regulation of RUVBL1 mRNA (Figure 3B) encoding a protein which binds to MYC . It is essential for cell proliferation. In addition, the RUVBL1 protein is associated with the activation of NuA4 histone acetyltransferase complex, which is associated to the de-regulated histone mRNAs mentioned above . Finally, SGK1 up-regulation is very interesting, as the encoded protein might counteract the cytotoxic activity of GA and activate cell survival processes. It deserves more detailed investigations in the future to elucidate the connection between KPNA2 and SGK1, as recognition by importin alpha of Sgk (protein of SGK1 gene) might be necessary for nuclear import of activated SGK1 .
Furthermore, there is another connection between SGK1 (Figure 3B) and FOXO3A (Figure 4A). FOXO3A is a pro-apoptotic protein which is regulated by SGK1 . FOXO3A has also an important role as transcription factor in oxidative stress reponse and cell protection . Its role in cellular response to GA needs further investigations, because the activity of FOXO3A protein is controlled by phosphorylation, which was not measured in our mRNA expression profiling approach. Another prominent member involved in cell death mechanisms is YWHAG, which codes for 14-3-3 gamma protein . 14-3-3 proteins are directly involved in apoptosis and cell survival regulation by inhibiting BAD and FOXO3A . In summary, the signaling network shown in (Figure 3B) depicts genes related to cell survival.
The overall results of the present investigation strengthened the cytotoxic potency, the effect on cell cycle distribution, apoptosis, angiogenesis of Guieranone A, and consequently give important information for the future investigation, that could lead to the potential use of this compound in cancer therapy.