Cis astaxanthin and especially 9-cis astaxanthin exhibits a higher antioxidant activity in vitro compared to the all-trans isomer

In recent years, a number of studies have implicated the potent antioxidant property of astaxanthin in various experimental systems; however, these studies employed only the all-trans isomer. On the other hand, it has been reported that all-trans natural astaxanthin is readily isomerized to cis-trans, especially 9-cis and 13-cis isomers, under certain conditions by chemical analysis; however, the biological activities of the cis isomers of astaxanthin are little known. In the present study, we investigated the antioxidant activity of 9-cis and 13-cis astaxanthin compared to the all-trans isomer in vitro. In a stable radical DPPH scavenging activity test and in rat microsome and rabbit erythrocyte ghost membrane lipid peroxidation systems induced by AAPH and t-BuOOH, respectively, the results apparently showed that cis-astaxanthin, especially 9-cis astaxanthin, exhibited a higher antioxidant effect than the all-trans isomer. In addition, during polyunsaturated fatty acid (PUFA) oxidation, both DHA and linoleic acid hydroperoxides formation were markedly inhibited by astaxanthin isomers addition in the order 9-cis >13-cis >all-trans. Furthermore, 9-cis also exhibited the most effective inhibition of the generation of ROS induced by 6-hydroxydopamine (6-OHDA) in human neuroblastoma SH-SY5Y cells among the astaxanthin isomers, as well as on the degradation of collagen type II induced by DHA and linoleic acid hydroperoxides. The above-mentioned results suggest, for the first time, that cis isomer astaxanthin, especially 9-cis astaxanthin, has a much higher antioxidant potency than that of the all-trans isomer.     

即早基因c-fos在THP-1单核巨噬细胞亚型极化中的差异表达*

目的:探讨即早基因c-fos在THP-1巨噬细胞亚型极化过程中的表达变化。方法:运用PMA刺激诱导THP-1单核细胞极化为巨噬细胞,观察c-fos在单核细胞极化过程中的表达变化;在PMA刺激的基础上,分别运用LPS和IL-4诱导THP-1巨噬细胞向M1及M2亚型极化,实时定量PCR及Western blot技术分析刺激24 h时,细胞亚型标记物CD274、CD86和CD163的表达变化,并动态观察诱导极化过程中,c-fos的表达情况。结果:c-fos在PMA刺激THP-1单核细胞分化为巨噬细胞过程中蛋白和mRNA水平显示上调;LPS诱导THP-1巨噬细胞极化为M1型过程中,c-fos蛋白和mRNA水平表达降低,其特异性标记物在24 h呈现出M1型极化的特点(CD86蛋白表达升高,CD274、CD163蛋白表达降低);IL-4诱导THP-1巨噬细胞极化为M2型过程中,c-fos蛋白和mRNA水平表达升高,其特异性标记物在24 h表现出M2型极化的特点(CD86蛋白表达降低,CD274、CD163蛋白表达升高)。结论:c-fos参与了THP-1单核细胞向巨噬细胞极化的过程,并且可能通过抑制巨噬细胞M1亚型形成,促进巨噬细胞向M2亚型极化的作用参与巨噬细胞的亚型极化及其功能调节中。     

Cis-trimethoxystilbene,exhibits higher genotoxic and antiproliferative effects than its isomer trans-trimethoxystilbene in MCF-7 and MCF-10A cell lines

Stilbenes are a class of natural compounds with a wide variety of biological effects, such as antitumor activity. The best-known stilbene is resveratrol, whose clinical application is limited due to its low bioavailability. Methoxylated derivatives of this stilbene, including cis-trimethoxystilbene (cis-TMS) and trans-trimethoxystilbene (trans-TMS) have demonstrated more pronounced cytotoxic and anti-proliferative effects than resveratrol. Thus, the objective of this study is to evaluate and compare the cytotoxicity and antiproliferative effects of cis- and trans-TMS in MCF-7 and its normal counterpart MCF-10A. Both compounds were cytotoxic, genotoxic, and induced G2-M accumulation and cell death in the two cell lines. These results suggested that the genotoxicity of cis- and trans-TMS is involved in the reduction of cellular proliferation of MCF-7 and MCF-10A cells, but notably, such antiproliferative effects are more pronounced for cis- than trans-TMS.     

Bid exhibits S phase checkpoint activation and plays a pro-apoptotic role in response to etoposide-induced DNA damage in hepatocellular carcinoma cells

Bid has multiple functions in apoptosis, survival, and proliferation. The role of Bid in etoposide-induced-DNA damage in HCC has not been investigated. Here, we report that p53-overexpression led to the notable up-regulation of the expression of Bid protein, whereas the acquired expression of Bid by PLC/PRF/5 cells dramatically decreased the p53 level. Upon the administration of a high dose of etoposide (causing irreparable damage), Bid sensitized cells to apoptosis. However, at a low dose of etoposide (repairable damage), Bid activated the S phase checkpoint through the up-regulation of p21 and p27, which are both p53-independent. While the unrepairable damage was being carried out, Bid was quickly translocated to the mitochondria to release cytochrome c into the cytosol, which activated caspases 9 and 3 and led to cell death. In conclusion, our study demonstrates that Bid both exhibits S phase checkpoint activation and plays a pro-apoptotic role in response to different degrees of etoposide-induced DNA damage in HCC cells. The elucidation of these intricate mechanisms of Bid points to the development of a possible therapeutic option that combines cytotoxic therapies to treat HCC.     

Natural product pectolinarigenin exhibits potent anti-metastatic activity in colorectal carcinoma cells in vitro and in vivo

Colorectal carcinoma (CRC) is one of the most common cancers with high metastatic potential, explaining why identifying new drug candidates that inhibit tumour metastasis is an urgent need. The aim of this study was to evaluate the biological activities of pectolinarigenin (PEC, a natural flavonoid present in Cirsium chanroenicum) in CRC in vitro and in vivo and to determine its underlying mechanism of action. Here, we observed that treatment with PEC could inhibit cell viability and induce apoptosis in cancer cells in a concentration- and time-dependent manner. The occurrence of apoptosis was associated with activation of caspase-3 and Bax and decreased expression of Bcl-2. In addition, PEC markedly impaired CRC cell migration and invasion by downregulating the expression of matrix metalloproteinase (MMP-9) and phosphorylated-Stat3^Tyr705. Moreover, our studies showed that PEC inhibited abdominal metastasis models of murine colorectal cancer. In addition, histological and immunohistochemical analyses revealed a decrease in Ki67-positive cells, MMP9-positive cells and p-Stat3^Tyr705 cells upon treatment with PEC compared to control samples. Furthermore, PEC reduced the number of myeloid-derived suppressor cells (MDSCs) in the blood and tumours, which was accompanied by the increased infiltration of CD8⁺T cells in the blood. Taken together, our findings suggested that PEC could be used as a natural drug to inhibit CRC metastasis.     

9-Phenyl acridine exhibits antitumour activity by inducing apoptosis in A375 cells

Several acridine derivatives have been screened for their therapeutic potential and some are already established as antiprotozoan, antibacterial or anticancer agents. However, phenyl derivative at C-9 position of acridine had remained unexplored for their biological activity so far. In this report, we present our findings with 9-phenyl acridine (ACPH) as an anticancer agent. Both A375 and HeLa, two human cancer cell lines, were more sensitive to ACPH than normal cells namely human lymphocytes and also Chinese hamster V79 cells. ACPH also led to regression of tumour volume in mice. In A375 cells, we have shown that it caused DNA damage and blocked cell cycle progression at G(2)-M phase. Treatment with ACPH led to lowering of mitochondrial potential, upregulation of bax, release of cytochrome C and activation of caspase 3. As a new agent showing lower toxicity to normal cells and greater sensitivity towards cancerous cell line, ACPH shows promise as an effective cancer chemotherapeutic agent. ACPH treatment resulted in apoptotic death of cells through mitochondria-mediated caspase-dependent pathway.     

1alpha, 25-dihydroxy-vitamin D3 alters Syk activation through FcgammaRII in monocytic THP-1 cells

In monocytes and macrophages, activation of the tyrosine kinase Syk is an essential step in the biochemical cascade linking aggregation of receptors for immunoglobulin G (FcgammaR) to initiation of effector functions. An increase in Syk activation during differentiation of myeloid cells by different agents has been reported. We studied the activation state of Syk in response to FcgammaRII crosslinking in monocytic cells before and after in vitro differentiation with 1alpha, 25-dihydroxy-vitamin D3. We show here that while in undifferentiated THP-1 cells clustering of FcgammaRII induces significant phosphorylation and activation of Syk, in THP-1 cells differentiated in vitro by 1alpha, 25-dihydroxy-vitamin D3, FcgammaRII crosslinking induced a decrease in Syk activity. In vitro differentiation did not induce changes in the expression of FcgammaRII isoforms. The observed effect on Syk activation though FcgammaRII could be mediated by differentiation-induced changes in the expression and basal activation level of Syk, as well as changes in the association of Syk with the tyrosine phosphatase SHP-1. These results suggest that the biochemical signaling pathways induced by FcgammaRII could be dependent on the differentiation state of the cell.     

In vitro and in vivo biological activity screening of Ru(III) complexes involving 6-benzylaminopurine derivatives with higher pro-apoptotic activity than NAMI-A

A series of novel octahedral ruthenium(III) complexes involving 6-benzylaminopurine (L) derivatives as N-donor ligands has been prepared by the reaction of [(DMSO)₂H][trans-RuCl₄(DMSO)₂] with the corresponding L derivative. The complexes 1-12 have the general compositions trans-[RuCl₄(DMSO)(n-Cl-LH)] ⋅ xSol (1-3), trans-[RuCl₄(DMSO)(n-Br-LH)] · xSol (4-6), trans-[RuCl₄(DMSO)(n-OMe-LH)] · xSol (7-9) and trans-[RuCl₄(DMSO)(n-OH-LH)] · xSol (10-12); n = 2, 3, and 4, x = 0-1.5; and Sol = H₂O, DMSO, EtOH and/or (Me)₂CO. The complexes have been thoroughly characterized by elemental analysis, UV-visible, FTIR, Raman, and EPR spectroscopy, ES + (positive ionization electrospray) mass spectrometry, thermal analysis, cyclic voltammetry, magnetic and conductivity measurements. The X-ray molecular structure of trans-[RuCl₄(DMSO)(3-Br-LH)] ⋅ (Me)₂CO (5) revealed the distorted octahedral coordination in the vicinity of the central atom, and also confirmed that the 3-Br-L ligand is present as the N3-protonated N7-H tautomer and is coordinated to Ru(III) through the N9 atom of the purine moiety. The tested complexes have been found to be in vitro non-cytotoxic against K562, G361, HOS and MCF7 human cancer cell lines with IC₅₀ > 100 μM in contrast to the moderate results regarding the antiradical activity with IC₅₀ ≈ 10^− 3 M. On the contrary, in vivo antitumor activity screening showed that the prepared Ru(III) complexes possess higher pro-apoptotic activity than NAMI-A. The reduction of Ru(III) to Ru(II) and Ru(II)-species formation in tumor tissues was confirmed by means of a simple method of detection and visualization of intracellular Ru(II) by fluorescence microscopy. The originality of this method is based on the preparation of a Ru(II)-bipyridine complex in situ.     

Simvastatin induces caspase-independent apoptosis in LPS-activated RAW264.7 macrophage cells

Macrophages participate in several inflammatory pathologies such as sepsis and arthritis. We examined the effect of simvastatin on the LPS-induced proinflammatory macrophage RAW264.7 cells. Co-treatment of LPS and a non-toxic dose of simvastatin induced cell death in RAW264.7 cells. The cell death was accompanied by disruption of mitochondrial membrane potential (MMP), genomic DNA fragmentation, and caspase-3 activation. Surprisingly, despite caspase-dependent apoptotic cascade being completely blocked by Z-VAD-fmk, a pan-caspase inhibitor, the cell death was only partially repressed. In the presence of Z-VAD-fmk, DNA fragmentation was blocked, but DNA condensation, disruption of MMP, and nuclear translocation of apoptosis inducing factor were obvious. The cell death by simvastatin and LPS was effectively decreased by both the FPP and GGPP treatments as well as mevalonate. Our findings indicate that simvastatin triggers the cell death of LPS-treated RAW264.7 cells through both caspase-dependent and -independent apoptotic pathways, suggesting a novel mechanism of statins for the severe inflammatory disease therapy.     

Curcumin induces pro-apoptotic endoplasmic reticulum stress in human leukemia HL-60 cells

Curcumin has been shown to induce apoptosis in many cancer cells. However, the molecular mechanism(s) responsible for curcumin-induced apoptosis is not well understood and most probably involves several pathways. In HL-60 cells, curcumin induced apoptosis and endoplasmic reticulum (ER) stress as evidenced by the survival molecules such as phosphorylated protein kinase-like ER-resident kinase, phosphorylated eukaryotic initiation factor-2alpha, glucose-regulated protein-78, and the apoptotic molecules such as caspase-4 and CAAT/enhancer binding protein homologous protein (CHOP). Inhibition of caspase-4 activity by z-LEVD-FMK, blockage of CHOP expression by small interfering RNA, and treatment with salubrinal, an ER inhibitor, significantly reduced curcumin-induced apoptosis. Removing two double bonds in curcumin, which was speculated to form Michael adducts with thiols in secretory proteins, resulted in a loss of the ability of curcumin to induce apoptosis as well as ER stress. Thus, the present study shows that curcumin-induced apoptosis is associated with its ability to cause ER stress.     

Isoforms of Jun kinase are differentially expressed and activated in human monocyte/macrophage (THP-1) cells

Ten isoforms of c-jun N-terminal kinase (JNK) have been described that arise by differential mRNA splicing of three genes. In that the relative expression and function of these different JNK proteins in human monocytic cells is not known, we have examined the JNK isoforms in THP-1 monocyte/macrophage cells. Differentiation of THP-1 cells by exposure to 10(-8) M PMA for 42-48 h enhances cellular responses to LPS, including enhanced activation of total JNK activity and increased phosphorylation of p54 JNK as well as p46 JNK. Examination of JNK proteins on Western blots reveals a predominance of p46 JNK1 and p54 JNK2 proteins. Clearing of lysates by immunoprecipitation of JNK1(99% effective) removes 46% of the JNK enzymatic activity (p < 0.01), whereas clearing of JNK1 plus JNK2 (70% effective) depletes the sample of 72% of the JNK activity (p < 0.01). Further analysis, undertaken with real-time RT-PCR, revealed that 98% of the JNK messages code for three isoforms: JNK1beta1, JNK2alpha1, and JNK2alpha2. The p54 JNK that is phosphorylated in LPS-stimulated, PMA-differentiated THP-1 cells is most likely JNK2alpha2 because 97% of the p54 JNK-encoding messages code for JNK2alpha2. By analogous reasoning, the p46 JNKs that are not heavily phosphorylated, but account for approximately half of the N-terminal c-jun kinase enzymatic activity, are most likely either JNK1beta1 or JNK2alpha1 because they account for 98% of the messages that can code for 46kDa JNKS:     

Interleukin-10 down-regulates oxLDL induced expression of scavenger receptor A and Bak-1 in macrophages derived from THP-1 cells

Here, we investigated the therapeutic potential of IL-10 by testing its effects on oxLDL-induced lipoprotein uptake and apoptosis by flow cytometry in THP-1-derived macrophages. The mRNA and protein expressions of lipid scavenger receptors (SR-A, CD36) and apoptosis-related proteins (Bcl-2, Bak-1) were also detected. Co-incubation of oxLDL with IL-10 reduced DiI-oxLDL uptake by 16.1 ± 3.8%, 35.2 ± 3.8% and 28.9 ± 1.8% at 6, 12 and 24 h of treatment, respectively. Furthermore, treatment with oxLDL for 24 h enhanced the SR-A mRNA and protein expressions by 89.3 ± 17.1% and 70.1 ± 17.6%, respectively. IL-10 abrogated the oxLDL-induced SR-A mRNA expression by 50.2 ± 3.9% and its protein by 45.6 ± 1.9%. Meanwhile IL-10 had no effect on the oxLDL-induced increase of CD36 expression. IL-10 inhibited the oxLDL-induced cell apoptosis in a time-dependent manner by 17.3 ± 3.3%, 36.4 ± 2.8% and 31.0 ± 4.3% at 6, 12 and 24 h, respectively. OxLDL increased Bak-1 mRNA and protein expressions by 38.4 ± 13.3% and 36.9 ± 12.1%, respectively. However co-stimulation of oxLDL with IL-10 for 24 h inhibited Bak-1 expression to 28.4 ± 7.2% (mRNA) and 25.7 ± 6.3% (protein). Meanwhile, IL-10 had no effect on the oxLDL-induced decrease of Bcl-2 expression. Our findings suggested that IL-10 reduced the oxLDL-induced lipoprotein uptake and apoptosis partly via down-regulating the oxLDL-induced expression of SR-A and Bak-1 in THP-1-derived macrophages.     

Beclin 1 augmented cis-diamminedichloroplatinum induced apoptosis via enhancing caspase-9 activity

Beclin 1, identified as a Bcl-2-interacting protein, is known to enhance autophagy. However, the effect of Beclin 1 on apoptotic signaling has remained unclear. Here, we show that overexpression of Beclin 1 in MKN28 human gastric cancer cells augmented cis-diamminedichloroplatinum (CDDP)-induced apoptosis. Conversely, "knockdown" of Beclin 1 by a small inhibitory RNA in MKN 1 cells attenuated this cytotoxicity. Furthermore, not only caspase-3/7 activities, but also caspase-9 activity was increased in Beclin 1 gene transfectants treated with CDDP, and caspase-9 inhibitor completely abolished augmentation of CDDP-induced apoptosis by Beclin 1 as did a caspase-3 inhibitor. Thus, Beclin 1 augments CDDP-induced apoptosis through enhancing caspase-9 activity and functions as a pro-apoptotic molecule.     

Potent inhibitory effect of trans9, trans11 isomer of conjugated linoleic acid on the growth of human colon cancer cells

This study compared the growth inhibitory effects of pure conjugated linoleic acid (CLA) isomers [cis(c)9,c11-CLA, c9,trans(t)11-CLA, t9,t11-CLA, and t10,c12-CLA] on human colon cancer cell lines (Caco-2, HT-29 and DLD-1). When Caco-2 cells were incubated up to 72 h with 200 μM, each isomer, even in the presence of 10% fetal bovine serum (FBS), cell proliferation was inhibited by all CLA isomers in a time-dependent manner. The strongest inhibitory effect was shown by t9,t11-CLA, followed by t10,c12-CLA, c9,c11-CLA and c9,t11-CLA, respectively. The strongest effect of t9,t11-CLA was also observed in other colon cancer cell lines (HT-29 and DLD-1). The order of the inhibitory effect of CLA isomer was confirmed in the presence of 1% FBS. CLA isomers supplemented in the culture medium were readily incorporated into the cellular lipids of Caco-2 and changed their fatty acid composition. The CLA contents in cellular lipids were 26.2±2.7% for t9,t11-CLA, 35.9±0.3% for c9,t11-CLA and 46.3±0.8% for t10,c12-CLA, respectively. DNA fragmentation was clearly recognized in Caco-2 cells treated with t9,t11-CLA. This apoptotic effect of t9,t11-CLA was dose- and time-dependent. DNA fragmentation was also induced by 9c,11t-CLA and t10,c12-CLA. However, fragmentation levels with both isomers were much lower than that with t9,t11-CLA. t9t11-CLA treatment of Caco-2 cells decreased Bcl-2 levels in association with apoptosis, whereas Bax levels remained unchanged. These results suggest that decreased expression of Bcl-2 by t9t11-CLA might increase the sensitivity of cells to lipid peroxidation and to programmed cell death, apoptosis.     

Sex determination and differentiation in organisms other than higher plants

The understanding of sex determination in general, but in particular in mammals, has been a subject of scientific speculation for a long time. It has been shown that in many vertebrate and invertebrate species, the sex of an individual is determined by the individual's chromosomal constitution. Initial studies of classical genetic searching for sex-transforming mutations and the scrupulous analyses of modified phenotypes have shed light on the mechanism(s) of sex-determination. They paved the road to successful studies at molecular level. After a brief review on sex determination in chosen model species, the “Drosophila system” is presented to exemplify a possible general principle for sex determinism.     

p38 MAPK and ERK1/2 pathways are involved in the pro-apoptotic effect of notoginsenoside Ft1 on human neuroblastoma SH-SY5Y cells

Aims

This study aims to investigate the effect and the mechanisms of notoginsenoside Ft1, a natural compound exclusively found in P. notoginseng, on the proliferation and apoptosis of human neuroblastoma SH-SY5Y cells.

Main methods

CCK-8 assay was used to assess the cell proliferation. Flow cytometry was performed to measure the cell cycle distribution and cell apoptosis. Hoechst 33258 staining was conducted to confirm the morphological changes of apoptotic cells. Protein expression was detected by western blot analysis and caspase 3 activity was measured by colorimetric assay kit.

Key findings

Among the saponins examined, Ft1 showed the best inhibitory effect on cell proliferation of SH-SY5Y cells with IC₅₀ of 45 μM. Ft1 not only arrested the cell cycle at S, G₂/M stages, but also promoted cell apoptosis, which was confirmed by Hoechst 33258 staining. Further studies demonstrated that Ft1 up-regulated the protein expressions of cleaved caspase 3, phospho-p53, p21, and cyclin B1, but down-regulated that of Bcl-2. Moreover, Ft1 enhanced the phosphorylation of ERK1/2, JNK and p38 MAPK. However, the phosphorylation of Jak2 and p85 PI3K was reduced by Ft1. Inhibitors of p38 MAPK and ERK1/2 but not JNK abrogated the up-regulated protein expressions of cleaved caspase 3, p21 and down-regulated protein expression of Bcl-2 as well as elevated caspase 3 activity induced by Ft1.

Significance

Ft1 arrested the proliferation and elicited the apoptosis of SH-SY5Y cells possibly via p38 MAPK and ERK1/2 pathways, which indicates the potential therapeutic effect of it on human neuroblastoma.