Mislocalization and inhibition of acetyl-CoA carboxylase 1 by a synthetic small molecule.

Chromeceptin is a synthetic small molecule that inhibits insulin-induced adipogenesis of 3T3-L1 cells and impairs the function of IGF2 (insulin-like growth factor 2). The molecular target of this benzochromene derivative is MFP-2 (multifunctional protein 2). The interaction between chromeceptin and MFP-2 activates STAT6 (signal transducer and activator of transcription 6), which subsequently induces IGF inhibitory genes. It was not previously known how the binding of chromeceptin with MFP-2 blocks adipogenesis and activates STAT6. The results of the present study show that the chromeceptin-MFP-2 complex binds to and inhibits ACC1 (acetyl-CoA carboxylase 1), an enzyme important for the de novo synthesis of malonyl-CoA and fatty acids. The formation of this ternary complex removes ACC1 from the cytosol and sequesters it in peroxisomes under the guidance of Pex5p (peroxisomal-targeting signal type 1 receptor). As a result, chromeceptin impairs fatty acid synthesis from acetate where ACC1 is a rate-limiting enzyme. Overexpression of malonyl-CoA decarboxylase or siRNA (small interfering RNA) knockdown of ACC1 results in STAT6 activation, suggesting a role for malonyl-CoA in STAT6 signalling. The molecular mechanism of chromeceptin may provide a new pharmacological approach to selective inhibition of ACC1 for biological studies and pharmaceutical development.

C1B domain peptide of protein kinase Cγ significantly suppresses growth of human colon cancer cells in vitro and in an in vivo mouse xenograft model through induction of cell cycle arrest and apoptosis.

Two peptides derived from the C1B domain of protein kinase Cγ (PKCγ) were shown to associate with classical PKC isozymes and modulate their activities. These C1B peptides are designated C1B1 (amino acid residues 101-112) and C1B5 (residues 141-151). Since PKC enzyme activity is shown to be involved in colon cancer development, the effect of C1B peptides on the growth of various human colon cancer cell lines was examined in vitro and in vivo. Sub-micromolar to micromolar levels of both C1B peptides induced approximately 60-70% growth attenuation in multiple colon cancer cell lines in a soft agar tumor colony assay; however, C1B5 peptide was not cytotoxic to normal colon epithelial cells in two dimensional culture. The effect of C1B5 peptide on colony growth of COLO205 cells was reversed by treatment with the PKCα/ß inhibitor, Ro-32-0432. C1B peptide treatment attenuated COLO205 cells via two mechanisms: 1) cell cycle arrest and 2) stimulation of apoptosis. This is evident in G 2 arrest and increases in levels of cleaved caspase 3 and p53 phosphorylated at serine 20. Intratumoral injection of C1B5 peptide (20 mg/kg/day, every three days) markedly attenuated the growth of subcutaneous xenografts of COLO205 cells in SCID mice by 76% compared with the control. Taken together, these results strongly suggest that C1B peptides have negligible effects on normal tissues but are potentially effective chemotherapeutic agents for colon cancer.

Topoisomerase I inhibitor SN-38 effectively attenuates growth of human non-small cell lung cancer cell lines in vitro and in vivo.

The effect of SN-38 was evaluated on multiple lung cancer cell lines. It inhibits anchorage-dependent and -independent growth as monitored by MTT and soft agar colony assay, respectively. SN-38 collapsed the mitochondrial membrane potential (MMP), arrested cells in S- and G2-phases of the cell cycle, and induced apoptosis via activation of caspase 3 and PARP. A single injection of 2 mg/kg body weight of SN-38 caused a significant reduction of lung cancer xenografts. These findings indicate that SN-38 induces apoptosis in the lung cancer cells effectively. Thus, SN-38 can potentially be an effective therapeutic agent against lung cancer.

Angiotensin II type 2 receptor signaling significantly attenuates growth of murine pancreatic carcinoma grafts in syngeneic mice.

BACKGROUND: Pancreatic cancer is one of the most aggressive human malignancies, with a very poor prognosis. To evaluate the effect of angiotensin II (Ang II) type 2 receptor (AT2) expression in the host's body on the growth of pancreatic carcinoma, we have investigated the growth of mouse pancreatic ductal carcinoma grafts in syngeneic wild type and AT2 receptor-deficient (AT2-KO) mice. METHODS: The role of AT2 receptor-signaling in stromal cells on the growth of murine pancreatic carcinoma cells (PAN02) was studied using various in vitro and in vivo assays. In vivo cell proliferation, apoptosis, and vasculature in tumors were monitored by Ki-67 immunostaining, TUNEL assay, and von Willebrand factor immunostaining, respectively. In the co-culture study, cell proliferation was measured by MTT cell viability assay. All the data were analyzed using t-test and data were treated as significant when p < 0.05. RESULTS: Our results show that the growth of subcutaneously transplanted syngeneic xenografts of PAN02 cells, mouse pancreatic ductal carcinoma cells derived from the C57/BL6 strain, was significantly faster in AT2-KO mice compared to control wild type mice. Immunohistochemical analysis of tumor tissue revealed significantly more Ki-67 positive cells in xenografts grown in AT2-KO mice than in wild type mice. The index of apoptosis is slightly higher in wild type mice than in AT2-KO mice as evaluated by TUNEL assay. Tumor vasculature number was significantly higher in AT2-KO mice than in wild type mice. In vitro co-culture studies revealed that the growth of PAN02 cells was significantly decreased when grown with AT2 receptor gene transfected wild type and AT2-KO mouse-derived fibroblasts. Faster tumor growth in AT2-KO mice may be associated with higher VEGF production in stromal cells. CONCLUSIONS: These results suggest that Ang II regulates the growth of pancreatic carcinoma cells through modulating functions of host stromal cells; Moreover, Ang II AT2 receptor signaling is a negative regulator in the growth of pancreatic carcinoma cells. These findings indicate that the AT2 receptor in stromal fibroblasts is a potentially important target for chemotherapy for pancreatic cancer.

Over-expression of angiotensin II type 2 receptor gene induces cell death in lung adenocarcinoma cells.

The endogenous angiotensin II (Ang II) type 2 receptor (AT 2) has been shown to mediate apoptosis in cardiovascular tissues. Thus, the aim of this study was to explore the anti-cancer effect of AT 2 over-expression on lung adenocarcinoma cells in vitro using adenoviral (Ad), FuGENE, and nanoparticle vectors. All three gene transfection methods efficiently transfected AT 2 cDNA into lung cancer cells but caused minimal gene transfection in normal lung epithelial cells. Ad-AT 2 significantly attenuated multiple human lung cancer cell growth (A549 and H358) as compared to the control viral vector, Ad-LacZ, when cell viability was examined by direct cell count. Examination of annexin V by flow cytometry revealed the activation of the apoptotic pathway via AT 2 over-expression. Western Blot analysis confirmed the activation of caspase-3. Similarly, poly (lactide-co-glycolic acid) (PLGA) biodegradable nanoparticles encapsulated AT 2 plasmid DNA were shown to be effectively taken up into the lung cancer cell. Nanoparticle-based AT 2 gene transfection markedly increased AT 2 expression and resultant cell death in A549 cells. These results indicate that AT 2 over-expression effectively attenuates growth of lung adenocarcinoma cells through intrinsic apoptosis. Our results also suggest that PLGA nanoparticles can be used as an efficient gene delivery vector for lung adenocarcinoma targeted therapy.

Naïve human umbilical cord matrix derived stem cells significantly attenuate growth of human breast cancer cells in vitro and in vivo.

The effect of un-engineered (naïve) human umbilical cord matrix stem cells (hUCMSC) on the metastatic growth of MDA 231 xenografts in SCID mouse lung was examined. Three weekly IV injections of 5x10(5) hUCMSC significantly attenuated MDA 231 tumor growth as compared to the saline-injected control. IV injected hUCMSC were detected only within tumors or in close proximity to the tumors. This in vivo result was corroborated by multiple in vitro studies such as colony assay in soft agar and [(3)H]-thymidine uptake. These results suggest that naïve hUCMSC may be a useful tool for cancer cytotherapy.

Rat umbilical cord stem cells completely abolish rat mammary carcinomas with no evidence of metastasis or recurrence 100 days post-tumor cell inoculation.

Genetically engineered stem cells efficiently deliver therapeutic proteins to cancer and other sites of inflammation. However, a major advantage would be realized if tumor-trafficking stem cells that have not been genetically modified exhibit an inherent antitumor effect, thus circumventing the necessity of the expression of exogenous genes by the cells. We transplanted Fisher 344 rat-derived mammary adenocarcinoma cells (Mat B III) orthotopically into syngeneic F344 rats with an intact immune system. Rat umbilical cord matrix stem (rUCMS) cells derived from Wharton's jelly were then administered intratumoral (i.t) or i.v. 4 days later. The tumor attenuation effect was significantly evident starting from day 14 in i.v. and i.t. rUCMS cell-transplanted rats compared with sham-transplanted rats. In addition, unmodified rUCMS cell-transplanted rats showed complete regression of tumors to undetectable levels by 34 to 38 days with no evidence of metastasis or recurrence 100 days post-tumor cell inoculation. Dye-loaded rUCMS cells were identified within tumors only 4 days after their i.v. transplantation. In vitro colony assays with rUCMS cells as feeder layers markedly reduced Mat B III colony size and number. Growth attenuation of Mat B III cells exposed to either rUCMS cells directly or to the conditioned medium derived from rUCMS cells was associated with apoptosis indicators, including increased activated caspase-3. In addition, rUCMS cells cocultured with Mat B III cells had a dose-dependent antiproliferative effect on Mat B III cells. These findings suggest that unmodified human UCMS cells could be used for targeted cytotherapy for breast cancer.

The synergistic induction of cyclooxygenase-2 in lung fibroblasts by angiotensin II and pro-inflammatory cytokines.

Although we have demonstrated that Angiotensin II (Ang II) signaling plays a role in colon and lung tumorigenesis, the precise mechanisms by which Ang II stimulates tumorigenesis remain unclear. The aim of this study was to investigate the synergistic induction of COX-2 by Ang II and pro-inflammatory cytokines in lung fibroblasts. We also compared the efficiencies of Ang II-dependent COX-2 induction in lung epithelial cells and stromal cells. Ang II induced COX-2 expression in lung fibroblasts in a dose-dependent manner (10(-9) to 10(-7) M) through the Ang II subtype 1 receptor (AT(1)). In addition, Ang II synergistically stimulated the induction of COX-2 by pro-inflammatory cytokines, IL-1beta, or TNF-alpha. Our results indicate that the pro-tumorigenic function of Ang II is attributable, in part, to its strong stimulatory effect of COX-2 expression in lung fibroblasts in which synergistic stimulation with pro-inflammatory cytokines was evident. It is also suggested that the AT(1) receptor in lung fibroblasts may be a rational target for chemoprevention of lung cancer.

Combination treatment of human umbilical cord matrix stem cell-based interferon-beta gene therapy and 5-fluorouracil significantly reduces growth of metastatic human breast cancer in SCID mouse lungs.

Umbilical cord matrix stem (UCMS) cells that were engineered to express interferon-beta (IFN-beta) were transplanted weekly for three weeks into MDA 231 breast cancer xenografts bearing SCID mice in combination with 5-fluorouracil (5-FU). The UCMS cells were found within lung tumors but not in other tissues. Although both treatments significantly reduced MDA 231 tumor area in the SCID mouse lungs, the combined treatment resulted in a greater reduction in tumor area than by either treatment used alone. These results indicate that a combination treatment of UCMS-IFN-beta cells and 5-FU is a potentially effective therapeutic procedure for breast cancer.

Proteomic analysis of hypoxia-induced tube breakdown of an in vitro capillary model composed of HUVECs: potential role of p38-regulated reduction of HSP27.

We recently reported that hypoxia could induce the breakdown of capillary-like tubes formed by human umbilical vein endothelial cells (HUVECs) and that this breakdown was regulated by p38 and not by a caspase cascade, although the exact molecular mechanisms remain unknown. The aim of this study was to identify proteins that regulated hypoxia-induced tube breakdown through p38-regulated and caspase-independent mechanisms. The involvement of adhesion proteins, integrins, VE-cadherin, PECAM-1, and occludin was first investigated. Although some of these proteins decreased after hypoxia, none of them met the conditions of being quantitatively restored by p38 inhibition but not by caspase inhibition. We then conducted 2-D DIGE coupled with MALDI-TOF/TOF-MS to identify altered protein expression. The differential proteomic analysis of tube-forming HUVECs treated with normoxia or hypoxia and treated with hypoxia in the presence or absence of SB203580, a specific p38 inhibitor, revealed the involvement of heat shock proteins in this tube breakdown. We also confirmed that the amount of HSP27 and HSP70 changed in a p38-regulated and caspase-independent manner during hypoxia. Knocking down HSP27 expression using RNAi further augmented hypoxia-induced tube breakdown. Taken together, it was shown that p38-regulated and caspase-independent reduction of HSP27 plays an important role in hypoxia-induced tube breakdown.

Angiotensin II bi-directionally regulates cyclooxygenase-2 expression in intestinal epithelial cells.

We previously demonstrated that angiotensin II (Ang II) receptor signaling is involved in azoxymethane-induced mouse colon tumorigenesis. In order to clarify the role of Ang II in COX-2 expression in the intestinal epithelium, the receptor subtype-specific effect on COX-2 expression in a rat intestinal epithelial cell line (RIE-1) has been investigated. Ang II dose- and time-dependently increased the expression of COX-2, but not COX-1 mRNA and protein. This stimulation was completely blocked by the AT(1) receptor antagonist but not the AT(2) receptor antagonist. Ang II and lipopolysaccharide (LPS) additively induced COX-2 protein in RIE-1 cells, whereas the LPS-induced COX-2 expression was significantly attenuated by low concentrations of Ang II or the AT(2) agonistic peptide CGP-42112A only in AT(2) over-expressed cells. These data indicate that Ang II bi-directionally regulates COX-2 expression via both AT(1) and AT(2) receptors. Control of COX-2 expression through Ang II signaling may have significance in cytokine-induced COX-2 induction and colon tumorigenesis.

Hypoxia-induced apoptosis and tube breakdown are regulated by p38 MAPK but not by caspase cascade in an in vitro capillary model composed of human endothelial cells.

In order to improve medical treatment of ischemic injury such as myocardial infarction, it is important to elucidate hypoxia-induced changes to endothelial cells. An in vitro blood vessel model, in which HUVECs are stimulated to form a network of capillary-like tubes, was used to analyze hypoxia-induced morphological and biochemical changes. When exposed to hypoxia, the network of capillary tubes broke down into small clusters. This tube breakdown was accompanied by chromatin condensation and cell nuclear fragmentation, morphological markers of apoptosis, and activation of two apoptotic signals, caspase-3 and p38. We investigated what roles caspase cascade and p38 play in hypoxia-induced apoptosis and tube breakdown by using zVAD-fmk and SB203580, specific inhibitors of these two apoptotic signals, respectively. Chromatin condensation and cell nuclear fragmentation and tube breakdown were effectively inhibited by SB203580, but not by zVAD-fmk. SB203580 caused dephosphorylation of p38, which indicates that p38 was autophosphorylated. Inhibition by zVAD-fmk caused slight MW increase in p17 and emergence of p19, which indicates that the inhibitor caused partial processing of caspase-3. Inhibition of p38 suppressed activation of caspase-3 but not vice versa. In addition, these two inhibitors were shown to differentially inhibit cleavage of so-called caspase substrates. SB203580 inhibited cleavage of PARP and lamin A/C, while zVAD-fmk inhibited cleavage of lamin A/C but not that of PARP. Taken together, these results show that p38 is located upstream of caspase cascade and that, although caspase-3 is activated, a p38-regulated caspase-independent pathway is crucial for the execution of hypoxia-induced apoptosis and tube breakdown.