Selective disruption of rb-raf-1 kinase interaction inhibits pancreatic adenocarcinoma growth irrespective of gemcitabine sensitivity.

Inactivation of the retinoblastoma (Rb) tumor suppressor protein is widespread in human cancers. Inactivation of Rb is thought to be initiated by association with Raf-1 (C-Raf) kinase, and here we determined how RRD-251, a disruptor of the Rb-Raf-1 interaction, affects pancreatic tumor progression. Assessment of phospho-Rb levels in resected human pancreatic tumor specimens by immunohistochemistry (n = 95) showed that increased Rb phosphorylation correlated with increasing grade of resected human pancreatic adenocarcinomas (P = 0.0272), which correlated with reduced overall patient survival (P = 0.0186). To define the antitumor effects of RRD-251 (50 µmol/L), cell-cycle analyses, senescence, cell viability, cell migration, anchorage-independent growth, angiogenic tubule formation and invasion assays were conducted on gemcitabine-sensitive and -resistant pancreatic cancer cells. RRD-251 prevented S-phase entry, induced senescence and apoptosis, and inhibited anchorage-independent growth and invasion (P < 0.01). Drug efficacy on subcutaneous and orthotopic xenograft models was tested by intraperitoneal injections of RRD-251 (50 mg/kg) alone or in combination with gemcitabine (250 mg/kg). RRD-251 significantly reduced tumor growth in vivo accompanied by reduced Rb phosphorylation and lymph node and liver metastasis (P < 0.01). Combination of RRD-251 with gemcitabine showed cooperative effect on tumor growth (P < 0.01). In conclusion, disruption of the Rb-Raf-1 interaction significantly reduces the malignant properties of pancreatic cancer cells irrespective of their gemcitabine sensitivity. Selective targeting of Rb-Raf-1 interaction might be a promising strategy targeting pancreatic cancer.

Enhanced anti-melanoma efficacy of interferon alfa-2b via inhibition of Shp2.

Interferon-α2b (IFN-α2b) is used to treat melanoma but there is a need to improve its efficacy. IFN-α2b signaling requires STAT1/STAT2 tyrosine phosphorylation and is subject to negative regulation by phosphatases. In this study, we determined whether inhibition of the protein tyrosine phosphatase Shp2 could enhance IFN-α2b responses in human melanoma cells. Shp2 knockdown increased IFN-α2b-stimulated STAT1 Tyr-701 phosphorylation and ISRE-luciferase activity even though it did not affect STAT2 Tyr-690 phosphorylation in A375 cells. In A375 tumor xenografts, Shp2 knockdown enhanced the anti-melanoma effect of IFN-α2b. Furthermore, the Shp2 inhibitor SPI-112Me increased the IFN-α2b-induced STAT1 activation and anti-proliferative response in A375 and SK-MEL-2 cells. These results demonstrate that inhibition of Shp2 can enhance the anti-melanoma activity of IFN-α2b.

Inhibition of cellular Shp2 activity by a methyl ester analog of SPI-112.

The protein tyrosine phosphatase (PTP) Shp2 (PTPN11) is an attractive target for anticancer drug discovery because it mediates growth factor signaling and its gain-of-function mutants are causally linked to leukemias. We previously synthesized SPI-112 from a lead compound of Shp2 inhibitor, NSC-117199. In this study, we demonstrated that SPI-112 bound to Shp2 by surface plasmon resonance (SPR) and displayed competitive inhibitor kinetics to Shp2. Like some other compounds in the PTP inhibitor discovery efforts, SPI-112 was not cell permeable, precluding its use in biological studies. To overcome the cell permeation issue, we prepared a methyl ester SPI-112 analog (SPI-112Me) that is predicted to be hydrolyzed to SPI-112 upon entry into cells. Fluorescence uptake assay and confocal imaging suggested that SPI-112Me was taken up by cells. Incubation of cells with SPI-112Me inhibited epidermal growth factor (EGF)-stimulated Shp2 PTP activity and Shp2-mediated paxillin dephosphorylation, Erk1/2 activation, and cell migration. SPI-112Me treatment also inhibited Erk1/2 activation by a Gab1-Shp2 chimera. Treatment of Shp2(E76K) mutant-transformed TF-1 myeloid cells with SPI-112Me resulted in inhibition of Shp2(E76K)-dependent cell survival, which is associated with inhibition of Shp2(E76K) PTP activity, Shp2(E76K)-induced Erk1/2 activation, and Bcl-XL expression. Furthermore, SPI-112Me enhanced interferon-gamma (IFN-gamma)-stimulated STAT1 tyrosine phosphorylation, ISRE-luciferase reporter activity, p21 expression, and the anti-proliferative effect. Thus, the SPI-112 methyl ester analog was able to inhibit cellular Shp2 PTP activity.

Induction of clusterin by AKT--role in cytoprotection against docetaxel in prostate tumor cells.

Clusterin (CLU), in its cytoplasmic form, is abundant in many advanced cancers and has been established to be cytoprotective against chemotherapeutic agents including docetaxel. However, little is known of the mechanism of its induction. Here, we provide evidence that AKT plays a critical role in upregulating cytoplasmic/secretory sCLU, which is responsible for docetaxel resistance. Western blot analysis indicated that docetaxel-resistant sublines derived from DU145 and PC3 prostate tumor cell lines displayed a markedly increased phospho-AKT level closely accompanied by heightened sCLU expression when compared with parental cells. To examine if AKT has a role in sCLU expression, AKT blockade was done by treatment with a specific inhibitor, API-2, or dominant-negative AKT transduction before analysis of sCLU gene expression. Loss of AKT function resulted in loss of sCLU and was accompanied by chemosensitization to docetaxel and increased cell death via a caspase-3-dependent pathway. To confirm that AKT affected resistance to docetaxel through sCLU and not through other mediators, tumor cells were first transfected with full-length CLU for overexpression and then treated with the AKT inhibitor API-2. We found that once sCLU was overexpressed, API-2 could not chemosensitize the tumor cells to docetaxel. Thus, the chemoresistance to docetaxel is mediated by sCLU and it can be induced by AKT. Lastly, AKT was found to mediate sCLU induction via signal transducer and activator of transcription 1 activation, which we have earlier shown to drive sCLU gene expression. These results identify a previously unrecognized pathway linking AKT to cytoprotection by sCLU in tumor cells.

A small molecule disruptor of Rb/Raf-1 interaction inhibits cell proliferation, angiogenesis, and growth of human tumor xenografts in nude mice.

Although it is well established that cyclin-dependent kinases phosphorylate and inactivate Rb, the Raf-1 kinase physically interacts with Rb and initiates the phosphorylation cascade early in the cell cycle. We have identified an orally active small molecule, Rb/Raf-1 disruptor 251 (RRD-251), that potently and selectively disrupts the Rb/Raf-1 but not Rb/E2F, Rb/prohibitin, Rb/cyclin E, and Rb/HDAC binding. The selective inhibition of Rb/Raf-1 binding suppressed the ability of Rb to recruit Raf-1 to proliferative promoters and inhibited E2F1-dependent transcriptional activity. RRD-251 inhibited anchorage-dependent and anchorage-independent growth of human cancer cells and knockdown of Rb with short hairpin RNA or forced expression of E2F1 rescued cells from RRD-251-mediated growth arrest. P.o. treatment of mice resulted in significant tumor growth suppression only in tumors with functional Rb, and this was accompanied by inhibition of angiogenesis, inhibition of proliferation, decreased phosphorylated Rb levels, and inhibition of Rb/Raf-1 but not Rb/E2F1 binding in vivo. Thus, selective targeting of Rb/Raf-1 interaction seems to be a promising approach for developing novel chemotherapeutic agents.

New Lewis-basic N-oxides as chiral organocatalysts in asymmetric allylation of aldehydes.

Allylation of aromatic and heteroaromatic aldehydes 1a-k with allyltrichlorosilane 2 can be catalyzed by the new heterobidentate, terpene-derived bipyridine N-monoxides 4, 6a,b, and 8-11 (

Synthesis of new chiral 2,2'-bipyridine ligands and their application in copper-catalyzed asymmetric allylic oxidation and cyclopropanation.

A series of modular bipyridine-type ligands 1 and 3-9 has been synthesized via a de novo construction of the pyridine nucleus. The chiral moieties of these ligands originate from the isoprenoid chiral pool, namely, beta-pinene (10 --> 1), 3-carene (14 --> 3 and 5), 2-carene (28 --> 4), alpha-pinene (43 --> 6-8), and dehydropregnenolone acetate (48 --> 9), respectively. Copper(I) complexes, derived from these ligands and (TfO)(2)Cu (1 mol %) upon an in situ reduction with phenylhydrazine, exhibit good enantioselectivity (up to 82% ee) and unusually high reaction rate (typicaly 30 min at room temperature) in allylic oxidation of cyclic olefins (52 --> 53). Copper-catalyzed cyclopropanation proceeded with < or =76% enantioselectivity and approximately 3:1 to 99:1 trans/cis-diastereoselectivity (54 --> 55 + 56). The level of the asymmetric induction is discussed in terms of the ligand architecture that controls the stereochemical environment of the coordinated metal.

Chiral 2,2'-bipyridine-type N-monoxides as organocatalysts in the enantioselective allylation of aldehydes with allyltrichlorosilane.

[reaction: see text] The Sakurai-Hosomi-type allylation of aromatic and heteroaromatic aldehydes can be catalyzed by the new heterobidenate bipyridine monoxide PINDOX with high enantioselectivities. The sterochemical outcome is mainly controlled by the axial chirality in PINDOX, which in turn is determined by the annulated terpene units.