Growth inhibition of mammalian cells by synthetic and natural photosensitising agents

A mammalian cell line, J774, was susceptible to both synthetic and natural photosensitising agents after irradiation with long-wave ultraviolet light. Both UV-A light and psoralen did not affect cell growth individually; a reduction in visual confluency was achieved only when psoralen and UV-A light were used in combination. The maximum visual confluency decreased by 55% when 50 ppm psoralen was added to a growing culture and irradiated with UV light for 3 min. Decreasing the UV-A exposure times from 3 min to 3 s did not greatly affect the maximum total visual confluence reached using different synthetic psoralen concentrations, but did affect the rate at which cell death occurred. The 3 min exposure time resulted in a rapid decrease in cell numbers in comparison to 3 s exposure time. Synthetic psoralen was found to have an increasing photosensitising activity with increasing concentration using a logarithmic shift between 0.5 ppm and 50 ppm. A visual confluency of 45% was achieved using concentrations of 50 ppm psoralen, and 70% visual confluency using 0.5 ppm. Natural mixtures of furanocoumarins containing psoralens, obtained from two separate parsley sources, were found to have greater efficacy at inhibiting the growth cycle of the cells when compared to the synthetic psoralen.

An in vitro growth inhibition test for measuring the potency of Leptospira spp. Sejroe group vaccine in buffaloes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

In vitro prostate cancer cell growth inhibition by Brazilian plant extracts

In the present study, 1220 plant extracts obtained from 352 plants belonging to 73 families that grow in the Amazon and Atlantic rain forests were screened for cytotoxicity against PC-3 prostate cancer cell lines. Extracts were tested in the single dose of 100 mu g/mL. Activity was observed in 17 aqueous or organic extracts belonging to Annonaceae, Apocynaceae, Araceae, Capparaceae, Commelinaceae, Flacourtiaceae, Lecythiclaceae, Leguminosae, Passifloraceae, Rutaceae, and Violaceae.

Anthocyanin incorporated dental copolymer: bacterial growth inhibition, mechanical properties, and compound release rates and stability by (1)h NMR

Objective. To evaluate bacterial growth inhibition, mechanical properties, and compound release rate and stability of copolymers incorporated with anthocyanin (ACY; Vaccinium macrocarpon). Methods. Resin samples were prepared (Bis-GMA/TEGDMA at 70/30 mol%) and incorporated with 2 w/w% of either ACY or chlorhexidine (CHX), except for the control group. Samples were individually immersed in a bacterial culture (Streptococcus mutans) for 24 h. Cell viability (n = 3) was assessed by counting the number of colony forming units on replica agar plates. Flexural strength (FS) and elastic modulus (E) were tested on a universal testing machine (n = 8). Compound release and chemical stability were evaluated by UV spectrophotometry and (1)H NMR (n = 3). Data were analyzed by one-way ANOVA and Tukey's test ( α = 0.05). Results. Both compounds inhibited S. mutans growth, with CHX being most effective (P < 0.05). Control resin had the lowest FS and E values, followed by ACY and CHX, with statistical difference between control and CHX groups for both mechanical properties (P < 0.05). The 24 h compound release rates were ACY: 1.33 μg/mL and CHX: 1.92 μg/mL. (1)H NMR spectra suggests that both compounds remained stable after being released in water. Conclusion. The present findings indicate that anthocyanins might be used as a natural antibacterial agent in resin based materials.

Drug-targeting in combined cancer chemotherapy: tumor growth inhibition in mice by association of paclitaxel and etoposide with a cholesterol-rich nanoemulsion

Lipid nanoemulsions (LDE) may be used as carriers of paclitaxel (PTX) and etoposide (ETP) to decrease toxicity and increase the therapeutic action of those drugs. The current study investigates the combined chemotherapy with PTX and ETP associated with LDE. Four groups of 10-20 B16F10 melanoma-bearing mice were treated with LDE-PTX and LDE-ETP in combination (LDE-PTX + ETP), commercial PTX and ETP in combination (PTX + ETP), single LDE-PTX, and single LDE-ETP. PTX and ETX doses were 9 mu mol/kg administered in three intraperitoneal injections on three alternate days. In two control groups mice were treated with saline solution or LDE alone. Tumor growth, metastasis presence, cell-cycle distribution, blood cell counts and histological data were analyzed. Toxicity of all treatments was evaluated in mice without tumors. Tumor growth inhibition was similarly strong in all treatment groups. However, there was a greater reduction in the number of animals bearing metastases in the LDE-PTX + ETP group (30 %) in comparison to the PTX + ETP group (82 %, p < 0.05). Reduction of cellular density, blood vessels and increase of collagen fibers in tumor tissues were observed in the LDE-PTX + ETP group but not in the PTX + ETP group, and in both groups reduced melanoma-related anemia and thrombocytosis were observed. Flow cytometric analysis suggested that LDE-PTX + ETP exhibited greater selectivity to neoplastic cells than PTX-ETP, showing arrest (65 %) in the G(2)/M phase of the cell cycle (p < 0.001). Toxicity manifested by weight loss and myelosuppression was markedly milder in the LDE-PTX + ETP than in the PTX + ETP group. LDE-PTX + ETP combined drug-targeting therapy showed markedly superior anti-cancer properties and reduced toxicity compared to PTX + ETP.

Development of a Tumour Growth Inhibition Model to Elucidate the Effects of Ritonavir on Intratumoural Metabolism and Anti-tumour Effect of Docetaxel in a Mouse Model for Hereditary Breast Cancer

In a mouse tumour model for hereditary breast cancer, we previously explored the anti-cancer effects of docetaxel, ritonavir and the combination of both and studied the effect of ritonavir on the intratumoural concentration of docetaxel. The objective of the current study was to apply pharmacokinetic (PK)-pharmacodynamic (PD) modelling on this previous study to further elucidate and quantify the effects of docetaxel when co-administered with ritonavir. PK models of docetaxel and ritonavir in plasma and in tumour were developed. The effect of ritonavir on docetaxel concentration in the systemic circulation of Cyp3a knock-out mice and in the implanted tumour (with inherent Cyp3a expression) was studied, respectively. Subsequently, we designed a tumour growth inhibition model that included the inhibitory effects of both docetaxel and ritonavir. Ritonavir decreased docetaxel systemic clearance with 8% (relative standard error 0.4%) in the co-treated group compared to that in the docetaxel only-treated group. The docetaxel concentration in tumour tissues was significantly increased by ritonavir with mean area under the concentration-time curve 2.5-fold higher when combined with ritonavir. Observed tumour volume profiles in mice could be properly described by the PK/PD model. In the co-treated group, the enhanced anti-tumour effect was mainly due to increased docetaxel tumour concentration; however, we demonstrated a small but significant anti-tumour effect of ritonavir addition (p value <0.001). In conclusion, we showed that the increased anti-tumour effect observed when docetaxel is combined with ritonavir is mainly caused by enhanced docetaxel tumour concentration and to a minor extent by a direct anti-tumour effect of ritonavir.

p21WAF1 is required for butyrate-mediated growth inhibition of human colon cancer cells

A diet high in fiber is associated with a decreased incidence and growth of colon cancers. Butyrate, a four-carbon short-chain fatty acid product of fiber fermentation within the colon, appears to mediate these salutary effects. We sought to determine the molecular mechanism by which butyrate mediates growth inhibition of colonic cancer cells and thereby to elucidate the molecular link between a high-fiber diet and the arrest of colon carcinogenesis. We show that concomitant with growth arrest, butyrate induces p21 mRNA expression in an immediate-early fashion, through transactivation of a promoter cis-element(s) located within 1.4 kb of the transcriptional start site, independent of p53 binding. Studies using the specific histone hyperacetylating agent, trichostatin A, and histone deacetylase 1 indicate that growth arrest and p21 induction occur through a mechanism involving histone hyperacetylation. We show the critical importance of p21 in butyrate-mediated growth arrest by first confirming that stable overexpression of the p21 gene is able to cause growth arrest in the human colon carcinoma cell line, HT-29. Furthermore, using p21-deleted HCT116 human colon carcinoma cells, we provide convincing evidence that p21 is required for growth arrest to occur in response to histone hyperacetylation, but not for serum starvation nor postconfluent growth. Thus, p21 appears to be a critical effector of butyrate-induced growth arrest in colonic cancer cells, and may be an important molecular link between a high-fiber diet and the prevention of colon carcinogenesis.

Transforming growth factor β-induced phosphorylation of Smad3 is required for growth inhibition and transcriptional induction in epithelial cells

Drosophila Mad proteins are intracellular signal transducers of decapentaplegic (dpp), the Drosophila transforming growth factor β (TGF-β)/bone morphogenic protein (BMP) homolog. Studies in which the mammalian Smad homologs were transiently overexpressed in cultured cells have implicated Smad2 in TGF-β signaling, but the physiological relevance of the Smad3 protein in signaling by TGF-β receptors has not been established. Here we stably expressed Smad proteins at controlled levels in epithelial cells using a novel approach that combines highly efficient retroviral gene transfer and quantitative cell sorting. We show that upon TGF-β treatment Smad3 becomes rapidly phosphorylated at the SSVS motif at its very C terminus. Either attachment of an epitope tag to the C terminus or replacement of these three serine residues with alanine abolishes TGF-β-induced Smad3 phosphorylation; these proteins act in a dominant-negative fashion to block the antiproliferative effect of TGF-β in mink lung epithelial cells. A Smad3 protein in which the three C-terminal serines have been replaced by aspartic acids is also a dominant inhibitor of TGF-β signaling, but can activate plasminogen activator inhibitor 1 (PAI-1) transcription in a ligand-independent fashion when its nuclear localization is forced by transient overexpression. Phosphorylation of the three C-terminal serine residues of Smad3 by an activated TGF-β receptor complex is an essential step in signal transduction by TGF-β for both inhibition of cell proliferation and activation of the PAI-1 promoter.

Expression of the telomerase catalytic subunit, hTERT, induces resistance to transforming growth factor β growth inhibition in p16INK4A(−) human mammary epithelial cells

Failures to arrest growth in response to senescence or transforming growth factor β (TGF-β) are key derangements associated with carcinoma progression. We report that activation of telomerase activity may overcome both inhibitory pathways. Ectopic expression of the human telomerase catalytic subunit, hTERT, in cultured human mammary epithelial cells (HMEC) lacking both telomerase activity and p16INK4A resulted in gaining the ability to maintain indefinite growth in the absence and presence of TGF-β. The ability to maintain growth in TGF-β was independent of telomere length and required catalytically active telomerase capable of telomere maintenance in vivo. The capacity of ectopic hTERT to induce TGF-β resistance may explain our previously described gain of TGF-β resistance after reactivation of endogenous telomerase activity in rare carcinogen-treated HMEC. In those HMEC that overcame senescence, both telomerase activity and TGF-β resistance were acquired gradually during a process we have termed conversion. This effect of hTERT may model a key change occurring during in vivo human breast carcinogenesis.

Two Genetically Separable Phases of Growth Inhibition Induced by Blue Light in Arabidopsis Seedlings1

High fluence-rate blue light (BL) rapidly inhibits hypocotyl growth in Arabidopsis, as in other species, after a lag time of 30 s. This growth inhibition is always preceded by the activation of anion channels. The membrane depolarization that results from the activation of anion channels by BL was only 30% of the wild-type magnitude in hy4, a mutant lacking the HY4 BL receptor. High-resolution measurements of growth made with a computer-linked displacement transducer or digitized images revealed that BL caused a rapid inhibition of growth in wild-type and hy4 seedlings. This inhibition persisted in wild-type seedlings during more than 40 h of continuous BL. By contrast, hy4 escaped from the initial inhibition after approximately 1 h of BL and grew faster than wild type for approximately 30 h. Wild-type seedlings treated with 5-nitro-2-(3-phenylpropylamino)-benzoic acid, a potent blocker of the BL-activated anion channel, displayed rapid growth inhibition, but, similar to hy4, these seedlings escaped from inhibition after approximately 1 h of BL and phenocopied the mutant for at least 2.5 h. The effects of 5-nitro-2-(3-phenylpropylamino)-benzoic acid and the HY4 mutation were not additive. Taken together, the results indicate that BL acts through HY4 to activate anion channels at the plasma membrane, causing growth inhibition that begins after approximately 1 h. Neither HY4 nor anion channels appear to participate greatly in the initial phase of inhibition.

Alterations in the Cytoskeleton Accompany Aluminum-Induced Growth Inhibition and Morphological Changes in Primary Roots of Maize1

Although Al is one of the major factors limiting crop production, the mechanisms of toxicity remain unknown. The growth inhibition and swelling of roots associated with Al exposure suggest that the cytoskeleton may be a target of Al toxicity. Using indirect immunofluorescence microscopy, microtubules and microfilaments in maize (Zea mays L.) roots were visualized and changes in their organization and stability correlated with the symptoms of Al toxicity. Growth studies showed that the site of Al toxicity was associated with the elongation zone. Within this region, Al resulted in a reorganization of microtubules in the inner cortex. However, the orientation of microtubules in the outer cortex and epidermis remained unchanged even after chronic symptoms of toxicity were manifest. Auxin-induced reorientation and cold-induced depolymerization of microtubules in the outer cortex were blocked by Al pretreatment. These results suggest that Al increased the stability of microtubules in these cells. The stabilizing effect of Al in the outer cortex coincided with growth inhibition. Reoriented microfilaments were also observed in Al-treated roots, and Al pretreatment minimized cytochalasin B-induced microfilament fragmentation. These data show that reorganization and stabilization of the cytoskeleton are closely associated with Al toxicity in maize roots.

Inhibition of activation of dsRNA-dependent protein kinase and tumour growth inhibition

Inhibition of dsRNA-activated protein kinase (PKR), not only attenuates muscle atrophy in a murine model of cancer cachexia (MAC16), but it also inhibits tumour growth. In vitro the PKR inhibitor maximally inhibited growth of MAC16 tumour cells at a concentration of 200 nM, which was also maximally effective in attenuating phosphorylation of PKR and of eukaryotic initiation factor (eIF)2 on the a-subunit. There was no effect on the growth of the MAC13 tumour, which does not induce cachexia, even at concentrations up to 1,000 nM. There was constitutive phosphorylation of PKR and eIF2a in the MAC16, but not in the MAC13 tumour, while levels of total PKR and eIF2a were similar. There was constitutive upregulation of nuclear factor-?B (NF-?B) in the MAC16 tumour only, and this was attenuated by the PKR inhibitor, suggesting that it arose from activation of PKR. In MAC16 alone the PKR inhibitor also attenuated expression of the 20S proteasome. The PKR inhibitor potentiated the cytotoxicity of both 5-fluorouracil and gemcitabine to MAC16 cells in vitro. These results suggest that inhibitors of PKR may be useful therapeutic agents against tumours showing increased expression of PKR and constitutive activation of NF-?B, and may also prove useful in sensitising tumours to standard chemotherapeutic agents.

Growth inhibition by tyrosine kinase inhibitors in mesothelioma cell lines.

Clinical outcome following chemotherapy for malignant pleural mesothelioma is poor and improvements are needed. This preclinical study investigates the effect of five tyrosine kinase inhibitors (PTK787, ZD6474, ZD1839, SU6668 and SU11248) on the growth of three mesothelioma cell lines (NCI H226, NCI H28 and MSTO 211H), the presence of growth factor receptors and inhibition of their downstream signalling pathways. GI50 values were determined: ZD6474 and SU11248, mainly VEGFR2 inhibitors, gave the lowest GI50 across all cell lines (3.5-6.9 microM) whereas ZD1839 gave a GI50 in this range only in H28 cells. All cell lines were positive for EGFR, but only H226 cells were positive for VEGFR2 by Western blotting. ZD6474 and ZD1839 inhibited EGF-induced phosphorylation of EGFR, AKT and ERK, whereas VEGF-induced phosphorylation of VEGFR2 was completely inhibited with 0.1 microM SU11248. VEGFR2 was detected in tumour samples by immunohistochemistry. VEGFR2 tyrosine kinase inhibitors warrant further investigation in mesothelioma.

Inhibition Effect on the Allium cepa L. Root Growth When Using Hexavalent Chromium-Doped River Waters

The effect of Cr(6+) on Allium cepa root length was studied using both clean and polluted river waters. Seven series of Cr(6+)-doped polluted and non-polluted river waters were used to grow onions. Chromium concentration (Cr(6+)) of 4.2 mg L(-1)(EC(50) value), doped in clean river water caused a 50% reduction of root length, while in organically polluted samples similar root growth inhibition occurred at 12.0 mg Cr(6+) L(-1). The results suggested that there was a dislocation to higher values in toxic chromium concentration in polluted river water due to the eutrophization level of river water.

In vitro characterization of intra-generic inhibition of growth in Salmonella typhimurium

The intra-generic inhibition of bacterial growth observed previously in vivo and in vitro with strains of Salmonella, Citrobacter and E. coli was studied in vitro using S. typhimurium strain F98. There was complete inhibition of multiplication of S. typhimurium when it was added to stationary-phase broth cultures of different Salmonella serotypes, but only partial inhibition when added to broth cultures of E. coli. The degree of inhibition between different mutants of F98 was affected by the numbers of bacteria of the inhibiting strain, but this was not the only factor, since exponential-phase bacterial cells were less inhibitory than stationary-phase cells. The inhibitory effect was produced at temperatures between 20°C and 40°C. The complete inhibition of growth observed between F98 mutants was abolished by ampicillin, rifampicin and streptomycin, but not by nalidixic acid. Inhibition was also prevented by separating the two cultures by a dialysis membrane. A Tnpho A Insertion mutant of F98 was produced which did not show inhibition in vitro but was still inhibitory in vivo. It is suggested that this complete inhibition of bacterial multiplication between organisms of the same genus, which is greater than that produced between organisms from different genera, is mediated by a cell surface protein.