Synthesis, proapoptotic screening, and structure-activity relationships of novel aza-lupane triterpenoids.

Apoptosis is a highly regulated process by which excessive cells are eliminated in order to maintain normal cell development and tissue homeostasis. Resistance to apoptosis often contributes to failure in cancer prevention and treatment. Apoptotic cell death regulators are considered important targets for discovery and development of new therapeutic agents in oncology research. A class of novel aza-lupane triterpenoids were designed, synthesized, and evaluated for antitumor activity against a panel of cancer cell lines of different histogenic origin and for ability to induce apoptosis. 3,30-Bis(aza) derivatives were identified not only to possess improved cytotoxicity compared to the natural product betulinic acid but also to affect cell death predominantly via apoptosis, whereas the mono(aza) derivatives apparently triggered cell death via different, non-apoptotic pathway(s).

Synthesis and cytotoxicity of 2-cyano-28-hydroxy-lup-1-en-3-ones.

New A-ring modified betulin and dihydrobetulin derivatives possessing the 2-cyano-1-en-3-one moiety were prepared and tested for cytotoxicity in seven cancer cell lines. The most active agent 9a synthesized in this account was further demonstrated to induce apoptosis and to activate caspases in malignant melanoma cells.

CRNK gene transfer improves function and reverses the myosin heavy chain isoenzyme switch during post-myocardial infarction left ventricular remodeling.

PYK2 is a Ca(2+)-dependent, nonreceptor protein tyrosine kinase that is involved in the induction of left ventricular hypertrophy (LVH) and its transition to heart failure. We and others have previously investigated PYK2's function in vitro using cultured neonatal and adult rat ventricular myocytes as model systems. However, the function of PYK2 in the in vivo adult heart remains unclear. Here we evaluate the effect of PYK2 inhibition following myocardial infarction (MI) using adenoviral (Adv) overexpression of the C-terminal domain of PYK2, known as CRNK. First we demonstrate that CRNK functions as a dominant-negative inhibitor of PYK2-dependent signaling, presumably by displacing PYK2 from focal adhesions and costameres. Then, male Sprague-Dawley rats (~300 g) underwent permanent left anterior descending coronary artery ligation. One wk post-MI, either Adv-GFP (n=34) or Adv-CRNK (n=28) was administered (10(10) pfu, 0.1 ml) via catheter-based, Optison-mediated gene transfer. LV structure and function were evaluated by echocardiography 1 and 3 wk after gene transfer, and LV tissue was analyzed by real-time RT-PCR and Western blotting. CRNK overexpression was readily detected by Western blotting 1 wk following gene transfer. Adv-CRNK improved overall survival (P=0.03; Logrank Test) and LV fractional shortening (23+/-2% vs. 31+/-2% for Adv-GFP vs. Adv-CRNK infected animals, respectively; P<0.05). Whereas MI hearts exhibited increased beta-, and decreased alpha-myosin heavy chain (MHC) mRNA expression characteristic of LVH, Adv-CRNK reversed the MHC isoenzyme switch (3.3+/-1.4 fold increase in alpha MHC; 0.4+/-0.1 fold decrease in beta MHC; P<0.05 for both). In summary, CRNK gene transfer improves survival, increases LV function, and alters MHC gene expression suggesting an attenuation of LV remodeling post-MI.

17ß-Estradiol inhibits HIV-1 by inducing a complex formation between ß-catenin and estrogen receptor α on the HIV promoter to suppress HIV transcription.

Human Immunodeficiency virus type 1 (HIV-1) disproportionately affects women, accounting for > 50% of new HIV infections in adults worldwide. While multiple mechanisms may contribute to a greater degree of HIV infection in women than men, we evaluated the direct effect of 17ß-estradiol, the most bioactive form of estrogen in women, on HIV replication in peripheral blood mononuclear cells (PBMCs). We demonstrate that 17ß-estradiol, in an ERα dependent manner, inhibits HIV replication by activating ß-catenin signaling. Specifically, we show for the first time that 17ß-estradiol induces a complex formation between ERα and ß-catenin which tether on the HIV LTR at -143nt site from +1 start site of HIV transcription to repress HIV promoter activity. These studies define a role of 17ß-estradiol in inhibiting HIV replication which may impact HIV pathogenesis in women and add to a growing list of viruses that are inhibited by 17ß-estradiol through ERα engagment.

Protein kinase Cepsilon-dependent MARCKS phosphorylation in neonatal and adult rat ventricular myocytes.

The myristoylated, alanine-rich protein kinase C substrate (MARCKS) is a cytoskeletal protein implicated in the regulation of cell spreading, stress fiber formation, and focal adhesion assembly in nonmuscle cells. However, its precise role in cardiomyocyte growth, and its PKC-dependent regulation have not been fully explored. In this report, we show that MARCKS is expressed and phosphorylated under basal conditions in cultured neonatal and adult rat ventricular myocytes (NRVM and ARVM, respectively). The PKC activators phenylephrine, angiotensin II, and endothelin-1 (ET) further increased MARCKS phosphorylation, with ET inducing the greatest response. To determine which PKC isoenzyme was responsible for agonist-induced MARCKS phosphorylation, NRVM and ARVM were infected with replication-defective adenoviruses (Adv) encoding wildtype (wt) and constitutively active (ca) mutants of PKCepsilon, PKCdelta, and PKCalpha. Only PKCepsilon increased phosphorylated MARCKS (pMARCKS). In contrast, Adv-mediated overexpression of a dominant-negative (dn) mutant of PKCepsilon reduced basal and ET-stimulated pMARCKS. dnPKCepsilon overexpression also prevented ET-induced, apparent co-localization of pMARCKS with f-actin staining structures. Adv-mediated overexpression of GFP-tagged, wtMARCKS (wtMARCKS-GFP) increased phosphorylation of focal adhesion kinase (FAK) and also increased NRVM surface area. In contrast, overexpression of a GFP-tagged, non-phosphorylatable (np) MARCKS mutant (npMARCKS-GFP) decreased basal and ET-induced endogenous MARCKS and FAK phosphorylation, and blocked the ET-induced increase in NRVM surface area. We conclude that MARCKS is expressed in cardiomyocytes, is phosphorylated by PKCepsilon, and participates in the regulation of FAK phosphorylation and cell spreading.

PYK2 regulates SERCA2 gene expression in neonatal rat ventricular myocytes.

The nonreceptor protein tyrosine kinase (PTK) proline-rich tyrosine kinase 2 (PYK2) has been implicated in cell signaling pathways involved in left ventricular hypertrophy and heart failure, but its exact role has not been elucidated. In this study, replication-defective adenoviruses (Adv) encoding green fluorescent protein (GFP)-tagged, wild-type (WT), and mutant forms of PYK2 were used to determine whether PYK2 overexpression activates MAPKs, and downregulates SERCA2 mRNA levels in neonatal rat ventricular myocytes (NRVM). PYK2 overexpression significantly decreased SERCA2 mRNA (as determined by Northern blot analysis and real-time RT-PCR) to 54 +/- 4% of Adv-GFP-infected cells 48 h after Adv infection. Adv-encoding kinase-deficient (KD) and Y(402)F phosphorylation-deficient mutants of PYK2 also significantly reduced SERCA2 mRNA (WT>KD>Y(402)F). Conversely, the PTK inhibitor PP2 (which blocks PYK2 phosphorylation by Src-family PTKs) significantly increased SERCA2 mRNA levels. PYK2 overexpression had no effect on ERK1/2, but increased JNK1/2 and p38(MAPK) phosphorylation from fourfold to eightfold compared with GFP overexpression. Activation of both "stress-activated" protein kinase cascades appeared necessary to reduce SERCA2 mRNA levels. Adv-mediated overexpression of constitutively active (ca)MKK6 or caMKK7, which activated only p38(MAPK) or JNKs, respectively, was not sufficient, whereas combined infection with both Adv reduced SERCA2 mRNA levels to 45 +/- 12% of control. WTPYK2 overexpression also significantly reduced SERCA2 promoter activity, as determined by transient transfection of a 3.8-kb SERCA2 promoter-luciferase construct. Thus a PYK2-dependent signaling cascade may have a role in abnormal cardiac Ca(2+) handling in left ventricular hypertrophy and heart failure via downregulation of SERCA2 gene transcription.

Protein kinase C-alpha-induced hypertrophy of neonatal rat ventricular myocytes.

Protein kinase C (PKC) isoenzymes play a critical role in cardiomyocyte hypertrophy. At least three different phorbol ester-sensitive PKC isoenzymes are expressed in neonatal rat ventricular myocytes (NRVMs): PKC-alpha, -delta, and -epsilon. Using replication-defective adenoviruses (AdVs) that express wild-type (WT) and dominant-negative (DN) PKC-alpha together with phorbol myristate acetate (PMA), which is a hypertrophic agonist and activator of all three PKC isoenzymes, we studied the role of PKC-alpha in signaling-specific aspects of the hypertrophic phenotype. PMA induced nuclear translocation of endogenous and AdV-WT PKC-alpha in NRVMs. WT PKC-alpha overexpression increased protein synthesis and the protein-to-DNA (P/D) ratio but did not affect cell surface area (CSA) or cell shape compared with uninfected or control AdV beta-galactosidase (AdV betagal)-infected cells. PMA-treated uninfected cells displayed increased protein synthesis, P/D ratio, and CSA and elongated morphology. PMA did not further enhance protein synthesis or P/D ratio in AdV-WT PKC-alpha-infected cells. To assess the requirement of PKC-alpha for these PMA-induced changes, AdV-DN PKC-alpha or AdV betagal-infected NRVMs were stimulated with PMA. Without PMA, AdV-DN PKC-alpha had no effects on protein synthesis, P/D ratio, CSA, or shape vs. AdV betagal-infected NRVMs. PMA increased protein synthesis, P/D ratio, and CSA in AdV betagal-infected cells, but these parameters were significantly reduced in PMA-stimulated AdV-DN PKC-alpha-infected NRVMs. Overexpression of DN PKC-alpha enhanced PMA-induced cell elongation. Neither WT PKC-alpha nor DN PKC-alpha affected atrial natriuretic factor gene expression. Insulin-like growth factor-1 also induced nuclear translocation of endogenous PKC-alpha. PMA but not WT PKC-alpha overexpression induced ERK1/2 activation. However, AdV-DN PKC-alpha partially blocked PMA-induced ERK activation. Thus PKC-alpha is necessary for certain aspects of PMA-induced NRVM hypertrophy.