Induction of apoptosis in tumor cells by naturally occurring sulfur-containing compounds

Chemoprevention is regarded as one of the most promising and realistic approaches in the prevention of human cancer. Among naturally occurring products, sulfur-containing compounds (OSCs), especially garlic compounds (GCs) and isothiocyanates (ITCs), represent two important and promising chemopreventive families because of their potent chemopreventive effects in various in vivo and in vitro models. In recent years, numerous investigations have shown that sulfur-containing compounds induce apoptosis in multiple cell lines and experimental animals. In the course of apoptosis induction by GCs and ITCs, multiple signal-transduction pathways and apoptosis intermediates are modulated. In particular, modulation of MAPKs and production of reactive oxygen species (ROS) seem to play pivotal roles in apoptosis induction by most GCs and ITCs. However, the role of P53 is still controversial. Based on present knowledge, GCs and ITCs may target not only the metabolism of carcinogens but also apoptosis signaling molecules. The effects of ITCs and GCs at multiple points of cancer development make these compounds highly promising candidates in cancer chemoprevention. However, the mechanisms of their anticancer effects are not fully understood, and further studies are required, especially to elucidate the role of cell-death receptors (the extrinsic pathway) and whether these agents induce apoptotic effects in non-tumor cells.     

beta1,4-N-Acetylglucosaminyltransferase III potentiates beta1 integrin-mediated neuritogenesis induced by serum deprivation in Neuro2a cells

Aspects of the biological significance of the bisecting N-acetylglucosamine (GlcNAc) structure on N-glycans introduced by beta1,4-N-acetylglucosaminyltransferase III (GnT-III) in Neuro2a cell differentiation are demonstrated. The overexpression of GnT-III in the cells led to the induction of axon-like processes with numerous neurites and swellings, in which beta1 integrin was localized, under conditions of serum starvation. This enhancement in neuritogenesis was suppressed by either the addition of a bisecting GlcNAc-containing N-glycan or erythroagglutinating phytohemagglutinin (E(4)-PHA), which preferentially recognizes the bisecting GlcNAc. GnT-III-promoted neuritogenesis was also significantly perturbed by treatment with a functional blocking anti-beta1 integrin antibody. In fact, beta1 integrin was found to be one of the target proteins of GnT-III, as confirmed by a pull-down assay with E(4)-PHA. These data suggest that N-glycans with a bisecting GlcNAc on target molecules, such as beta1 integrin, play important roles in the regulation of neuritogenesis.     

Characterization of naturally occurring auxotrophic mammalian cells.

In a previous study, several cultured cell lines were detected which are naturally occurring auxotrophs. In this investigation, the enzyme deficienceis involved are described. It is demonstrated that the Chinese hamster cell lines CHO(K1), YH21, RJK-36, and CHW-1102 are deficient in cystathionase and argininosuccinate synthetase. In addition, CHO (K1) and CHW-1102 were found to lack argininosuccinate lyase. CHW-1102 cells were also found to be unable to proliferate in medium containing branched-chain alpha-keto acids in place of the corresponding L-amino acids since CHW-1102 cells lack branched-chain aminotransferase.     

Myricetin, a naturally occurring flavonol, ameliorates insulin resistance induced by a high-fructose diet in rats

The present study was conducted to explore the effects of myricetin on insulin resistance in rats fed for 6 weeks with a diet containing 60% fructose. Repeated intravenous (i.v.) injection of myricetin (1 mg/kg per injection, 3 times daily) for 14 days was found to significantly decrease the high glucose and triglyceride levels in plasma of fructose chow-fed rats. Also, the higher degree of insulin resistance in fructose chow-fed rats as measured by homeostasis model assessment of basal insulin resistance was significantly decreased by myricetin treatment. Myricetin increased the whole-body insulin sensitivity in fructose chow-fed rats, as evidenced by the marked elevation of composite whole-body insulin sensitivity index during the oral glucose tolerance test. Myricetin was found to reverse the defect in expression of insulin receptor substrate-1 (IRS-1) and the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) in soleus muscle of fructose chow-fed rats under the basal state, despite the protein expression of insulin receptor (IR). Increased basal phosphorylation of IR and IRS-1 as well as Akt was observed in parallel. The reduced level of insulin action on phosphorylation of IR, IRS-1 and Akt in soleus muscle of fructose chow-fed rats was reversed by myricetin treatment. Furthermore, myricetin treatment improved the defective insulin action on the translocation of glucose transporter subtype 4 (GLUT 4) in insulin-resistant soleus muscle. These findings indicate that myricetin improves insulin sensitivity through the enhancement of insulin action on IRS-1-associated PI 3-kinase and GLUT 4 activity in soleus muscles of animals exhibiting insulin resistance.     

Sulfated proteoglycans synthesized by Neuro 2a neuroblastoma cells: Comparison between cells with and without ganglioside-induced neurites

Mouse neuroblastoma Neuro 2a cells are known to extend neurite-like processes in response to gangliosides added to the culture medium. We compared the structural features of proteoglycans (PG) synthesized by conventional Neuro 2a cells with those of neurite-bearing cells. Two different proteoglycans labeled with [³⁵S]sulfate, namely, chondroitin sulfate proteoglycan (CS-PG) and heparan sulfate proteoglycan (HS-PG), were found both in the cell layer and in the culture medium of the conventional cells. CS-PG isolated from the cell layer had a K_av value of 0.38 on Sepharose CL-6B, and had CS side chains with Mr of 27,000. HS-PG in the cell layer was slightly larger (K_av of 0.33) in terms of hydrodynamic size than CS-PG, and the apparent Mr of the heparan sulfate side chains was 10,000. The structural parameters of CS-PG and HS-PG isolated from the medium were almost identical to those of the PGs in the cell layer. In addition to these PGs, single-chain HS, with an average Mr of 2,500, was observed only in the cell layer and this component was the major sulfated component in the cell layers of both control and ganglioside treated cells. The neurite-bearing cells also synthesized both CS-PG and HS-PG which were very similar in hydrodynamic size to those synthesized by the conventional cells, but the size of HS side chains was greater. Radioactivity, as³⁵S, of each sulfated component from the gangliosideteated culture seemed to be slightly less than that of the corresponding component from the control culture. These findings indicate that the marked morphological change in Neuro 2a cells, induced by gangliosides is not accompanied by major changes in the synthesis of PGs.     

Homocysteine-induced toxicity increases TG2 expression in Neuro2a cells

High levels of homocysteine promote cell damage mainly through induction of oxidative stress, endoplasmic reticulum (ER) stress, and activation of pro-inflammatory factors. The effects of homocysteine were here examined in the continuously dividing neuroblastoma cell line Neuro2a. Cell treatment with homocysteine (100–500 μM) for 4 h increased ROS production while reducing cell viability in a dose-dependent manner. Cell exposure to 250 μM homocysteine was able to induce transglutaminase 2 up-regulation and increased in situ transglutaminase activity. These effects were prevented by the incubation with the transglutaminase activity inhibitor cystamine. Homocysteine also induced NF-κB activation that seemed associated with transglutaminase 2 up-regulation since the specific NF-κB inhibition by SN50 was able to reduce transglutaminase expression and activity levels. In the light of these observations, it may be postulated that TG2 up-regulation is involved in cell response to homocysteine-induced stress, in which NF-κB activation plays also a pivotal role.     

Identification of myeloid derived suppressor cells in dogs with naturally occurring cancer

Dogs with naturally occurring cancer represent an important large animal model for drug development and testing novel immunotherapies. However, poorly defined immunophenotypes of canine leukocytes have limited the study of tumor immunology in dogs. The accumulation of myeloid derived suppressor cells (MDSCs) is known to be a key mechanism of immune suppression in tumor-bearing mice and in human patients. We sought to identify MDSCs in the blood of dogs with cancer. Peripheral blood mononuclear cells (PBMCs) from dogs with advanced or early stage cancer and from age-matched healthy controls were analyzed by flow cytometry and microscopy. Suppressive function was tested in T cell proliferation and cytokine elaboration assays. Semi-quantitative RT-PCR was used to identify potential mechanisms responsible for immunosuppression. PBMCs from dogs with advanced or metastatic cancer exhibited a significantly higher percentage of CD11b(+)CD14(-)MHCII(-) cells compared to dogs diagnosed with early stage non-metastatic tumors and healthy dogs. These CD11b(+) CD14(-)MHCII(-) cells constitute a subpopulation of activated granulocytes that co-purify with PBMCs, display polymorphonuclear granulocyte morphology, and demonstrate a potent ability to suppress proliferation and IFN-γ production in T cells from normal and tumor-bearing donors. Furthermore, these cells expressed hallmark suppressive factors of human MDSC including ARG1, iNOS2, TGF-β and IL-10. In summary our data demonstrate that MDSCs accumulate in the blood of dogs with advanced cancer and can be measured using this three-marker immunophenotype, thereby enabling prospective studies that can monitor MDSC burden.     

Stereochemical aspects of synthetic and naturally occurring 2-hydroxy fatty acids. Their absolute configurations and assays of optical purity

d- and l-Cerebronic acid⁵, ⁶ methyl esters were obtained from a dl-mixture by converting the racemic ester to the l-acetylmandelates and separating the diastereomers by thin-layer chromatography; the diasteromers were then decomposed with mild acid methanolysis. The optical purities of the d- and l-enantiomers were determined as described below and found to be 82.5% and 87.1%, respectively. Absolute configurations of the enantiomers were confirmed by their ORD spectra; specific rotations at the peak of the anomalous curves at 223 nm of the d- and l-isomers were ?989 ° and +1120 °, respectively. Considering their optical purities, the actual maximum rotations of these enantiomers were calculated as ?1520 ° and +1509 °, respectively. The conditions for the reaction of l-acetylmandelyl chloride and 2-hydroxy fatty acid esters were modified to be suitable for the optical assay of 2-hydroxy fatty acid methyl esters. The method was also shown to be applicable to the assay of 3-hydroxy isomers and possibly to other positional isomers. The absolute configuration of 2-hydroxy fatty acid methyl esters obtained from calf brain cerebrosides and from yeast cerebrins were determined to be d(?), confirming previous assignments. Methyl cerebronate obtained from its less soluble strychnine salt was found to be the l(+)-enantiomer, contrary to a previous assignment. Methyl d-cerebronate was hydrolyzed with tetrahydrofuran-HCl to obtain D-cerebronic acid without racemization.     

灵芝肽诱导人肝癌HepG2细胞凋亡的研究

研究了灵芝肽(GLP)在体外对人肝癌HepG2细胞凋亡的影响,并初步探讨了其作用机制。结果显示,透射电镜下可见细胞染色质浓缩、聚集于核边缘成块状,形成典型的凋亡小体;GLP使HepG2细胞阻滞于G0/G1期,随着GLP浓度升高,其G0/G1期的细胞比例随之增加;同时细胞的早期、晚期和总的凋亡率亦均随之增加,存在剂量-效应关系;Western blotting检测结果显示,抑制凋亡基因bcl-2和survivin表达下调,而促凋亡基因p53表达上调,并且都存在剂量依赖性;细胞凋亡的关键蛋白酶caspase-3被激活,并且caspase-3酶活性与GLP浓度亦有剂量依赖性。提示GLP体外可诱导人肝癌HepG2细胞凋亡,其作用机制可能与bcl-2和survivin表达下调、p53表达上调及Caspase-3被激活有关。     

Up-regulation of heme biosynthesis during differentiation of Neuro2a cells

Heme is an iron-containing tetrapyrrole molecule that functions as a prosthetic group for proteins such as mitochondrial respiratory enzymes. Several studies have suggested that heme has essential functions in the construction and maintenance of the nervous system. In this study, the contents of three biologically important forms of heme (types a, b, and c) and the expression of heme biosynthetic enzymes were examined in differentiating Neuro2a cells. During neuronal differentiation, there were increases in the cellular heme levels and increases in the mRNA levels for the rate-limiting enzymes of heme biosynthesis, such as aminolevulinic acid synthase (ALAS; EC 2.3.1.37) and coproporphyrinogen oxidase (EC 1.3.3.3). With respect to heme contents, heme b increased in the late phase of differentiation, but no apparent increase in heme a or b was observed in the early phase. In contrast, heme c (cytochrome c) markedly increased during the early phase of differentiation. This change preceded the increase in heme b and the up-regulation of the mRNA levels for heme biosynthetic enzymes. This study suggests the up-regulation of heme biosynthesis and differential regulation of the heme a, b, and c levels during neuronal differentiation.     

Stabilization of human haemoglobin by naturally occurring osmolytes

The influence of the naturally occurring osmolytes xylitol, glycine and betaine on the thermal stability of human haemoglobin was investigated. Experiments were made in the temperature range of 55–70 °C, adding up to 30% w/w of osmolytes to the protein solution. All the additives stabilized haemoglobin, with xylitol and glycine appearing more effective. A kinetic analysis based on the Lumry–Eyring inactivation scheme showed that the denaturation process can be described by a second-order rate expression, with an apparent activation energy ranging from 45 to 82 kcal/mol.     

Purification of naturally occurring peptides by reversed-phase HPLC

Reversed-phase high performance liquid chromatography (HPLC) has become the method of choice for the purification of peptides and small proteins (M(r) < 10,000 Da) from natural sources. The technique combines high resolution and recovery with ease and speed of operation and is applicable to a wide range of peptides with different physicochemical properties. This protocol describes procedures for (1) the extraction of a biologically active peptide from animal tissue, (2) concentration of the extracts and partial purification on Sep-Pak cartridges, and (3) purification to near homogeneity on a range of silica-based HPLC columns. Standard operating procedures involve acetonitrile as organic modifier, trifluoroacetic acid as ion-pairing reagent and sequential chromatographies on octadecyl (C18), butyl (C4) and diphenyl wide-pore (300 A) columns under gradient elution conditions. The limiting factor in the time taken to isolate a peptide is usually the speed at which assays to detect the peptide can be performed, but purifications can generally be accomplished within 1 or 2 weeks.     

Apoptosis in granulose cells induced by intrafollicular peptide

Ovarian follicular fluid peptide (OFFP) purified from sheep ovaries has been earlier shown to induce degeneration of ovarian follicles in mice. In the present study, whether the effect of OFFP on granulosa cells was similar to apoptosis was studied using three parameters. Immature mice injected with pregnant mare serum gonadotropin on day 0 were administered with 10 or 20 μg of OFFP on day 1 and autopsied on day 2. The granulosa cells were collected from the ovarian follicles. The presence of apoptotic bodies were observed by staining the cells with acridine orange. DNA profiles of DAPI-stained cells analysed by flow cytometry also revealed apoptotic response to OFFP. Furthermore, agarose gel electrophoresis of low molecular weight DNA fraction extracted from the cells of OFFP-treated animals confirmed ladder formation and induction of apoptosis and not necrosis in granulosa cells. In conclusion, all the three parameters indicated apoptotic changes in granulosa cells of ovarian follicles in .mice treated with OFFP. The effect of OFFP seems to be exerted directly on the granulosa cells showing its autocrine role in the process of follicular atresia. This is discussed in the light of other intra/extra ovarian factors.