Colon Adenocarcinoma Cell Line HT-29 Research Articles

Cytotoxic, antioxidant and antimicrobial activities of Nerium oleander collected in Morocco

Objective: To investigate the cytotoxic, antioxidant and antibacterial activities of ethanolic and aqueous crude extracts of Nerium oleander (N. oleander) leaves. Methods: Cytotoxic activities were evaluated by WST-1 bioassay on two human cancer cell lines, namely human colon adenocarcinoma cell line HT-29 and human breast cancer cell line MDA-MB-231. The antioxidant property of N. oleander extracts was assessed by DPPH scavenging and β -carotene bleaching tests. The agar disc diffusion method was used for the determination of antimicrobial activity against different strains. Results: Using cell viability indices, the WST-1 test revealed that both extracts reduced cell viability in both cell lines. Our results also showed that aqueous extract was more active than ethanolic extract, with IC50 values of (1.67±0.22) μg/mL and (2.36±0.44) μg/mL on MDA-MB-231 cells, and (2.89±0.35) μg/mL and (5.09±0.52) μg/mL on HT29 cells, respectively. The study of the antioxidant activity showed that N. oleander extracts had a considerable scavenging capacity and exerted a significant preventive effect against the oxidation of β -carotene by the peroxide radicals. However, the antibacterial test showed that both ethanolic and aqueous extracts of N. oleander had a moderate antibacterial effect limited only to Gram-positive bacteria. Conclusions: Our results shows that N. oleander aqueous and ethanolic extracts have significant cytotoxic activities against tumor cell lines and possesses a strong antioxidant capacity, suggesting the presence of active compounds in N. oleander leaves that could be a potential source of phytochemicals with high medicinal value to be used in cancer treatment and prevention.

E-Cadherin in Colorectal Cancer: Relation to Chemosensitivity

BackgroundThe conventional chemotherapy of colorectal cancer with irinotecan, 5-fluorouracil, and oxaliplatin remains one of the front-line treatments worldwide. However, its efficacy is quite low. Recently studies of the epithelial–mesenchymal transition (EMT) have become the focus of investigations into the cause of chemoresistance in several types of cancer, including colorectal cancer. The data about the role of EMT in chemosensitivity are controversial. Materials and MethodsHuman colon adenocarcinoma cell lines HT29 and HCT116 and 14 primary short-term cultures established from patient tumors were used. The chemosensitivity to irinotecan, 5-fluorouracil, and oxaliplatin was assessed using the (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. Immunocytochemistry, immunohistochemistry, and Western blot test were used to investigate the E-cadherin expression, the loss of which is a major hallmark of EMT. ResultsElevated chemosensitivity of the cell line with EMT phenotype, HCT116, was demonstrated. Increased chemosensitivity was revealed in HT29 cell line upon EMT induction. E-cadherin–positive short-term cultures were more resistant to all the drugs tested, whereas each of E-cadherin–negative cultures showed sensitivity to at least one drug. The statistically significant dependency of cells viability on the E-cadherin expression (P < .04) was demonstrated on the short-term cultures using 2 concentrations of each drug. ConclusionThe data obtained may serve as a basis for the analysis of colon cancer chemosensitivity using short-term cultures and the assay of E-cadherin expression.

Indonesian Pasuruan propolis extract does not exert antiproliferation and pro-apoptotic effect on human colon carcinoma cell line HT-29

Purpose : To evaluate the anti-cancer activity of Indonesian Pasuruan propolis extract (PPE) against HT-29 human colon carcinoma cell line. Methods : HT-29 cells were cultured and treated in different concentrations of PPE (50, 100, 200, 400 μg/mL) for 24 h. The cells were evaluated by several indicators such as cell proliferation and apoptosis as well as the expression of protein Ki67, p53, cyclin D1, and Bcl-xL. Results : Administration of PPE inhibited cell proliferation of HT-29 in concentration-dependent manner but the effect was not significant (p = 0.842); the results were similar with regard to the expression of Ki67 in HT-29 cells (p = 0.953). Administration of 400 μg/mL PPE insignificantly decreased cyclin D1 expression (p = 0.149). The concentration of PPE at 50, 100, and 200 μg/mL induced cell apoptosis of HT-29 cells initially, but the level of apoptosis subsequently decreased (p = 0.416). Furthermore, the expressions of p53 and Bcl-xL decreased following treatment with PPE at 50, 100 and 200 μg/mL but increased for the PPE 400 μg/mL group (p = 0.000). Conclusion : PPE reduced the expressions of p53, Ki67, cyclin D1, and Bcl-xL insignificantly as it it generally failed inhibit cell proliferation and promote cell apoptosis of HT-29 cells. Keywords : Bcl-xL, Colon cancer, Cyclin D1, Human colon adenocarcinoma cell line HT-29, Proliferation marker Ki67, Tumor protein p53

An in vitro and in silico study on the antioxidant and cell culture-based study on the chemoprotective activities of fish muscle protein hydrolysates obtained from European seabass and gilthead seabream

European seabass (Dicentrarchus labrax, Linnaeus, 1758) (L) and gilthead seabream (Sparus aurata, Linnaeus, 1758) (C) muscles were hydrolysated by Alcalase (Lalc, Calc) and Chymotrypsin (Lch, Cch) then hydrolysates were examined and their peptide profiles obtained. A total of 765, 794, 132 and 232 peptides were identified in Calc, Lalc, Cch and Lch, respectively. Although, Lch and Cch were expected to have more antioxidant capacity because of their peptide profiles, Alcalase hydrolysates observed in vitro, were slightly higher (TEAC assay for Calc: 848.11 ± 60.78 μmol TE/g protein). Maximum inhibition of oxidative stress was determined for Lalc (12.8% ± 4.5%) in MDCK1 cell lines. Highest proliferative capacity observed for Calc (147.0% ± 3.1%) at MTT assay in MDCK1 cell culture. Lch showed the highest chemopreventive effect with a 40–60% decrease for human colon adenocarcinoma cell line HT-29. This research points out the importance of aquatic sources as raw materials for peptide researches.

PO-058 Unravelling the protective role of androgens/androgen receptorin breast cancer: when BAD goes good

IntroductionThe transcription factor STAT6 is strongly expressed in various tumours with highest expression in malignant lymphomas and pancreatic, colorectal, prostate and breast cancers. STAT6 expression in colorectal cancer is associated with an increased malignancy and a poor prognosis. Incidence of colorectal cancer is approximately 1,361,000 patients per annum worldwide and approximately 60% show STAT6 expression. Patients with colorectal cancer expressing STAT6 also show poor survival rates. The 5 year relative survival rate for patients with stage IIIC and IV colon cancer is about 53% and 11% respectively. Techniques aimed at reducing or blocking STAT6 expression may be useful in treating those cancers with STAT6 expression.Material and methodsCelixir owns a patent covering four STAT6 specific small interfering RNA (siRNA) sequences. We tested these four sequences in vitro using the human colon adenocarcinoma cell line HT-29. The four sequences were introduced individually and in combination into HT-29 cells by cationic lipid-mediated transfer at different concentrations (10 to 200 nM). STAT6 mRNA and protein levels were analysed to confirm the transfection was successful. The proliferation of cells was analysed using cell counting at various times (using a NucleoCounter) and the apoptosis of cells was analysed using Annexin V and propidium iodide (PI) staining.Results and discussions100 nM siRNA concentration was the most effective and all four individual sequences knocked-down STAT6 mRNA and protein by more than 50%. All individual sequences were capable of significantly inhibiting cell proliferation, with sequences #1 and #4 reducing the number of cells to ~50% compared with control 7 days after treatment. STAT6 silencing also significantly induced late apoptotic events. Despite using several combinations of all four sequences at the same time and serially, the best results were obtained with sequences #1 and #4 individually.ConclusionThesein vitro resultsdemonstrate that the STAT6 siRNA sequences are capable of inhibiting theproliferation of and inducing late apoptosis of HT-29 colon cancer cells and,in some instances, halving the number of cancer cells. We propose to repeatthese experiments in vivousing immunocompromised mice containing human colon cancer xenografts and totest the siRNA sequences in other cancers in which STAT6 is expressed.

PO-055 STAT6 SIRNA reduces proliferation of and induces apoptosis in the human colon adenocarcinoma cell line HT-29

IntroductionGerm-cell-tumours (GCTs) are clinically and pathologically heterogeneous neoplasms that arise at gonadal (testicular/ovarian) and extra-gonadal sites. They occur across a wide age-range, from early childhood to late adulthood. Poor-risk patients have <50% survival and the chemotherapy burden results in substantial long-term side-effects. Identifying common molecular changes and therapeutic targets in GCTs is of major clinical importance.Material and methodsFour representative Malignant Germ Cell Tumour (mGCT) cell lines (TCam2, 1411 hour, GCT44, 2102Ep), were co-transfected using 16 nM mimics (8.3 nM miR-99a-5p/100–5p+8.3 nM miR-125b-5p) in order to assess the functional relevance of the miRNAs in cancer progression. Transfected cells were then analysed using Illumina mRNA arrays to assess the genes mostly affected by the treatment.Results and discussionsWe previously showed that miR-100–5 p/99a-5p/125b-5p are among the most frequently under-expressed microRNAs in malignant GCTs.The mature form of miR-125b-5p is the product of two genomic loci, which cluster with either miR-99a-5p on chromosome21, or miR-100–5 p on chromosome11. MiR-100–5 p/99a-5p share identical seed regions, which determine their mRNA targets, indicating functional overlap. Linear regression analysis of qRT-PCR data reveals a strong positive correlation between miR-100–5 p/99a-5p and miR-125b-5p (R2=0.9) in malignant GCTs, suggesting co-regulation.After mimics transfection, a significant decrease in cell growth was seen in 1411 hour (p<0.01) and TCam2 (p<0.03) at day 7. We used Illumina mRNA arrays to analyse the genes that were mostly affected by mimic treatment at days 2 and 7 and observed an enrichment in genes involved in cell proliferation/apoptosis, in particular TRIM71, FGFR3, E2F7 and LIN28A. Restoring miR-100–5 p/99a-5p/125b-5p in TCam2 cells also resulted in G0-G1 accumulation from day 3.Previous work in our lab demonstrated that LIN28A knockdown resulted in de-repression of let-7 (a class of microRNAs that are highly downregulated in GCTs). Its downregulation led to de-repression of let-7 implying a contribution of miR-125b-5p in this regulatory pathway.ConclusionThese data support a significant role of miR99a-100–5 p/125b-5p in GCT progression. We discovered that replenishment of these miRNAs can also reduce downregulation of other miRNAs, opening the possibility that multiple benefit can be achieved with a therapeutic replenishment approach for treating these and potentially other tumours.

Overview of the Biological Activities of a Methanol Extract from Wild Red Belt Conk, Fomitopsis pinicola (Agaricomycetes), Fruiting Bodies from Central Italy.

Fomitopsis pinicola (Sw.) P. Karst. (Fomitopsidaceae) is a medicinal mushroom with a variety of healthy properties. In this study we tested the radical scavenging activity and antimicrobial and anticancer potential of methanol extracts of F. pinicola from central Italy. Molecular identification confirmed that the samples were F. pinicola; a Basic Local Alignment Search Tool search showed a close match (99% sequence identity) with European isolates of this species. The free radical scavenging capacities, measured by DPPH assay, showed that the extract activity was 3.5% that of Trolox. The MTT test, evaluated after 72 hours of treatment with increasing doses of extract (5-500 μg · mL-1), considerably inhibited proliferation in a dose-dependent manner in 2 human tumor cell lines. This reduction was coupled with a relevant induction of apoptosis in the human leukemia THP-1 cell line after 24 hours of treatment, but a relevant toxic effect occurred in the human colon adenocarcinoma HT29 cell line. The genotoxic potential of the methanol extracts was studied by single-cell gel electrophoresis of normal human leukocytes exposed to 20 μg extract at 37°C for 30 minutes; no DNA damage was observed. The F. pinicola methanol extract was found to have varying degrees of antifungal effects against the pathogenic fungi tested (minimum inhibitory concentration from 23.63 to 66.81 μg · mL-1). The results show that the tested F. pinicola extract has strong antimicrobial and chemo-preventive activities, but is a poor antioxidant.

The secondary Fusarium metabolite aurofusarin induces oxidative stress, cytotoxicity and genotoxicity in human colon cells

Aurofusarin (AURO), a dimeric naphthoquinone, is produced by Fusarium fungi. Although frequently found in food and feed, toxicological studies are limited. Hence, the in vitro toxicity of AURO was investigated in the colon adenocarcinoma cell line HT29 and the non-tumorigenic colon cells HCEC-1CT. Cytotoxic effects were found at concentrations ≥1 μM by evaluating mitochondrial activity (WST-1) and cellular proliferation (sulforhodamine B assay). 10 μM of AURO induced a decrease of cells in the S-phase, measured by flow cytometry. Confocal microscopy revealed AURO-mediated increase of intracellular p53 protein. In accordance, DNA-damage was seen in the comet assay (≥1 μM) together with enhanced levels of formamidopyrimidine-DNA-glycosylase (fpg)-sensitive sites, indicative for oxidative stress. An increase of intracellular reactive oxygen species was observed in the dichlorofluorescein (DCF) assay (≥5 μM). The GSSG/GSH ratio was elevated, but no impact on redox-sensitive Nrf2-dependent genes (Nrf2, γ-GCL, NQO1) was found at the gene expression level. However, induction of cytochrome P450 monooxygenase (CYP) 1A1 was measured at the gene expression and protein level. In conclusion, these in vitro data suggest that, when co-occurring, AURO might be considered as a potential contributor to the overall toxicity of respective Fusarium mycotoxin mixtures.

Isolation of Petrocidin A, a New Cytotoxic Cyclic Dipeptide from the Marine Sponge-Derived Bacterium Streptomyces sp. SBT348.

A new cyclic dipeptide, petrocidin A (1), along with three known compounds—2,3-dihydroxybenzoic acid (2), 2,3-dihydroxybenzamide (3), and maltol (4)—were isolated from the solid culture of Streptomyces sp. SBT348. The strain Streptomyces sp. SBT348 had been prioritized in a strain collection of 64 sponge-associated actinomycetes based on its distinct metabolomic profile using liquid chromatography/high-resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance (NMR). The absolute configuration of all α-amino acids was determined by HPLC analysis after derivatization with Marfey’s reagent and comparison with commercially available reference amino acids. Structure elucidation was pursued in the presented study by mass spectrometry and NMR spectral data. Petrocidin A (1) and 2,3-dihydroxybenzamide (3) exhibited significant cytotoxicity towards the human promyelocytic HL-60 and the human colon adenocarcinoma HT-29 cell lines. These results demonstrated the potential of sponge-associated actinomycetes for the discovery of novel and pharmacologically active natural products.

Mertensene, a Halogenated Monoterpene, Induces G2/M Cell Cycle Arrest and Caspase Dependent Apoptosis of Human Colon Adenocarcinoma HT29 Cell Line through the Modulation of ERK-1/-2, AKT and NF-κB Signaling.

Conventional treatment of advanced colorectal cancer is associated with tumor resistance and toxicity towards normal tissues. Therefore, development of effective anticancer therapeutic alternatives is still urgently required. Nowadays, marine secondary metabolites have been extensively investigated due to the fact that they frequently exhibit anti-tumor properties. However, little attention has been given to terpenoids isolated from seaweeds. In this study, we isolated the halogenated monoterpene mertensene from the red alga Pterocladiella capillacea (S.G. Gmelin) Santelices and Hommersand and we highlight its inhibitory effect on the viability of two human colorectal adenocarcinoma cell lines HT29 and LS174. Interestingly, exposure of HT29 cells to different concentrations of mertensene correlated with the activation of MAPK ERK-1/-2, Akt and NF-κB pathways. Moreover, mertensene-induced G2/M cell cycle arrest was associated with a decrease in the phosphorylated forms of the anti-tumor transcription factor p53, retinoblastoma protein (Rb), cdc2 and chkp2. Indeed, a reduction of the cellular level of cyclin-dependent kinases CDK2 and CDK4 was observed in mertensene-treated cells. We also demonstrated that mertensene triggers a caspase-dependent apoptosis in HT29 cancer cells characterized by the activation of caspase-3 and the cleavage of poly (ADP-ribose) polymerase (PARP). Besides, the level of death receptor-associated protein TRADD increased significantly in a concentration-dependent manner. Taken together, these results demonstrate the potential of mertensene as a drug candidate for the treatment of colon cancer.

16 - Identification of Aberrant Splicing Events in Myelodysplastic Syndrome Patients with Splicing Factor Gene Mutations

The aim of this study was to evaluate the performance of the overall antioxidant of Lactobacillus fermentum LF31 bacterium with prebiotic supplement in human colon cultured cells.The antioxidant capability of L. fermentum LF31 has been assayed in vitro on human colon adenocarcinoma HT-29 cell line using the oxygen radical absorbance capacity method.The analysis revealed that the interaction of probiotic strain cells supplemented with a prebiotic exerts a remarkable antioxidant capacity.The L. fermentum used in the present study exhibited significant in vitro antioxidant capacity, increasing the total antioxidant potential.

Construction and immunogenic properties of a chimeric protein comprising CfaE, CfaB and LTB against Enterotoxigenic Escherichia coli

ETEC (Enterotoxigenic Escherichia coli) is a major cause of diarrhea in developing countries and children. ETEC has two virulence factors including colonization factors antigen (CFA) and labile enterotoxins (LTs). CFA/I consists the major pilin subunit CfaB and a minor adhesive subunit, CfaE. In this study a tripartite fusion protein containing CfaB, CfaE and LTB was designed. In silico analysis of the tertiary structure of the chimeric protein showed a protein with three main domains linked together with linkers. Linear and conformational B-cell epitopes were identified. A chimera consisting cfaB, cfaE and ltB(BET)was then synthesized with E. coli codon bias in pUC57 and sub cloned into pET32 vector. Recombinant protein was expressed and purified by affinity chromatography and confirmed by western blotting. Mice were immunized with recombinant protein and the antibody titer and specificity of the sera were analyzed by ELISA. The efficiency of the immune sera against ETEC was evaluated by binding assay and GM1-ELISA. VaxiJen analysis of the protein showed high antigenicity. Post-immune sera contained high titers of anti-BET IgG. Pretreatment of ETEC cells with sera from immunized mice decreased their ability to adhere to cells of the human colon adenocarcinoma cell line HT29.

Abstract 3684: Proteome-wide triple negative breast cancer mutant p53 association index identifies chromatin unwinding for precision therapeutics

Abstract Over 80% of TNBCs express mutant p53 (mtp53) proteins. We coupled cell fractionation with stable isotope labeling with amino acids in cell culture (SILAC) and inducible knockdown of endogenous mtp53 to determine the mtp53 driven proteome in the cytoplasm and chromatin of triple negative breast cancer (TNBC) cells. Using SILAC coupled to tandem mass spectrometry (LC-MS/MS) we identified that R273H mtp53 expression in MDA-MB-468 breast cancer cells both up and down-regulated multiple proteins and metabolic pathways. We sequenced 73,154 peptide pairs that corresponded to 3010 proteins detected under reciprocal labeling conditions in the cytoplasm and 48,825 peptide pairs that corresponded to 5195 proteins in the chromatin fraction. Pathway enrichment analysis ranked the DNA unwinding pathway as the highest chromatin associated pathway regulated by mtp53. Moreover, to summarize and quantify the degree of under- or over-expression of a protein from two reciprocal experiments, a mutant p53 association index (mPAI) was defined as the log (of base 2) ratio of two readings from the two reciprocal experiments. Values of mPAI were normally distributed with a mean close to zero, consistent with the fact that levels of the majority of proteins were not affected by mtp53 knockdown. Standard deviation of mPAI is close to one and mtp53 itself shows mPAI values of greater than 2.0 (z-score &amp;gt; 2.0) in both cytosol and chromatin fractions, consistent with the expectation that its levels are significantly reduced by knockdown experiments. We thereby identified proteins and pathways significantly affected by mtp53 knockdowns as those with mPAI &amp;gt; 1.0 (indicating positive association) or mPAI &amp;lt; -1.0 (indicating negative association). A pathway that was positively associated in both the cytoplasm and the chromatin was the DNA unwinding pathway, which was represented by associated changes for all the minichromosome maintenance protein complex proteins 2 through 7 (MCM2-7). This hexomeric protein ring structure complex is the eukaryotic DNA helicase complex required for DNA replication and elongation of the replication fork. We validated that the MCM4 protein level is positively associated with mtp53 R273H in the TNBC cell line MDA-MB-468 and in the colon adenocarcinoma cell line HT-29. This suggests that targeting inhibition of the MCM hexomeric complex may be a good precision medicine approach for TNBCs. Further studies are in progress to determine the efficacy of targeting inhibition of MCMs 2-7 as a mechanism to block TNBC. Citation Format: Wei-Gang Qiu, Alla Polotskaia, Gu Xiao, Lia Di, John Philip, Ronald C. Hendrikson, Jill Bargonetti. Proteome-wide triple negative breast cancer mutant p53 association index identifies chromatin unwinding for precision therapeutics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3684.

β-III tubulin modulates the behavior of Snail overexpressed during the epithelial-to-mesenchymal transition in colon cancer cells

Class III β-tubulin (TUBB3) is a marker of drug resistance expressed in a variety of solid tumors. Originally, it was described as an important element of chemoresistance to taxanes. Recent studies have revealed that TUBB3 is also involved in an adaptive response to a microenvironmental stressor, e.g. low oxygen levels and poor nutrient supply in some solid tumors, independently of the microtubule targeting agent. Furthermore, it has been demonstrated that TUBB3 is a marker of biological aggressiveness associated with modulation of metastatic abilities in colon cancer. The epithelial-to-mesenchymal transition (EMT) is a basic cellular process by which epithelial cells lose their epithelial behavior and become invasive cells involved in cancer metastasis. Snail is a zinc-finger transcription factor which is able to induce EMT through the repression of E-cadherin expression. In the presented studies we focused on the analysis of the TUBB3 role in EMT-induced colon adenocarcinoma cell lines HT-29 and LS180. We observed a positive correlation between Snail presence and TUBB3 upregulation in tested adenocarcinoma cell lines. The cellular and behavioral analysis revealed for the first time that elevated TUBB3 level is functionally linked to increased cell migration and invasive capability of EMT induced cells. Additionally, the post-transcriptional modifications (phosphorylation, glycosylation) appear to regulate the cellular localization of TUBB3 and its phosphorylation, observed in cytoskeleton, is probably involved in cell motility modulation.

A novel bispecific diabody targeting both vascular endothelial growth factor receptor 2 and epidermal growth factor receptor for enhanced antitumor activity.

Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2) are receptor tyrosine kinases known to play critical roles in the development and progression of tumors. Based on the cross-talk between EGFR and VEGFR2 signal pathways, we designed and produced a bispecific diabody (bDAb) targeting both EGFR and VEGFR2 simultaneously. The bispecific molecule (EK-02) demonstrated that it could bind to HUVEC (VEGFR2 high-expressing) and A431 (EGFR overexpressing) cells. Additionally, similar to the parental antibodies, it was able to inhibit proliferation and migration, and induced apoptosis in these cells (HUVECs and A431), demonstrating that it had retained the functional properties of its parental antibodies. Furthermore, the efficacy of EK-02 was evaluated using the human colon adenocarcinoma cell line HT29 (VEGFR2 and EGFR coexpressing). In vitro assay showed that EK-02 could bind to HT29 cells, restrain cell growth and migration, and induce apoptosis with enhanced efficacy compared to both parental antibodies. Further, it inhibited the neovascularization and tumor formation on an HT29 cell bearing chicken chorioallantoic membrane (CAM) tumor model in vivo. In conclusion, these data suggest that the novel bDAb (EK-02) has antiangiogenesis and antitumor capacity both in vitro and in vivo, and can possibly be used as cotargeted therapy for the treatment of EGFR and VEGFR2 overexpressing tumors. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:294-302, 2016.

Development and characterization of bio-derived polyhydroxyalkanoate nanoparticles as a delivery system for hydrophobic photodynamic therapy agents.

In this study, we developed and investigated nanoparticles of biologically-derived, biodegradable polyhydroxyalkanoates (PHAs) as carriers of a hydrophobic photosensitizer, 5,10,15,20-Tetrakis(4-hydroxy-phenyl)-21H, 23H-porphine (pTHPP) for photodynamic therapy (PDT). Three PHA variants; polyhydroxybutyrate, poly(hydroxybutyrate-co-hydroxyvalerate) or P(HB-HV) with 12 and 50% HV were used to formulate pTHPP-loaded PHA nanoparticles by an emulsification-diffusion method, where we compared two different poly(vinyl alcohol) (PVA) stabilizers. The nanoparticles exhibited nano-scale spherical morphology under TEM and hydrodynamic diameters ranging from 169.0 to 211.2 nm with narrow size distribution. The amount of drug loaded and the drug entrapment efficiency were also investigated. The in vitro photocytotoxicity was evaluated using human colon adenocarcinoma cell line HT-29 and revealed time and concentration dependent cell death, consistent with a gradual release pattern of pTHPP over 24 h. This study is the first demonstration using bacterially derived P(HB-HV) copolymers for nanoparticle delivery of a hydrophobic photosensitizer drug and their potential application in PDT.

Apelin as a marker for monitoring the tumor vessel normalization window during antiangiogenic therapy.

Antiangiogenic agents transiently normalize tumor vessel structure and improve vessel function, thereby providing a window of opportunity for enhancing the efficacy of chemotherapy or radiotherapy. Currently, there are no reliable predictors or markers reflecting this vessel normalization window during antiangiogenic therapy. Apelin, the expression of which is regulated by hypoxia, and which has well-described roles in tumor progression, is an easily measured secreted protein. Here, we show that apelin can be used as a marker for the vessel normalization window during antiangiogenic therapy. Mice bearing s.c. tumors resulting from inoculation of the colon adenocarcinoma cell line HT29 were treated with a single injection of bevacizumab, a mAb neutralizing vascular endothelial growth factor. Tumor growth, vessel density, pericyte coverage, tumor hypoxia, and small molecule delivery were determined at four different times after treatment with bevacizumab (days 1, 3, 5, and 8). Tumor growth and vessel density were significantly reduced after bevacizumab treatment, which also significantly increased tumor vessel maturity, and improved tumor hypoxia and small molecule delivery between days 3 and 5. These effects abated by day 8, suggesting that a time window for vessel normalization was opened between days 3 and 5 during bevacizumab treatment in this model. Apelin mRNA expression and plasma apelin levels decreased transiently at day 5 post-treatment, coinciding with vessel normalization. Thus, apelin is a potential indicator of the vessel normalization window during antiangiogenic therapy.

In Vitro Evidence for the Use of Astragali Radix Extracts as Adjuvant against Oxaliplatin-Induced Neurotoxicity.

The repeated exposure to the anticancer drug oxaliplatin induces a disabling, painful neuropathy. The current pharmacological treatments are unsatisfactory and unable to modify the complex nervous damage induced by the platin derivative. Recently, we described a system of cellular measures of oxidative stress as a method for studying features of oxaliplatin neurotoxicity and screening new compounds able to reduce oxaliplatin-induced neuropathy. Based on this experimental design, the protective properties of Astragali radix were studied comparing aqueous and two hydroalcoholic root extracts. Aqueous and the 20 % hydroalcoholic (20 % water) extract were prepared from plant material, while the 50 % hydroalcoholic (50 % water) extract was a commercial one. All of the extracts were characterized in terms of drug extract ratio and content of typical isoflavonoids, Astragaloside IV, and related saponins. Furthermore, the molecular weight of the polysaccharide fraction was evaluated by light scattering analysis. Oxaliplatin increased the superoxide anion production both in the neuronal-derived cell line SH-SY5Y and in primary cultures of rat cortical astrocytes. Aqueous and the 50 % hydroalcoholic extract (50 µg/mL) showed significant antioxidant effects. In astrocytes, aqueous and the 50 % hydroalcoholic extract showed protective effects against oxaliplatin-induced lipid peroxidation (malonyl dialdehyde levels), protein (carbonylated proteins), and DNA oxidation (8-OH-2-dG levels). The 50 % hydroalcoholic extract was the most active in preventing the activation of the apoptotic enzyme caspase-3 and it was the only able to stimulate astrocyte viability. None of the tested extracts interfered with the toxicity elicited by oxaliplatin in the human colon adenocarcinoma cell line HT-29. The pharmacological profile of Astragali radix extracts, in particular, the aqueous and 50 % hydroalcoholic extracts, makes these natural products candidates as therapeutic adjuvant agents against oxaliplatin neurotoxicity.

OPTIMIZATION OF PHYSICO–CHEMICAL PARAMETERS FOR THE EXTRACTION OF CHEBULINIC ACID FROM MEDICINAL HERBS

Chebulinic acid is a phenolic compound found in the fruits of Terminalia chebula (Haritaki), Phyllanthus emblica (Amla) and seeds of Dimocarpus longan (Longan) species etc. It showed many pharmacological activities including inhibition of cancer cell growth like human leukemia K562 cells, colon adenocarcinoma HT-29 cell lines, anti-neisseria gonorrhoeae activity etc. The present studies on optimization of physico-chemical parameters like effects of different solvents, soaking time, extraction time with hexane, particle size, different solvent percentages, different volumes of hexane with ethanol and methanol as solvents, pH and different weight dosages for the extraction of Chebulinic acid from Medicinal herbs. The highest chebulinic acid concentration for optimized conditions of Terminalia chebula fruit, Amla fruit and Longan seeds were 6.2 mg/mL, 4.8 mg/mL and 5.7 mg/mL respectively.

The effect of cell surface components on adhesion ability of Lactobacillus rhamnosus.

The aim of this study was to analyze the cell envelope components and surface properties of two phenotypes of Lactobacillus rhamnosus isolated from the human gastrointestinal tract. The ability of the bacteria to adhere to human intestinal cells and to aggregate with other bacteria was determined. L. rhamnosus strains E/N and PEN differed with regard to the presence of exopolysaccharides (EPS) and specific surface proteins. Transmission electron microscopy showed differences in the structure of the outer cell surface of the strains tested. Bacterial surface properties were analyzed by Fourier transform infrared spectroscopy, fatty acid methyl esters and hydrophobicity assays. Aggregation capacity and adhesion of the tested strains to the human colon adenocarcinoma cell line HT29 was determined. The results indicated a high adhesion and aggregation ability of L. rhamnosus PEN, which possessed specific surface proteins, had a unique fatty acid content, and did not synthesize EPS. Adherence of L. rhamnosus was dependent on specific interactions and was promoted by surface proteins (42–114 kDa) and specific fatty acids. Polysaccharides likely hindered bacterial adhesion and aggregation by masking protein receptors. This study provides information on the cell envelope constituents of lactobacilli that influence bacterial aggregation and adhesion to intestinal cells. This knowledge will help to understand better their specific contribution in commensal–host interactions and adaptation to this ecological niche.