Terms Of Growth Inhibition Research Articles

Combined modality doxorubicin-based chemotherapy and chitosan-mediated p53 gene therapy using double-walled microspheres for treatment of human hepatocellular carcinoma

The therapeutic efficiency of combined chemotherapy and gene therapy on human hepatocellular carcinoma HepG2 cells was investigated using double-walled microspheres that consisted of a poly(d,l-lactic-co-glycolic acid) (PLGA) core surrounded by a poly(l-lactic acid) (PLLA) shell layer and fabricated via the precision particle fabrication (PPF) technique. Here, double-walled microspheres were used to deliver doxorubicin (Dox) and/or chitosan-DNA nanoparticles containing the gene encoding the p53 tumor suppressor protein (chi-p53), loaded in the core and shell phases, respectively. Preliminary studies on chi-DNA nanoparticles were performed to optimize gene transfer to HepG2 cells. The transfection efficiency of chi-DNA nanoparticles was optimal at an N/P ratio of 7. In comparison to the 25-kDa branched polyethylenimine (PEI), chitosan showed no inherent toxicity towards the cells. Next, the therapeutic efficiencies of Dox and/or chi-p53 in microsphere formulations were compared to free drug(s) and evaluated in terms of growth inhibition, and cellular expression of tumor suppressor p53 and apoptotic caspase 3 proteins. Overall, the combined Dox and chi-p53 treatment exhibited enhanced cytotoxicity as compared to either Dox or chi-p53 treatments alone. Moreover, the antiproliferative effect was more substantial when cells were treated with microspheres than those treated with free drugs. High p53 expression was maintained during a five-day period, and was largely due to the controlled and sustained release of the microspheres. Moreover, increased activation of caspase 3 was observed, and was likely to have been facilitated by high levels of p53 expression. Overall, double-walled microspheres present a promising dual anticancer delivery system for combined chemotherapy and gene therapy.

4-Methyl-7-hydroxycoumarin antifungal and antioxidant activity enhancement by substitution with thiosemicarbazide and thiazolidinone moieties

According to literature data, thiosemicarbazide and thiazolidinone moieties should enhance biological properties of coumarin. Antioxidant, metal-chelating and antifungal activities of all compounds were investigated and compared to the activity of the starting material, 7-hydroxy-4-methylcoumarin, and were proven to possess potent antioxidant and antifungal activity. In general, thiosemicarbazides showed higher scavenging activity towards DPPH and galvinoxyl radicals than did 4-thiazolidinones and some of them had the same or even better activity than had ascorbic acid itself, depending on the free radical used. In antifungal activity tests towards four foodborne mycotoxigenic fungi, Aspergillus flavus, Aspergillus ochraceus. Fusarium graminearum and Fusarium verticillioides, coumarin derivatives were proven to possess a very high activity in terms of growth inhibition, depending on the fungi investigated. In general, 4-thiazolidinones showed better antifungal activity than did thiosemicarbazides. F. graminearum was the most susceptible to the compounds investigated and F. verticillioides was proven to be the most resistant. Two compounds, both coumarinyl thiosemicarbazides, were found to possess both antifungal and antioxidant activity which could be useful for applications in medicine, food industry and agriculture.

A-ring oxygenation modulates the chemistry and bioactivity of caged Garcinia xanthones

Natural products of the caged Garcinia xanthones (CGX) family are characterized by a unique chemical structure, potent bioactivities and promising pharmacological profiles. We have developed a Claisen/Diels-Alder reaction cascade that, in combination with a Pd(0)-catalyzed reverse prenylation, provides rapid and efficient access to the CGX pharmacophore, represented by the structure of cluvenone. To further explore this pharmacophore, we have synthesized various A-ring oxygenated analogues of cluvenone and have evaluated their bioactivities in terms of growth inhibition, mitochondrial fragmentation, induction of mitochondrial-dependent cell death and Hsp90 client inhibition. We found that installation of an oxygen functionality at various positions of the A-ring influences significantly both the site-selectivity of the Claisen/Diels-Alder reaction and the bioactivity of these compounds, due to remote electronic effects.

Abstract 496: Isolation and characterization of a highly invasive subpopulation from MDA-MB-231 breast cancer cells

Abstract The acquisition of an invasive phenotype is a pre-requisite for metastasis. We set out to develop cellular systems that can mirror transient and stbale molecular alterations that confer an invasive phenotype to breast cancer cells. We observed that it is possible to isolate invasive subpopulations from moderately invasive cancer cell lines. Enrichment of invasive sub-populations of MDA-MB-231 breast cancer cells in three successive preparative invasion assays in Matrigel covered Boyden chambers yielded a highly invasive cell line. Prolonged cultivation of these cells did not abolish the invasive phenotype although not all phenotypic changes acquired during selection are maintained by long term cultures. Genetic analyses of these cells by cytogenetics and array based comparative genome hybridization revealed many genetic alterations including increased ploidy. The flow cytometric DNA Index (DI) changes from 1.28 to 2.28. Cells with DI 2.28 constitute 1.4% of the parental cell line. Whole genome SNP analysis shows that the two populations are genetically related excluding any cell contamination. The invasive cells proliferate and undergo apoptosis similar to the parental cells. Commitment to apoptosis is increased since invasive cells respond more strongly to curcumin or peroxide induced apoptosis. Invasive cells show relative resistance to the cytotoxic, alkylating agent Doxorubicin and increased sensitivity to the anti-mitotic drugs Vincristine and Taxol. Increased resistance to the topoisomerase II inhibitor Mitoxantrone is observed only transiently in invasive cells and lost in long term cultures. Similarly, the chemokines CXCL1 and -2 are transiently upregulated. Response to the anti-diabetic drug Metformin showed a reduced sensibility for invasive cells in terms of growth inhibition (IC50). Gene expression profiling shows complex alterations in gene expression. Many of the genes that are differentially expressed in highly versus moderately invasive cells are differentially expressed in human breast cancer cases with and without distant metastasis and correlate with disease free survival. The invasive phenotype is not related to stem cell features nor to epithelial mesenchymal transition. These cells constitute a novel model for tumor progression. Tumorigenicity and metastatic potential in vivo are currently being tested. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 496. doi:1538-7445.AM2012-496

The relation between the uptake of cetyltrimethylammonium bromide by Escherichia coli and its effects on cell growth and viability.

Abstract The effects of CTAB uptake on cells of E. coli have been examined in terms of growth inhibition and decrease in cell viability. Different thresholds of CTAB uptake exist below which no effect on cell growth or viability could be detected and there was a distinct separation of the two effects. It is suggested that both reflect all or none responses by individual cells. A mechanism for the mode of antibacterial action of CTAB against E. coli is postulated.

Definition of Ubiquitination Modulator COP1 as a Novel Therapeutic Target in Human Hepatocellular Carcinoma

The development of targeted therapeutics for hepatocellular carcinoma (HCC) remains a major challenge. The ubiquitination modulator COP1 regulates p53 activity by ubiquitination and it is frequently overexpressed in human HCC. In this study, we tested the hypothesis that COP1 blockade by short interfering RNA (siRNA)-mediated inhibition could affect the course of HCC progression. The COP1 isoform COP1-1 was selected as the most effective target for siRNAs in terms of growth inhibition and apoptotic induction in several HCC cell lines. Growth inhibition occurred in HCC cells that retained wild-type p53 or expressed mutant p53 (Y220C or R249S), whereas p53-null Hep3B cells were resistant. Microarray expression analysis revealed that the antiproliferative effects of COP1 blockade were driven by a common subset of molecular alterations including a p53-associated functional network. In an orthotopic mouse xenograft model of HCC, systemic delivery of a modified COP1 siRNA by stable nucleic acid-lipid particles suppressed neoplastic growth in liver without unwanted immune responses. Our findings offer a first proof of principle that COP1 can be a promising target for systemic therapy of HCC.

Weekly Administration of Docetaxel and Epirubicin as First-Line Treatment for Hormone-Refractory Prostate Carcinoma

Androgen-independent prostate carcinoma (AICP) is one of the tumors that continue to respond poorly to chemotherapy. Recently, protocols based on the use of docetaxel have significantly improved survival for patients in this disease. In other types of neoplastic disease, combined therapy with taxanes and anthracycline derivatives has been shown to produce additive effects in terms of growth inhibition, and superior tolerability when associated with weekly administration schedules. These findings prompted us to examine the tolerability and efficacy of weekly treatment of AICP with docetaxel (DOX) plus epirubicin (EPI). We enrolled 35 chemotherapy-naive men with AICP (mean age 72 years, range 68-77) and normal hepatic, renal, and cardiac function. The chemotherapy protocol provided for the IV administration of DOX (30 mg/m2) and EPI (30 mg/m2) on days 1, 8, and 15 every 28 days. Treatment was continued for 6 months or until disease progression and/or unacceptable toxicity was observed. Serum levels of prostate-specific antigen (PSA) were monitored in all patients, and reductions from baseline values of >50% were considered indicative of positive responses to treatment. Thirty-four patients were included in the analysis of toxicity, and objective responses to treatment were assessed in the 28 patients with measurable lesions. Nineteen patients (56%) experienced PSA reductions of >50% that persisted for more than 4 weeks. The response to therapy was classified as complete in 1 of the 28 patients (4%) with measurable disease (at the lymph node level). Thirteen others (13/28, 46%) had partial responses, in nine (32%) the disease remained unchanged, and progression was observed in the remaining five (18%); overall response rate was 50% (CR + PR). Of the 27 patients with pain at the time of enrollment, 16 (59%) experienced pain reduction during treatment. The median time to disease progression was 11.7 months (95% CI: 7.7-15.7) while the median survival time was 18.7 months (95% CI: 12.3-25.1). During the study, four patients developed grade 3 anemia and leukopenia, which was reversible in all cases. Lower grades of asthenia, nausea, vomiting, diarrhea, and peripheral edema were also observed. There were no cases of cardiotoxic effects. Alopecia was frequent but reversible in all cases. The results of this preliminary study indicate that the combined administration of DOX and EPI for treatment of AIPC is effective and well tolerated. The weekly administration of the drug combination appears to be a promising approach to the treatment of these tumors.

Relationship Between Lipophilicity and Inhibitory Activity Against Cancer Cell Growth of Nine Kinds of Alk(en)yl Trisulfides With Different Side Chains

Of the compounds contained in or derived from garlic (Allium sativum L.), alk(en)yl sulfides are known to be responsible for most of the physiological or neutraceutical functions of garlic. We previously found that diallyl trisulfide (DATS) is a potent inhibitor of tubulin polymerization and cancer cell growth, and an effective stimulator of the hepatic detoxification system. Here, we synthesized nine trisulfides having different aliphatic side chains, and determined their log P, a parameter for lipophilicity of nonionized solutes, and inhibitory activities, IC50 (microM), toward cancer cell growth. The log P values of these trisulfides ranged from the lowest, 2.72, for dimethyl trisulfide, to the highest, 7.62, for dipentyl trisulfide. The relationship between the IC50 and log P of the nine trisulfides was parabolic in nature, in which dibutenyl- and dipropyl-compounds, determined to have a log P of approximately 5, were located at the minimum point of the parabola, indicating the maximum potency. The reason why DATS, having a log P of about 4, was excessively stronger than diethyl trisulfide, with a similar log P, is not fully understood; but perhaps it can be explained by a higher reactivity of the diallyl compound in nucleophilic substitution. The compounds with 3-carbon chains were stronger in terms of growth inhibition than the others; but weaker compounds, those with 4- or 5-carbon chains, showed higher activity if a double bond was introduced into them to reduce their log P to the effective range. In this study, we confirmed the superiority of trisulfides with 3-carbon chains [i.e., DATS and dipropyl trisulfide (DPTS)]. In addition, we observed for the first time that dibutenyl trisulfide, a compound not found in garlic, is one of the potent structures among alk(en)yl trisulfides.

NSC606985 induces apoptosis, exerts synergistic effects with cisplatin, and inhibits hypoxia-stabilized HIF-1α protein in human ovarian cancer cells

The camptothecins, which target the intranuclear enzyme topoisomerase I, have advanced to the forefront of several areas of developmental chemotherapy of cancers. In the present study, we investigated the potential anti-human ovarian cancer effects of NSC606985, a novel and rarely studied camptothecin analog, and its combination with cisplatin (CDDP). Human ovarian cancer cell line COC1 cells were treated with different nanomolar of NSC606985 with or without CDDP, and cell growth and apoptosis were evaluated, respectively, by MTT assay and annexin-V assay on flow cytometry. Chou–Talalay analysis was used to evaluate combined effect of NSC606985 and CDDP. Western blot was used to detect protein kinase Cδ (PKCδ), caspase-3 and hypoxia-inducible factor-1α (HIF-1α) proteins. Our results showed that NSC606985 at nanomolar concentration induced apoptosis with the activation of PKCδ in COC1 cells. Especially, NSC606985 presented the significant combined effects on COC1 cells in terms of growth inhibition and apoptosis induction. In addition, NSC606985 significantly antagonized the accumulation of HIF-1α stabilized by hypoxia or hypoxia-mimetic agent. These results suggest that NSC606985 and its combination with CDDP present the therapeutic potential on ovarian cancer, and deserve further preclinical and clinical studies.

The Combination of a Histone Deacetylase (HDAC) Inhibitor with the BCL-2 Inhibitor GX15-070 Has Synergistic Antileukemia Effect by Inducing Both Apoptotic and Autophagic Pathways.

HDAC inhibitors have limited single agent clinical activity in leukemia, a probable effect of dysregulated proapoptotic pathways. We hypothesized that the combination of a HDAC inhibitor with a Bcl-2 inhibitor may result in synergistic antileukemia activity. To test this concept, we modeled in vitro the combination of MGCD0103, a class 1 specific HDAC inhibitor, with GX15-070 (Obatoclax), a novel Bcl-2 homology domain-3 (BH3) mimetic The cytotoxic potential of the combination was first assessed measuring cell viability using trypan blue extraction assays in leukemia cell lines HL60, U937, THP1, and Molt4. Mathematical modeling analysis indicated a strong synergistic effect of the combination of MGCD0103 and GX15-070 in terms of growth inhibition. Inhibitory effect of the combination at concentrations of MGCD0103 600 nM and GX15-070 200 nM was 64 fold the effect of single agent MGCD0103 and 16 fold that of GX15-070. Analysis of sequence of treatment indicated that the optimal sequence was addition of GX15-070 first followed by MGCD0103. We then analyzed the effect of the combination on the induction of apoptosis using Annexin V flow assays. The combination also showed a marked synergistic effect in terms of apoptosis induction (the combination was 217% more potent than single agent MGCD0103 and 79% more than GX15-070). Western blot analysis indicated that the use of the combination substantially enhanced the cleavage/activation of caspase-3 and the subsequent cleavage of caspase-3 substrate PARP. As demonstrated in AML and other cell types previously, we showed that GX15-070 dissociated the pro-apoptotic protein BAK from MCL-1. The release of BAK has been demonstrated to play an important role in caspase activation. Western blot analysis also demonstrated that while anti-apoptotic protein XIAP, the major inhibitor of caspase 3, was reduced in MGCD0103 treated cells, the reduction was enhanced with the combination. These results suggest that GX15-070 may sensitize XIAP to the down-regulation effect of MGCD0103, which in turn further activates caspase 3. The combinatory effect of BAK release by GX-15-070, with the suppression of XIAP by MGCD0103, that is potentiated by GX15-070, may play a critical role in the synergistic effect of these two agents on caspase activation, which then results in increased induction of apoptosis. Because in initial experiments we evidenced a discrepancy between cell viability and apoptosis, we also studied the effect of the combination on the induction of autophagy using electron microscopy: non-apoptotic cells after MGCD0103 treatment did not show increase autophagic characteristics compared to control cells, while more non-apoptotic cells under GX15-070 evidenced swollen endoreticulum, a feature of early autophagy. Most non-apoptotic cells treated with MGCD0103 and GX15-070 combination showed accumulation of macro-autophagosomes, a marker of late autophagy, indicating that more severe autophagy was induced by the combination. Furthermore, induction of LC3-II, a marker of autophagy, was observed after combination. We confirmed these results (both in terms of apoptosis and autophagy) ex vivo in primary cells (n=8) obtained from patients with relapsed/refractory AML. In conclusion, the combination of an HDAC inhibitor with GX15-070 has synergistic antileukemia activity and should be studied in human clinical trials.

In Vitro Characterization of Wnt/β-Catenin Pathway Inhibition in Multiple Myeloma Cell Lines Using a Novel Class of Small Molecules.

Multiple Myeloma (MM) is a fatal form of hematologic cancer characterized by the clonal expansion of plasma cells in the bone marrow. Recent finding by several research laboratories have shown that Wnt/β-Catenin signaling pathway is activated in MM leading to the translocation of β-Catenin to the nucleus where it binds to T cell factor (TCF) and drives transcription of genes involved in the progression of cancer (Derksen et. Al., PNAS 2004). The mechanism by which Wnt/β-Catenin signaling is activated in MM is not well understood, however, β-Catenin is expressed in the majority of MM cell lines and inhibition of the pathway with a dominant negative form of TCF4 or the small molecule PKF115-584 have shown anti-proliferative effects in in vitro and in vivo cell line and xenograft models making inhibition of Wnt/β-Catenin signaling a promising modality for the treatment of MM (Sukhdeo et al., PNAS 2007). To this end, Avalon Pharmaceuticals has developed a series of small molecule compounds, lead candidate series-363 (LC-363), with potent inhibitory effects on Wnt/β-Catenin signaling and anti-proliferative effects in colon cancer cell lines and in vivo models (EORTC, 2007). LC-363 compounds down regulate expression of the noted β-Catenin/TCF transcriptional targets c-jun, fra-1, and PPARδ as well as increase expression of the Dickkopf family of secreted proteins 1 and 3 which inhibit Wnt signaling. In this study, we report on the in vitro characteristics of LC-363 in MM cell line models. We show that LC-363 compounds have broad and potent growth inhibitory and cytotoxic effects on MM cell lines. Additionally, these effects are mediated through a G1 phase cell cycle block, apoptosis, and decrease in cytoplasmic levels of β-Catenin. Importantly, we show the relationship between expression level of β-Catenin protein and sensitivity to LC-363 compounds in terms of growth inhibition and apoptosis. Lastly, we describe the gene expression signatures induced by LC-363 in MM cell models across multiple time and dose studies to detail their dynamic effects on expression of genes within the Wnt/β-Catenin signaling pathway. In conclusion, we believe that inhibition of Wnt/β-Catenin signaling is a viable therapeutic alternative for treatment of MM patients with activated Wnt/β-Catenin signaling and to that end; LC-363 compounds are promising candidates currently in pre-clinical development for treatment of cancers with activated Wnt/β-Catenin signaling including MM.

Antileukemia activity of the combination of 5-aza-2′-deoxycytidine with valproic acid

Aberrant DNA methylation of promoter associated CpG islands is a common phenomenon in human leukemias and cooperates with histone code changes in the control of gene expression. 5-Aza-2′-deoxycytidine (DAC) is a hypomethylating agent with significant antileukemia activity in humans. Recently, valproic acid (VPA) has been shown to be a histone deacetylase inhibitor and to have potential antineoplastic activity. In this report, we study the in vitro effects of the combination of DAC and VPA on the leukemic cell lines HL-60 and MOLT4. DAC alone induced growth inhibition and apoptosis at doses of 1 μM, an effect observed with VPA at doses of 1 mM. Each drug alone had the capacity to induce the expression of p57KIP2 and p21CIP1. DAC mediated hypomethylation of p57KIP2 was not required to induce p57KIP2 gene expression, and treatment with DAC resulted in the induction of p21CIP1. VPA induced global histone acetylation, an effect enhanced by the addition of DAC. The combination of DAC and VPA had a synergistic effect in terms of growth inhibition, induction of apoptosis and reactivation of p57KIP2 and p21CIP1. These results suggest that the combination of DAC and VPA could have significant antileukemia activity in vivo.

Mutagenicity and DNA repair of glycidamide-induced adducts in mammalian cells

Glycidamide (GA)-induced mutagenesis in mammalian cells is not very well understood. Here, we investigated mutagenicity and DNA repair of GA-induced adducts utilizing Chinese hamster cell lines deficient in base excision repair (BER), nucleotide excision repair (NER) or homologous recombination (HR) in comparison to parent wild-type cells. We used the DRAG assay in order to map pathways involved in the repair of GA-induced DNA lesions. This assay utilizes the principle that a DNA repair deficient cell line is expected to be affected in growth and/or survival more than a repair proficient cell. A significant induction of mutations by GA was detected in the hprt locus of wild-type cells but not in BER deficient cells. Cells deficient in HR or BER were three or five times, respectively, more sensitive to GA in terms of growth inhibition than were wild-type cells. The results obtained on the rate of incisions in BER and NER suggest that lesions induced by GA are repaired by short patch BER rather than long patch BER or NER. Furthermore, a large proportion of the GA-induced lesions gave rise to strand breaks that are repaired by a mechanism not involving PARP. It is suggested that these strand breaks, which might be the results from alkylation of the backbone phosphate, are misrepaired by HR during replication thereby leading to a clastogenic rather than a mutagenic pathway. The type of lesion responsible for the mutagenic effect of GA cannot be concluded from the results presented in this study.

Enhancement of therapeutic effect in combination treatment with imatinib and anticancer drug in gastric carcinoma

4163 Background: Imatinib (STI571, Gleevec) is a novel molecular targeting agent, which inhibits the activity of c-kit and PDGFR in gastrointestinal stromal tumors. We conducted preclinical evaluation of therapeutic effect of imatinib and potential synergism in combination with imatinib and anticancer drug in gastric carcinoma cells, in terms of growth inhibition by targeting PDGF/PDGFR signaling pathway. Materials and Methods: MKN-45 gastric carcinoma cells were employed, and the cells were transplanted into nude mice. When the tumor size was reached approximately 100 mm3 after transplantation, the mice were arbitrarily divided in four groups (n=8/group), according to no treatment, treatment with anticancer drug alone, treatment with imatinib alone, and combination treatment with imatinib and anticancer drug, respectively. Imatinib was administered intraperitoneally at 50 mg/kg for consecutive 28 days, whereas anticancer drug was administered at LD50/3 four times in qwk. Results: The treatment with imatinib of MKN-45 gastric carcinoma xenograft showed antitumor effect to a greater extent than the treatment with 5-fluorouracil (5-FU) or docetaxel (TXT) alone. Further, antitumor effect was significantly enhanced in the combination treatment with imatinib and 5-FU or TXT, compared to the treatment with imatinib, 5-FU or TXT alone (P<0.01, Student t-test). Enhanced antitumor effect in combination treatment with imatinib and anticancer drug was associated with both induction of apoptosis and inhibition of tumor angiogenesis, which was explained with the decreased expression in pAkt, and PDGF-betaR, and decreased phosphorylation of PDGF-betaR. Conclusions: Combination treatment with imatinib and anticancer drugs such as 5-FU and TXT may a new strategy for enhancing therapeutic effect in the treatment of gastric carcinoma, targeting inhibition of PDGFR-mediated paracrine tumor growth. No significant financial relationships to disclose.

Enhancement of therapeutic effect in combination treatment with imatinib and anticancer drug in gastric carcinoma

4163 Background: Imatinib (STI571, Gleevec) is a novel molecular targeting agent, which inhibits the activity of c-kit and PDGFR in gastrointestinal stromal tumors. We conducted preclinical evaluation of therapeutic effect of imatinib and potential synergism in combination with imatinib and anticancer drug in gastric carcinoma cells, in terms of growth inhibition by targeting PDGF/PDGFR signaling pathway. Materials and Methods: MKN-45 gastric carcinoma cells were employed, and the cells were transplanted into nude mice. When the tumor size was reached approximately 100 mm3 after transplantation, the mice were arbitrarily divided in four groups (n=8/group), according to no treatment, treatment with anticancer drug alone, treatment with imatinib alone, and combination treatment with imatinib and anticancer drug, respectively. Imatinib was administered intraperitoneally at 50 mg/kg for consecutive 28 days, whereas anticancer drug was administered at LD50/3 four times in qwk. Results: The treatment with imatinib of MKN-45 gastric carcinoma xenograft showed antitumor effect to a greater extent than the treatment with 5-fluorouracil (5-FU) or docetaxel (TXT) alone. Further, antitumor effect was significantly enhanced in the combination treatment with imatinib and 5-FU or TXT, compared to the treatment with imatinib, 5-FU or TXT alone (P<0.01, Student t-test). Enhanced antitumor effect in combination treatment with imatinib and anticancer drug was associated with both induction of apoptosis and inhibition of tumor angiogenesis, which was explained with the decreased expression in pAkt, and PDGF-betaR, and decreased phosphorylation of PDGF-betaR. Conclusions: Combination treatment with imatinib and anticancer drugs such as 5-FU and TXT may a new strategy for enhancing therapeutic effect in the treatment of gastric carcinoma, targeting inhibition of PDGFR-mediated paracrine tumor growth. No significant financial relationships to disclose.

Resistance to TGF-beta-induced apoptosis in regenerating hepatocytes.

Treatment with transforming growth factor beta (TGF-beta) of hepatocytes from two different proliferative conditions, such as fetal development and adult liver regeneration, shows that regenerating cells respond to this cytokine in terms of growth inhibition, but are less sensitive than the fetal ones to the apoptosis induced by this factor. Regenerating TGF-beta treated cells show higher cell viability and lower percentage of apoptotic cells than the fetal treated ones. Furthermore, TGF-beta treated regenerating hepatocytes maintain a well-preserved parenchyma-like organization. Treatment with TGF-beta induces the loss of mitochondrial transmembrane potential in fetal but not in regenerating hepatocytes and activation of caspase-3 is lower in regenerating than in fetal cells. Regenerating hepatocytes show higher intracellular levels of some antiapoptotic proteins, such as Bcl-x(L) and c-IAP-1 and, interestingly, they present higher intracellular glutathione levels, which might provide of mechanisms to avoid potential dangerous effects of the oxidative stress-mediated apoptosis induced by TGF-beta. In fact, treatment with BSO (a glutathione synthesis inhibitor) restores the response of regenerating hepatocytes to TGF-beta in terms of cell death. In conclusion, increased levels of Bcl-x(L) and cIAP-1 and higher intracellular glutathione levels could confer resistance to the apoptosis induced by TGF-beta during liver regeneration.

Heart and liver defects and reduced transforming growth factor beta2 sensitivity in transforming growth factor beta type III receptor-deficient embryos.

The type III transforming growth factor beta (TGFbeta) receptor (TbetaRIII) binds both TGFbeta and inhibin with high affinity and modulates the association of these ligands with their signaling receptors. However, the significance of TbetaRIII signaling in vivo is not known. In this study, we have sought to determine the role of TbetaRIII during development. We identified the predominant expression sites of TbetaRIII mRNA as liver and heart during midgestation and have disrupted the murine TbetaRIII gene by homologous recombination. Beginning at embryonic day 13.5, mice with mutations in TbetaRIII developed lethal proliferative defects in heart and apoptosis in liver, indicating that TbetaRIII is required during murine somatic development. To assess the effects of the absence of TbetaRIII on the function of its ligands, primary fibroblasts were generated from TbetaRIII-null and wild-type embryos. Our results indicate that TbetaRIII deficiency differentially affects the activities of TGFbeta ligands. Notably, TbetaRIII-null cells exhibited significantly reduced sensitivity to TGFbeta2 in terms of growth inhibition, reporter gene activation, and Smad2 nuclear localization, effects not observed with other ligands. These data indicate that TbetaRIII is an important modulator of TGFbeta2 function in embryonic fibroblasts and that reduced sensitivity to TGFbeta2 may underlie aspects of the TbetaRIII mutant phenotype.

Tamarindus indica L. leaf is a source of allelopathic substance

The allelopathic potential of the Tamarindus indica L. leaf was investigated through bioassay guided studies using several weed and edible crop species. Both radicle and hypocotyl growth of all the plant species tested was strongly inhibited by the tamarind leaf using a sandwich method. The growth of weed species was reduced more than that of edible crop species. Among the weed species, barnyard grass followed by white clover, and in the edible crop species, lettuce followed by radish ranked top in terms of growth inhibition. Different concentrations of tamarind leaf crude water-soluble extract exhibited a strong inhibition in all the plant species tested and, by contrast, the magnitude of inhibition in the weed species was higher than in edible crop species and ranged from 30–75%. The 10% concentration of the tamarind leaf crude water-soluble extract was most potent against growth of seedlings. The concentrations of the nutrient components were linearly correlated with an increase in the concentration of tamarind leaf crude water-soluble extract. No significant changes in either pH or EC were found in the variations of different concentrations of tamarind leaf crude water-soluble extracts. As compared to control, growth of both radicle and hypocotyl in weed (barnyard grass and white clover) and in edible crop (lettuce and radish) species were significantly reduced when blended tamarind leaves at different concentrations were incorporated into the growth medium. The inhibitory magnitude increased with an increase in the concentration of the tamarind leaf. In terms of growth inhibition, among these tested plants, weed species particularly barnyard grass were most sensitive to the allelochemicals exuded from blended tamarind leaves. When the blended tamarind leaves were removed from the growth medium, all the seedlings grew quickly and the percentage of recovery was between 76–97% of the corresponding controls. Reduction in the fresh and dry weight of these 4 plant species was observed under the experimental conditions, and ranged between 33–42% and 40–53% in the radicle and hypocotyl, respectively. The fresh and dry weight, and total chlorophyll content declined significantly in the incorporated tamarind leaf treatments. Compared to the control, the highest drop in the chlorophyll content of 60% in barnyard grass was observed with the 10% concentration of the leaf treatment. These results clearly indicate that the tamarind leaf contains one or more strong biologically active allelochemical(s) that function as true growth regulator(s) and is involved in plant growth regulation, particularly in weed species.

Allelopathic competence of Tamarindus indica L. root involved in plant growth regulation

The allelopathic competence of tamarind root was evaluated using several weed and edible crop species under both laboratory and greenhouse conditions. Bio-assay guided studies using agar and soil medium revealed that the growth of both radicle and hypocotyl were strongly inhibited under both conditions. Accelerated root exudation observed with an increase in the age of tamarind seedlings caused a high magnitude of growth inhibition of the plant species tested by the plant-box method. Tamarind seedlings at 21-DAG (days after germination) exerted the strongest inhibitory effect (85.0–95.1%) on the growth of the plant species tested. Root dry weight of tamarind seedlings in the plant-box method experiment was highly correlated (R 2 values more than 0.92) with the percentage of growth inhibition. The growth of species grown in the soil under the tamarind tree was inhibited by 85.3–97.1% in the greenhouse. The percentage of growth inhibition declined by 18.4–22.0% (as compared to the natural soil condition) when autoclaved soil of the same trees was used for bio-assay of plant species by the soil-agar sandwich method. This indicates that ca. a 20% increase in response was associated with the allelopathic activity of tamarind root exuded into the natural soil and was due to the effects of soil microbes and soil texture. In terms of growth inhibition of the plant species tested, the root zone soil of the tamarind tree showed stronger inhibitory effects (80.1–94.2%) than the rhizosphere soil, as determined by the soil-agar sandwich method. In all cases, growth inhibition especially in the radicle was higher in the weed species than the edible crop species. Our observations clearly indicate that tamarind root exudate has allelochemical competence and this contributes to a weed free environment around the tamarind tree.

Activation of the Jak-Stat- and MAPK-pathways by oncostatin M is not sufficient to cause growth inhibition of human glioma cells

We have recently described that oncostatin M (OSM), a member of the IL-6 family of cytokines, induces the differentiation of human glioma cells in culture. In order to extend this studies, we analyzed the effect of OSM on other human glioma cell lines including A172, U343-MG and T98G. All of these cell lines express the receptor components of OSM and leukemia inhibitory factor (LIF) gp130, LIFR and the OSM specific OSMRβ. Therefore, we expected these cell lines to respond to OSM and LIF. Using specific antibodies recognizing proteins of the janus kinase (Jak-)/signal transducers and activator of transcription (Stat-) signaling cascade that has been shown to transduce the signals of the IL-6 cytokines to the nucleus, we could show that Jak1, Jak2 and Tyk2, as well as the Stat proteins Stat1, Stat3 and Stat5b were phosphorylated in all three cell lines by OSM and, at least in part, by LIF. Activation of the Stat proteins was also detected by EMSA which revealed complex formation on the Stat3 DNA-binding element and on a Stat5 binding site. Consistent with our recent findings, OSM treatment also induced the activation of the MAPK erk2 and the tyrosine phosphatase SHP-2 in cells of the A172, T98G and U343-MG cell lines. Although this activation pattern was very close to what we had observed in the GOS3 glioma cells, only T98G showed a growth inhibition in response to OSM while the A172 and the U343-MG cell lines did not respond to OSM treatment in terms of growth inhibition.