Targeting the yin and the yang: combined inhibition of the tyrosine kinase c-Src and the tyrosine phosphatase SHP-2 disrupts pancreatic cancer signaling and biology in vitro and tumor formation in vivo.

OBJECTIVES: Although c-Src (Src) has emerged as a potential pancreatic cancer target in preclinical studies, Src inhibitors have not demonstrated a significant therapeutic benefit in clinical trials. The objective of these studies was to examine the effects of combining Src inhibition with inhibition of the protein tyrosine phosphatase SHP-2 in pancreatic cancer cells in vitro and in vivo. METHODS: SHP-2 and Src functions were inhibited by siRNA or small molecule inhibitors. The effects of dual Src/SHP-2 functional inhibition were evaluated by Western blot analysis of downstream signaling pathways; cell biology assays to examine caspase activity, viability, adhesion, migration, and invasion in vitro; and an orthotopic nude mouse model to observe pancreatic tumor formation in vivo. RESULTS: Dual targeting of Src and SHP-2 induces an additive or supra-additive loss of phosphorylation of Akt and ERK-1/2 and corresponding increases in expression of apoptotic markers, relative to targeting either protein individually. Combinatorial inhibition of Src and SHP-2 significantly reduces viability, adhesion, migration, and invasion of pancreatic cancer cells in vitro and tumor formation in vivo, relative to individual Src/SHP-2 inhibition. CONCLUSIONS: These data suggest that the antitumor effects of Src inhibition in pancreatic cancer may be enhanced through simultaneous inhibition of SHP-2.

Unique dual targeting of thymidylate synthase and topoisomerase1 by FdUMP[10] results in high efficacy against AML and low toxicity.

Acute myeloid leukemia (AML) is an aggressive malignancy that leads to marrow failure and death. There is a desperate need for new therapies. The novel fluoropyrimidine, FdUMP[10], was highly active against both human AML cell lines, (IC(50) values, 3.4nM-21.5nM) and murine lines (IC(50) values, 123.8pM-131.4pM). In all cases, the IC(50) of FdUMP[10] was lower than for cytarabine and ∼ 1000 times lower than 5-fluorouracil (5-FU). FdUMP[10] remained effective against cells expressing the Flt3 internal tandem duplication, BCR-ABL, MN1, and an shRNA against p53. It had activity against patient samples at concentrations that did not affect normal hematopoietic cells. FdUMP[10] inhibited thymidylate synthase (TS) and trapped topoisomerase I cleavage complexes (Top1CCs), leading to DNA damage and apoptosis. All cell lines and nearly all primary AML samples examined expressed both TS and Top1. In vivo, FdUMP[10] was active against a syngeneic AML model with a survival advantage equivalent to doxorubicin plus cytarabine. 5-FU treatment was toxic and did not improve survival. FdUMP[10] was better tolerated than 5-FU or cytarabine plus doxorubicin and did not affect normal HSCs, while 5-FU dramatically impaired their ability to engraft. In summary, FdUMP[10] was highly efficacious and better tolerated than standard therapies.

Increased heating efficiency and selective thermal ablation of malignant tissue with DNA-encased multiwalled carbon nanotubes.

Nanoparticles, including multiwalled carbon nanotubes (MWNTs), strongly absorb near-infrared (nIR) radiation and efficiently convert absorbed energy to released heat which can be used for localized hyperthermia applications. We demonstrate for the first time that DNA-encasement increases heat emission following nIR irradiation of MWNTs, and DNA-encased MWNTs can be used to safely eradicate a tumor mass in vivo. Upon irradiation of DNA-encased MWNTs, heat is generated with a linear dependence on irradiation time and laser power. DNA-encasement resulted in a 3-fold reduction in the concentration of MWNTs required to impart a 10 degrees C temperature increase in bulk solution temperature. A single treatment consisting of intratumoral injection of MWNTs (100 microL of a 500 microg/mL solution) followed by laser irradiation at 1064 nm, 2.5 W/cm(2) completely eradicated PC3 xenograft tumors in 8/8 (100%) of nude mice. Tumors that received only MWNT injection or laser irradiation showed growth rates indistinguishable from nontreated control tumors. Nonmalignant tissues displayed no long-term damage from treatment. The results demonstrate that DNA-encased MWNTs are more efficient at converting nIR irradiation into heat compared to nonencased MWNTs and that DNA-encased MWNTs can be used safely and effectively for the selective thermal ablation of malignant tissue in vivo.

p38 MAPK as a negative regulator of VEGF/VEGFR2 signaling pathway in serum deprived human SK-N-SH neuroblastoma cells.

Evidence suggests that vascular endothelial growth factor (VEGF) mediates neuroprotection to prevent an apoptotic cell death. The p38 mitogen-activated protein kinase (MAPK) pathway is implicated as an important mediator of neuronal apoptosis but its role in VEGF-mediated neuroprotection is unclear. Herein, we show that treatments with the p38 MAPK inhibitor, SB202190, enhanced VEGF-mediated survival in serum deprived SK-N-SH neuroblastoma cells by decreasing caspase-3/7 activation while increasing the phosphorylation of the extracellular signal-regulated kinase (ERK1/2) and Akt signaled through the VEGF receptor, VEGFR2. A blockade of VEGFR2 signaling with a selective inhibitor, SU1498 or gene silencing with VEGFR2 siRNA in SB202190 treated cells abrogated this prosurvival response and induced high activation levels of caspase-3/7. These findings suggested that the protection elicited by p38 MAPK inhibition in serum starved cells was dependent on a functional VEGF/VEGFR2 pathway. However, p38 MAPK inhibition attenuated caspase-3 cleavage in SU1498/SB202190 treated cells, indicating that p38 MAPK and caspase-3 only contributed in part to the total levels of caspase-3/7 induced by VEGFR2 inhibition. Pretreatments with the pan caspase inhibitor, z-VAD-fmk, prevented the apoptosis induced by VEGFR2 inhibition and promoted survival in serum starved cells irrespective of p38 MAPK inhibition. Collectively, our findings suggest that p38 MAPK exerts a negative effect on VEGF-mediated signaling through VEGFR2 in serum starved neuroblastoma cells. Furthermore, VEGF signals protection against a caspase-mediated cell death that is regulated by p38 MAPK-dependent and -independent mechanisms.

The VEGFR2 and PKA pathways converge at MEK/ERK1/2 to promote survival in serum deprived neuronal cells.

Identifying prosurvival mechanisms in stressed neuronal cells would provide protective strategies to hinder neurodegeneration. Recent evidence shows that vascular endothelial growth factor (VEGF), a well-established mitogen in endothelial cells, can mediate neuroprotection against damaging insults through the activation of its cognate receptor VEGFR2. In addition, growth factor receptor signaling pathways have been shown to crosstalk with cAMP-dependent Protein Kinase A (PKA) to protect neuronal cells from harmful stimuli. Whether a relationship exists between VEGFR2 and PKA in mediating neuroprotection under stressful conditions is unknown. Using SK-N-SH neuronal cells as a model system, we show that serum deprivation induces an upregulation in VEGF and VEGFR2 that concomitantly serves as a prosurvival signaling pathway. Inhibitor studies revealed that PKA functioned concurrently with VEGFR2 pathway to signal the activation of the extracellular signal-regulated protein kinases (ERK1/2) as protection against caspase-3/7 activation and a subsequent cell death. The loss in cell viability induced by VEGFR2 and PKA inhibition was prevented by caspase inhibition or overexpression of ERK1. Overexpression of the antiapoptotic protein Bcl-xL also promoted survival when VEGFR2 function was blocked. However, the protection elicited by all three treatments were prevented by the inclusion of a selective inhibitor of mitogen-activated protein kinase kinase (MEK), the upstream kinase that activates ERK1/2. Taken together, these findings suggested that PKA and VEGFR2 converge at the MEK/ERK1/2 pathway to protect serum starved neuronal cells from a caspase-dependent cell death.

Reactive oxygen species induced by proteasome inhibition in neuronal cells mediate mitochondrial dysfunction and a caspase-independent cell death.

While increasing evidence shows that proteasome inhibition triggers oxidative damage, mitochondrial dysfunction and death in neuronal cells, the regulatory relationship among these events is unclear. Using mouse neuronal cells we show that the cytotoxicity induced by mild (0.25 microM) and potent (5.0 microM) doses of the proteasome inhibitor, N-Benzyloxycarbonyl-Ile-Glu (O-t-butyl)-Ala-leucinal, (PSI) involved a dose-dependent increase in caspase activation, overproduction of reactive oxygen species (ROS) and a mitochondrial dysfunction manifested by the translocation of the proapoptotic protein, Bax, from the cytoplasm to the mitochondria, membrane depolarization and the release of cytochrome c and the apoptosis inducing factor (AIF) from mitochondria to the cytoplasm and nucleus, respectively. Whereas caspase or Bax inhibition failed to prevent mitochondrial membrane depolarization and neuronal cell death, pretreatments with the antioxidant N-acetyl-L-cysteine (NAC) or overexpression of the antiapoptotic protein Bcl-xL abrogated these events in cells exposed to mild levels of PSI. These findings implicated ROS as a mediator of PSI-induced cytotoxicity. However, depletions in glutathione and Bcl-xL with potent proteasome inhibition exacerbated this response whereupon survival required the cooperative protection of NAC with Bcl-xL overexpression. Collectively, ROS induced by proteasome inhibition mediates a mitochondrial dysfunction in neuronal cells that culminates in death through caspase- and Bax-independent mechanisms.

Myc stabilization in response to estrogen and phospholipase D in MCF-7 breast cancer cells.

Estrogen, which has been strongly implicated in breast cancer, suppresses apoptosis in estrogen receptor (ER) positive MCF-7 breast cancer cells. Phospholipase D (PLD), which is commonly elevated in ER negative breast cancer cells, also suppresses apoptosis. Survival signals generated by both estrogen and PLD are dependent upon elevated Myc expression. We report here that estrogen- and PLD-induced increases in Myc expression are due to reduced turnover of Myc protein. Estrogen and PLD suppressed phosphorylation of Myc at Thr58--a site that targets Myc for degradation by the proteasome. The data provide a mechanism for elevated Myc expression in hormone-dependent and hormone-independent breast cancer.

Redox regulates COX-2 upregulation and cell death in the neuronal response to cadmium.

We reported previously that cadmium, an oxidative stressor, induced cyclooxygenase-2 (COX-2) upregulation in mouse neuronal cells that culminated in cell death. Herein, we show that cadmium induces reactive oxygen species (ROS) that activate c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) and their substrates, activating transcription factor 2 (ATF-2), CRE-binding protein (CREB) and c-Jun. This response is accompanied by induction of heme-oxygenase-1 (HO-1), poly(ADP-ribose) polymerase cleavage and a caspase-independent cell death. Inhibition of p38 MAPK, but not JNK, suppressed COX-2 protein expression and the cytotoxic response induced by cadmium. Selective inhibitors of phosphatidylinositol-3-kinase (PI3-K), LY294002, and flavoproteins, dipheneylene iodonium chloride (DPI), attenuated cadmium-induced ROS and stress kinase activation, suggesting that ROS can signal the COX-2 upregulation and neuronal cell death mediated by p38 MAPK. Collectively, these findings implicate PI3-K, a flavoprotein, p38 MAPK and COX-2 in a neuronal redox-regulated pathway that mediates cadmium-induced oxidative stress.