Adrenomedullin improves fertility and promotes pinopodes and cell junctions in the peri-implantation endometrium.

Implantation is a complex event demanding contributions from both embryo and endometrium. Despite advances in assisted reproduction, endometrial receptivity defects persist as a barrier to successful implantation in women with infertility. We previously demonstrated that maternal haploinsufficiency for the endocrine peptide adrenomedullin (AM) in mice confers a subfertility phenotype characterized by defective uterine receptivity and sparse epithelial pinopode coverage. The strong link between AM and implantation suggested the compelling hypothesis that administration of AM prior to implantation may improve fertility, protect against pregnancy complications, and ultimately lead to better maternal and fetal outcomes. Here, we demonstrate that intrauterine delivery of AM prior to blastocyst transfer improves the embryo implantation rate and spacing within the uterus. We then use genetic decrease-of-function and pharmacologic gain-of-function mouse models to identify potential mechanisms by which AM confers enhanced implantation success. In epithelium, we find that AM accelerates the kinetics of pinopode formation and water transport and that, in stroma, AM promotes connexin 43 expression, gap junction communication, and barrier integrity of the primary decidual zone. Ultimately, our findings advance our understanding of the contributions of AM to uterine receptivity and suggest potential broad use for AM as therapy to encourage healthy embryo implantation, for example, in combination with in vitro fertilization.

H11 kinase/heat shock protein 22 deletion impairs both nuclear and mitochondrial functions of STAT3 and accelerates the transition into heart failure on cardiac overload.

BACKGROUND: Cardiac overload, a major cause of heart failure, induces the expression of the heat shock protein H11 kinase/Hsp22 (Hsp22). METHODS AND RESULTS: To determine the specific function of Hsp22 in that context, a knockout mouse model of Hsp22 deletion was generated. Although comparable to wild-type mice in basal conditions, knockout mice exposed to pressure overload developed less hypertrophy and showed ventricular dilation, impaired contractile function, increased myocyte length and accumulation of interstitial collagen, faster transition into heart failure, and increased mortality. Microarrays revealed that hearts from knockout mice failed to transactivate genes regulated by the transcription factor STAT3. Accordingly, nuclear STAT3 tyrosine phosphorylation was decreased in knockout mice. Silencing and overexpression experiments in isolated neonatal rat cardiomyocytes showed that Hsp22 activates STAT3 via production of interleukin-6 by the transcription factor nuclear factor-κB. In addition to its transcriptional function, STAT3 translocates to the mitochondria where it increases oxidative phosphorylation. Both mitochondrial STAT3 translocation and respiration were also significantly decreased in knockout mice. CONCLUSIONS: This study found that Hsp22 represents a previously undescribed activator of both nuclear and mitochondrial functions of STAT3, and its deletion in the context of pressure overload in vivo accelerates the transition into heart failure and increases mortality.

Lats2 is a negative regulator of myocyte size in the heart.

Mammalian sterile 20-like kinase (Mst)1 plays an important role in mediating apoptosis and inhibiting hypertrophy in the heart. Because Hippo, a Drosophila homolog of Mst1, forms a signaling complex with Warts, a serine/threonine kinase, which in turn stimulates cell death and inhibits cell proliferation, mammalian homologs of Warts, termed Lats1 and Lats2, may mediate the function of Mst1. We here show that Lats2, but not Lats1, dose-dependently increased apoptosis in cultured cardiac myocytes. Lats2 also dose-dependently reduced [(3)H]phenylalanine incorporation and cardiac myocyte size, whereas dominant negative Lats2 (DN-Lats2) increased them, suggesting that endogenous Lats2 negatively regulates myocyte growth. DN-Lats2 significantly attenuated induction of apoptosis and inhibition of hypertrophy by Mst1, indicating that Lats2 mediates the function of Mst1 in cardiac myocytes. Cardiac specific overexpression of Lats2 in transgenic mice significantly reduced the size of left and right ventricles, whereas that of DN-Lats2 caused hypertrophy in both ventricles. Overexpression of Lats2 reduced left ventricular systolic and diastolic function without affecting baseline levels of myocardial apoptosis. Expression of endogenous Lats2 was significantly upregulated in response to transverse aortic constriction. Overexpression of DN-Lats2 significantly enhanced cardiac hypertrophy and inhibited cardiac myocyte apoptosis induced by transverse aortic constriction. These results suggest that Lats2 is necessary and sufficient for negatively regulating ventricular mass in the heart. Although Lats2 is required for cardiac myocyte apoptosis in response to pressure overload, it was not sufficient to induce apoptosis at baseline. In conclusion, Lats2 affects both growth and death of cardiac myocytes, but it primarily regulates the size of the heart and acts as an endogenous negative regulator of cardiac hypertrophy.

Sirt1 regulates aging and resistance to oxidative stress in the heart.

Silent information regulator (Sir)2, a class III histone deacetylase, mediates lifespan extension in model organisms and prevents apoptosis in mammalian cells. However, beneficial functions of Sir2 remain to be shown in mammals in vivo at the organ level, such as in the heart. We addressed this issue by using transgenic mice with heart-specific overexpression of Sirt1, a mammalian homolog of Sir2. Sirt1 was significantly upregulated (4- to 8-fold) in response to pressure overload and oxidative stress in nontransgenic adult mouse hearts. Low (2.5-fold) to moderate (7.5-fold) overexpression of Sirt1 in transgenic mouse hearts attenuated age-dependent increases in cardiac hypertrophy, apoptosis/fibrosis, cardiac dysfunction, and expression of senescence markers. In contrast, a high level (12.5-fold) of Sirt1 increased apoptosis and hypertrophy and decreased cardiac function, thereby stimulating the development of cardiomyopathy. Moderate overexpression of Sirt1 protected the heart from oxidative stress induced by paraquat, with increased expression of antioxidants, such as catalase, through forkhead box O (FoxO)-dependent mechanisms, whereas high levels of Sirt1 increased oxidative stress in the heart at baseline. Thus, mild to moderate expression of Sirt1 retards aging of the heart, whereas a high dose of Sirt1 induces cardiomyopathy. Furthermore, although high levels of Sirt1 increase oxidative stress, moderate expression of Sirt1 induces resistance to oxidative stress and apoptosis. These results suggest that Sirt1 could retard aging and confer stress resistance to the heart in vivo, but these beneficial effects can be observed only at low to moderate doses (up to 7.5-fold) of Sirt1.

Cardiac-specific overexpression of AT1 receptor mutant lacking G alpha q/G alpha i coupling causes hypertrophy and bradycardia in transgenic mice.

Ang II type 1 (AT1) receptors activate both conventional heterotrimeric G protein-dependent and unconventional G protein-independent mechanisms. We investigated how these different mechanisms activated by AT1 receptors affect growth and death of cardiac myocytes in vivo. Transgenic mice with cardiac-specific overexpression of WT AT1 receptor (AT1-WT; Tg-WT mice) or an AT1 receptor second intracellular loop mutant (AT1-i2m; Tg-i2m mice) selectively activating G(alpha)q/G(alpha)i-independent mechanisms were studied. Tg-i2m mice developed more severe cardiac hypertrophy and bradycardia coupled with lower cardiac function than Tg-WT mice. In contrast, Tg-WT mice exhibited more severe fibrosis and apoptosis than Tg-i2m mice. Chronic Ang II infusion induced greater cardiac hypertrophy in Tg-i2m compared with Tg-WT mice whereas acute Ang II administration caused an increase in heart rate in Tg-WT but not in Tg-i2m mice. Membrane translocation of PKCepsilon, cytoplasmic translocation of G(alpha)q, and nuclear localization of phospho-ERKs were observed only in Tg-WT mice while activation of Src and cytoplasmic accumulation of phospho-ERKs were greater in Tg-i2m mice, consistent with the notion that G(alpha)q/G(alpha)i-independent mechanisms are activated in Tg-i2m mice. Cultured myocytes expressing AT1-i2m exhibited a left and upward shift of the Ang II dose-response curve of hypertrophy compared with those expressing AT1-WT. Thus, the AT1 receptor mediates downstream signaling mechanisms through G(alpha)q/G(alpha)i-dependent and -independent mechanisms, which induce hypertrophy with a distinct phenotype.

Immunological tolerance and tumor rejection in embryo-aggregated chimeric mice - lessons for tumor immunity.

BACKGROUND: Rejection of transplanted tumors by the immune system is a rare event in syngeneic hosts, and is considered to be dependent on the local interaction of defensive immune reactions and tumor tolerance mechanisms. Here, we have enlisted the aid of a unique set of embryo-aggregated lineage chimeric mice derived from C57/BL6 and FVB donors to study the interplay between local and systemic tumor immunity and tolerance in rejection of mouse B16 melanoma cells, syngeneic to the C57/BL6 donor strain. METHODS: Two variants of embryo-aggregated chimeric mice with either variable or no contribution of C57-derived cells to their skin were generated by the fusion of different ratios of morula stage blastomers. Chimeric mice were analyzed for s.c. growth of B16 tumors in comparison to their respective donor strains as well as normal F1 hybrids, and the relative frequencies of cellular components of the immune system by FACS analysis of peripheral blood or lymph node cells. RESULTS: B16 tumors grew significantly faster in mice with full chimerism in their skin as compared to syngeneic C57 or semi-syngeneic C57 x FVB F1 hosts. In contrast, s.c. tumor growth was either absent or significantly reduced in chimeric mice lacking C57-derived cells in their skin, but tolerant to C57 tissue in other organs. Comparison of the relative frequencies of various immune cells in the periphery via FACS-analysis did not reveal any significant differences between the two types of chimeric mice with respect to their donor strains. CONCLUSION: Our data suggest a complex interplay between mechanisms of local peripheral tolerance and innate antitumor mechanisms possibly involving NK cell allorecognition as a basis for the differential growth or rejection of B16 tumors in these unique chimeric mice, which we suggest to constitute a valuable new model system for the study of immune-mediated tumor rejection.

An angiotensin II type 1 receptor mutant lacking epidermal growth factor receptor transactivation does not induce angiotensin II-mediated cardiac hypertrophy.

We have shown previously that tyrosine 319 in a conserved YIPP motif in the C terminus of angiotensin II (Ang II) type 1 receptors (AT(1)Rs) is essential for transactivation of epidermal growth factor receptor (EGFR) in vitro. We hypothesized that the signaling mechanism mediated through the specific amino acid sequence in the G protein-coupled receptor plays an important role in mediating cardiac hypertrophy in vivo. Transgenic mice with cardiac-specific overexpression of wild-type AT(1)R (Tg-WT) and an AT(1)R with a mutation in the YIPP motif (Tg-Y319F) were studied. Tg-Y319F mice developed no significant cardiac hypertrophy, in contrast to the significant development of hypertrophy in Tg-WT mice. Expression of fetal-type genes, such as atrial natriuretic factor, was also significantly lower in Tg-Y319F than in Tg-WT mice. Infusion of Ang II caused an enhancement of hypertrophy in Tg-WT mice but failed to induce hypertrophy in Tg-Y319F mice. Left ventricular myocardium in Tg-Y319F mice developed significantly less apoptosis and fibrosis than that in Tg-WT mice. EGFR phosphorylation was significantly inhibited in Tg-Y319F mice, confirming that EGFR was not activated in Tg-Y319F mouse hearts. In contrast, activation/phosphorylation of protein kinase C, STAT3, extracellular signal-regulated kinase, and Akt and translocation of Galphaq/11 to the cytosolic fraction were maintained in Tg-Y319F hearts. Furthermore, a genetic cross between Tg-WT and transgenic mice with cardiac-specific overexpression of dominant negative EGFR mimicked the phenotype of Tg-Y319F mice. In conclusion, overexpression of AT(1)-Y319F in cardiac myocytes diminished EGFR transactivation and inhibited a pathological form of cardiac hypertrophy. The YIPP motif in the AT(1)R plays an important role in mediating cardiac hypertrophy in vivo.

Inhibition of endogenous thioredoxin in the heart increases oxidative stress and cardiac hypertrophy.

Thioredoxin 1 (Trx1) has redox-sensitive cysteine residues and acts as an antioxidant in cells. However, the extent of Trx1 contribution to overall antioxidant mechanisms is unknown in any organs. We generated transgenic mice with cardiac-specific overexpression of a dominant negative (DN) mutant (C32S/C35S) of Trx1 (Tg-DN-Trx1 mice), in which the activity of endogenous Trx was diminished. Markers of oxidative stress were significantly increased in hearts from Tg-DN-Trx1 mice compared with those from nontransgenic (NTg) mice. Tg-DN-Trx1 mice exhibited cardiac hypertrophy with maintained cardiac function at baseline. Intraperitoneal injection of N-2-mercaptopropionyl glycine, an antioxidant, normalized cardiac hypertrophy in Tg-DN-Trx1 mice. Thoracic aortic banding caused greater increases in myocardial oxidative stress and enhanced hypertrophy in Tg-DN-Trx1 compared with NTg mice. In contrast, transgenic mice with cardiac-specific overexpression of wild-type Trx1 did not show cardiac hypertrophy at baseline but exhibited reduced levels of hypertrophy and oxidative stress in response to pressure overload. These results demonstrate that endogenous Trx1 is an essential component of the cellular antioxidant mechanisms and plays a critical role in regulating oxidative stress in the heart in vivo. Furthermore, inhibition of endogenous Trx1 in the heart primarily stimulates hypertrophy, both under basal conditions and in response to pressure overload through redox-sensitive mechanisms.

The role of human prolactin and its antagonist, G129R, in mammary gland development and DMBA-initiated tumorigenesis in transgenic mice.

Human prolactin (hPRL) has been implicated to have a pathological role in breast cancer and play a critical role in mammary gland development. The hPRL antagonist, G129R, has been shown to induce breast cancer cell apoptosis. 9,10-Dimethyl-1,2-benzanthracene (DMBA), a potent mammary gland carcinogen, induces hormone responsive mammary tumor formation in rodents. To investigate the effects of hPRL and its counterpart, G129R, on mammary gland development and tumorigenesis, transgenic mice that express hPRL or G129R under the regulation of the metallothionein (Mt) promoter were generated. Mammary glands from virgin female transgenic mice at the ages of 12, 24, and 36 weeks were used to compare the effect of hPRL and G129R in various developmental stages. Mammary gland whole mount comparisons between transgenic mice and their littermates revealed a significant increase in ductal branching and lobular bud formation in hPRL transgenic mice; whereas a drastic decrease in ductal branching and lobular bud formation was observed in the mammary glands of G129R transgenic mice. In addition, total RNA isolated from the mammary glands of transgenic mice at the three different ages was analyzed on Affymetrix GeneChip Mouse Expression 430A chips (MOE430A). Microarray data revealed alteration to the gene expression levels, greatest at 12 and 36 weeks. Furthermore, hPRL and G129R transgenic mice, as well as their littermates, were treated with multiple doses of DMBA and the rate of mammary tumor formation and survival were compared. The tumor rates in the G129R transgenic mice were significantly reduced (18% at 28 weeks) as compared to that of either NTG (39%) or hPRL (40%). On the other hand, the tumor appearance is significantly earlier in the PRL transgenic group as compared to that of controls. Taken together, the data further confirmed the inhibitory effects of G129R in mammary gland development, which translates to a resistance to DMBA-initiated breast tumorigenesis.

Bcl-2 targeting siRNA expressed by a T7 vector system inhibits human tumor cell growth in vitro.

A T7 promoter driven siRNA expression vector system (Bcl-2/T7) that targets Bcl-2 mRNA in MCF-7 human cancer cells was designed in the present study. In the presence of prebound T7 RNA polymerase, successful expression of Bcl-2 siRNA as well as its function was demonstrated via cell proliferation assays, Bcl-2 Elisa, and TUNEL assay. MCF-7 breast cancer cells transfected with Bcl-2/T7 show decreased levels of Bcl-2 expression at the protein level as well as decreased cell proliferation. Also, the number of apoptotic cells was increased in cells expressing Bcl-2 siRNA. Previous studies have shown that Bcl-2 levels are increased in a large number of different types of cancer. Therefore, the ability of Bcl-2/T7 to produce functional Bcl-2 siRNA in breast cancer cells suggests a potential role for this delivery system in cancer gene therapy.

A matrix metalloproteinase 2 cleavable melittin/avidin conjugate specifically targets tumor cells in vitro and in vivo.

Extracellular matrix breakdown as well as increased expression in cancer cells and tumor microvascular endothelial cells make matrix metalloproteinase 2 (MMP2) an attractive target for cancer treatment. By taking advantage of MMP2's properties, an MMP2 cleavable melittin/avidin conjugate was designed. Melittin alone is extremely toxic to cells and induces immediate cell lysis, but becomes inactive when coupled with avidin. The incorporation of the MMP2 target sequence into the peptide was used as a means for targeting tumor cells. In vitro, the melittin/avidin conjugate showed strong cytolytic activity against cancer cells with high MMP2 activity; DU 145 prostate cancer cells and SK-OV-3 ovarian cancer cells. The conjugate exhibited very little cytolytic activity against normal L-cells that displayed low MMP2 activity. These data demonstrate the MMP2 specificity of the melittin/avidin conjugate. In vivo, the size of tumors injected with the melittin/avidin conjugate was significantly smaller as compared to untreated tumors. Therefore, due to its tumor targeting capabilities as well as its cytolytic properties in vitro and in vivo, the melittin/avidin conjugate displays the potential for use in cancer therapy.

In vitro targeted killing of human endothelial cells by co-incubation of human serum and NGR peptide conjugated human albumin protein bearing alpha (1-3) galactose epitopes.

The NGR/alpha1,3Gal-HSA peptide was designed to specifically target CD13 positive cells and induce cell lysis. NGR is the targeting component of the peptide in that it binds the CD13 isoform (aminopeptidase) that is expressed in tumor vessels. Galactose alpha1,3-galactose terminal carbohydrate epitope (alpha1,3Gal) induces a strong antibody reaction in human and Old World Monkeys and in vivo, this reaction leads to organ rejection. The human serum albumin (HSA) bearing alpha1,3Gal epitope was therefore used to lyse cells. In the present study, we were able to demonstrate that NGR/alpha1,3Gal-HSA binds CD13 positive human umbilical vein endothelial cells (HUVEC). We also found by live/dead fluorescent staining that NGR/alpha1,3Gal-HSA was able to induce lysis of HUVECs upon incubation with human serum. Therefore, by conjugating NGR to HSA bearing alpha1,3Gal epitopes, we are able to specifically target and lyse cells expressing CD13. This strategy may be potentially useful in tumor anti-angiogenesis therapy.

Efficient translocation and apoptosis induction by adenovirus encoded VP22-p53 fusion protein in human tumor cells in vitro.

BACKGROUND: p53 mutations are one of the most frequent genetic alterations in cancer. Various mechanisms of delivering p53 protein into tumor cells, such as plasmids, retroviruses and adenoviruses, have been widely used in experimental studies. Although these methods are relatively successful, the transduction rate into surrounding cells is still a limiting factor. Recent studies have shown that fusing VP22 (important for intercellular transport) to p53 and delivering this protein to cells in the form of an adenovirus is a very efficient method of getting p53 into cells. RESULTS: In the present study, the effect of adenovirus encoded VP22-p53 fusion protein on p53 negative human cancer cells (LNCaP, SK-OV-3, OVCAR-3, DU 145) was investigated. A functional VP22-p53 fusion protein was produced as verified by immunofluorescence and analysis of p21 expression. Induction of p21 expression (target gene for p53) confirmed p53 was functional and immunofluorescence staining using p53 antiserum demonstrated the intercellular trafficking ability of the VP22 portion of the fusion protein. CONCLUSION: In conclusion, VP22-p53 showed efficient translocation into tumor cells, inhibition of tumor cell proliferation and induction of apoptosis. These characteristics make the fusion protein an attractive method for introducing p53 into human cells as well as a potential candidate for gene therapy.

Combined HSV-TK/GCV and secondary lymphoid tissue chemokine gene therapy inhibits tumor growth and elicits potent antitumor CTL response in tumor-bearing mice.

Suicide gene therapy in combination with pro-drugs represents an attractive approach to the treatment of cancer. Unfortunately this approach is limited by difficulty in targeting all tumor cells, especially those at the distant metastases associated with the most complex tumors. For this reason, attempts to stimulate global anti-tumor immune responses at the sites of effective suicide gene/pro-drug-mediated tumor cell destruction are appealing. Here we show that, by including a gene coding for secreted secondary lymphoid tissue chemokine (SLC) along with the herpes simplex virus thymide kinase (HSV-TK) gene in a bicistronic vector for anti-tumor gene therapy in conjunction with the pro-drug ganciclovir (GCV), we are able to mediate a greatly enhanced anti-tumor effect in the murine B16 melanoma tumor model. The data presented suggests that this enhanced antitumor effect is the result of a strong induced CTL immune response resulting from the recruitment of immune cells to the site of HSV-TK/GCV-induced tumor destruction by the potent chemokine SLC.

Efficient genetic manipulation of the NOD-Rag1-/-IL2RgammaC-null mouse by combining in vitro fertilization and CRISPR/Cas9 technology.

Humanized mouse models have become increasingly important and widely used in modeling human diseases in biomedical research. Immunodeficient mice such as NOD-Rag1-/-IL2RgammaC-null (NRG) or NOD-SCID-IL2RgammaC-null (NSG) mice are critical for efficient engraftment of human cells or tissues. However, their genetic modification remains challenging due to a lack of embryonic stem cells and difficulty in the collection of timed embryos after superovulation. Here, we report the generation of gene knockout NRG mice by combining in vitro fertilization (IVF) and CRISPR/Cas9 technology. Sufficient numbers of fertilized embryos were produced through IVF, and a high rate of Fah gene targeting was achieved with microinjection of Cas9 mRNA, gRNA and single strand oligonucleotide DNA (ssDNA) into the embryos. The technology paves the way to construct NRG or NSG mutant mice to facilitate new humanized mouse models. The technology can also be readily adapted to introduce mutations in other species such as swine and non-human primates.

Lentiviral-mediated RNAi knockdown yields a novel mouse model for studying Cyp2b function.

There are few in vivo knockout models available to study the function of Cyp2 members involved in the metabolism of endogenous and exogenous chemicals. These models may help provide insight into the cytochrome P450s (CYPs) responsible for the detoxification and activation of drugs, environmental toxicants, and endobiotics. The aim of this work is to produce a potent Cyp2b-knockdown (KD) mouse for subsequent study of Cyp2b function. We made a quintuple Cyp2b-KD mouse using lentiviral-promoted short hairpin RNA (shRNA) homologous to all five murine Cyp2b subfamily members (Cyp2b9, 2b10, 2b13, 2b19, and 2b23). The Cyp2b-KD mice are viable, fertile, and without obvious gross abnormalities except for an increase in liver weight. Expression of the three hepatic Cyp2b members, 2b9, 2b10, and 2b13, is significantly repressed as demonstrated by quantitative real-time PCR and Western blotting. The constitutive androstane receptor activator, 1,4-Bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), was used to determine if shRNA-mediated Cyp2b10 repression could be outcompeted by Cyp2b10 induction. TCPOBOP-treated Cyp2b-KD mice show 80-90% less Cyp2b protein expression than TCPOBOP-treated wild-type (WT) mice, demonstrating that Cyp induction does not outcompete the repressive function of the shRNA. Untreated and TCPOBOP-treated Cyp2b-KD mice are poor metabolizers of parathion compared with WT mice. Furthermore, Cyp2b-KD mice are sensitive to parathion, an organophosphate insecticide primarily metabolized by Cyp2b enzymes, when compared with WT mice. In summary, we designed an shRNA construct that repressed the expression and activity of multiple Cyp2b enzymes. We foresee that this novel Cyp2b-KD mouse model will significantly improve our understanding of the role of Cyp2b enzymes in chemical sensitivity and drug metabolism.

In vitro- and in vivo-targeted tumor lysis by an MMP2 cleavable melittin-LAP fusion protein.

Chemotherapy is one of the main treatment options for cancer, but the effectiveness of chemotherapeutic drugs is severely limited due to their systemic toxicity. Therefore, the need for a more targeted approach in tumor treatment is obvious. A tumor-activated agent would decrease systemic toxicity as well as increase the efficacy of the treatment. It has previously been shown that the latency of pro-TGF-beta is conferred by dimerization of two latency-associated peptides (LAP) that form a protective shield, which is cleaved off upon activation by matrix metalloproteinases (MMPs). It has also been shown that the fusion of this LAP peptide with other cytokines can confer their latency. In the present study, a recombinant adenovirus with a fusion gene encoding a tumor-activated pro-cytolytic peptide was made in which the LAP domain of TGF-beta was fused with melittin, a potent cytolytic toxin, with an MMP2 cleavage site in between the two. In vitro studies show that the melittin-MMP2-LAP recombinant adenovirus can be activated by MMP2 which leads to the release of free melittin to lyse the target cells. In vivo studies show approximately a 70% decrease in B16 tumor volume in melittin-MMP2-LAP recombinant adenovirus-treated mice as compared to control mice. No significant systemic toxicity was observed in the treated mice.

In vivo targeted killing of prostate tumor cells by a synthetic amoebapore helix 3 peptide modified with two γ-linked glutamate residues at the COOH terminus.

We previously designed a pro-cytolytic peptide to target prostate-specific membrane antigen (PSMA)-positive prostate tumor cells. The backbone of the peptide was derived from the cell lytic amoebapore H-3 domain, which becomes completely inactive upon modification by two glutamate residues linked to the ε-amide group of the COOH-terminal lysine through γ-linkages (H-3Glu2). This modified H-3 domain regains its lytic activity against PSMA-positive cells (LNCaP) after the γ-linked glutamate residues are cleaved by PSMA. Our previous in vitro results demonstrate that the modified amoebapore peptide has strong cytolytic activity towards PSMA-positive cells and very little activity towards PSMA-negative cells. In the present study, the in vivo efficacy of this modified peptide was examined in human LNCaP prostate tumor xenografts in nude mice. The results showed significantly decreased tumor size and PSA levels in treated mice as compared to control mice. As well, 5/12 of the treated mice were tumor-free. Peptide distribution studies showed that peptide levels in the prostate tumors maintained a steady concentration for approximately 6 hours. Single-dose toxicity studies showed no toxic effects of the peptide when administered intraperitoneally or intravenously at a dose of 30 mg/kg.

Glycogen synthase kinase-3alpha reduces cardiac growth and pressure overload-induced cardiac hypertrophy by inhibition of extracellular signal-regulated kinases.

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase having multiple functions and consisting of two isoforms, GSK-3alpha and GSK-3beta. Pressure overload increases expression of GSK-3alpha but not GSK-3beta. Despite our wealth of knowledge about GSK-3beta, the function of GSK-3alpha in the heart is not well understood. To address this issue, we made cardiac-specific GSK-3alpha transgenic mice (Tg). Left ventricular weight and cardiac myocyte size were significantly smaller in Tg than in non-Tg (NTg) mice, indicating that GSK-3alpha inhibits cardiac growth. After 4 weeks of aortic banding (transverse aortic constriction (TAC)), increases in left ventricular weight and myocyte size were significantly smaller in Tg than in NTg, indicating that GSK-3alpha inhibits cardiac hypertrophy. More severe cardiac dysfunction developed in Tg after TAC. Increases in fibrosis and apoptosis were greater in Tg than in NTg after TAC. Among signaling molecules screened, ERK phosphorylation was decreased in Tg. Adenovirus-mediated overexpression of GSK-3alpha, but not GSK-3beta, inhibited ERK in cultured cardiac myocytes. Knockdown of GSK-3alpha increased ERK phosphorylation, an effect that was inhibited by PD98059, rottlerin, and protein kinase Cepsilon (PKCepsilon) inhibitor peptide, suggesting that GSK-3alpha inhibits ERK through PKC-MEK-dependent mechanisms. Knockdown of GSK-3alpha increased protein content and reduced apoptosis, effects that were abolished by PD98059, indicating that inhibition of ERK plays a major role in the modulation of cardiac growth and apoptosis by GSK-3alpha. In conclusion, up-regulation of GSK-3alpha inhibits cardiac growth and pressure overload-induced cardiac hypertrophy but increases fibrosis and apoptosis in the heart. The anti-hypertrophic and pro-apoptotic effect of GSK-3alpha is mediated through inhibition of ERK.