Akt regulates the expression of MafK, synaptotagmin I, and syntenin-1, which play roles in neuronal function

<p>Abstract</p> <p>Background</p> <p>Akt regulates various cellular processes, including cell growth, survival, and metabolism. Recently, Akt's role in neurite outgrowth has also emerged. We thus aimed to identify neuronal function-related genes that are regulated by Akt.</p> <p>Methods</p> <p>We performed suppression subtractive hybridization on two previously established PC12 sublines, one of which overexpresses the wild-type (WT) form and the other, the dominant-negative (DN) form of Akt. These sublines respond differently to NGF's neuronal differentiation effect.</p> <p>Results</p> <p>A variety of genes was identified and could be classified into several functional groups, one of which was developmental processes. Two genes involved in neuronal differentiation and function were found in this group. v-Maf musculoaponeurotic fibrosarcoma oncogene homolog K (MafK) induces the neuronal differentiation of PC12 cells and immature telencephalon neurons, and synaptotagmin I (SytI) is essential for neurotransmitter release. Another gene, <it>syntenin-1 </it>(<it>Syn-1</it>) was also recognized in the same functional group into which <it>MafK </it>and <it>SytI </it>were classified. Syn-1 has been reported to promote the formation of membrane varicosities in neurons. Quantitative reverse transcription polymerase chain reaction analyses show that the transcript levels of these three genes were lower in PC12 (WT-Akt) cells than in parental PC12 and PC12 (DN-Akt) cells. Furthermore, treatment of PC12 (WT-Akt) cells with an Akt inhibitor resulted in the increase of the expression of these genes and the improvement of neurite outgrowth. These results indicate that dominant-negative or pharmacological inhibition of Akt increases the expression of <it>MafK</it>, <it>SytI</it>, and <it>Syn-1 </it>genes. Using lentiviral shRNA to knock down endogenous Syn-1 expression, we demonstrated that Syn-1 promotes an increase in the numbers of neurites and branches.</p> <p>Conclusions</p> <p>Taken together, these results indicate that Akt negatively regulates the expression of <it>MafK</it>, <it>SytI</it>, and <it>Syn-1 </it>genes that all participate in regulating neuronal integrity in some way or another.</p

Deep RNA Sequencing Reveals Novel Cardiac Transcriptomic Signatures for Physiological and Pathological Hypertrophy

Although both physiological hypertrophy (PHH) and pathological hypertrophy (PAH) of the heart have similar morphological appearances, only PAH leads to fatal heart failure. In the present study, we used RNA sequencing (RNA-Seq) to determine the transcriptomic signatures for both PHH and PAH. Approximately 13–20 million reads were obtained for both models, among which PAH showed more differentially expressed genes (DEGs) (2,041) than PHH (245). The expression of 417 genes was barely detectable in the normal heart but was suddenly activated in PAH. Among them, Foxm1 and Plk1 are of particular interest, since Ingenuity Pathway Analysis (IPA) using DEGs and upstream motif analysis showed that they are essential hub proteins that regulate the expression of downstream proteins associated with PAH. Meanwhile, 52 genes related to collagen, chemokines, and actin showed opposite expression patterns between PHH and PAH. MAZ-binding motifs were enriched in the upstream region of the participating genes. Alternative splicing (AS) of exon variants was also examined using RNA-Seq data for PAH and PHH. We found 317 and 196 exon inclusions and exon exclusions, respectively, for PAH, and 242 and 172 exon inclusions and exclusions, respectively for PHH. The AS pattern was mostly related to gains or losses of domains, changes in activity, and localization of the encoded proteins. The splicing variants of 8 genes (i.e., Fhl1, Rcan1, Ndrg2, Synpo, Ttll1, Cxxc5, Egfl7, and Tmpo) were experimentally confirmed. Multilateral pathway analysis showed that the patterns of quantitative (DEG) and qualitative (AS) changes differ depending on the type of pathway in PAH and PHH. One of the most significant changes in PHH is the severe downregulation of autoimmune pathways accompanied by significant AS. These findings revealed the unique transcriptomic signatures of PAH and PHH and also provided a more comprehensive understanding at both the quantitative and qualitative levels

비장의 과오종(2증례)

Two cases of splenic hamartoma are described. Both cases occurred in women in their fifties and presented as vague abdominal symptoms. Case 1 was a large solitary hamartoma, 6.5 cm in size and hypervascular mass angiographically. No white pulp with germinal centers or central arterioles were seen in the mass. Instead, a mass of red pulp with well-formed sinuses, often exhibiting active endothelial cells, were the predominent feature, scattered with lymphoid nodules. Extramedullary hemopoieses was meager. Case 2 was an example of multiple (3) hamartomas that showed extraordinary extramedullary hemopoiesis particularly in the mass itself. The proliferative megakaryocytes were quite atypical, and in areas they mimicked the neoplastic process. There was evidence of compression of normal parenchyma by the hamartomatous masses both grossly and microscopically. It seems true that the histological features of splenic hamartoma can vary from case to case and that one should pay particular attention to these lesions so that they are not misinterpreted as true neoplastic processes

Hemin inhibits cyclin D1 and IGF-1 expression via STAT5b under hypoxia in ER alpha-negative MDA-MB 231 breast cancer cells

Cychn D1 and insulin-like growth factor 1 receptor (IGE-IR) are key regulators of cell proliferation that are overexpressed in most breast cancers The purpose of the present study was to investigate the molecular mechanism by which hemin exerts its inhibitory effects on aggressive breast cancer cells We found that hemin regulates cychn DI and ICE-I R proteins and insulin-like growth factor-1 gene expression through STAT5b in breast cancer cells. We confirmed that STAT5b. cyclin DI. and IGE-1R is up-regulated by hypoxia. and the increased sTAT5b binds strongly to the STAT5-binding sites contained within the distal 5-flanking region of IGE-1 acne in breast cancer cells EMSA studies showed that S1`AT5 binding activity to the IGE-1 and cychn DI promoter was distinctly decreased by hemin in STAT5b-transfected COS-7 or MDA-MB 231 cells. IGE-1 gene expression was also decreased by hemin in mammary epithelial cells STAT5b expression was inhibited in sr RNA experiments and by hermit. leadma to decreased levels of IGE-1 These results provide a basis for molecular targets in cancer treatment via the STAT5b/IGE-1 or /cychn DI pathway in solid tumor cells These data indicate that hem in inhibits the cyclin D1 and IGE-1 expression via STAT5b under hypoxia in ER alpha-negative breast cancer cells These findings are valuable toward understanding the role of hemin-induced inhibition of cyclin D1 and IGF-1 expression under hypoxia in Invasive and metastatic breast cancerBernaciak TM, 2009, BREAST CANCER RES, V11, DOI 10.1186/bcr2341Joung YH, 2008, INT J ONCOL, V33, P477, DOI 10.3892/ijo_00000030Diorio C, 2008, BREAST CANCER RES, V10, DOI 10.1186/bcr2093Tian XB, 2007, J NUCL MED, V48, P1699, DOI 10.2967/jnumed.107.042499Tworoger SS, 2007, CANCER CAUSE CONTROL, V18, P743, DOI 10.1007/s10552-007-9017-5Cui Y, 2007, CANCER EPIDEM BIOMAR, V16, P1682, DOI 10.1158/1055-9965.EPI-07-0187Joung YH, 2007, BIOCHEM BIOPH RES CO, V358, P733, DOI 10.1016/j.bbrc.2007.04.201Weaver AM, 2007, BREAST CANCER RES, V9, DOI 10.1186/bcr1794Tsiftsoglou AS, 2006, PHARMACOL THERAPEUT, V111, P327, DOI 10.1016/j.pharmthera.2005.10.017Carroll VA, 2006, CANCER RES, V66, P6264, DOI 10.1158/0008-5472.CAN-05-2519Pouyssegur J, 2006, NATURE, V441, P437, DOI 10.1038/nature04871Lee MY, 2006, BREAST, V15, P187, DOI 10.1016/j.breast.2005.05.005Hwang PH, 2005, EXP MOL MED, V37, P391Joung YH, 2005, EXP MOL MED, V37, P353Wang Y, 2005, J BIOL CHEM, V280, P10955, DOI 10.1074/jbc.M412808200Lang D, 2005, INT IMMUNOL, V17, P155, DOI 10.1093/intimm/dxh196Kim YS, 2005, FREE RADICAL BIO MED, V38, P85, DOI 10.1016/j.freeradbiomed.2004.09.031GABEI K, 2005, J NATL CANCER I, V97, P790Buitenhuis M, 2004, INT J BIOCHEM CELL B, V36, P2120, DOI 10.1016/j.biocel.2003.11.008Ibrahim YH, 2004, GROWTH HORM IGF RES, V14, P261, DOI 10.1016/j.ghir.2004.01.005Gonzalez-Michaca L, 2004, AM J PHYSIOL-RENAL, V286, pF370, DOI 10.1152/ajprenal.00300.2003Woelfle J, 2003, J BIOL CHEM, V278, P51261, DOI 10.1074/jbc.M309486200Joung YH, 2003, EXP MOL MED, V35, P350Bando H, 2003, BIOMED PHARMACOTHER, V57, P333, DOI 10.1016/S0753-3322(03)00098-2Nakahira K, 2003, BIOCHEM PHARMACOL, V66, P1091, DOI 10.1016/S0006-2952(03)00444-1Richardson DR, 2003, J LAB CLIN MED, V141, P289, DOI 10.1016/S0022-2143(03)00003-9Amon M, 2003, FASEB J, V17, P175Wilson SJ, 2003, VASC PHARMACOL, V40, P109, DOI 10.1016/S1537-1891(02)00340-3Ghattas MH, 2002, INT J BIOCHEM CELL B, V34, P1619Martin MB, 2002, J NUTR, V132, p3799SDuan C, 2002, J ENDOCRINOL, V175, P41Schaaf GJ, 2002, FREE RADICAL RES, V36, P835, DOI 10.1080/1071576021000005267Suliman HB, 2002, FREE RADICAL BIO MED, V32, P246OSHEA JJ, 2002, CELL S1, V109, P21Yamashita H, 2001, MOL CELL ENDOCRINOL, V183, P151Davey HW, 2001, ENDOCRINOLOGY, V142, P3836Yu QY, 2001, NATURE, V411, P1017Bienvenu F, 2001, J BIOL CHEM, V276, P16840Park JH, 2000, J BIOCHEM MOL BIOL, V33, P396Ariyoshi K, 2000, J BIOL CHEM, V275, P24407Clark JE, 2000, BIOCHEM J, V348, P615Bowman T, 2000, ONCOGENE, V19, P2474Yamada N, 1999, AM J RESP CELL MOL, V21, P428Kazansky AV, 1999, J BIOL CHEM, V274, P22484Grimley PM, 1999, CYTOKINE GROWTH F R, V10, P131Matsumura I, 1999, EMBO J, V18, P1367Teglund S, 1998, CELL, V93, P841Rund D, 1998, ACTA HAEMATOL-BASEL, V100, P82Udy GB, 1997, P NATL ACAD SCI USA, V94, P7239, DOI 10.1073/pnas.94.14.7239Liu XW, 1997, GENE DEV, V11, P179Onishi M, 1996, EXP HEMATOL, V24, P324JONES JI, 1995, ENDOCR REV, V16, P3BALLA J, 1993, P NATL ACAD SCI USA, V90, P9285BICHELL DP, 1992, MOL ENDOCRINOL, V6, P1899MACAULAY VM, 1992, BRIT J CANCER, V65, P311DAUGHADAY WH, 1989, ENDOCR REV, V10, P68GUTTERIDGE JMC, 1988, BIOCHEM J, V256, P861AFT RL, 1984, J BIOL CHEM, V259, P301BENZ EJ, 1980, P NATL ACAD SCI-BIOL, V77, P3509RUTHERFORD TR, 1979, NATURE, V280, P164

<i>Hwanggeumchal sorghum</i> Induces Cell Cycle Arrest, and Suppresses Tumor Growth and Metastasis through Jak2/STAT Pathways in Breast Cancer Xenografts

<div><p>Background</p><p>Cancer is one of the highly virulent diseases known to humankind with a high mortality rate. Breast cancer is the most common cancer in women worldwide. Sorghum is a principal cereal food in many parts of the world, and is critical in folk medicine of Asia and Africa. In the present study, we analyzed the effects of HSE in metastatic breast cancer.</p><p>Methodology/Principal Findings</p><p>Preliminary studies conducted on MDA-MB 231 and MCF-7 xenograft models showed tumor growth suppression by HSE. Western blotting studies conducted both <i>in vivo</i> and <i>in vitro</i> to check the effect of HSE in Jak/STAT pathways. Anti-metastatic effects of HSE were confirmed using both MDA-MB 231 and MCF-7 metastatic animal models. These studies showed that HSE can modulate Jak/STAT pathways, and it hindered the STAT5b/IGF-1R and STAT3/VEGF pathways not only by down-regulating the expression of these signal molecules and but also by preventing their phosphorylation. The expression of angiogenic factors like VEGF, VEGF-R2 and cell cycle regulators like cyclin D, cyclin E, and pRb were found down-regulated by HSE. In addition, it also targets Brk, p53, and HIF-1α for anti-cancer effects. HSE induced G1 phase arrest and migration inhibition in MDA-MB 231 cells. The metastasis of breast cancer to the lungs also found blocked by HSE in the metastatic animal model.</p><p>Conclusions/Significance</p><p>Usage of HS as a dietary supplement is an inexpensive natural cancer therapy, without any side effects. We strongly recommend the use of HS as an edible therapeutic agent as it possesses tumor suppression, migration inhibition, and anti-metastatic effects on breast cancer.</p></div

Suppression of STAT3/DNA binding activity leads to the crackdown of target gene products.

<p>A, western blotting analysis of whole cell lysates from MDA MB-231, MCF-7 and SKBR-3 cells treated with increasing concentration of HSE. B, RT-PCR analysis of MDA-MB 231, MCF-7, and SKBR-3 cells treated with different concentration of HSE showed transcriptional regulation of IGF-1R, Cyclin D1 and VEGF mRNA levels. 18S RNA was used as a control.</p

HSE might block the metastasis of breast cancer to lungs in animal model.

<p>A, analysis of the effect of HSE on the experimental lung metastases established by MDA-MB 231 and MCF-7 cells. Mice were injected with MDA-MB 231 cells (1×10⁷ in 200 µL PBS per mouse) and MCF-7 cells (1×10⁷ in 200 µL PBS per mouse). They are randomized into two groups for each cell model. One group of mice (n = 6) was injected with HSE for 30 days, and the other group of mice (n = 6) was injected with water using the same schedule. Lungs were collected on day 30 after the start of treatment and formalin-fixed paraffin-embedded lung tissues were stained with hematoxylin and eosin. Representative images are shown (A, magnification: ×200, scale bar = 200 µm). Arrow points the area of metastasis. B, quantitative analysis of tumor areas shown in A. The area of metastatic nodules in each section was quantified from six fields (magnification: ×200). The values are means ± S.E (n = 6) after normalization to vehicle (internal control). Asterisks indicate a statistically significant decrease by t-test (***p<0.001). C, HSE inhibited the migration of MDA-MB 231 cells treated with HSE (10 µg/ml for 24 h). MDA-MB 231 cells were plated into DMEM medium separately. After 24 hours, medium was replaced with and without HSE (10 µg/ml). Live cell images were acquired 24 hours after media changes (i and ii; pre-confluent), (iii and iv; confluent). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040531#pone.0040531.s001" target="_blank">Movie S1</a>, MDA-MB 231 cells without HSE (control) and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040531#pone.0040531.s002" target="_blank">Movie S2</a>, MDA-MB 231 cells treated by HSE.</p

HSE suppressed the binding activities of STAT5b to the IGF-1R and STAT3 to the VEGF promoter sites.

<p>A, electrophoretic mobility shift assay (EMSA) showed, HSE suppressed the DNA-binding activity of STAT5b to the IGF-1R (i) and STAT3 to the VEGF (ii) binding sites in MDA-MB 231 cells treated with HSE for 24 h. B, nuclear protein extracts (NE) were separated and blotted onto nitrocellulose membrane showing decrease in the level of p-STAT5, p-STAT3, VEGF and IGF-1R. C and D, shows the activation of STAT5b/IGF-1R promoter and STAT3/VEGF promoter in COS-7 cells by HSE. COS-7 cells were transiently co-transfected with STAT5b and IGF-1R (C) and STAT3 and VEGF genes (D). Data represent means of at least three separate experiments. Asterisks indicate a statistically significant decrease by t-test (***p<0.001) in the crosstalks of STAT5b/IGF-1R and STAT3/VEGF respectively.</p

HSE down-regulated the STAT5b/IGF-1R and STAT3/VEGF pathways in time and dose dependent manner.

<p>A, human breast cancer cells MDA-MB 231, MCF-7, and SKBR-3 were exposed to different incubation time with HSE (10 µg/ml). B, MDA-MB 231, MCF-7, and SKBR-3 cells were exposed to various concentrations of HSE. The protein extracts (10 µg) were separated by 10% SDS-PAGE, and western blots were performed as described in experimental procedures. β-actin was used as a control for protein loading.</p