Evaluation of Cyclic Peptide Inhibitors of the Grb7 Breast Cancer Target: Small Change in Cargo Results in Large Change in Cellular Activity.

Grb7 is an adapter protein, overexpressed in HER2+ve breast and other cancers, and identified as a therapeutic target. Grb7 promotes both proliferative and migratory cellular pathways through interaction of its SH2 domain with upstream binding partners including HER2, SHC, and FAK. Here we present the evaluation of a series of monocyclic and bicyclic peptide inhibitors that have been developed to specifically and potently target the Grb7 SH2-domain. All peptides tested were found to inhibit signaling in both ERK and AKT pathways in SKBR-3 and MDA-MB-231 cell lines. Proliferation, migration, and invasion assays revealed, however, that the second-generation bicyclic peptides were not more bioactive than the first generation G7-18NATE peptide, despite their higher in vitro affinity for the target. This was found not to be due to steric hindrance by the cell-permeability tag, as ascertained by ITC, but to differences in the ability of the bicyclic peptides to interact with and penetrate cellular membranes, as determined using SPR and mass spectrometry. These studies reveal that just small differences to amino acid composition can greatly impact the effectiveness of peptide inhibitors to their intracellular target and demonstrate that G7-18NATE remains the most effective peptide inhibitor of Grb7 developed to date.

Discovery, Development, and Cellular Delivery of Potent and Selective Bicyclic Peptide Inhibitors of Grb7 Cancer Target.

Grb7 is a signaling protein with critical roles in tumor cell proliferation and migration and an established cancer therapeutic target. Here we explore chemical space to develop a new bicyclic peptide inhibitor, incorporating thioether and lactam linkers that binds with affinity (KD = 1.1 µM) and specificity to the Grb7-SH2 domain. Structural analysis of the Grb7-SH2/peptide complex revealed an unexpected binding orientation underlying the binding selectivity by this new scaffold. We further incorporated carboxymethylphenylalanine and carboxyphenylalanine phosphotyrosine mimetics and arrived at an optimized inhibitor that potently binds Grb7-SH2 (KD = 0.13 µM) under physiological conditions. X-ray crystal structures of these Grb7-SH2/peptide complexes reveal the structural basis for the most potent and specific inhibitors of Grb7 developed to date. Finally, we demonstrate that cell permeable versions of these peptides successfully block Grb7 mediated interactions in a breast cancer cell line, establishing the potential of these peptides in the development of novel therapeutics targeted to Grb7.

Aging alters histone H3 lysine 4 methylation in mouse germinal vesicle stage oocytes.

Decreasing oocyte competence with maternal aging is a major factor in mammalian infertility. One of the factors contributing to this infertility is changes to chromatin modifications, such as histone acetylation in old MII stage oocytes. Recent studies indicate that changes in histone acetylation at MII arise at the germinal vesicle (GV) stage. We hypothesised that histone methylation could also change in old GV oocytes. To test this hypothesis, we examined mono-, di- and trimethylation of histone H3 lysine 4 (H3K4 me1, me2 and me3, respectively) in young and older oocytes from 6-8- and 42-44-week-old mice, respectively. We found that H3K4 me2 and me3 decreased in older compared with young GV oocytes (100% vs. 81% and 100% vs. 87%, respectively; P<0.05). H3K4 me2 later increased in older MII oocytes (21% vs. 56%; P<0.05). We also examined the expression of genes encoding the H3K4 demethylases lysine (K)-specific demethylase 1A (Kdm1a) and retinol binding protein 2 (Rbp2). Expression of Kdm1a increased at both the mRNA and protein levels in older GV oocytes, but decreased in older MII oocytes (P<0.05), and was negatively correlated with H3K4 me2 levels. Conversely, expression of Rbp2 mRNA and protein decreased in older GV oocytes (P<0.05), and this was not correlated with H3K4 me3 levels. Finally, we showed that inhibition of Kdm1a of older oocytes at the GV stage restored levels of H3K4 me2 at the MII stage to those seen in 'young' oocytes (41% vs. 38%; P>0.05). These results suggest that changes in expression of H3K4 me2 and Kdm1a in older GV oocytes may represent a molecular mechanism underlying human infertility caused by aging.

Expression of histone H3 lysine 4 methylation and its demethylases in the developing mouse testis.

Histone H3 lysine 4 methylation (H3K4me) is an epigenetic modification associated with gene activation and is dynamically regulated by histone methylases and demethylases. To date, the expression patterns of H3K4me and its demethylases in the developing testis remain unclear. The present study was designed to detect the expression of H3K4me1/2/3 and its demethylases LSD1, RBP2 and SMCX in 21-, 40- and 60-day-old mouse testes by using immunohistochemistry, quantitative real-time polymerase chain reaction (PCR) and Western blot. The immunohistochemical results demonstrated that the expression patterns of the same protein were similar in testes at different ages and that the positive staining cell types were mainly Leydig cells, type A and B spermatogonia, leptotene spermatocytes and spermatids for H3K4me1/2/3, Leydig cells, type A spermatogonia, zygotene and pachytene spermatocytes, spermatids, and Sertoli cells for LSD1 and type A and B spermatogonia for RBP2. Immunostaining for SMCX was not detected in testes. Quantitative real-time PCR and Western blot showed that the amounts of LSD1, RPB2 and SMCX mRNA and protein were age-dependent, were significantly reduced with increasing age and exhibited a negative correlation with the protein levels of H3K4me1/2/3. Thus, H3K4me, which is modified by its demethylases, probably plays a role in male spermatogenesis and testis development.

Dynamic patterns of histone H3 lysine 4 methyltransferases and demethylases during mouse preimplantation development.

Extensive and dynamic chromatin remodeling occurs after fertilization, including DNA methylation and histone modifications. These changes underlie the transition from gametic to embryonic chromatin and are thought to facilitate early embryonic development. Histone H3 lysine 4 methylation (H3K4me) is an important epigenetic mechanism that associates with gene-specific activation and functions in development. However, dynamic regulation of H3K4me during early embryonic development remains unclear. Herein, the authors examined the dynamic changes of H3K4me and its key regulators (Ash1l, Ash2l, Kmt2a, Kmt2b, Kmt2c, Setd1a, Setd7, Kdm1a, Kdm1b, Kdm5a, Kdm5b, Kdm5c, and Kdm5d) in mouse oocytes and preimplantation embryos. An increase in levels of H3K4me2 and me3 was observed at the one- to two-cell stages (P < 0.05), corresponding to the period of embryonic genome activation (EGA). Subsequently, the H3K4me2 level dramatically decreased at the four-cell stage and remained at low level until the blastocyst stage (P < 0.05), whereas the H3K4me3 level transiently decreased in the four-cell embryos but steadily increased to the peak in the blastocysts (P < 0.05). The high level of H3K4me2 during the EGA was coinciding with a peak expression of its methyltransferase, ASH2L, which may stabilize this methylation level during this period. Correspondingly, a concomitant decrease in levels of its demethylases, KDM5B and KDM1A, was observed. H3K4me3 was correlated to the expression of its methyltransferase (KMT2B) and demethylase (KDM5A). Thus, these enzymes may function for the EGA and the first lineage segregation in preimplantation mouse embryos.

PKG II inhibits EGF/EGFR-induced migration of gastric cancer cells.

BACKGROUND: Our previous research results showed that Type II cGMP dependent protein kinase (PKG II) could block the activation of epidermal growth factor receptor (EGFR) and consequently inhibit the proliferation and the related MAPK/ERK-mediated signal transduction of gastric cancer cell line BGC-823, suggesting that PKG II might inhibit other EGFR-triggered signal transduction pathways and related biological activities of gastric cancer cells. This paper was designed to investigate the potential inhibition of PKG II on EGF/EGFR-induced migration activity and the related signal transduction pathways. METHODOLOGY/PRINCIPAL FINDINGS: In gastric cancer cell line AGS, expression and activity of PKG II were increased by infecting the cells with adenoviral construct encoding PKG II cDNA (Ad-PKG II) and treating the cells with cGMP analogue 8-pCPT-cGMP. Phosphorylation of proteins was detected by Western Blotting and active small G protein Ras and Rac1 was measured by "Pull-down" method. Cell migration activity was detected with trans-well equipment. Binding between PKG II and EGFR was detected with Co-IP. The results showed EGF stimulated migration of AGS cell and the effect was related to PLCγ1 and ERK-mediated signal transduction pathways. PKG II inhibited EGF-induced migration activity and blocked EGF-initiated signal transduction of PLCγ1 and MAPK/ERK-mediated pathways through preventing EGF-induced Tyr 992 and Tyr 1068 phosphorylation of EGFR. PKG II bound with EGFR and caused threonine phosphorylation of it. CONCLUSION/SIGNIFICANCE: Our results systemically confirms the inhibition of PKG II on EGF-induced migration and related signal transduction of PLCγ1 and MAPK/ERK-mediated pathways, indicating that PKG II has a fargoing inhibition on EGF/EGFR related signal transduction and biological activities of gastric cancer cells through phosphorylating EGFR and blocking the activation of it.

Type II, but not type I, cGMP-dependent protein kinase reverses bFGF-induced proliferation and migration of U251 human glioma cells.

Previous data have shown that the type II cGMP­dependent protein kinase (PKG II) inhibits the EGF­induced MAPK signaling pathway. In order to thoroughly investigate PKG, it is necessary to elucidate the function of another type of PKG, PKG I. The aim of this study was to investigate the possible inhibitory effect of PKG II and PKG I activity on the basic fibroblast growth factor (bFGF)­induced proliferation and migration of U251 human glioma cells and the possible underlying mechanisms. U251 cells were infected with adenoviral constructs encoding cDNA of PKG I (Ad­PKG I) or PKG II (Ad­PKG II) to increase the expression levels of PKG I or PKG II and then treated with 8­Br­cGMP and 8­pCPT­cGMP, respectively, to activate the enzyme. An MTT assay was used to detect the proliferation of the U251 cells. The migration of the U251 cells was analyzed using a Transwell migration assay. Western blot analysis was used to detect the phosphorylation/activation of the fibroblast growth factor receptor (FGFR), MEK and ERK and the nuclear distribution of p-ERK. The results showed that bFGF treatment increased the proliferation and migration of U251 cells, accompanied by increased phosphorylation of FGFR, MEK and ERK. Furthermore, the nuclear distribution of p-ERK increased following bFGF treatment. Increasing the activity of PKG II through infection with Ad-PKG II and stimulation with 8-pCPT-cGMP significantly attenuated the aforementioned effects of the bFGF treatment, while increased PKG I activity did not inhibit the effects of bFGF treatment. These data suggest that increased PKG II activity attenuates bFGF­induced proliferation and migration by inhibiting the MAPK/ERK signaling pathway, whereas PKG I does not.

Type II cGMP-dependent protein kinase inhibits activation of key members of the RTK family in gastric cancer cells.

Receptor tyrosine kinases (RTKs) are key to the regulation of biological cell activities, particularly of tumor cells. In a previous study, we demonstated that type II cGMP-dependent protein kinase (PKG II) may inhibit the activation of epidermal growth factor receptor (EGFR), a key member of the RTK family, and inhibit the consequent signal transduction in gastric cancer cells. Since RTKs exhibit a high level of conservation in their molecular structure, we hypothesized that PKG II may exert a similar inhibitory effect on the activation of other members of the RTK family. The aim of the present study was to investigate the potential inhibitory effect of PKG II on the activation of vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR) and insulin-like growth factor-1 receptor (IGF-1R). The AGS gastric cancer cell line was transfected with adenoviral vector encoding PKG II cDNA (Ad-PKG II) to increase the expression of PKG II and incubated with 8-pCPT-cGMP to activate the kinase. The cells were then stimulated with VEGF, PDGF and IGF-1, and the phosphorylation/activation of relative RTKs was detected by western blot analysis. The results demonstrated that stimulating cells with VEGF-C (100 ng/ml), PDGF-BB (100 ng/ml) and IGF-1 (100 ng/ml) for 5 min led to a clear increase of phosphorylation of VEGFR2 (Tyr 951), PDGFRß (Tyr 751) and IGF-1R (Tyr 1161), respectively. Pre-infection of the cells with Ad-PKG II with a multiplicity of infection (MOI) of 100% overnight and pre-incubation of cells with 8-pCPT-cGMP (100 and 250 µM) for 1 h efficiently inhibited the ligand-binding-induced phosphorylation/activation of the RTKs. PKG II also inhibited the MAPK/ERK- and PI3K-mediated signal transductions induced by VEGF-C, PDGF-BB and IGF-1. The results demonstrated that PKG II may exert a wide range of inhibitory effects on the activation of RTKs and provided further evidence to confirm PKG II as a tumor suppressor.

Endogenous cGMP-dependent protein kinase reverses EGF-induced MAPK/ERK signal transduction through phosphorylation of VASP at Ser239.

In our previous study, we demonstrated that type II cGMP-dependent protein kinase (PKG II) was expressed at lower levels in different human cancer cell lines and that exogenous PKG II inhibited epidermal growth factor (EGF)-induced MAPK/ERK signaling. In order to investigate its functions further in this signaling pathway, it is necessary to elucidate whether endogenous PKG has the same effect or not. This study aimed to investigate the possible inhibitory effect of endogenous PKG activity on EGF-induced MAPK/ERK signal transduction in human lung cancer cells and its mechanism. Human small cell lung carcinoma cells (SCLCs) were treated with the PKG-selective cGMP analog 8-pCPT-cGMP to activate endogenous PKG, EGF and cGMP followed by EGF, respectively. The results showed that increased endogenous PKG activity inhibited the EGF-induced phosphorylation of the epidermal growth factor receptor (EGFR) and the binding between Sos1 and Grb2. In addition, EGF-triggered Ras activation was reversed by increased endogenous PKG activity. While the EGF-induced phosphorylation of MEK and ERK were inhibited by increased endogenous PKG activity, there was a significant increase of phosphorylated vasodilator-stimulated phosphoprotein (p-VASP) at Ser239. Furthermore, we investigated whether endogenous PKG exerted its effects on EGF-induced MAPK/ERK signaling through phosphorylation of VASP at Ser239. Downregulation of the levels of p-VASP Ser239 by point mutation blocked the effects of endogenous PKG on EGF-induced MAPK/ERK signal transduction. The data shown here suggest that endogenous PKG reverses the EGF-induced MAPK/ERK signaling pathway by phosphorylating VASP at Ser239.

Type II cGMP-dependent protein kinase inhibits ERK/JNK-mediated activation of transcription factors in gastric cancer cells.

A previous study has shown that type II cGMP­dependent protein kinase (PKG II) inhibits the proliferation of gastric cancer cells through blocking EGF-triggered MAPK/ERK signal transduction, indicating that the kinase may be a potential anticancer factor. In the present study, the role of PKG II in the EGF-induced activation of transcription factors in the MAPK/ERK signal transduction pathway was investigated. BGC-823 human gastric cancer cells were infected with adenoviral constructs encoding the cDNA of PKG II (pAd­PKG II) to increase the expression of PKG II and treated with 8-pCPT­cGMP to activate the enzyme. Using luciferase reporter assays, it was revealed that PKG II markedly suppressed the EGF-induced transcriptional activities of AP-1 and Elk1. Consistent with the inhibitory effect of PKG II on AP-1 activity, the expression levels of c-Jun and c-Fos, components of AP-1, were also inhibited. Co-immunoprecipitation analysis demonstrated that EGF treatment increased the AP-1 content through inducing the formation of p-c-Jun-c-Jun homodimers and p-c-Jun-c-Fos heterodimers. However, this combination was efficiently blocked by activated PKG II. While pretreatments with MAPK inhibitors suppressed the EGF-induced transcriptional activities of AP-1 and Elk1, PKG II prevented the EGF-induced phosphorylation/activation of ERK and JNK, but not the phosphorylation of p38MAPK induced by EGF. These data suggest that PKG II inhibits the EGF-triggered proliferation of gastric cancer cells through suppressing ERK-/JNK-, but not p38MAPK, -mediated AP-1 and Elk1 transactivation.

[Effect of monoamine oxidase inhibitor on the differentiation of malignant glioma cell].

To investigate the effect of monoamine oxidase inhibitor tranylcypromine (TCP) on the differentiation of human U251 glioma cells, we treated U251 cells with TCP and/or 100 nmol/L histone deacetylase inhibitor trychostatin A (TSA). The differentiation of U251 cells was observed with inverted microscopy. The cell proliferation and cell cycle distribution were determined by MTT assay and flow cytometry, respectively. Apoptosis was observed by Hoechst 33258 staining. The levels of differentiation-related genes were assessed by real-time PCR and Western blotting. TCP-induced differentiation was characterized by typical morphological changes, inhibition of cellular proliferation, accumulation of cells in the G1 phase of the cell cycle, decreased expression of the pluripotency transcription factors Oct4 and Sox2, and increased expression of glial fibrillary acid protein (GFAP). The combination of TCP and TSA treatment also triggered an over-expression of GFAP. These findings suggest that TCP may induce differentiation of U251 glioma cells, and the differentiation process may be promoted by histone deacetylase inhibitor TSA.

Type II cGMP-dependent protein kinase inhibits EGF-induced MAPK/JNK signal transduction in breast cancer cells.

Our previous research data showed that type II cGMP-dependent protein kinase (PKGII) inhibited EGF-induced MAPK/ERK-mediated signal transduction through blocking the phosphorylation of EGFR caused by EGF. Since EGFR also mediates other MAPK-mediated signal transduction pathways, this study was designed to investigate whether PKGII inhibits EGF-induced MAPK/c-Jun N-terminal kinase (JNK) signal transduction. MCF-7 human breast cancer cells were infected with adenoviral constructs encoding the cDNA of PKGII (pAd-PKGII) to increase the expression of PKGII and treated with 8-pCPT-cGMP to activate the enzyme. Western blotting was applied to detect the phosphorylation/activation of EGFR, JNK, MKK7 and c-Jun. The Pull-down method was used to detect the activation of Ras protein. Co-IP was used to analyze the binding between Grb2 and Sos1. TUNEL staining was used to detect the apoptosis of MCF-7 cells. The results showed that EGF treatment increased the phosphorylation of EGFR, the binding between Grb2 and Sos1, the activation of Ras, and the phosphorylation/activation of MKK7, JNK and c-Jun, but decreased the apoptosis of the cells. Increase of PKGII activity through infection with pAd-PKGII and stimulation with 8-pCPT-cGMP efficiently reversed the above changes caused by EGF. The results suggest that PKGII also inhibits EGF-induced MAPK/JNK-mediated signal transduction and further confirmed that PKGII can block the activation of EGFR.

Type II cGMP-dependent protein kinase inhibits EGF-triggered signal transduction of the MAPK/ERK-mediated pathway in gastric cancer cells.

Our previous study found that Type II cGMP-dependent protein kinase (PKG II) is expressed at lower levels in human gastric cancer tissues and cell lines and increasing the expression and activity of PKG II inhibited the proliferation of cancer cell line BGC-823. However, the mechanism through which PKG II inhibits proliferation of gastric cancer cells is still not clear. Herein, we show that PKG II can inhibit EGF-induced MAPK signal transduction. In the gastric cancer cell line BGC-823, the expression and activity of PKG II were increased by infecting the cells with adenoviral construct encoding PKG II cDNA and treating the cells with the cGMP analogue 8-pCPT-cGMP. We found that PKG II inhibited the EGF-induced dual phosphorylation of ERK, a key component of the MAPK signal transduction pathway. Upstream of ERK, PKG II inhibited the phosphorylation of MEK1/2, the phosphorylation/activation of Raf-1, the activation of Ras, and the binding between adaptor protein Grb2 and GTP exchange factor Sos1 induced by EGF. Of note, PKG II inhibited the tyrosine phosphorylation of EGFR induced by EGF. Downstream of ERK, the EGF-induced nuclear translocation of phospho-ERK was also inhibited by PKG II. The results suggest that PKG II inhibits the proliferation of gastric cancer cells through blocking EGF-triggered MAPK signal transduction and the key blocking point is the tyrosine phosphorylation of the EGF receptor.

Nitric oxide inhibits gastric cancer cell growth through the modulation of the Akt pathway.

Nitric oxide (NO) is involved in a number of physiological and pathological processes. As an important biological mediator, NO has been the focus of cancer study for its function in tumorigenesis, tumor progression and death. The effects of NO on tumor cells are multifaceted, but many details underlying these effects are not yet well understood. In this study, we demonstrate that NO directly suppresses the growth of BGC-823 cells by inducing G0/G1 phase arrest in a dose- and time-dependent manner. We also reveal that G0/G1 arrest results from the NO-induced disruption of the cell cycle balance, which is mediated by the up-regulation of p21waf1/cip1 and the down-regulation of cyclin D1 and E and PCNA. Exposure of BGC-823 cells to various sodium nitroprusside (SNP) concentrations for 24 h or to 1 mmol/l SNP for various times resulted in a marked decrease in the level of phospho-Akt and increase in the level of phospho-ERK. In brief, the NO-induced cell growth suppression and G0/G1 arrest are mediated through the regulation of cell cycle-related proteins, which may depend on the inactivation of Akt signaling. This may be one mechanism through which NO inhibits gastric cancer cell growth.

RhoC protein stimulates migration of gastric cancer cells through interaction with scaffold protein IQGAP1.

The scaffold protein IQGAP1 is closely related to certain Rho GTPases. Research has revealed that IQGAP1 acts as an effector of Cdc42 and Rac1 in the regulation of cell activity such as proliferation and migration. However, whether IQGAP1 is associated with RhoC, another important Rho GTPase, is unclear. Previous results from our laboratory indicated that IQGAP1 and RhoC are highly expressed in gastric cancer tissues and cells. This study was designed to investigate the possible interaction between IQGAP1 and RhoC in the regulation of the migration of cancer cells. The expression of IQGAP1 and RhoC in gastric cancer tissues and cell lines was detected by Western blotting. siRNAs targeting IQGAP1 or RhoC were transfected into gastric cancer cells to knock down the expression of the proteins. Adenoviral constructs encoding full length IQGAP1, the C­terminal fragment of IQGAP1, and the constitutively active RhoC gene were used to infect gastric cancer cells to increase the expression of the proteins. The migratory activity of a gastric cancer cell line was measured by a transwell migration assay. Western blotting revealed that the IQGAP1 and RhoC proteins were highly expressed in gastric cancer tissues and cells. Spearman's rank correlation analysis indicated that the increases in the expression of IQGAP1 and RhoC were closely correlated. The transwell migration assay revealed that both IQGAP1 and RhoC stimulated the migration activity of the gastric cancer cell line AGS. The knockdown of IQGAP1 expression by siRNA blocked the migration­stimulating activity of RhoC, while the knockdown of RhoC expression had no effect on the migration-stimulatory activity of IQGAP1. Co-IP results showed that RhoC and IQGAP1 bound to each other. These results reveal a previously unrecognized interaction between IQGAP1 and RhoC, and demonstrate that IQGAP1 is a downstream effector of RhoC in the regulation of the migration activity of gastric cancer cells.

[Histone demethylase LSD1 and its biological functions].

Discovery of histone lysine specific demethylase 1 (LSD1) indicates that even histone methylation is reversible. Structural analysis shows that LSD1 is a flavin-dependent amine oxidase, which is able to catalyze the specific removal of methyl groups from mono- and dimethylated Lys4 and Lys9 of histone H3. Functional studies demonstrate that LSD1 regulates activation and inhibition of gene transcription in the nucleus, which is known as the innermost gene switch of cells. LSD1 plays important roles in embryonic development and tumorigenesis. Here, we review recent insights into the structure and chemical mechanism of LSD1, and its regulatory roles in development and cancer.

Effect of nitric oxide on the proliferation of AGS gastric cancer cells.

BACKGROUND AND OBJECTIVE: Nitric oxide (NO) is involved in many physiologic and pathologic processes. As an important biologic mediator, NO has been the focus of cancer study for its function in tumorigenesis, tumor progression, and death. This study investigated the effect of NO donor sodium nitroprusside (SNP) on the growth and proliferation of gastric cancer cell line AGS. METHODS: The growth inhibition of AGS cells was analyzed using MTT assay. The cell cycle was measured using flow cytometry. The changes of mRNA expression of proliferating cell nuclear antigen (PCNA) and caspase-3 were examined using reverse transcriptase polymerase chain reaction (RT-PCR), and the protein expressions of PCNA and caspase-3 were analyzed using Western blot. RESULTS: Dose-dependent SNP inhibited cell growth and proliferation. When the AGS cells were treated with SNP at 100, 500, 1000, 1500, and 2000 mumol/L for 24 h, the growth inhibition rates were (2.02 +/- 2.96)%, (10.82 +/- 2.21)%, (18.95 +/- 3.35)%, (26.88 +/- 2.54)%, and (42.57 +/- 1.27)%, respectively (P < 0.05). SNP altered the cell cycle in AGS cells. Compared with the control group, treatment with SNP at 100, 500, 1000, 1500, and 2000 mumol/L for 24 h reduced the number of cells in the S phase by 2.29%, 7.8%, 11.34%, 20.49%, and 23.6%, respectively, and enhanced the number of cells in the G1/G0 phases by 3.33%, 9.3%, 13.46%, 21.37%, and 24.73%, respectively (P < 0.05). With the increasing concentration and action time of SNP, the expressions of PCNA mRNA and protein decreased. The expression of caspase-3 mRNA remained unchanged, but procaspase-3 was activated. CONCLUSION: NO not only inhibits cell growth and proliferation, but also induces apoptosis in gastric cancer cells, and such effects of NO showed significant dose-dependent activity.

Type II cGMP-dependent protein kinase inhibits proliferation of the gastric cancer cell line BGC-823.

Our previous studies have demonstrated that the expression and activity of protein kinase G (PKG) II are significantly lower in human gastric cancer cell lines than in normal cells. This study was designed to investigate the effect of PKG II activation on the proliferation of human cultured BGC-823 gastric cancer cells. An adenoviral construct encoding the PKG II gene (Ad-PKG II) was used to infect BGC-823 cells, and the activity of the enzyme was induced by cGMP analogue 8-pCPT-cGMP. The proliferation-inhibitory effect of PKG II was analyzed by the MTT assay, BrdU incorporation assay and detection of proliferating cell nuclear antigen (PCNA) expression. Colony formation in soft agarose was performed to analyze the effect of PKG II on the anchorage-independent growth of the cells. The effect of PKG II in vivo was investigated in an immunocompromised nude mice model, and its effect on the cell cycle was analyzed by flow cytometry. The results showed that Ad-PKG II infection increased the expression of PKG II in BGC-823 cells. The activation of PKG II by 8-pCPT-cGMP caused a significant decrease in the number of live cells and inhibited DNA synthesis in individual cells. PKG II activation inhibited the EGF-induced increase in PCNA expression. The activation of PKG II also caused a significant inhibition of colony formation in soft agarose and significantly suppressed the in vivo growth of BGC-823 cells in immunocompromised nude mice. There was substantial cell arrest at the G1 phase and a decrease in the number of S phase cells in the Ad-PKG II/8-pCPT-cGMP-treated cells. These data indicate that the activation of PKG II by 8-pCPT-cGMP inhibits the proliferation of human gastric cancer cells.

Phosphorylation of vasodilator stimulated phosphoprotein is correlated with cell cycle progression in HeLa cells.

Vasodilator stimulated phosphoprotein (VASP) is known as an actin-binding protein. The phosphorylation of VASP plays an important role in its function. In a previous study, serine 157 phosphorylated VASP (p-VASP S157) was shown to be co-localized with α-tubulin on the spindle of SGC-7901 cells. In the present study, we demonstrated that the level of p-VASP S157 increases and has a peak which coincides with serine 10 phosphorylated histone 3 (p-H3 S10) during mitotic progression in a human cervical cancer cell line (HeLa cells). Application of protein kinase A inhibitor H89, protein kinase G inhibitor KT5823 and protein kinase C inhibitor Go6983, or a combination of these inhibitors, caused a partial decrease in p-VASP S157 and a delay in G2/M progression. Depletion of p-VASP S157 by VASP siRNA resulted in an increase in binucleated cells and x4n cells, a further delay in G2/M progression and the inhibition of HeLa cell proliferation. These results suggest that p-VASP S157 may play an important role in the G2/M transition and the completion of cytokinesis in HeLa cells.

Nitric oxide reduces hydrogen peroxide accumulation involved in water stress-induced subcellular anti-oxidant defense in maize plants.

Nitric oxide (NO) is a bioactive molecule involved in many biological events, and has been reported as pro-oxidant as well as anti-oxidant in plants. In the present study, the sources of NO production under water stress, the role of NO in water stress-induced hydrogen peroxide (H2O2) accumulation and subcellular activities of anti-oxidant enzymes in leaves of maize (Zea mays L.) plants were investigated. Water stress induced defense increases in the generation of NO in maize mesphyll cells and the activity of nitric oxide synthase (NOS) in the cytosolic and microsomal fractions of maize leaves. Water stress-induced defense increases in the production of NO were blocked by pretreatments with inhibitors of NOS and nitrate reductase (NR), suggesting that NO is produced from NOS and NR in leaves of maize plants exposed to water stress. Water stress also induced increases in the activities of the chloroplastic and cytosolic anti-oxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR), and the increases in the activities of anti-oxidant enzymes were reduced by pretreatments with inhibitors of NOS and NR. Exogenous NO increases the activities of water stress-induced subcellular anti-oxidant enzymes, which decreases accumulation of H2O2. Our results suggest that NOS and NR are involved in water stress-induced NO production and NOS is the major source of NO. The potential ability of NO to scavenge H2O2 is, at least in part, due to the induction of a subcellular anti-oxidant defense.