Molecular network profiling of U373MG human glioblastoma cells following induction of apoptosis by novel marine-derived anti-cancer 1,2,3,4-tetrahydroisoquinoline alkaloids

Glioblastoma, World Health Organization (WHO) grade IV, is the most common and aggressive form of primary malignant brain tumors affecting the adult human brain. Glioblastoma is categorized into two distinct subtypes that develop through different genetic and molecular pathways, designated as primary glioblastoma and secondary glioblastoma [1]. Primary glioblastoma that develops without precursor lesions shows an amplification of the epidermal growth factor receptor (EGFR) gene more frequently than secondary glioblastoma that generates via the stepwise progression from the pre-existing low-grade glioma. EGFR amplification in primary glioblastoma is often associated with the expression of EGFRvIII, a ligand-independent constitutively active mutant of EGFR, capable of persistently activating the phosphatidylinositol 3-kinase (PI3K)/v-akt murine thymoma viral oncogene homolog (AKT) signaling pathway that promotes the survival of glioma cells [2]. The genetic mutations of phosphatase and tensin homolog (PTEN), a negative regulator of AKT signaling pathway, are more frequent found in primary glioblastoma. In contrast, secondary glioblastoma more often shows tumor protein p53 (TP53) mutations, and fairly consistently exhibits the genetic mutation of isocitrate dehydrogenase 1 (IDH1), a negative regulator of hypoxia-inducible factor 1-alpha (HIFA), whereas IDH1 mutations are barely detectable in primary glioblastoma [1].

Despite the multidisciplinary therapy that combines maximal surgical resection, radiation and chemotherapy, the median survival period does not usually exceed 2 years in the patients with glioblastoma owing to the high incidence of recurrence, invasion, and progression of the tumors [3]. The current drugs that specifically target the tyrosine kinase activity of EGFR or selectively inhibit the mammalian target of rapamycin (mTOR), a PI3K/AKT downstream signal transducer showed little efficacy in treatment of primary glioblastoma [3]. These observations suggest that the best way to eradicate glioblastoma requires the concurrent inhibition of multiple signaling pathways and target molecules that play a pivotal role in the survival and progression of glioma cells.

After the completion of the Human Genome Project, the global analysis of genome, transcriptome, proteome, and metabolome, collectively termed omics, promotes us to characterize the genome-wide molecular basis of the diseases, and helps us to identify disease-specific molecular signatures and biomarkers for diagnosis, classification, and prediction of prognosis. Because omics studies usually produce high-throughput experimental data at one time, it is often difficult to find out the meaningful biological implications from such a huge dataset. Recent advances in bioinformatics and systems biology have made major breakthroughs by illustrating the cell-wide map of complex molecular interactions with the aid of the literature-based knowledgebase of molecular pathways [4]. The logically arranged molecular networks construct the whole system characterized by robustness, which maintains the proper function of the system in the face of genetic and environmental perturbations. In the scale-free molecular networks, targeted disruption of limited numbers of critical components designated hubs, on which the biologically important molecular connections concentrate, could disturb the whole cellular function by destabilizing the networks [5]. Based on these views, the integration of omics data derived from glioma cells with underlying molecular networks provides a useful approach not only to characterize the glioma-relevant pathways but also to identify the network-based effective drug targets.

Various tetrahydroisoquinoline alkaloids with anti-cancer activities were isolated from marine invertebrates. They are classified into two major categories, ecteinascidins and renieramycins and their related compounds. The most potent bioactive member of ecteinascidins is ecteinascidin-743 (ET-743; the compound 1b in Figure 1, Trabectedin, Yondelis, CID 108150) isolated from the Carribean tunicate Ecteinascidia turbinata. The European Commission has approved ET-743 for the first-line therapy in the patients with unresectable soft tissue sarcoma refractory to the conventional chemotherapy [6]. ET-743 has a unique mechanism of anti-cancer activity in that it binds to the minor groove of the DNA double helix and alkylates the N2 of guanine to interfere with cell division, DNA repair, and transcriptional activation, leading to apoptosis of target cells [7‐9].

Previously, we purified and identified a series of biologically active compounds from Thai marine invertebrates. Among them, ecteinascidin-770 (ET-770; the compound 1a in Figure 1, CID 16728498) is a stabilized derivative of ET-743 that maintained anti-cancer activities, obtained on a large scale from Thai tunicate Ecteinascidia thurstoni by pretreatment with potassium cyanide in buffer solution [10]. Recently, we prepared nitrogen-containing heterocyclic derivatives of ET-770, and we found that a 2’-N-4”-pyridinecarbonyl derivative of ET-770 (the compound 3 in Figure 1) shows the greater cytotoxicity to human colon, lung and prostate cancer cells than the compound 1a [11]. We also isolated renieramycin M (RM; the compound 2a in Figure 1), a major bis-1,2,3,4-tetrahydroisoquinolinequinone alkaloid from the Thai marine sponge Xestospongia sp., which exhibits a potent cytotoxicity to human colon and breast cancer cells at the nanomolar concentrations [12]. Transcriptome analysis of the compound 2a-treated cancer cells suggested that the downregulation of protein tyrosine phosphatase receptor type K (PTPRK) serves as a biomarker for monitoring anti-tumor effects of RM [13]. Furthermore, RM induces apoptosis through the p53-dependent pathway and inhibits progression and metastasis of lung cancer cells at low sublethal concentrations [14].

The present study attempted to characterize and elucidate the molecular pathways responsible for cytotoxic effects of three distinct marine-derived anti-cancer 1,2,3,4-tetrahydroisoquinoline alkaloids 1a, 2a, and 3 on a human glioblastoma cell line U373MG by investigating the genome-wide gene expression profile and the relevant molecular networks.

To identify the molecular pathways responsible for cytotoxic effects of novel 1,2,3,4-tetrahydroisoquinoline alkaloids 1a, 2a, and 3 on U373MG cells, we studied the genome-wide gene expression profile and molecular networks by using pathway analysis tools of bioinformatics. All of these compounds induced apoptosis of U373MG cells at physiologically relevant nanomolar concentrations. The compound 3 reduced the expression of 417 genes and elevated the levels of 84 genes, while the compound 1a downregulated 426 genes and upregulated 45 genes. The compound 2a decreased the expression of 274 genes and increased the expression of 9 genes. Importantly, the set of 196 downregulated genes and 6 upregulated genes showed an overlap among all of these compounds, suggesting an existence of the common pathways involved in induction of apoptosis. By molecular network analysis, we identified the ErbB (EGFR) signaling pathway, in addition to axonal guidance and cell adhesion pathways, as those of the significant pathways in downregulated genes shared among all the compounds. Importantly, a recent study by combining genome-wide screening of altered genes and molecular network analysis identified axonal guidance and cell adhesion pathways as those enriched in a variety of cellular processes deregulated in human glioblastoma [18]. We found that the ErbB (EGFR) signaling pathway is composed of FAK/PTK2, AKT3, and GSK3B, serving as key molecules involved in cell movement and the nervous system development. In contrast, the set of upregulated genes by the compounds 1a and 3 showed the most significant relationship with the cell cycle pathway, where CDC25A acts as a hub molecule. Finally, we found that a GSK3B-specific inhibitor induced apoptosis of U373MG cells, supporting an anti-apoptotic role of GSK3B. These observations indicate that molecular network analysis is a useful approach not only to characterize the glioma-relevant pathways but also to identify the network-based effective drug targets.

Being consistent with our observations, downregulation of AKT3 expression by RNA interference reduces the expression of the phosphorylated form of Bad, resulting in induction of the caspase-dependent apoptosis of glioma cells [19]. AKT3 is required for anchorage-independent growth of human glioma cells [20]. GSK3 is a serine/threonine kinase that regulates in an integrated manner Wnt/β-catenin, Hedgehog, and receptor tyrosine kinase (RTK) signaling pathways involved in a wide range of cellular functions, such as glycogen metabolism, cell differentiation, proliferation, and apoptosis [21]. Interference with GSK3β activity by siRNA inhibits cell migration and induces apoptosis of glioma cells by activating c-Myc and inactivating nuclear factor-κB (NF-κB) activities [22, 23].

The cell cycle progression is positively and negatively regulated by the complex checkpoint mechanism that involves cyclins A, B, D, and E, along with cyclin-dependent kinases (CDKs) and CDK inhibitors (CDKIs) of both the Cip/Kip and Ink4 families. The hypophosphorylated Rb protein interacts with the E2F family transcription factors E2F1, E2F2, and E2F3, and activates the expression of the genes pivotal for cell cycle progression, whereas the Rb protein, hyperphosphorylated by cyclin D1-CDK4 and cyclin E1-CDK2 complexes, releases E2Fs, and represses the expression of cell cycle genes [24]. Thus, the Rb/E2F pathway constitutes a molecular switch deciding either progression or arrest of the cell cycle. By using KeyMolnet, we found that the molecular network, composed of the combined set of downregulated and upregulated genes in U373MG cells shared between the compounds 1a and 3, shows the most significant relationship with transcriptional regulation by Rb/E2F transcription factors. These observations suggest that both of the structurally-related tetrahydroisoquinoline alkaloids act as a DNA-alkylating agent that interferes with cell division, leading to apoptosis of target cells via the mechanisms very similar to those of ET-743-mediated cytotoxicity [7].

CDC25A encodes a protein phosphatase that activates cyclin/CDK complexes by removing inhibitory phosphates from the conserved threonine and tyrosine residues on CDK proteins. A previous study showed that inhibition of CDC25A expression induces apoptosis of human glioma cells [25], seemingly contradictory to upregulation of CDC25A during induction of apoptosis of U373MG cells in our study. However, a recent study showed that CDC25A, by activating cyclin B1 and Cdc2, acts as a proapoptotic mediator that enhances staurosporine-induced apoptosis, supporting our observations [26].

We identified ELMO1 as one of top 10 downregulated genes and OSR2 as one of top 10 upregulated genes in U373MG cells exposed to all three compounds. Importantly, ELMO1, by forming the complex with dedicator of cytokinesis 180 (Dock180), serves as a Rac1 guanine nucleotide exchange factor that stimulates glioma cell migration and invasion [27]. OSR2 acts as a transcription factor for expression of the genes involved in the epithelial and mesenchymal interaction during craniofacial development and cellular differentiation [28].

Molecular network analysis suggested that novel marine-derived anti-cancer 1,2,3,4-tetrahydroisoquinoline alkaloids 1a, 2a, and 3 induced apoptosis of glioma cells through the shared molecular mechanisms involving multiple pathways and targets that play a pivotal role in the survival and invasion of glioma cells.