Systems analysis of cancer cell heterogeneity in caspase-dependent apoptosis subsequent to mitochondrial outer membrane permeabilization.

Deregulation of apoptosis is a hallmark of carcinogenesis. We here combine live cell imaging and systems modeling to investigate caspase-dependent apoptosis execution subsequent to mitochondrial outer membrane permeabilization (MOMP) in several cancer cell lines. We demonstrate that, although most cell lines that underwent MOMP also showed robust and fast activation of executioner caspases and apoptosis, the colorectal cancer cell lines LoVo and HCT-116 Smac(-/-), similar to X-linked inhibitor of apoptosis protein (XIAP)-overexpressing HeLa (HeLa XIAP(Adv)) cells, only showed delayed and often no caspase activation, suggesting apoptosis impairment subsequent to MOMP. Employing APOPTO-CELL, a recently established model of apoptosis subsequent to MOMP, this impairment could be understood by studying the systemic interaction of five proteins that are present in the apoptosis pathway subsequent to MOMP. Using APOPTO-CELL as a tool to study detailed molecular mechanisms during apoptosis execution in individual cell lines, we demonstrate that caspase-9 was the most important regulator in DLD-1, HCT-116, and HeLa cells and identified additional cell line-specific co-regulators. Developing and applying a computational workflow for parameter screening, systems modeling identified that apoptosis execution kinetics are more robust against changes in reaction kinetics in HCT-116 and HeLa than in DLD-1 cells. Our systems modeling study is the first to draw attention to the variability in cell specific protein levels and reaction rates and to the emergent effects of such variability on the efficiency of apoptosis execution and on apoptosis impairment subsequent to MOMP.

Cell survival or apoptosis: rooperol's role as anticancer agent.

Nontoxic hypoxoside, isolated from Hypoxis, is converted to cytotoxic rooperol in the presence of beta-glucosidase. In this study, we investigated rooperol's mechanism of action. IC50 values of hypoxoside and rooperol were determined against the HeLa, HT-29, and MCF-7 cancer cell lines, and peripheral blood mononuclear cells. DNA cell cycle arrest occurred in late G1 and/or early S phases, associated with increased p21(Waf1/Cip1) levels. Apoptosis was shown by caspase-3 and/or caspase-7 activation, phosphatidylserine translocation, DNA fragmentation, cell blebbing, and apoptotic body formation. Increased phospho-Akt, phospho-Bcl-2, and p21(Waf1/Cip1) proteins, and cell size correspond to cell survival strategies (associated with endoreduplication).

The apoptotic and autophagic properties of two natural occurring prodrugs, hyperoside and hypoxoside, against pancreatic cancer cell lines.

Pancreatic cancer is only the 12th most common cancer, but the fourth leading cause of cancer-related deaths in the world. This is due to late prognosis, poor response to chemotherapy and early metastases. Natural prodrugs may play an important role in the treatment of pancreatic cancer. The main aim of this study was to determine the cytotoxicity of five natural prodrugs, namely harpagoside, hyperoside, hypoxoside, oleuropein and polydatin, by investigating apoptosis and autophagy as possible mechanism/s of action. Hypoxoside and hyperoside have shown selective cytotoxicity at IC50 values of ∼25 and 50µM against INS-1 and MIA PaCa-2 pancreatic cancer cells, respectively. Hypoxoside and hyperoside induced G2/M phase arrest and caspase-3 activation in INS-1 and MIA PaCa-2 cells, respectively. Hoechst/phalloidin staining confirmed morphological changes, including condensed chromatin multinucleation, membrane blebbing and loss of cytoskeletal arrangement in INS-1 and MIA PaCa-2 cells. Acridine orange staining was absent in INS-1 (hypoxoside) and MIA PaCa-2 (hyperoside) treated cells, whereas LC3B expression was not significantly increased. INS-1 and MIA PaCa-2 treated cells favour the cell death pathway, apoptosis, over the cell survival pathway, autophagy.

Systems analysis of BCL2 protein family interactions establishes a model to predict responses to chemotherapy.

Apoptotic desensitization is a hallmark of cancer cells, but present knowledge of molecular systems controlling apoptosis has yet to provide significant prognostic insights. Here, we report findings from a systems study of the intrinsic pathway of apoptosis by BCL2 family proteins and clinical translation of its findings into a model with applications in colorectal cancer (CRC). By determining absolute protein quantifications in CRC cells and patient tumor samples, we found that BAK and BAX were expressed more highly than their antiapoptotic inhibitors. This counterintuitive finding suggested that sole inhibition of effector BAX and BAK could not be sufficient for systems stability in nonstressed cells. Assuming a model of direct effector activation by BH3-only proteins, we calculated that the amount of stress-induced BH3-only proteins required to activate mitochondrial apoptosis could predict individual death responses of CRC cells to 5-fluorouracil/oxaliplatin. Applying this model predictor to protein profiles in tumor and matched normal tissue samples from 26 patients with CRCs, we found that differences in protein quantities were sufficient to model the increased tumor sensitivity to chemotherapy compared with normal tissue. In addition, these differences were sufficient to differentiate clinical responders from nonresponders with high confidence. Applications of our model, termed DR_MOMP, were used to assess the impact of apoptosis-sensitizing drugs in lowering the necessary dose of state-of-the-art chemotherapy in individual patients. Together, our findings offer a ready clinical tool with the potential to tailor chemotherapy to individual patients.

Rooperol as an antioxidant and its role in the innate immune system: an in vitro study.

ETHNOPHARMACOLOGICAL RELEVANCE: Biologically active rooperol is formed when the glucose subunits of the nontoxic glycoside, hypoxoside, are cleaved by ß-glucosidase. Hypoxoside is isolated from Hypoxis, a medicinal plant genus frequently used by the indigenous people of South Africa as an immune system booster. The aim of this study was to investigate rooperol's antioxidant and anti-inflammatory properties using the ferric reducing ability of plasma (FRAP) assay, NO and ROS production, and phagocytosis. MATERIALS AND METHODS: Differentiation of human promonocytic U937 leukemia cells to monocyte-macrophages was induced using 10-100 nM 1,25(OH)(2)D(3) and PMA over 72 h. Differentiation was confirmed by light microscopy and flow cytometry. Undifferentiated and/or differentiated cells were treated with DMSO (0.25 v/v%, vehicle control), hypoxoside (50 µg/mL), rooperol (20 µg/mL) or PMA (10/20 nM, positive control). ROS production was measured in undifferentiated and differentiated monocyte-macrophages using DCFH-DA and flow cytometry. Phagocytosis of pHrodo™ Escherichia coli BioParticles(®) was measured using pre-treated monocyte-macrophage differentiated U937 cells. NO production was measured in monocyte-macrophage differentiated U937 cells using DAF-2 DA and flow cytometry. RESULTS: Rooperol was shown to have similar or greater antioxidant potential than ascorbic acid. Differentiation of human promonocytic U937 leukemia cells to monocyte-macrophages were confirmed morphologically (cell attachment, clump- and pseudopodia-formation) and biochemically (CD11b and CD14 cell surface marker expression). Rooperol significantly increased ROS and NO production, and phagocytosis in undifferentiated and/or differentiated human promonocytic U937 leukemia cells. Hypoxoside had no or very little effect on ROS and NO production, and phagocytosis. CONCLUSION: This study confirms previous reports that hypoxoside has to be converted to rooperol to be biologically active. The FRAP assay confirms the antioxidant capacity of rooperol seen in previous studies, whereas rooperol's induction of ROS and NO production, and phagocytosis constitute novel findings. Possible mode(s) of action for the in vitro anti-inflammatory activities of rooperol may be explained by ROS and NO production, and phagocytosis.