Alexander Yemelyanov

Effects of IKK inhibitor PS1145 on NF-κB function, proliferation, apoptosis and invasion activity in prostate carcinoma cells

A key antiapoptotic transcription factor, nuclear factor kappa-B (NF-κB), is known to be critically important for tumor cell growth, angiogenesis and development of metastatic lesions. We and others showed previously that NF-κB transcription factor was constitutively activated in androgen-independent prostate carcinoma (PC) cell lines due to the upregulated activity of inhibitor of NF-κB kinases (IKK). In this work, using luciferase assay, electrophoretic mobility shift assay and Northern blot analysis of expression of endogenous κB-responsive genes, we demonstrate that a novel highly specific small-molecule IKK inhibitor, PS1145, efficiently inhibited both basal and induced NF-κB activity in PC cells. We found that PS1145 induced caspase 3/7-dependent apoptosis in PC cells and significantly sensitized PC cells to apoptosis induced by tumor necrosis factor alpha. We also showed that PS1145 inhibited PC cell proliferation. Effects of PS1145 on proliferation and apoptosis correlated with inhibition of interleukin (IL)-6, cyclin D1, D2, inhibitor of apoptosis (IAP)-1 and IAP-2 gene expression and decreased IL-6 protein level. In addition, we found that incubation with PS1145 inhibited the invasion activity of highly invasive PC3-S cells in invasion chamber assay in a dose-dependent manner. Overall, this study provides the framework for development of a novel therapeutic approach targeting NF-κB transcription factor to treat advanced PC.

Tumor suppressor activity of glucocorticoid receptor in the prostate.

Glucocorticoids are extensively used in combination chemotherapy of advanced prostate cancer (PC). Little is known, however, about the status of the glucocorticoid receptor (GR) in PC. We evaluated over 200 prostate samples and determined that GR expression was strongly decreased or absent in 70–85% of PC. Similar to PC tumors, some PC cell lines, including LNCaP, also lack GR. To understand the role of GR, we reconstituted its expression in LNCaP cells using lentiviral approach. Treatment of LNCaP-GR cells with the glucocorticoids strongly inhibited proliferation in the monolayer cultures and blocked anchorage-independent growth. This was accompanied by upregulation of p21 and p27, down-regulation of cyclin D1 expression and c-Myc phosphorylation. Importantly, the activation of GR resulted in normalized expression of PC markers hepsin, AMACR, and maspin. On the signaling level, GR decreased expression and inhibited activity of the MAP-kinases (MAPKs) including p38, JNK/SAPK, Mek1/2 and Erk1/2. We also found that activation of GR inhibited activity of numerous transcription factors (TF) including AP-1, SRF, NF-κB, p53, ATF-2, CEBPα, Ets-1, Elk-1, STAT1 and others, many of which are regulated via MAPK cascade. The structural analysis of hepsin and AMACR promoters provided the mechanistic rationale for PC marker downregulation by glucocorticoids via inhibition of specific TFs. Our data suggest that GR functions as a tumor suppressor in prostate, and inhibits multiple signaling pathways and transcriptional factors involved in proliferation and transformation.

Paracrine activation of WNT/β-catenin pathway in uterine leiomyoma stem cells promotes tumor growth

Significance Stem cells and the ovarian steroids estrogen and progesterone are essential for leiomyoma tissue growth. The underlying mechanisms are unknown, particularly because leiomyoma stem cells are deficient in estrogen and progesterone receptors. Expression of these receptors is much higher in surrounding mature myometrial or leiomyoma smooth muscle cells. Here, we demonstrate that wingless-type (WNT) acts as a paracrine signal from estrogen/progesterone receptor-rich mature cells to activate the canonical β-catenin pathway in leiomyoma stem cells. Our findings suggest a paracrine role for the canonical WNT pathway in the growth of leiomyoma tumor. Uterine leiomyomas are extremely common estrogen and progesterone-dependent tumors of the myometrium and cause irregular uterine bleeding, severe anemia, and recurrent pregnancy loss in 15–30% of reproductive-age women. Each leiomyoma is thought to arise from a single mutated myometrial smooth muscle stem cell. Leiomyoma side-population (LMSP) cells comprising 1% of all tumor cells and displaying tumor-initiating stem cell characteristics are essential for estrogen- and progesterone-dependent in vivo growth of tumors, although they have remarkably lower estrogen/progesterone receptor levels than mature myometrial or leiomyoma cells. However, how estrogen/progesterone regulates the growth of LMSP cells via mature neighboring cells is unknown. Here, we demonstrate a critical paracrine role of the wingless-type (WNT)/β-catenin pathway in estrogen/progesterone-dependent tumorigenesis, involving LMSP and differentiated myometrial or leiomyoma cells. Estrogen/progesterone treatment of mature myometrial cells induced expression of WNT11 and WNT16, which remained constitutively elevated in leiomyoma tissues. In LMSP cells cocultured with mature myometrial cells, estrogen-progesterone selectively induced nuclear translocation of β-catenin and induced transcriptional activity of its heterodimeric partner T-cell factor and their target gene AXIN2, leading to the proliferation of LMSP cells. This effect could be blocked by a WNT antagonist. Ectopic expression of inhibitor of β-catenin and T-cell factor 4 in LMSP cells, but not in mature leiomyoma cells, blocked the estrogen/progesterone-dependent growth of human tumors in vivo. We uncovered a paracrine role of the WNT/β-catenin pathway that enables mature myometrial or leiomyoma cells to send mitogenic signals to neighboring tissue stem cells in response to estrogen and progesterone, leading to the growth of uterine leiomyomas.

Vitamin A in the cerebrospinal fluid of patients with and without idiopathic intracranial hypertension.

We quantified vitamin A in the cerebrospinal fluid of patients with idiopathic intracranial hypertension, elevated intracranial pressure of other causes and normal intracranial pressure. Vitamin A could be detected by high‐pressure liquid chromatography in most of the specimens. There was a significantly higher level of vitamin A in the cerebrospinal fluid of some patients with idiopathic intracranial hypertension. Vitamin A toxicity may play a role in the pathogenesis of idiopathic intracranial hypertension.

Novel Steroid Receptor Phyto-Modulator Compound A Inhibits Growth and Survival of Prostate Cancer Cells

Androgen receptor (AR)- and glucocorticoid receptor (GR)- mediated signaling play opposite roles in prostate tumorigenesis: AR promotes prostate carcinoma (PC) development, whereas GR acts as a tumor suppressor. Compound A (CpdA) is a stable analogue of an aziridine precursor from the African shrub Salsola tuberculatiformis Botschantzev. It was shown recently that, in model cells, CpdA inhibits AR function and strongly enhances anti-inflammatory function of GR. We determined the effects of CpdA in prostate cells with different AR/GR status: (a) RWPE-1 cells (AR(low)/GR(low)), (b) PC3 and DU145 cells (GR(+)/AR(-)), (c) LNCaP cells (GR(-)/AR(+)), and (d) LNCaP-GR cells expressing both receptors. Similar to steroid hormones, CpdA induces nuclear translocation of both receptors in prostate cells. Despite this, CpdA inhibits DNA-binding and transactivation potential of AR. In addition, CpdA inhibits GR-mediated transactivation but induces GR transrepression via inhibition of several transcription factors, including nuclear factor-kappaB, AP-1, Ets-1, Elk-1, SRF, CRE/ATF, and NFATc. CpdA strongly decreases growth and induces caspase-dependent apoptosis in highly malignant PC3 and DU145 cells and in other AR/GR-expressing PC cells. The cytostatic effect of CpdA is receptor dependent: down-regulation of GR or AR expression drastically attenuates CpdA-induced PC cell growth inhibition. Finally, virtual docking analysis indicates that CpdA shares binding cavities in AR and GR ligand-binding domains with corresponding hormones and forms hydrogen bonds (H-bond) with the same amino acids that are involved in H-bond formation during steroid binding. Overall, our data suggest that CpdA is a unique dual-target steroid receptor modulator that has a high potential for PC therapy.

Androgen receptor targets NFκB and TSP1 to suppress prostate tumor growth in vivo

The androgen role in the maintenance of prostate epithelium is subject to conflicting opinions. While androgen ablation drives the regression of normal and cancerous prostate, testosterone may cause both proliferation and apoptosis. Several investigators note decreased proliferation and stronger response to chemotherapy of the prostate cancer cells stably expressing androgen receptor (AR), however no mechanistic explanation was offered. In this paper we demonstrate in vivo anti‐tumor effect of the AR on prostate cancer growth and identify its molecular mediators. We analyzed the effect of AR on the tumorigenicity of prostate cancer cells. Unexpectedly, the AR‐expressing cells formed tumors in male mice at a much lower rate than the AR‐negative controls. Moreover, the AR‐expressing tumors showed decreased vascularity and massive apoptosis. AR expression lowered the angiogenic potential of cancer cells, by increasing secretion of an anti‐angiogenic protein, thrombospondin‐1. AR activation caused a decrease in RelA, a subunit of the pro‐survival transcription factor NFκB, reduced its nuclear localization and transcriptional activity. This, in turn, diminished the expression of its anti‐apoptotic targets, Bcl‐2 and IL‐6. Increased apoptosis within AR‐expressing tumors was likely due to the NFκB suppression, since it was restricted to the cells lacking nuclear (active) NFκB. Thus we for the first time identified combined decrease of NFκB and increased TSP1 as molecular events underlying the AR anti‐tumor activity in vivo. Our data indicate that intermittent androgen ablation is preferable to continuous withdrawal, a standard treatment for early‐stage prostate cancer.

Targeting transcription factor NFκB: comparative analysis of proteasome and IKK inhibitors

Nuclear factor-κB (NF-κB) plays a critical role in cancer development and progression. Thus, the NF-κB signaling pathway provides important targets for cancer chemoprevention and anti-cancer chemotherapy. The central steps in NF-κB activation are phosphorylation and proteasome-dependent degradation of its inhibitory proteins termed IκBs. Consequently, the major pharmacological approaches to target NF-κB include (i) repression of IκB kinases (IKKs) and (ii) blocking the degradation of IκBs by proteasome inhibitors. We quantitatively compared the efficacy of various proteasome inhibitors (MG132, lactacystin and epoxomicin) and IKK inhibitors (BAY 11-7082 and PS1145) to block NF-κB activity induced by TNFα or TPA and to sensitize LNCaP prostate carcinoma cells to apoptosis. Our studies revealed significant differences between these two classes of NF-κB inhibitors. We found that proteasome inhibitors epoxomicin and MG132 attenuated NF-κB induction much more effectively than the IKK inhibitors. Furthermore, in contrast to IKK inhibitors, all studied proteasome inhibitors specifically blocked TPA-induced generation de novo of NF-κB p50 homodimers – (p50/p50). These results suggest that the proteasome plays a dominant role in TPA-induced formation of functional p50 homodimers, while IKK activity is less important for this process. Interestingly, profound attenuation of p50/p50 DNA-binding does not reduce the high potency of proteasome inhibitors to suppress NF-κB-dependent transcription. Finally, proteasome inhibitors were much more effective in sensitizing LNCaP cells to TNFα-induced apoptosis compared to IKK inhibitors at the concentrations when both types of agents similarly attenuated NF-κB activity. We conclude that this remarkable pro-apoptotic potential of proteasome inhibitors is partially mediated through NF-κB-independent mechanism.

Differential targeting of androgen and glucocorticoid receptors induces ER stress and apoptosis in prostate cancer cells: A novel therapeutic modality

Androgen (AR) and glucocorticoid (GR) receptor signaling play opposing roles in prostate tumorigenesis: in prostate, AR acts as an oncogene, and GR is a tumor suppressor. Recently, we found that non-steroidal phyto-chemical Compound A (CpdA) is AR/GR modulator acting as anti-inflammatory anti-androgen. CpdA inhibits AR and prevents GR transactivation while enhancing GR transrepression. GR and AR are controlled by proteasomal degradation. We found that prolonged exposure of LNCaP, LNCaP-GR, DU145 and PC3 prostate carcinoma (PCa) cells to proteasome inhibitor Bortezomib (BZ) caused AR degradation and GR accumulation. BZ enhanced CpdA ability to inhibit AR and to augment GR transrepression. We also found that CpdA+BZ differentially regulated GR/AR to cooperatively suppress PCa cell growth and survival and to induce endoplasmic reticulum stress (ERS). Importantly, CpdA+BZ differentially regulated GR-responsive genes. CpdA+BZ blocked activation of glucocorticoid-responsive pro-survival genes, including SGK1, but activated BZ-induced ERS-related genes BIP/HSPA5 and CHOP /GADD153. Using ChIP, we showed that SGK1, BIP/HSPA5 and CHOP regulation was due to effects of CpdA and CpdA+BZ on GR loading on their promoters. We also found that AR and GR are abundant in advanced PCa from patients treated by androgen ablation and/or chemotherapy: 56% of carcinomas from treated patients expressed both receptors, and the other 27% expressed either GR or AR. Overall, our data validate the concept of dual AR/GR targeting in prostate cancer (PC) and suggest that BZ combination with dual-target steroid receptor modulator CpdA has high potential for PC therapy.

Photophysical properties of xanthophylls in carotenoproteins from human retina

Abstract The macula of the human retina contains high amounts of the xanthophyll carotenoids lutein and zeaxanthin [a mixture of (3R,3′R)-zeaxanthin and (3R,3′S-meso)-zeaxanthin]. Recently, it was shown that the uptake and the stabilization of zeaxanthin and lutein into the retina are likely to be mediated by specific xanthophyll-binding proteins (XBP). Here, we have used femtosecond pump–probe spectroscopy to study the dynamics of the S1 state of these xanthophylls in xanthophyll-enriched and native XBP. The results from the native XBP and the enriched XBP were then compared with those for carotenoids in organic solvents and in detergent micelles. Steady-state and transient absorption spectra show that the incorporation of xanthophylls into the protein causes a redshift of the spectra, which is stronger for lutein than for zeaxanthin. The transient absorption spectra further indicate that a part of the xanthophylls remains unbound in the xanthophyll-enriched XBP. The transient absorption spectra of the native XBP prove the presence of both xanthophylls in native XBP. Although the S1 lifetime of lutein does not exhibit any changes when measured in solution, micelles or XBP, we have observed the influence of the environment on the S1 lifetime of meso-zeaxanthin, which has a longer (12 ps) lifetime in XBP than in solution (9 ps). The most pronounced effect was found for vibrational relaxation in the S1 state, which is significantly slower for xanthophylls in XBP compared with micelles and solution. This effect is more pronounced for meso-zeaxanthin, suggesting a specific site of binding of this carotenoid to XBP.

The mechanisms of tumor suppressor effect of glucocorticoid receptor in skin

Glucocorticoid hormones exert a tumor suppressor effect in different experimental models, including mouse skin carcinogenesis. The glucocorticoid control of cellular functions is mediated via the glucocorticoid receptor (GR), a well‐known transcription factor that regulates genes by DNA‐binding dependent transactivation, and DNA‐binding independent transrepression through negative interaction with other transcription factors. In this perspective, we analyze known mechanisms that underlie the anticancer effect of GR signaling, including effects on cell growth, differentiation, apoptosis, and angiogenesis. We also discuss a novel mechanism for the tumor suppressor effect of the GR in skin: through the regulation of the number and status of follicular epithelial stem cells (SC), which are a target cell population for skin carcinogenesis. Our studies on keratin5.GR transgenic animals that are resistant to skin carcinogenesis, demonstrated that the GR diminishes the number of follicular epithelial SCs, reduces their proliferative and survival potential and affects the expression of follicular SC “signature” genes. The analysis of global effect of the GR on gene expression in follicular epithelial SCs, basal keratinocytes, and mouse skin tumors provided an unexpected evidence that gene transrepression by GR plays an important role in the maintenance of SC and in inhibition of skin carcinogenesis by this steroid hormone receptor. It is known that antiinflammatory effect of glucocorticoids is chiefly mediated by GR transrepression. Thus, our findings suggest the similarity between the mechanisms of antiinflammatory and anticancer effects of the GR signaling. We discuss the potential clinical applications of our findings in light of drug discovery programs focused on the development of selective GR modulators that preferentially induce GR transrepression.