Rhizochalinin Exhibits Anticancer Activity and Synergizes with EGFR Inhibitors in Glioblastoma In Vitro Models.

Rhizochalinin (Rhiz) is a recently discovered cytotoxic sphingolipid synthesized from the marine natural compound rhizochalin. Previously, Rhiz demonstrated high in vitro and in vivo efficacy in various cancer models. Here, we report Rhiz to be highly active in human glioblastoma cell lines as well as in patient-derived glioma-stem like neurosphere models. Rhiz counteracted glioblastoma cell proliferation by inducing apoptosis, G2/M-phase cell cycle arrest, and inhibition of autophagy. Proteomic profiling followed by bioinformatic analysis suggested suppression of the Akt pathway as one of the major biological effects of Rhiz. Suppression of Akt as well as IGF-1R and MEK1/2 kinase was confirmed in Rhiz-treated GBM cells. In addition, Rhiz pretreatment resulted in a more pronounced inhibitory effect of γ-irradiation on the growth of patient-derived glioma-spheres, an effect to which the Akt inhibition may also contribute decisively. In contrast, EGFR upregulation, observed in all GBM neurospheres under Rhiz treatment, was postulated to be a possible sign of incipient resistance. In line with this, combinational therapy with EGFR-targeted tyrosine kinase inhibitors synergistically increased the efficacy of Rhiz resulting in dramatic inhibition of GBM cell viability as well as a significant reduction of neurosphere size in the case of combination with lapatinib. Preliminary in vitro data generated using a parallel artificial membrane permeability (PAMPA) assay suggested that Rhiz cannot cross the blood brain barrier and therefore alternative drug delivery methods should be used in the further in vivo studies. In conclusion, Rhiz is a promising new candidate for the treatment of human glioblastoma, which should be further developed in combination with EGFR inhibitors.

Anticancer Activity of the Marine Triterpene Glycoside Cucumarioside A2-2 in Human Prostate Cancer Cells.

Despite recent advances in the treatment of metastatic castration-resistant prostate cancer (CRPC), treatment is inevitably hampered by the development of drug resistance. Thus, new drugs are urgently needed. We investigated the efficacy, toxicity, and mechanism of action of the marine triterpene glycoside cucumarioside A2-2 (CA2-2) using an in vitro CRPC model. CA2-2 induced a G2/M-phase cell cycle arrest in human prostate cancer PC-3 cells and caspase-dependent apoptosis executed via an intrinsic pathway. Additionally, the drug inhibited the formation and growth of CRPC cell colonies at low micromolar concentrations. A global proteome analysis performed using the 2D-PAGE technique, followed by MALDI-MS and bioinformatical evaluation, revealed alterations in the proteins involved in cellular processes such as metastatic potential, invasion, and apoptosis. Among others, the regulation of keratin 81, CrkII, IL-1ß, and cathepsin B could be identified by our proteomics approach. The effects were validated on the protein level by a 2D Western blotting analysis. Our results demonstrate the promising anticancer activity of CA2-2 in a prostate cancer model and provide insights on the underlying mode of action.

Salvage Chemotherapy with Cisplatin, Ifosfamide, and Paclitaxel in Aggressive Variant of Metastatic Castration-Resistant Prostate Cancer.

Significant progress has been achieved in the treatment of metastatic castration-resistant prostate cancer (mCRPC). However, results in patients with aggressive variant prostate cancer (AVPC) have been disappointing. Here, we report retrospectively collected data from intensively pretreated AVPC patients (n = 17; 88.2% visceral metastases; 82% elevation of neuroendocrine markers) treated with salvage chemotherapy consisting of cisplatin, ifosfamide, and paclitaxel (TIP). At the interim analysis, 60% of patients showed radiographic response or stable disease (PFS = 2.5 months; OS = 6 months). In men who responded to chemotherapy, an OS > 15 months was observed. Preclinical analyses confirmed the high activity of the TIP regimen, especially in docetaxel-resistant prostate cancer cells. This effect was primarily mediated by increased cisplatin sensitivity in the emergence of taxane resistance. Proteomic and functional analyses identified a lower DNA repair capacity and cell cycle machinery deficiency to be causative. In contrast, paclitaxel showed inconsistent effects, partially antagonizing cisplatin and ifosfamide in some AVPC models. Consequently, paclitaxel has been excluded from the TIP combination for future patients. In summary, we report for the first time the promising efficacy of TIP as salvage therapy in AVPC. Our preclinical data indicate a pivotal role for cisplatin in overcoming docetaxel resistance.

Study of Structure-Activity Relationships of the Marine Alkaloid Fascaplysin and Its Derivatives as Potent Anticancer Agents.

Marine alkaloid fascaplysin and its derivatives are known to exhibit promising anticancer properties in vitro and in vivo. However, toxicity of these molecules to non-cancer cells was identified as a main limitation for their clinical use. Here, for the very first time, we synthesized a library of fascaplysin derivatives covering all possible substituent introduction sites, i.e., cycles A, C and E of the 12H-pyrido[1-2-a:3,4-b']diindole system. Their selectivity towards human prostate cancer versus non-cancer cells, as well as the effects on cellular metabolism, membrane integrity, cell cycle progression, apoptosis induction and their ability to intercalate into DNA were investigated. A pronounced selectivity for cancer cells was observed for the family of di- and trisubstituted halogen derivatives (modification of cycles A and E), while a modification of cycle C resulted in a stronger activity in therapy-resistant PC-3 cells. Among others, 3,10-dibromofascaplysin exhibited the highest selectivity, presumably due to the cytostatic effects executed via the targeting of cellular metabolism. Moreover, an introduction of radical substituents at C-9, C-10 or C-10 plus C-3 resulted in a notable reduction in DNA intercalating activity and improved selectivity. Taken together, our research contributes to understanding the structure-activity relationships of fascaplysin alkaloids and defines further directions of the structural optimization.

Cabazitaxel overcomes cisplatin resistance in germ cell tumour cells.

BACKGROUND: Cisplatin-based chemotherapy is highly effective in metastasized germ cell tumours (GCT). However, 10-30 % of patients develop resistance to cisplatin, requiring salvage therapy. We investigated the in vitro activity of paclitaxel and the novel taxane cabazitaxel in cisplatin-sensitive and -resistant GCT cell lines. METHODS: In vitro activity of paclitaxel and cabazitaxel was determined by proliferation assays, and mode of action of cabazitaxel was assessed by western blotting and two screening approaches, i.e. whole proteome analysis and a human apoptosis array. RESULTS: Activity of paclitaxel and cabazitaxel was not affected by cisplatin resistance, suggesting that there is no cross-resistance between these agents in vitro. Cabazitaxel treatment showed a strong inhibitory effect on colony formation capacity. Cabazitaxel induced classical apoptosis in all cell lines, reflected by cleavage of PARP and caspase 3, without inducing specific changes in the cell cycle distribution. Using the proteomic and human apoptosis array screening approaches, differential regulation of several proteins, including members of the bcl-2 family, was found, giving first insights into the mode of action of cabazitaxel in GCT. CONCLUSION: Cabazitaxel shows promising in vitro activity in GCT cells, independent of levels of cisplatin resistance.

Proteome analysis of the effects of all-trans retinoic acid on human germ cell tumor cell lines.

We analysed the effects of all-trans retinoic acid (ATRA) on proliferation and changes in the global proteome of the nullipotent human embryonal carcinoma cell line 2102Ep and the pluripotent cell line NTERA2 cl.D1 (NT2). Differentially expressed proteins were assessed by 2D-PAGE and mass spectrometry, followed by verification and analysis of protein modifications of proteins of the retinoid pathway. We established a proteome map of the germ cell tumor (GCT) cell line NT2 showing neuronal differentiation under ATRA treatment for 7days. Using bioinformatic analyses, we identified functional groups of altered proteins and potentially involved pathways, of which changes to the organization of the cytoskeleton and anti-apoptotic effects were the most prominent. Changes observed in the expression of factors involved in the retinoid pathway under ATRA, namely an upregulation of CRBP and CRABP2, were also reflected in GCT tissues of different histologies, providing further insight into factors involved in the differentiation of these pluripotent tumors. BIOLOGICAL SIGNIFICANCE: Treatment of NT2 germ cell tumor cells with all-trans retinoic acid (ATRA) is a model to investigate differentiation. We analysed differentially expressed proteins by 2D-PAGE and mass spectrometry and provide a proteome map of NT2 cells under 7days of ATRA. By bioinformatic analyses, functional groups of proteins and involved pathways like changes to the cytoskeleton and anti-apoptotic effects were identified. Factors involved in the retinoid pathway, in particular upregulation of CRBP, CRABP1 and CRABP2, also showed differential expression in tumors with different histological subtypes, which provides insight into gene regulation under induced and spontaneous differentiation in germ cell tumors.

Comparative proteome, transcriptome, and genome analysis of a gonadal and an extragonadal germ cell tumor cell line.

Whereas clinical differences between testicular and extragonadal germ cell tumors (GCT), like reduced cisplatin sensitivity of extragonadal tumors, are well-established, little is known about underlying tumor biology. A combined approach using global proteome analysis and RT-PCR to assess mRNA levels of selected proteins on the one hand, and array comparative genomic hybridization (array-CGH), on the other hand, was used to compare two germ cell tumor (GCT) cell lines showing embryonal carcinoma histology, one of testicular, and one of extragonadal origin. Overall, the two cell lines show remarkably similar protein profiles. In total, 66 proteins were found to be differentially expressed in an at least 2-fold manner. Of these, 35 proteins (53%) could be positively identified by peptide mass fingerprinting and database search. mRNA levels of 27 differentially expressed proteins were analyzed by RT-PCR. In 17/27 genes (63%), differences in mRNA expression corresponded with differences detected on protein level, suggesting that these proteins are mainly regulated through transcription. Interestingly, no close correlation was found between proteomic and genomic analysis: 13/30 genes (43%) with higher protein levels in one cell line showed higher copy numbers of the respective gene loci in array-CGH analysis. Corresponding differences from proteome, transcriptome, and mRNA analyses were found in 9 of 27 proteins (33%). Several proteins potentially involved in cisplatin resistance were identified in the extragonadal cell line, suggesting that the cisplatin-resistant phenotype of this cell line is multifactorial. Furthermore, our data demonstrate that a combined approach of proteome, transcriptome, and genome analysis is a promising tool to gain information on gene regulation in human tumors.