Polygalacin D inhibits the growth of hepatocellular carcinoma cells through BNIP3L-mediated mitophagy and endogenous apoptosis pathways.

Platycodon grandiflorum (Jacq.) A. DC. is a famous medicinal plant commonly used in East Asia. Triterpene saponins isolated from P. grandiflorum are the main biologically active compounds, among which polygalacin D (PGD) has been reported to be an anti-tumor agent. However, its anti-tumor mechanism against hepatocellular carcinoma is unknown. This study aimed to explore the inhibitory effect of PGD in hepatocellular carcinoma cells and related mechanisms of action. We found that PGD exerted significant inhibitory effect on hepatocellular carcinoma cells through apoptosis and autophagy. Analysis of the expression of apoptosis-related proteins and autophagy-related proteins revealed that this phenomenon was attributed to the mitochondrial apoptosis and mitophagy pathways. Subsequently, using specific inhibitors, we found that apoptosis and autophagy had mutually reinforcing effects. In addition, further analysis of autophagy showed that PGD induced mitophagy by increasing BCL2 interacting protein 3 like (BNIP3L) levels.In vivo experiments demonstrated that PGD significantly inhibited tumor growth and increased the levels of apoptosis and autophagy in tumors. Overall, our findings showed that PGD induced cell death of hepatocellular carcinoma cells primarily through mitochondrial apoptosis and mitophagy pathways. Therefore, PGD can be used as an apoptosis and autophagy agonist in the research and development of antitumor agents.

Polygalacin D inhibits the growth of hepatocellular carcinoma cells through BNIP3L-mediated mitophagy and endogenous apoptosis pathways / 中国天然药物

Platycodon grandiflorum (Jacq.) A. DC. is a famous medicinal plant commonly used in East Asia. Triterpene saponins isolated from P. grandiflorum are the main biologically active compounds, among which polygalacin D (PGD) has been reported to be an anti-tumor agent. However, its anti-tumor mechanism against hepatocellular carcinoma is unknown. This study aimed to explore the inhibitory effect of PGD in hepatocellular carcinoma cells and related mechanisms of action. We found that PGD exerted significant inhibitory effect on hepatocellular carcinoma cells through apoptosis and autophagy. Analysis of the expression of apoptosis-related proteins and autophagy-related proteins revealed that this phenomenon was attributed to the mitochondrial apoptosis and mitophagy pathways. Subsequently, using specific inhibitors, we found that apoptosis and autophagy had mutually reinforcing effects. In addition, further analysis of autophagy showed that PGD induced mitophagy by increasing BCL2 interacting protein 3 like (BNIP3L) levels.In vivo experiments demonstrated that PGD significantly inhibited tumor growth and increased the levels of apoptosis and autophagy in tumors. Overall, our findings showed that PGD induced cell death of hepatocellular carcinoma cells primarily through mitochondrial apoptosis and mitophagy pathways. Therefore, PGD can be used as an apoptosis and autophagy agonist in the research and development of antitumor agents.

Human recombinant RNASET2-induced inflammatory response and connective tissue remodeling in the medicinal leech.

In recent years, several studies have demonstrated that the RNASET2 gene is involved in the control of tumorigenicity in ovarian cancer cells. Furthermore, a role in establishing a functional cross-talk between cancer cells and the surrounding tumor microenvironment has been unveiled for this gene, based on its ability to act as an inducer of the innate immune response. Although several studies have reported on the molecular features of RNASET2, the details on the mechanisms by which this evolutionarily conserved ribonuclease regulates the immune system are still poorly defined. In the effort to clarify this aspect, we report here the effect of recombinant human RNASET2 injection and its role in regulating the innate immune response after bacterial challenge in an invertebrate model, the medicinal leech. We found that recombinant RNASET2 injection induces fibroplasias, connective tissue remodeling and the recruitment of numerous infiltrating cells expressing the specific macrophage markers CD68 and HmAIF1. The RNASET2-mediated chemotactic activity for macrophages has been further confirmed by using a consolidated experimental approach based on injection of the Matrigel biomatrice (MG) supplemented with recombinant RNASET2 in the leech body wall. One week after injection, a large number of CD68+ and HmAIF-1+ macrophages massively infiltrated MG sponges. Finally, in leeches challenged with lipopolysaccharides (LPS) or with the environmental bacteria pathogen Micrococcus nishinomiyaensis, numerous macrophages migrating to the site of inoculation expressed high levels of endogenous RNASET2. Taken together, these results suggest that RNASET2 is likely involved in the initial phase of the inflammatory response in leeches.

Subrepellent doses of Slit1 promote Netrin-1 chemotactic responses in subsets of axons.

BACKGROUND: Axon pathfinding is controlled by guidance cues that elicit specific attractive or repulsive responses in growth cones. It has now become clear that some cues such as Netrin-1 can trigger either attraction or repulsion in a context-dependent manner. In particular, it was recently found that the repellent Slit1 enables the attractive response of rostral thalamic axons to Netrin-1. This finding raised the intriguing possibility that Netrin-1 and Slit1, two essential guidance cues, may act more generally in an unexpected combinatorial manner to orient specific axonal populations. To address this major issue, we have used an innovative microfluidic device compatible not only with dissociated neuronal cultures but also with explant cultures to systematically and quantitatively characterize the combinatorial activity of Slit1 and Netrin-1 on rostral thalamic axons as well as on hippocampal neurons. RESULTS: We found that on rostral thalamic axons, only a subthreshold concentration of the repellent Slit1 triggered an attractive response to a gradient of Netrin-1. On hippocampal neurons, we similarly found that Slit1 alone is repulsive and a subthreshold concentration of Slit1 triggered a potent attractive or repulsive behavioral response to a gradient of Netrin-1, depending on the nature of the substrate. CONCLUSIONS: Our study reveals that at subthreshold repulsive levels, Slit1 acts as a potent promoter of both Netrin-1 attractive and repulsive activities on distinct neuronal cell types, thereby opening novel perspectives on the role of combinations of cues in brain wiring.

A novel tumor suppressor function of glycine N-methyltransferase is independent of its catalytic activity but requires nuclear localization.

Glycine N-methyltransferase (GNMT), an abundant cytosolic enzyme, catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to glycine generating S-adenosylhomocysteine and sarcosine (N-methylglycine). This reaction is regulated by 5-methyltetrahydrofolate, which inhibits the enzyme catalysis. In the present study, we observed that GNMT is strongly down regulated in human cancers and is undetectable in cancer cell lines while the transient expression of the protein in cancer cells induces apoptosis and results in the activation of ERK1/2 as an early pro-survival response. The antiproliferative effect of GNMT can be partially reversed by treatment with the pan-caspase inhibitor zVAD-fmk but not by supplementation with high folate or SAM. GNMT exerts the suppressor effect primarily in cells originated from malignant tumors: transformed cell line of non-cancer origin, HEK293, was insensitive to GNMT. Of note, high levels of GNMT, detected in regenerating liver and in NIH3T3 mouse fibroblasts, do not produce cytotoxic effects. Importantly, GNMT, a predominantly cytoplasmic protein, was translocated into nuclei upon transfection of cancer cells. The presence of GNMT in the nuclei was also observed in normal human tissues by immunohistochemical staining. We further demonstrated that the induction of apoptosis is associated with the GNMT nuclear localization but is independent of its catalytic activity or folate binding. GNMT targeted to nuclei, through the fusion with nuclear localization signal, still exerts strong antiproliferative effects while its restriction to cytoplasm, through the fusion with nuclear export signal, prevents these effects (in each case the protein was excluded from cytosol or nuclei, respectively). Overall, our study indicates that GNMT has a secondary function, as a regulator of cellular proliferation, which is independent of its catalytic role.

The SSRI citalopram affects fetal thalamic axon responsiveness to netrin-1 in vitro independently of SERT antagonism.

Serotonin (5-hydroxytryptamine, 5-HT) signaling is thought to modulate nervous system development. Genetic and pharmacological studies support the idea that altered 5-HT signaling during development can have enduring consequences on brain function and behavior. Recently, we discovered that 5-HT can modulate thalamic axon guidance in vitro and in vivo. Embryonic thalamic axons transiently express the 5-HT transporter (SERT; Slc6a4) and accumulate 5-HT, suggesting that the SERT activity of these axons may regulate 5-HT-modulated guidance cues. We tested whether pharmacologically blocking SERT using selective 5-HT reuptake inhibitors (SSRIs) would impact the action of 5-HT on thalamic axon responses to netrin-1 in vitro. Surprisingly, we observed that two high-affinity SSRIs, racemic citalopram ((RS)-CIT) and paroxetine, affect the outgrowth of embryonic thalamic axons, but differ with respect to their dependence on SERT blockade. Using a recently developed 'citalopram insensitive' transgenic mouse line and in vitro pharmacology, we show that the effect of (RS)-CIT effect is SERT independent, but rather arises from R-CIT activation of the orphan sigma-1 receptor(σ1, Oprs1). Our results reveal a novel σ1 activity in modulating axon guidance and a 5-HT independent action of a widely prescribed SSRI. By extension, (RS)-CIT and possibly other structurally similar SSRIs may have other off-target actions that can impact neural development and contribute to therapeutic efficacy or side effects.

Kisspeptin resets the hypothalamic GnRH clock in men.

CONTEXT: Reproduction in all mammals is controlled by a hypothalamic clock that produces periodic secretory pulses of GnRH, but how the timing of these pulses is determined is poorly understood. The neuropeptide kisspeptin potently and selectively stimulates the secretion of GnRH. Although this property of kisspeptin is well described, the effects of kisspeptin on endogenous GnRH pulse generation remain largely unexplored. OBJECTIVE: The objective of the study was to detail the effects of kisspeptin on GnRH secretion, as reflected by LH secretion, in men. PARTICIPANTS: Thirteen healthy adult men participated in the study. INTERVENTION: The intervention was the administration of a single iv bolus of the C-terminal decapeptide of kisspeptin (amino acids 112-121 of the parent protein). RESULTS: Kisspeptin induced an immediate LH pulse, regardless of the timing of the previous endogenous pulse. The kisspeptin-induced pulses were on average larger than endogenous pulses (amplitude 5.0 ± 1.0 vs. 2.1 ± 0.3 mIU/ml, P = 0.02). Comparison of the morphology of kisspeptin-induced LH pulses in healthy men with that of GnRH-induced LH pulses in men with isolated GnRH deficiency suggests that a single i.v. bolus of kisspeptin triggered sustained GnRH release lasting approximately 17 min. Furthermore, kisspeptin reset the GnRH pulse generator, as it not only induced an immediate LH pulse but also delayed the next endogenous pulse by an interval approximating the normal interpulse interval. CONCLUSIONS: As the first known agent capable of resetting the hypothalamic GnRH pulse generator, kisspeptin can be used as a physiological tool for studying GnRH pulse generation and opens a door to understanding the mechanisms of biological clocks in general.

Anticancer activity of the PR domain of tumor suppressor RIZ1.

Human tumor suppressor gene RIZ encodes two protein products, tumor suppressor RIZ1 and proto-oncoprotein RIZ2, which regulate cellular functions in a Yin-Yang fashion. The only structural difference between them is that RIZ2 lacks the N-terminal PR domain. In this study, we showed that RIZ1 mRNA expression level was elevated in stage IV of eight different types of cancer (stage III for prostate cancer), indicating that RIZ1 might play an important role in tumor metastasis, and the PR domain alone possessed anticancer activity.

Kisspeptin-10 elicits triphasic cytosolic calcium responses in immortalized GT1-7 GnRH neurones.

Kisspeptins, which are alternatively called as metastin since they were originally identified as products of metastasis suppressor gene KiSS-1, are the natural ligands for the G protein-coupled receptor 54 (GPR54). Kisspeptins are the most potent activators of hypothalamic-pituitary-gonadal (HPG) axis reported to date. The pulsatile pattern of GnRH release, which results in the intermittent release of gonadotropic hormones from the pituitary, has a critical importance for reproductive function but the factors responsible from this release pattern are not known. Therefore, the pattern of kisspeptin-induced intracellular signaling and the role of PKC in the intracellular signaling cascade were investigated by fluorescence calcium imaging using the immortalized GnRH-secreting GT1-7 hypothalamic neurons. Kisspeptin-10 caused a triphasic change characterized by an initial small increase followed by a significant decrease and increase in intracellular free calcium concentrations ([Ca(2+)](i)). The changes in [Ca(2+)](i) were significantly attenuated by pre-treatment with protein kinase C inhibitor. The compatibility of appeared mirrored-patterns of kisspeptin-10-induced changes in [Ca(2+)](i) concentrations in these neurons and GnRH secretion confirm the importance of intracellular calcium flux downstream from GPR54 through PKC signaling pathway.

Kisspeptin directly regulates neuropeptide Y synthesis and secretion via the ERK1/2 and p38 mitogen-activated protein kinase signaling pathways in NPY-secreting hypothalamic neurons.

Kisspeptin is a key component of reproduction that directly stimulates GnRH neurons. However, recent studies indicate that kisspeptin can indirectly stimulate GnRH neurons through unidentified afferent networks. Neuropeptide Y (NPY) is another key reproductive hormone that is an afferent stimulator of GnRH neurons. Herein, we report kisspeptin receptor Kiss1r mRNA expression in native NPY neurons FAC-sorted from NPY-GFP transgenic mice. Thus, we hypothesized that kisspeptin indirectly stimulates GnRH neurons through direct regulation of NPY neurons. Using hypothalamic NPY-secreting cell lines, we determined that kisspeptin stimulates NPY mRNA expression and secretion in the mHypoE-38 cells, but not the mHypoE-42 cells, using quantitative RT-PCR and enzyme immunoassays. Furthermore, agouti-related peptide, ghrelin, neurotensin, or Kiss1r mRNA expression was not changed upon exposure to kisspeptin in either cell line. These results concur with our previous work identifying the mHypoE-38 cell line as a putative reproductive NPY neuron and the mHypoE-42 cell line as a potential feeding-related NPY neuron. In the mHypoE-38 cells, kisspeptin activated the ERK1/2 and p38 MAPK kinases as shown by Western blot analysis. Moreover, inhibiting the ERK1/2 and p38 pathways with U0126 and SB239063, respectively, prevented kisspeptin induction of NPY mRNA expression and secretion. Altogether, we find that kisspeptin directly regulates NPY synthesis and secretion via the ERK1/2 and p38 MAPK pathways in a NPY-secreting cell line, and we propose NPY neurons as an afferent network by which kisspeptin indirectly stimulates GnRH secretion.

Kisspeptin: a novel regulator of reproductive function.

The UK and international neuroendocrine community was deeply shocked and saddened the unbelievably premature death of Michael Harbuz in Bristol in 2006. Mick was a superb friend and colleague, and played a huge part in the development and activities of the British Neuroendocrine Group/British Society for Neuroendocrinology (BSN), serving as both Membership Secretary and Treasurer between 1999 and 2004. Mick was a leader in the field of neuroendocrine-immune interactions, and brought a great deal of charisma, humour and ability to meetings and conferences. He was also a passionate and committed supporter of the progress of young researchers and of their participation in neuroendocrine events. He recognised that today's postgraduate students and postdoctoral research fellows are tomorrow's neuroendocrine researchers, be it in academia, the health services or industry. To recognise Mick's great commitment to and enthusiasm for postgraduate education both in the University of Bristol and in the BSN, we decided to honour and remember him by instituting the 'Michael Harbuz Young Investigator Prize Lecture' to be delivered annually. Dr Waljit Dhillo from Imperial College London was the inaugural recipient of this award, and presented his lecture at the Annual Meeting of the BSN in Nottingham in September 2007, upon which this review is based. Recent evidence demonstrates that the neuropeptide kisspeptin and its receptor, GPR54, have a fundamental role in initiating the onset of puberty and are important in regulating reproductive function. This review discusses the evidence available from animals and humans demonstrating that kisspeptin potently stimulates the release of gonadotrophins by stimulating the release of gonadotrophin-releasing hormone and that a lack of kisspeptin or GPR54 results in reproductive failure.

KiSS-1 peptide induces release of LH by a direct effect on the hypothalamus of ovariectomized ewes.

The objectives of this study were: to determine if peripheral administration of the ovine C-terminal decapeptide (oKiSS-1) increases secretion of LH and FSH in ovariectomized (OVX) ewes; to evaluate the effect of ovarian steroid hormones on the predicted increase in gonadotropin secretion induced by oKiSS-1; to establish if the stimulatory effect of oKiSS-1 is due to a direct action on the hypothalamus or was mediated directly at the anterior pituitary gland. Intravenous injection of 3mg oKiSS-1 to OVX ewes (n=6) increased (P<0.01) basal and mean serum concentrations of LH. The amplitude and the area under the curve (AUC) of LH induced by oKiSS-1 was greater (P<0.01) than the highest pulse of LH detected before oKiSS-1. Pre-treatment with steroid hormones (1mg progesterone plus 50 microg estradiol-17beta for 3 days) in OVX ewes (n=6) reduced (P<0.1) the magnitude of the stimulatory effect of oKiSS-1 on LH secretion. In these animals, oKiSS-1 increased (P<0.1) basal and mean concentrations of LH, and the amplitude of the pulse of LH induced by oKiSS-1, but not the AUC (P>0.1). In our hands, oKiSS-1 failed to increase serum concentration of FSH at the dose used in this study. Administration of GnRH antiserum completely suppressed the secretory patterns of LH and prevented the increase in secretion of LH induced by oKiSS-1. We conclude that the rapid increase in secretion of LH induced by peripheral administration of oKiSS-1 is the result of a direct action on the hypothalamus.

Cloning, tissue expression pattern and genomic organization of latexin, a human homologue of rat carboxypeptidase A inhibitor.

Latexin, a carboxypeptidase A inhibitor, is expressed in a cell type-specific manner in both central and peripheral nervous systems in the rat. It is used as a molecular marker for the regional specification of the neocortex. In this study, a cDNA was isolated from a human fetal brain cDNA library. The cDNA (LXN) contains an open reading frame encoding 222 amino acids. The comparison between the deduced amino acid sequences of LXN and latexins of rat and mouse revealed high sequence identity (84.2 and 84.7%, respectively). Northern blot analysis showed that LXN was expressed as a transcript of 1.3 kb in 15 out of 16 tissues examined, except in peripheral blood leukocyte. The expression levels were high in heart, prostate, ovary, kidney, pancreas, and colon, moderate or low in other tissues including brain. It is noteworthy that the tissue distribution of human LXN differs greatly to that of its homologue in the model animal, rat latexin. In addition, the LXN gene contains at least 6 exons and spans 5.9 kb according to the genomic sequence of the clone RP11-79M21 and the gap sequence cloned in this paper. LXN was assigned to 3q25-q26.2 according to the position of the marker SHGC-35682 found adjacent to LXN gene.