Mitomycin C potentiates TRAIL-induced apoptosis through p53-independent upregulation of death receptors: evidence for the role of c-Jun N-terminal kinase activation.

The discovery of the molecular targets of chemotherapeutic medicines and their chemical footprints can validate and improve the use of such medicines. In the present report, we investigated the effect of mitomycin C (MMC), a classical chemotherapeutic agent on cancer cell apoptosis induced by TRAIL. We found that MMC not only potentiated TRAIL-induced apoptosis in HCT116 (p53-/-) colon cancer cells but also sensitized TRAIL-resistant colon cancer cells HT-29 to the cytokine both in vitro and in vivo. MMC also augmented the pro-apoptotic effects of two TRAIL receptor agonist antibodies, mapatumumab and lexatumumab. At a mechanistic level, MMC downregulated cell survival proteins, including Bcl2, Mcl-1 and Bcl-XL, and upregulated pro-apoptotic proteins including Bax, Bim and the cell surface expression of TRAIL death receptors DR4 and DR5. Gene silencing of DR5 by short hairpin RNA reduced the apoptosis induced by combination treatment of MMC and TRAIL. Induction of DR4 and DR5 was independent of p53, Bax and Bim but was dependent on c-Jun N terminal kinase (JNK) as JNK pharmacological inhibition and siRNA abolished the induction of the TRAIL receptors by MMC.

Overcoming hypoxia-induced apoptotic resistance through combinatorial inhibition of GSK-3ß and CDK1.

Tumor hypoxia is an inherent impediment to cancer treatment that is both clinically significant and problematic. In this study, we conducted a cell-based screen to identify small molecules that could reverse the apoptotic resistance of hypoxic cancer cells. Among the compounds, we identified were a structurally related group that sensitized hypoxic cancer cells to apoptosis by inhibiting the kinases GSK-3ß and cyclin-dependent kinase (CDK) 1. Combinatorial inhibition of these proteins in hypoxic cancer cells and tumors increased levels of c-Myc and decreased expression of c-IAP2 and the central hypoxia response regulator hypoxia-inducible factor (HIF) 1α. In mice, these compounds augmented the hypoxic tumor cell death induced by cytotoxic chemotherapy, blocking angiogenesis and tumor growth. Taken together, our findings suggest that combinatorial inhibition of GSK-3ß and CDK1 augment the apoptotic sensitivity of hypoxic tumors, and they offer preclinical validation of a novel and readily translatable strategy to improve cancer therapy.

Utility of dual-modality bioluminescence and MRI in monitoring stem cell survival and impact on post myocardial infarct remodeling.

RATIONALE AND OBJECTIVES: Firefly luciferase (Fluc) reporter gene is an authentic marker for surviving stem cells. However, it is unable to visualize the intramyocardial delivery of stem cells or their impact on cardiac function. The investigators demonstrate that bioluminescence imaging (BLI) combined with magnetic resonance imaging (MRI) allows better assessment of cell delivery and the impact on post-myocardial infarction remodeling. MATERIALS AND METHODS: Murine embryonic stem cells (0.3 million) were double-labeled with Fluc and superparamagnetic iron oxide particles and injected into the infarct border zone of athymic rat hearts. BLI and MRI were performed serially up to 2 months after injection, followed by immunohistochemistry. RESULTS: Dual-modality imaging was able to verify the initial intramyocardial delivery of the cells and their survival status. Over time, BLI signal increased in seven of nine hearts and disappeared in the other two hearts. The divergence of BLI signal over time was supported by MRI findings. Left ventricular ejection fraction and fractional shortening estimated by MRI suggested that cell engraftment mediated a positive impact on post-myocardial infarction remodeling. Two months after intramyocardial injection, superparamagnetic iron oxide-associated signals facilitated the localization of the injection site. CONCLUSIONS: Dual-modality imaging has the unique ability to monitor cell delivery, survival status, graft morphology, and impact on post-myocardial infarction remodeling.

The myc-miR-17~92 axis blunts TGF{beta} signaling and production of multiple TGF{beta}-dependent antiangiogenic factors.

c-Myc stimulates angiogenesis in tumors through mechanisms that remain incompletely understood. Recent work indicates that c-Myc upregulates the miR-17∼92 microRNA cluster and downregulates the angiogenesis inhibitor thrombospondin-1, along with other members of the thrombospondin type 1 repeat superfamily. Here, we show that downregulation of the thrombospondin type 1 repeat protein clusterin in cells overexpressing c-Myc and miR-17∼92 promotes angiogenesis and tumor growth. However, clusterin downregulation by miR-17∼92 is indirect. It occurs as a result of reduced transforming growth factor-ß (TGFß) signaling caused by targeting of several regulatory components in this signaling pathway. Specifically, miR-17-5p and miR-20 reduce the expression of the type II TGFß receptor and miR-18 limits the expression of Smad4. Supporting these results, in human cancer cell lines, levels of the miR-17∼92 primary transcript MIR17HG negatively correlate with those of many TGFß-induced genes that are not direct targets of miR-17∼92 (e.g., clusterin and angiopoietin-like 4). Furthermore, enforced expression of miR-17∼92 in MIR17HG(low) cell lines (e.g., glioblastoma) results in impaired gene activation by TGFß. Together, our results define a pathway in which c-Myc activation of miR-17∼92 attenuates the TGFß signaling pathway to shut down clusterin expression, thereby stimulating angiogenesis and tumor cell growth.

Cell cycle dependent and schedule-dependent antitumor effects of sorafenib combined with radiation.

The antineoplastic drug sorafenib (BAY 43-9006) is a multikinase inhibitor that targets the serine-threonine kinase B-Raf as well as several tyrosine kinases. Given the numerous molecular targets of sorafenib, there are several potential anticancer mechanisms of action, including induction of apoptosis, cytostasis, and antiangiogenesis. We observed that sorafenib has broad activity in viability assays in several human tumor cell lines but selectively induces apoptosis in only some lines. Sorafenib was found to decrease Mcl-1 levels in most cell lines tested, but this decrease did not correlate with apoptotic sensitivity. Sorafenib slows cell cycle progression and prevents irradiated cells from reaching and accumulating at G2-M. In synchronized cells, sorafenib causes a reversible G1 delay, which is associated with decreased levels of cyclin D1, Rb, and phosphorylation of Rb. Although sorafenib does not affect intrinsic radiosensitivity using in vitro colony formation assays, it significantly reduces colony size. In HCT116 xenograft tumor growth delay experiments in mice, sorafenib alters radiation response in a schedule-dependent manner. Radiation treatment followed sequentially by sorafenib was found to be associated with the greatest tumor growth delay. This study establishes a foundation for clinical testing of sequential fractionated radiation followed by sorafenib in gastrointestinal and other malignancies.

Reduction of TRAIL-induced Mcl-1 and cIAP2 by c-Myc or sorafenib sensitizes resistant human cancer cells to TRAIL-induced death.

Cells expressing oncogenic c-Myc are sensitized to TNF superfamily proteins. c-Myc also is an important factor in determining whether a cell is sensitive to TRAIL-induced apoptosis, and it is well established that the mitochondrial pathway is essential for apoptosis induced by c-Myc. We investigated whether c-Myc action on the mitochondria is required for TRAIL sensitivity and found that Myc sensitized cells with defective intrinsic signaling to TRAIL. TRAIL induced expression of antiapoptotic Mcl-1 and cIAP2 through activation of NF-kappaB. Both Myc and the multikinase inhibitor sorafenib block NF-kappaB. Combining sorafenib with TRAIL in vivo showed dramatic efficacy in TRAIL-resistant tumor xenografts. We propose the combination of TRAIL with sorafenib holds promise for further development.