Normal Human Fibroblast Cell Line Research Articles

Phenotype-specific apoptosis induced by three new triterpenoid saponins from Albizia glaberrima (Schumach. & Thonn.) Benth

As part of our search of new bioactive saponins from Cameroonian medicinal plants, phytochemical investigation of the roots of Albizia glaberrima led to the isolation of three new oleanane-type saponins, named glaberrimosides A–C (1–3). Their structures were established by direct interpretation of their spectral data, mainly HRESIMS, 1D NMR (1H, 13C NMR, and DEPT) and 2D NMR (COSY, ROESY, HSQC and HMBC) as 3-O-[α-l-arabinopyranosyl-(1→6)-[β-d-glucopyranosyl-(1→2)]-β-d-glucopyranosyl]-28-O-[β-d-glucopyranosyl-(1→6)-[β-d-glucopyranosyl-(1→2)]-β-d-glucopyranosyl]-oleanolic acid (1), 3-O-[α-l-arabinopyranosyl-(1→6)-[β-d-glucopyranosyl-(1→2)]-β-d-glucopyranosyl]-28-O-[β-d-glucopyranosyl-(1→6)-β-d-glucopyranosyl]-oleanolic acid (2), and 3-O-[β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl]-28-O-[β-d-glucopyranosyl-(1→6)-[β-d-fucopyranosyl-(1→2)]-β-d-glucopyranosyl]-oleanolic acid (3). The pro-apoptotic effect of the three saponins was evaluated on three human cell lines (pancreatic carcinoma AsPC-1, hematopoietic monocytic THP-1, and human fibroblast cell line BJ). Saponins 1–3 specifically induced apoptosis of pancreatic carcinoma cell (AsPC-1) in a dose-dependent manner. More interestingly, there were inactive on monocytic (THP-1) and normal human fibroblast (BJ) cell lines.

Impaired ATP6V0A2 expression contributes to Golgi dispersion and glycosylation changes in senescent cells.

Many genes and signaling pathways have been found to be involved in cellular senescence program. In the present study, we have identified 16 senescence-associated genes by differential proteomic analysis of the normal human diploid fibroblast cell line, TIG-1, and focused on ATP6V0A2. The aim of this study is to clarify the role of ATP6V0A2, the causal gene for ARCL2, a syndrome of abnormal glycosylation and impaired Golgi trafficking, in cellular senescence program. Here we showed that ATP6V0A2 is critical for cellular senescence; impaired expression of ATP6V0A2 disperses the Golgi structure and triggers senescence, suggesting that ATP6V0A2 mediates these processes. FITC-lectin staining and glycoblotting revealed significantly different glycosylation structures in presenescent (young) and senescent (old) TIG-1 cells; reducing ATP6V0A2 expression in young TIG-1 cells yielded structures similar to those in old TIG-1 cells. Our results suggest that senescence-associated impaired expression of ATP6V0A2 triggers changes in Golgi structure and glycosylation in old TIG-1 cells, which demonstrates a role of ATP6V0A2 in cellular senescence program.

Discovery of indeno[1,2-b]quinoxaline derivatives as potential anticancer agents

We have synthesized certain indeno[1,2-b]quinoxaline derivatives for antiproliferative evaluation. Among them, 11-{[3-(dimethylamino)propoxy]imino}-N-[3-(dimethylamino) propyl]-11H-indeno[1,2-b]quinoxaline-6-carboxamide (10a) was active against the growth of MDA-MB231, PC-3, and Huh-7 with IC50 values of 0.87 (selectivity index, SI = 36.22), 0.82 (SI = 38.43), and 0.64 μM (SI = 49.23) respectively. Compound 10a was inactive against the growth of normal human fetal lung fibroblast cell line (MRC-5) with an IC50 value of 31.51 μM. Its analogs, 10b and 10c, were also active against the growth of MB231, PC-3, and Huh-7 with IC50 values of <1.0 μM in each case. Our results have also indicated compounds 10a–10c exhibited comparable inhibitory activities against topo I and topo II with the positive compound 2 at a concentration of 10 μM. Mechanism studies indicated that compound 10a induced cell cycle arrest at S phase via activation of caspase-3, -7 and an increase in the protein expression of Bad and Bax but a decrease in expression of Bcl-2 and PARP, which consequently cause cell death. In addition, compound 10a attenuated the levels of phosphorylated Src, Akt-1, and Akt-2 protein levels but did not affect the total protein expression of Akt. We have also implanted human hepatocellular carcinoma cells into the yolk sac of zebrafish larvae and incubated larvae with various concentrations of 10a. Our results of the zebrafish xenograft assay confirmed the anti-tumor effect of 10ain vivo.

Design, Synthesis and In Vitro Activity of Anticancer Styrylquinolines. The p53 Independent Mechanism of Action.

A group of styrylquinolines were synthesized and tested for their anti-proliferative activity. Anti-proliferative activity was evaluated against the human colon carcinoma cell lines that had a normal expression of the p53 protein (HCT116 p53+/+) and mutants with a disabled TP53 gene (HCT116 p53-/-) and against the GM 07492 normal human fibroblast cell line. A SAR study revealed the importance of Cl and OH as substituents in the styryl moiety. Several of the compounds that were tested were found to have a marked anti-proliferative activity that was similar to or better than doxorubicin and were more active against the p53 null than the wild type cells. The cellular localization tests and caspase activity assays suggest a mechanism of action through the mitochondrial pathway of apoptosis in a p53-independent manner. The activity of the styrylquinoline compounds may be associated with their DNA intercalating ability.

Cell-penetrating and endoplasmic reticulum-locating TAT-IL-24-KDEL fusion protein induces tumor apoptosis.

Interleukin-24 (IL-24) is a unique IL-10 family cytokine that could selectively induce apoptosis in cancer cells without harming normal cells. Previous research demonstrated that intracellular IL-24 protein induces an endoplasmic reticulum (ER) stress response only in cancer cells, culminating in apoptosis. In this study, we developed a novel recombinant fusion protein to penetrate into cancer cells and locate on ER. It is composed of three distinct functional domains, IL-24, and the targeting domain of transactivator of transcription (TAT) and an ER retention four-peptide sequence KDEL (Lys-Asp-Glu-Leu) that link at its NH2 and COOH terminal, respectively. The in vitro results indicated that TAT-IL-24-KDEL inhibited growth in bladder cancer cells, as well as in non-small cell lung cancer cell line and breast cancer cell line, but the normal human lung fibroblast cell line was not affected, indicating the cancer specificity of TAT-IL-24-KDEL. Western blot analysis showed that apoptosis activation was induced by TAT-IL-24-KDEL through the ER stress-mediated cell death pathway. Treatment with TAT-IL-24-KDEL significantly inhibited the growth of human H460 xenografts in nude mice, and the tumor growth inhibition was correlated with increased hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. These findings suggest that the artificially designed recombinant fusion protein TAT-IL-24-KDEL may be highly effective in cancer therapy and worthy of further evaluation and development.

Different patterns of nuclear and mitochondrial penetration by the G3 PAMAM dendrimer and its biotin-pyridoxal bioconjugate BC-PAMAM in normal and cancer cells in vitro.

The intracellular localization and colocalization of a fluorescently labeled G3 amine-terminated cationic polyamidoamine (PAMAM) dendrimer and its biotin–pyridoxal (BC-PAMAM) bioconjugate were investigated in a concentration-dependent manner in normal human fibroblast (BJ) and squamous epithelial carcinoma (SCC-15) cell lines. After 24 hours treatment, both cell lines revealed different patterns of intracellular dendrimer accumulation depending on their cytotoxic effects. Cancer cells exhibited much higher (20-fold) tolerance for native PAMAM treatment than fibroblasts, whereas BC-PAMAM was significantly toxic only for fibroblasts at 50 µM concentration. Fibroblasts accumulated the native and bioconjugated dendrimers in a concentration-dependent manner at nontoxic range of concentration, with significantly lower bioconjugate loading. After reaching the cytotoxicity level, fluorescein isothiocyanate-PAMAM accumulation remains at high, comparable level. In cancer cells, native PAMAM loading at higher, but not cytotoxic concentrations, was kept at constant level with a sharp increase at toxic concentration. Mander’s coefficient calculated for fibroblasts and cancer cells confirmed more efficient native PAMAM penetration as compared to BC-PAMAM. Significant differences in nuclear dendrimer penetration were observed for both cell lines. In cancer cells, PAMAM signals amounted to ~25%–35% of the total nuclei area at all investigated concentrations, with lower level (15%–25%) observed for BC-PAMAM. In fibroblasts, the dendrimer nuclear signal amounted to 15% at nontoxic and up to 70% at toxic concentrations, whereas BC-PAMAM remained at a lower concentration-dependent level (0.3%–20%). Mitochondrial localization of PAMAM and BC-PAMAM revealed similar patterns in both cell lines, depending on the extracellular dendrimer concentration, and presented significantly lower signals from BC-PAMAM, which correlated well with the cytotoxicity.

The effects of Malaysian propolis and Brazilian red propolis on connective tissue fibroblasts in the wound healing process.

BackgroundTo evaluate and compare the effects of ethanolic extracts of Malaysian propolis and Brazilian red propolis at different concentrations on the migration and proliferation of fibroblast cells.MethodsMalaysian and Brazilian red propolis crude samples were extracted using ethanol. Their wound healing effects were tested in vitro on the normal human fibroblast cell line CRL-7522. Cell migration and proliferation assays were carried out using propolis concentrations of 1, 10, 100, 250, 500 and 1000 μg/mL. The data were analyzed using one-way ANOVA and post hoc Bonferroni tests (α = 0.05).ResultsMalaysian and Brazilian red propolis followed a concentration-dependent increasing and decreasing trend. Malaysian propolis showed the fastest migration rate at 250 μg/mL which was statistically significant (p < 0.05) and maximum proliferation at 500 μg/mL with no significant difference (p > 0.05) compared to control. Brazilian red propolis showed a slight increase in migration and proliferation at 10 and 100 μg/mL, respectively with no significant difference (p > 0.05) compared to control, while concentrations above these conferred inhibitory effects.ConclusionMalaysian and Brazilian red propolis show potential to assist in wound healing, depending on their concentration.

Microbial Metabolism of Atovaquone and Cytotoxicity of the Produced Phase I Metabolite.

Atovaquone is a hydroxynaphthoquinone with selective action in the mitochondrial respiratory chain of malaria parasite. It is employed for both the treatment and prevention of malaria, in a combination with proguanil. The aim of this study was to elucidate the in vitro metabolites from atovaquone and to evaluate their cytotoxic activities. The biotransformation of atovaquone was performed using Mucor rouxii NRRL 1894, Cunninghamella echinulata var. elegans ATCC 8688a and C. elegans ATCC 10028b, which have been reported as microbial models of mammalian drug metabolism. Experiments were also carried out with two probiotic strains from the human intestinal tract: Bifidobacterium sp. and Lactobacillus acidophilus. The phase I metabolite was isolated, its chemical structure was elucidated and its toxicity was evaluated using the neoplastic cell line SKBR-3 derived from human breast cancer and normal human fibroblast cell line GM07492-A. Cell cytotoxicity assays were also carried out with atovaquone. Only the fungi were able to convert atovaquone to metabolite trans-3-[4'-(4″-chlorophenyl)cyclohexyl)-1,2-dioxo-dihydro-1H-indene-3-carboxylic acid. The metabolite displayed 50 % inhibitory concentration (IC50) values of 110.20±2.2 and 108.80±1.5µmol/L against breast cancer cell line SKBR-3 and fibroblasts cell line GM07492-A, respectively. The IC50 values of atovaquone were 282.30±1.8 and 340.50±1.4µmol/L against breast cancer and normal fibroblasts cell lines, respectively. The produced metabolite was more toxic than atovaquone and was not selective to normal or cancer cell lines. The present study is the first to report the production of atovaquone metabolite.

Reduced mtDNA copy number increases the sensitivity of tumor cells to chemotherapeutic drugs.

Many cancer drugs are toxic to cells by activating apoptotic pathways. Previous studies have shown that mitochondria have key roles in apoptosis in mammalian cells, but the role of mitochondrial DNA (mtDNA) copy number variation in the pathogenesis of tumor cell apoptosis remains largely unknown. We used the HEp-2, HNE2, and A549 tumor cell lines to explore the relationship between mtDNA copy number variation and cell apoptosis. We first induced apoptosis in three tumor cell lines and one normal adult human skin fibroblast cell line (HSF) with cisplatin (DDP) or doxorubicin (DOX) treatment and found that the mtDNA copy number significantly increased in apoptotic tumor cells, but not in HSF cells. We then downregulated the mtDNA copy number by transfection with shRNA-TFAM plasmids or treatment with ethidium bromide and found that the sensitivity of tumor cells to DDP or DOX was significantly increased. Furthermore, we observed that levels of reactive oxygen species (ROS) increased significantly in tumor cells with lower mtDNA copy numbers, and this might be related to a low level of antioxidant gene expression. Finally, we rescued the increase of ROS in tumor cells with lipoic acid or N-acetyl-L-cysteine and found that the apoptosis rate decreased. Our studies suggest that the increase of mtDNA copy number is a self-protective mechanism of tumor cells to prevent apoptosis and that reduced mtDNA copy number increases ROS levels in tumor cells, increases the tumor cells' sensitivity to chemotherapeutic drugs, and increases the rate of apoptosis. This research provides evidence that mtDNA copy number variation might be a promising new therapeutic target for the clinical treatment of tumors.

Preparation and characterization of chitosan–hydroxyapatite–glycopolymer/Cloisite 30 B nanocomposite for biomedical applications

A new composite was synthesized from chitosan/hydroxyapatite and glycopolymer grafted onto a Cloisite 30B clay surface (CS/HAP/clay-Gh) in aqueous media via a solvent casting and evaporation method. Another composite containing chitosan/hydroxyapatite and pristine Cloisite 30B (CS/HAP/clay) was prepared and compared with the (CS/HAP/clay-Gh). The resulting composites were characterized using various analytical tools such as Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), thermal gravimetric analyses (TGA) and scanning electron microscopy (SEM). Water uptake capability or swelling was tested for each composite. The cytotoxicity of the resulting tri-component composites was tested against a breast carcinoma cell line (MCF-7), liver carcinoma cell line (HepG-2) and a normal human skin fibroblast cell line (BJ-1) by MTT [3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide] as well as LDH (lactate dehydrogenase) assay. The obtained preliminary results showed good properties for CS/HAP/clay-Gh on the way to be used in the future in the biomedical applications.

Antioxidative and cytotoxic potential of some Chenopodium L. species growing in Poland

The cytotoxic and antioxidant properties of lipophilic compounds extracted from different parts of four Chenopodium L. (Chenopodium album, Chenopodium hybridum, Chenopodium rubrum and Chenopodium urbicum) species were evaluated. The highest phenolic content was found in herb and seeds of all examined plants. Large amounts of free polyphenols were observed in herb extracts of C. album (3.36mg/g DW), seeds of C. urbicum (3.87mg/g DW) and roots of C. urbicum (1.52mg/g DW). The cytotoxic activities of the extracts were assessed against human lung carcinoma A-549 and ovarian carcinoma TOV-112D and normal human fibroblast cell lines. Our study demonstrated that the extracts from the herb of C. rubrum and C. urbicum had the best antioxidant effect of all the extracts analyzed. Most of the extracts tested exhibited low cytotoxicity. However, the extracts from herb and seeds of C. album and C. hybridum showed the significant antiproliferative effect on the TOV-112 cell line.It can be concluded that antioxidant activity and phenolic composition differ mainly between plant parts and are quite similar between the plants, when the same plant part is analyzed. Thus, the Chenopodium extracts could be used as a readily accessible source of natural antioxidants, and may be used in the pharmaceutical industry and for food supplements production.

Induction of Apoptosis in the Human Prostate Cancer Cell Line DU-145 by a Novel Micronutrient Formulation

Prostate cancer, the most frequently diagnosed cancer in men, primarily affects males aged 55 and older and is more common in African Americans than Caucasians. Once the cancer has metastasized, current treatments are generally ineffective. We have identified a novel anti-neoplastic agent, a specifically designed nutrient mixture (NM), containing ascorbic acid, lysine, proline and green tea extract that demonstrates a broad spectrum of anti-tumor activity against a number of human cancer cell lines. In a previous study NM significantly inhibited prostate tumor in nude mice. In this study, we tested whether the formulation exerts its anti-tumor effects through induction of apoptosis on prostate cancer cell line DU-145. The effect of the nutrient mixture (NM) on cell growth inhibition in DU-145 cells was examined by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Morphological changes and caspase activation associated with apoptosis induction was checked by H&E staining and Live Green Caspase assay, respectively. The NM was found to be slightly toxic to DU-145 cells at 100 μg/ml, but significantly toxic at 500 μg/ml and 1000 μg/ml. Percentage of cells undergoing apoptosis also increased from 6% at 100 μg/ml to 49% at 500 μg/ml and 83% at 1000 μg/ml, with greater number of cells showing morphological changes such as condensed nuclei and an acidophilic cytoplasm at higher concentrations. For the purpose of comparison, NM was also tested on a normal human dermal fibroblast (NHDF) cell line which exhibited far less apoptosis induction as compared to DU-145 cells. The percentage of cells undergoing apoptosis in case of NHDF cells was 7% at 100 μg/ml, 25.6% at 500 μg/ml and 76.5% at 1000 μg/ml. Our results demonstrate that the NM is effective in inhibiting cancer cell viability and inducing apoptosis in prostate cancer DU-145 cells and can thus be used as an effective treatment for prostate cancer.

Xanthohumol induces apoptosis and S phase cell cycle arrest in A549 non-small cell lung cancer cells.

Background:Xanthohumol, a major prenylated chalcone found in female hop plant, Humulus lupulus, was reported to have various chemopreventive and anti-cancer properties. However, its apoptotic effect on human alveolar adenocarcinoma cell line (A549) of non-small cell lung cancer (NSCLC) was unknown.Objective:This study aimed to investigate the effects of xanthohumol on apoptosis in A549 human NSCLC cells.Materials and Methods:A549 cell proliferation was determined by sulforhodamine B assay. Morphological changes of the cells were studied via phase contrast and fluorescent microscopy. Induction of apoptosis was assessed by Annexin-V fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) staining, DNA fragmentation (TUNEL) assay mitochondrial membrane potential assay, cell cycle analysis, and caspase activity studies.Results:Xanthohumol was found to decrease cell proliferation in A549 cells but had relatively low cytotoxicity on normal human lung fibroblast cell line (MRC-5). Typical cellular and nuclear apoptotic features were also observed in A549 cells treated with xanthohumol. Onset of apoptosis in A549 cells was further confirmed by externalization of phosphatidylserine, changes in mitochondrial membrane potential, and DNA fragmentation in the cells after treatment. Xanthohumol induced accumulation of cells in sub G1 and S phase based on cell cycle analysis and also increased the activities of caspase-3, -8, and -9.Conclusion:This work suggests that xanthohumol as an apoptosis inducer, may be a potent therapeutic compound for NSCLC.

Development of Rhamnolipid Based White board cleaner

In the present study biosurfactant based white board cleaner was formulated, using column purified rhamnolipid produced by Pseudomonas aeruginosa. A rhamnolipid concentration of 0.01% was finalized, as it was efficiently able to remove fresh and aged white board marker stains on white board. The rhamnolipid formulation was found to be stable over a tested span of 6 months and it did not exhibit any cytotoxicity on Human Lung Epithelial Cell Line (L132) and Normal Human Skin Fibroblast (NHSF) cell line. The cleaning efficiency was equivalent to conventional synthetic surfactants, but the biosurfactant based approach is more environment friendly and biocompatible. DOI: http://dx.doi.org/10.3126/ijasbt.v2i4.11589 Int J Appl Sci Biotechnol, Vol. 2(4): 570-573

A novel human interleukin-24 peptide created by computer-guided design contributes to suppression of proliferation in esophageal squamous cell carcinoma Eca-109 cells.

Based on the three-dimensional modeling structure of human interleukin-24 (hIL-24) and its most likely active position predicted by solvent accessibility and apparent electrostatic properties, a novel hIL-24 peptide M1 was created by computer-guided molecular design. The cytotoxicity and cell selectivity of M1 were examined in three human carcinoma cell lines and one normal human embryo lung fibroblast cell line (HEL). MTT assay showed that M1 induced growth arrest in two IL-20 receptor complex-positive cancer cell lines (the esophageal squamous cell carcinoma cell line Eca-109 and the melanoma cell line A375), and antibodies against IL-24 or IL-20 receptor complexes significantly neutralized the inhibitory activity. Moreover, M1 had almost no cytotoxicity on the lung cancer A549 cell line, which lacks a full complement of the IL-20 receptor complexes, or on HEL cells that express the IL-20 receptor complexes. These findings demonstrate that M1 could act as an excellent candidate for the induction of growth arrest on receptor complex-positive cancer cells. In summary, the M1 peptide may represent a novel anticancer agent for esophageal squamous cell carcinoma therapy due to its cancer cell selectivity and its relatively low cytotoxicity to normal cells.

Apoptotic potential role of Agave palmeri and Tulbaghia violacea extracts in cervical cancer cells.

Cervical cancer, a gynaecological malignant disorder, is a common cause of death in females in Sub-Saharan Africa, striking nearly half a million of lives each year worldwide. Currently, more than 50 % of all modern drugs in clinical use are of natural products, many of which have an ability to control cancer cells (Madhuri and Pandey, Curr Sci 96:779-783, 2009; Richter, Traditional medicines and traditional healers in South Africa, 2003). In South Africa, plants used to treat cancer are rare even though majority of our population continue to put their trust in traditional medicine. In this study we aimed to screen Agave palmeri (AG) and Tulbaghia violacea (TV) for potential role in inducing cell death in cervical cancer cell lines HeLa and ME-180, and in normal human fibroblast cell line KMST-6 cell lines. To achieve this, AG and TV crude extracts were utilized to screen for apoptosis induction, inhibition of cell proliferation followed by elucidation of the role of Bax, Bcl-2, p53, Rb, RBBP and Mdm2 genes in cervical cancer. In brief, plant leaves and roots were collected, crushed and methanolic extracts obtained. Different concentrations of the stock extracts were used to treat cancer cells and measure cell death using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay and flow cytometry. Western blot was applied to measure gene expression at protein level using RBBP6, p53, Mdm2, Rb, Bax, Bcl-2 and β-actin mouse monoclonal primary antibodies (IgG) and goat anti mouse coupled with horseradish peroxidase secondary antibody from Santa Cruz Biotechnology and real time-PCR was used for mRNA expression level. Plant extracts of AG and TV were time (24 h) and dose (50, 100, 150 μg/ml) dependent in their induction of cell death with an IC50 ~ 150 μg/ml. A further mixed respond by several genes was observed following treatment with the two plant extracts where RBBP6 was seen to be spliced in cancer cells while Bax was induced and Bcl-2 was inhibited with the levels of p53 remaining the same. The two plant extracts do induce cell death, in a p53 independent manner.

Essential oil content of the rhizome of Curcuma purpurascens Bl. (Temu Tis) and its antiproliferative effect on selected human carcinoma cell lines.

Curcuma purpurascens Bl., belonging to the Zingiberaceae family, is known as temu tis in Yogyakarta, Indonesia. In this study, the hydrodistilled dried ground rhizome oil was investigated for its chemical content and antiproliferative activity against selected human carcinoma cell lines (MCF7, Ca Ski, A549, HT29, and HCT116) and a normal human lung fibroblast cell line (MRC5). Results from GC-MS and GC-FID analysis of the rhizome oil of temu tis showed turmerone as the major component, followed by germacrone, ar-turmerone, germacrene-B, and curlone. The rhizome oil of temu tis exhibited strong cytotoxicity against HT29 cells (IC50 value of 4.9 ± 0.4 μg/mL), weak cytotoxicity against A549, Ca Ski, and HCT116 cells (with IC50 values of 46.3 ± 0.7, 32.5 ± 1.1, and 35.0 ± 0.3 μg/mL, resp.), and no inhibitory effect against MCF7 cells. It exhibited mild cytotoxicity against a noncancerous human lung fibroblast cell line (MRC5), with an IC50 value of 25.2 ± 2.7 μg/mL. This is the first report on the chemical composition of this rhizome's oil and its selective antiproliferative effect on HT29. The obtained data provided a basis for further investigation of the mode of cell death.

Cytotoxic effect of Alpinia scabra (Blume) Náves extracts on human breast and ovarian cancer cells

BackgroundAlpinia scabra, locally known as 'Lengkuas raya’, is an aromatic, perennial and rhizomatous herb from the family Zingiberaceae. It is a wild species which grows largely on mountains at moderate elevations in Peninsular Malaysia, but it can also survive in the lowlands like in the states of Terengganu and Northern Johor. The present study reports the cytotoxic potential of A. scabra extracts from different parts of the plant.MethodsThe experimental approach in the present study was based on a bioassay-guided fractionation. The crude methanol and fractionated extracts (hexane, chloroform and water) from different parts of A. scabra (leaves, rhizomes, roots and pseudo stems) were prepared prior to the cytotoxicity evaluation against human ovarian (SKOV-3) and hormone-dependent breast (MCF7) carcinoma cells. The identified cytotoxic extracts were then subjected to chemical investigations in order to identify the active ingredients. A normal human lung fibroblast cell line (MRC-5) was used to determine the specificity for cancerous cells. The cytotoxic extracts and fractions were also subjected to morphological assessment, DNA fragmentation analysis and DAPI nuclear staining.ResultsThe leaf (hexane and chloroform) and rhizome (chloroform) extracts showed high inhibitory effect against the tested cells. Ten fractions (LC1-LC10) were yielded after purification of the leaf chloroform extract. Fraction LC4 which showed excellent cytotoxic activity was further purified and resulted in 17 sub-fractions (VLC1-VLC17). Sub-fraction VLC9 showed excellent cytotoxicity against MCF7 and SKOV-3 cells but not toxic against normal MRC-5 cells. Meanwhile, eighteen fractions (RC1-RC18) were obtained after purification of the rhizome chloroform extract, of which fraction RC5 showed cytotoxicity against SKOV-3 cells with high selectivity index. There were marked morphological changes when observed using phase-contrast inverted microscope, DAPI nuclear staining and also DNA fragmentations in MCF7 and SKOV-3 cells after treatment with the cytotoxic extracts and fractions which were indicative of cell apoptosis. Methyl palmitate and methyl stearate were identified in the hexane leaf extract by GC-MS analysis.ConclusionsThe data obtained from the current study demonstrated that the cell death induced by cytotoxic extracts and fractions of A. scabra may be due to apoptosis induction which was characterized by apoptotic morphological changes and DNA fragmentation. The active ingredients in the leaf sub-fraction VLC9 and rhizome fraction RC5 may lead to valuable compounds that have the ability to kill cancer cells but not normal cells.

Potential treatments to reduce phorbol esters levels in jatropha seed cake for improving the value added product

Jatropha seed cake contains high amounts of protein and other nutrients, however it has a drawback due to toxic compounds. The aim of this study was to investigate the methods applied to detoxify the main toxin, phorbol esters in jatropha seed cake, to a safe and acceptable level by maintaining the nutritional values. Phorbol esters are tetracyclic diterpenoids-polycyclic compounds that are known as tumor promoters and hence exhibited the toxicity within a broad range of species. Mismanagement of the jatropha waste from jatropha oil industries would lead to contamination of the environment, affecting living organisms and human health through the food chain, so several methods were tested for reducing the toxicity of the seed cake. The results from this investigation showed that heat treatments at either 120°C or 220°C for 1 hour and then mixing with adsorbing bentonite (10%), nanoparticles of zinc oxide (100 μg/g) plus NaHCO3 at 4%, followed by a 4-week incubation period yielded the best final product. The remaining phorbol esters concentration (0.05–0.04 mg/g) from this treatment was less than that reported for the nontoxic jatropha varieties (0.11–0.27 mg/g). Nutritional values of the seed cake after treatment remained at the same levels found in the control group and these values were crude protein (20.47–21.40 + 0.17–0.25%), crude lipid (14.27–14.68 + 0.13–0.14%) and crude fiber (27.33–29.67 + 0.58%). A cytotoxicity test conducted using L929 and normal human dermal fibroblast cell lines confirmed that most of the toxic compounds, especially phorbol esters, were shown as completely eliminated. The results suggested that the detoxification of phorbol esters residues in the jatropha seed cake was possible while it also retained nutritional values. Therefore, the methods to detoxify phorbol esters are necessary to minimize the toxicity of jatropha seed cake. Further, it is essential to reduce the possible environmental impacts that may be generated throughout the jatropha waste-handling process. However additional tests such as digestibility as well as acceptability of the treated jatropha seed cake should be conducted using both in vivo and in vitro studies before recommending the jatropha seed cake as a source of renewable animal feed and other value-added products.

Degradation of p12 Subunit by CRL4Cdt2 E3 Ligase Inhibits Fork Progression after DNA Damage

After acute DNA damage, the cell arrests S-phase progression by inhibiting origin initiation and fork progression to repair damaged DNA. The intra-S-phase checkpoint kinase Chk1 phosphorylates Cdc25A to target the latter for degradation by CRL1(β-TrCP) and so inhibit origin firing. The mechanism for inhibiting fork progression, however, has not been identified. Here, we show that degradation of p12, the fourth subunit of DNA polymerase δ, is critical for inhibiting fork progression. CRL4(Cdt2) is an E3 ligase that ubiquitinates and degrades p12 after UV treatment. Cells expressing a stable form of p12 exhibit UV-resistant DNA synthesis. DNA fiber assay and alkaline-sucrose gradient assay demonstrate that the impairment of fork progression after DNA damage requires p12 degradation. These results suggest that ubiquitination of p12 through CRL4(Cdt2) and subsequent degradation form one mechanism by which a cell responds to DNA damage to inhibit fork progression.