Thalidomide up-regulates prostate-specific antigen secretion from LNCaP cells

Thalidomide has been shown to have species- and metabolic-dependent antiangiogenic activity in vitro and in vivo, suggesting its potential in treating human angiogenesis-dependent pathologies such as solid tumors. Based on promising preclinical studies, thalidomide has entered phase II clinical trials for prostate, brain, breast cancer, and Kaposi's sarcoma. However, the antiangiogenic mechanism of action is largely unresolved, as are its effects on tumor-associated gene expression, cytokine secretion, etc. We have investigated the effects of thalidomide on: 1) the secretion of prostate-specific antigen (PSA) in a human androgen-dependent prostate cell line; 2) growth and viability of human prostate cells; and 3) differential gene expression profiles of thalidomide-treated vs untreated human prostate cells. A human androgen-dependent prostate carcinoma cell line (LNCaP) and a human androgen-independent prostate carcinoma cell line (PC-3) were incubated with thalidomide 0.6, 6, or 60 7g/mL for 5-6 days. Secreted PSA from LNCaP cells was measured using a commercial enzyme-linked immunosorbant assay. Cell viability studies were conducted in both LNCaP and PC-3 cells using the same thalidomide concentrations. Furthermore, the differential gene expression of thalidomide-treated LNCaP cells was compared to that of untreated control cells using a commercially available human cancer cDNA expression array system. Thalidomide-treated LNCaP cells demonstrated increased PSA/cell levels at all concentrations tested compared to untreated control cells. Thalidomide demonstrated a cytostatic effect in LNCaP cells but had no appreciable effect on PC-3 cell viability compared to untreated control cells. Comparison of cDNA expression arrays hybridized with thalidomide-treated LNCaP cDNA probes suggests that thalidomide may up- or downregulate expression of angiogenesis-related genes, i.e., vitronectin, but these differential effects require further verification. Thalidomide over a range of doses has demonstrated nontoxic, cytostatic activity in LNCaP cells and significant upregulation of LNCaP cell PSA secretion in vitro. Furthermore, preliminary data from cDNA nucleic acid arrays of thalidomide-treated LNCaP cells suggest that thalidomide upregulates a potential angiogenic modulatory protein, the vitronectin precursor, which may eventually link thalidomide's antiangiogenic activity with modulation of angiogenic vascular integrin pathways.     

Anti-proliferative effect of retinoids and interferon-α-2a on vaginal cell lines derived from squamous intra-epithelial lesions

A panel of retinoids (all-trans-, 13-cis-, 19-cis retinoic acid and acitretin), and interferon-α-2a was tested for the capacity to modulate the proliferation of UT-DEC-1 (HPV-33-positive) and UT-DEC-2 (HPV-16-positive) cell lines derived from vaginal intra-epithelial neoplasias (VAIN). At concentrations 10⁻⁶ to 10⁻⁸ M, all retinoids inhibited the growth of early-passage UT-DEC cell lines, but also of normal vaginal keratinocytes and fibroblasts. The inhibition was significantly reduced in late-passage UT-DEC cells. The effect on proliferation was essentially equal for all retinoids in high (1.8 mM)-Ca²⁺ medium, but decreased markedly in low (0.09 mM)-Ca²⁺ medium. Interferon-α-2a at 1000 IU/ml had an additive growth-inhibitory effect in the low- and in the high-Ca²⁺ medium. No consistent decrease in HPV E6-E7 mRNA levels could be associated either with retinoid or with interferon effect in either cell line. The expression of TGFβ1 and TGFβ2 mRNA increased 2- to 3-fold by 10⁻⁶ M 13-cis-RA treatment in early- and in late-passage cells of both cell lines. TGFβ1 at 0.1 to 1.0 ng/ml also inhibited the proliferation of both cell lines, and was more effective at early passage, but the inhibition was not dependent on calcium concentration. Neutralizing anti-TGFβ antibodies partially relieved the proliferation inhibition by 13-cis-RA. The results show that the calcium-associated regulation of growth by the tested retinoids was seen in normal vaginal cells and in early pre-neoplastic cells, but was significantly reduced in cells with higher-grade phenotype, while also suggesting that the loss of responsiveness to retinoids and TGFβ may play a role in the progression of squamous intra-epithelial neoplasia. Int. J. Cancer 78:338–345, 1998.© 1998 Wiley-Liss, Inc.     

Differentiating agents in pediatric malignancies: Retinoids in neuroblastoma

Retinoids are derivatives of vitamin A that include all-trans-retinoic acid (ATRA), 13-cis-retinoic acid, (13-cis-RA), and fenretinide (4-HPR). High levels of either ATRA or 13-cis-RA can cause arrest of cell growth and morphologic differentiation of human neuroblastoma cell lines. Phase I trials have shown that higher and more sustained drug levels were obtained with 13-cis-RA relative to ATRA. A phase III randomized trial showed that high-dose pulse therapy with 13-cis-RA given after completion of intensive chemoradiotherapy (with or without autologous bone marrow transplantation) significantly improves event-free survival in high-risk neuroblastoma. Because 4-HPR achieves multi-log cell kills in neuroblastoma cell lines that are resistant to ATRA and 13-cis-RA, a pediatric phase I trial is in progress to determine the maximum tolerated dose of 4-HPR, with a view toward giving 4-HPR after completion of myeloablative therapy and 13-cis-RA.     

The Effect of Curcumin on Proliferation and Apoptosis in LNCaP Prostate Cancer Cells

OBJECTIVE To observe the effect of curcumin on proliferation and apop-tosis in the prostate cancer LNCaP cell line. METHODS The AXSYMTM system luciferase method was used to examine the effect of various concentratious of curcumin on the content of prostate specific antigen (PSA) in prostate cancer LNCaP cells. A pGL3-PSA luciferase expression vector, containing 640 bp DNA of the PSA gene 5' -promoter region was constructed and transfected into the LNCaP cells with lipofectin. By measuring luciferase activity, the effect of 10 μmol/L, 20 μmol/L, 30 and 40 μmol/L curcumin on the promoter was studied. Effects on cell growth and apoptosis were analyzed by microscopy, the MTT colorimetric assay and flow cytometry. Western-blotting was used to measure expression of the androgen receptor (AR) in the LNCaP cells treated with different concentrations of curcumin. RESULTS The results showed that the expression of PSA was inhibited as curcumin reduced the activity of luciferase. Curcumin also caused a sigificant concentration-dependent decrease in AR expession measured by Western -blotting. Cell growth was inhibited and apoptosis was induced. CONCLUSION By inhibiting AR expression, curcumin reduced the function of the PSA promoter and inhibited PSA protein expression. Curcumin decreased the cellular proliferation and induced apoptosis in LNCaP cells in a concention-dependent manner.     

Genistein affects androgen-responsive genes through both androgen- and estrogen-induced signaling pathways

This study examined the mechanisms by which the prostate cancer chemopreventive agent genistein modulates gene expression in LNCaP human prostate cancer cells. Expression of androgen- and estrogen-regulated genes was measured in LNCaP cells cultured in the presence or absence of hormonal stimulation and the presence or absence of genistein. Genistein strongly suppressed basal expression of androgen-responsive gene (ARG) mRNAs, including prostate-specific antigen (PSA) and Ste20-related proline-alanine-rich kinase (SPAK). However, genistein had little or no effect on basal expression of two other ARGs, beta2-microglobulin (B2M) or selenoprotein P (SEPP1). Culturing LNCaP cells in the presence of the synthetic androgen R1881-induced increases in PSA, SPAK, B2M, and SEPP1 mRNA levels. The R1881-induced expression of these genes was uniformly blocked by genistein. For PSA and SPAK, genistein also blocked or downregulated 17beta-estradiol-induced increases in mRNA expression. These results indicate that genistein selectively alters expression of ARG mRNAs in LNCaP cells through modulation of both androgen- and estrogen-induced signaling pathways.     

O-glycosylation regulates LNCaP prostate cancer cell susceptibility to apoptosis induced by galectin-1

Resistance to apoptosis is a critical feature of neoplastic cells. Galectin-1 is an endogenous carbohydrate-binding protein that induces death of leukemia and lymphoma cells, breast cancer cells, and the LNCaP prostate cancer cell line, but not other prostate cancer cell lines. To understand the mechanism of galectin-1 sensitivity of LNCaP cells compared with other prostate cancer cells, we characterized glycan ligands that are important for conferring galectin-1 sensitivity in these cells, and analyzed expression of glycosyltransferase genes in galectin-1-sensitive, prostate-specific antigen-positive (PSA(+)) LNCaP cells compared with a galectin-1-resistant PSA(-) LNCaP subclone. We identified one glycosyltransferase, core 2 N-acetylglucosaminyltransferase, which is down-regulated in galectin-1-resistant PSA(-) LNCaP cells compared with galectin-1-sensitive PSA(+) LNCaP cells. Intriguingly, this is the same glycosyltransferase required for galectin-1 susceptibility of T lymphoma cells, indicating that similar O-glycan ligands on different polypeptide backbones may be common death trigger receptors recognized by galectin-1 on different types of cancer cells. Blocking O-glycan elongation by expressing alpha2,3-sialyltransferase 1 rendered LNCaP cells resistant to galectin-1, showing that specific O-glycans are critical for galectin-1 susceptibility. Loss of galectin-1 susceptibility and synthesis of endogenous galectin-1 has been proposed to promote tumor evasion of immune attack; we found that galectin-1-expressing prostate cancer cells killed bound T cells, whereas LNCaP cells that do not express galectin-1 did not kill T cells. Resistance to galectin-1-induced apoptosis may directly contribute to the survival of prostate cancer cells as well as promote immune evasion by the tumor.     

Tissue prostate-specific antigen facilitates refractory prostate tumor progression via enhancing ARA70-regulated androgen receptor transactivation

Despite being well recognized as the best biomarker for prostate cancer, pathophysiologic roles of prostate-specific antigen (PSA) remain unclear. We report here that tissue PSA may be involved in the hormone-refractory prostate cancer progression. Histologic analyses show that the increased tissue PSA levels are correlated with lower cell apoptosis index and higher cell proliferation rate in hormone-refractory tumor specimens. By stably transfecting PSA cDNA into various prostate cancer cell lines, we found that PSA could promote the growth of androgen receptor (AR)-positive CWR22rv1 and high-passage LNCaP (hormone-refractory prostate cancer cells) but not that of AR-negative PC-3 and DU145 cells. Surprisingly, the protease activity of PSA is not crucial for PSA to stimulate growth and promote AR transactivation. We further showed that increased PSA could enhance ARA70-induced AR transactivation via modulating the p53 pathway that results in the decreased apoptosis and increased cell proliferation in prostate cancer cells. Knockdown of PSA in LNCaP and CWR22rv1 cells causes cell apoptosis and cell growth arrest at the G(1) phase. In vitro colony formation assay and in vivo xenografted tumor results showed the suppression of prostate cancer growth via targeting PSA expression. Collectively, our findings suggest that, in addition to being a biomarker, PSA may also become a new potential therapeutic target for prostate cancer. PSA small interfering RNA or smaller molecules that can degrade PSA protein may be developed as alternative approaches to treat the prostate cancer.     

Role of p115RhoGEF in the regulation of extracellular Ca2+‐induced choline kinase activation and prostate cancer cell proliferation

Ca²⁺ is a ubiquitous cellular signal which plays a central role in the regulation of cell function. To understand aberrant signaling through the Ca²⁺‐sensing receptor (CaR) in prostate cancer cells, we compared expression of CaR signaling components in human nonmalignant prostate epithelial cells and several prostate cancer cell lines, as well as normal human prostate and prostate tumor specimens. We found that levels of the CaR, Gα₁₂ and p115RhoGEF expression are significantly up‐regulated in more tumorigenic prostate cancer cells and prostate tumor specimens. By silencing CaR, Gα₁₂, p115RhoGEF or choline kinase (ChoK) expression, analyzing the change in lipid profiles, blocking signaling pathways using chemical inhibitors, and co‐immunoprecipitating the relevant signaling proteins, we demonstrate that p115RhoGEF, a regulator of G protein signaling (RGS) with GAP activity for Gα_12/13 and with guanine nucleotide exchange activity for the small G protein Rho, plays an important role in the regulation of Ca‐induced ChoK activation and cell proliferation in more tumorigenic prostate cancer cell lines. The results demonstrate an important role of p115RhoGEF in prostate tumorigenesis and provide a potential target of cancer therapeutics.     

Combinational effect of PPARγ agonist and RXR agonist on the growth of SGC7901 gastric carcinoma cells in vitro

In order to investigate the inhibitory effects and mechanisms of troglitazone (TGZ), a peroxisome proliferator-activated receptor γ (PPARγ) agonist, and retinoid X receptor (RXR) agonist (9-cis-retinoic acid (RA)) on gastric carcinoma cells SGC7901, SGC7901 cells were treated with TGZ and 9-cis-RA, respectively, or in combination. Then, the cell growth, apoptosis, morphological changes, and the expression of PPARγ, RXRγ, Bcl-2, and Bax were detected by MTT assay, flow cytometry, HE staining, immunocytochemistry staining, and Western blot assay, respectively. Our results showed that the growth of SGC7901 cells was inhibited and the cells got sparser at the concentrations of 50 μmol/L TGZ, 20 μmol/L 9-cis-RA, or combination of TGZ (25 μmol/L) and 9-cis-RA (10 μmol/L). Immunocytochemistry and Western blot showed that after 72 h, the expression of PPARγ, RXRγ, and Bax were upregulated; Bcl-2 was downregulated compared with the negative control group. These data indicated that PPARγ agonist and RXR agonist could inhibit the proliferation of SGC7901 cells via inducing the apoptosis, which involved the increase in the level of Bax/Bcl-2. The combination of RXR agonist and PPARγ agonist could induce the maximal inhibitory effects on tumor growth and apoptosis via promoting the formation of RXR/PPARγ heterodimer.     

Correlation between overexpression of EpCAM in prostate tissues and genesis of androgen-dependent prostate cancer

The objective of this study was to investigate the role of epithelial cell adhesion molecule (EpCAM) in the genesis and the progress of prostate cancer, especially of castration-resistant prostate cancer. Protein expression of EpCAM in ten pairs of prostate cancer tissues and normal adjacent tissues, plus three cell lines, was examined. Short hairpin RNA (shRNA) interference technique was employed to silence the expression of EpCAM in prostate cancer cell LNCaP and construct a stable transfected cell line. In vitro assay was conducted to analyze the effect of EpCAM expression on the expressions of Androgen receptor (AR), Prostate specific antigen (PSA), and cellular proliferation and invasion. EpCAM was found significantly expressed higher in prostate cancer tissues than in normal adjacent tissues. In three cell lines (DU-145, PC-3, and LNCaP), the expression of EpCAM in LNCaP, androgen-dependent prostate cancer cells, was significantly higher than that in the other two. As EpCAM was silenced in LNCaP, the expression levels of AR and PSA obviously descended, and cellular abilities of proliferation and invasion were obviously inhibited.The overexpression of EpCAM has correlation with the genesis of prostate cancer, especially androgen-dependent prostate cancer. As the expression of AR is facilitated, prostate cancer cells’ abilities to proliferate and invade are consequently enhanced.     

Effect of calcitriol on prostate-specific antigen in vitro and in humans.

BACKGROUND: Calcitriol, the natural ligand for the vitamin D receptor, has significant potential in prostate cancer treatment. Measurement of its antineoplastic activity in prostate cancer clinical trials may be complicated by effects of calcitriol on prostate-specific antigen (PSA) production. We examined the effects of calcitriol at similar concentration on cell proliferation, androgen receptor (AR) expression, and PSA production in vitro and on PSA concentrations in prostate cancer patients. EXPERIMENTAL DESIGN: LNCaP prostate cancer cell proliferation was examined by cell counts 6 days after exposure to a range of concentrations of calcitriol. AR and PSA protein was quantified in LNCaP cells over 96 hours after exposure to 1 nmol/L calcitriol. Serum PSA and free PSA was serially measured by immunoassay over a period of 8 days in patients with hormone-na?ve prostate cancer after a single dose of 0.5 microg/kg calcitriol. RESULTS: Calcitriol treatment resulted in dose-dependent growth inhibition of LNCaP with approximately 50% growth inhibition at the clinically achievable concentration of 1 nmol/L. Time-dependent up-regulation of AR expression and of PSA production in LNCaP cells was shown at the same concentration. No significant change in serum PSA or free PSA over 8 days was seen in eight subjects treated with a single dose of 0.5 microg/kg calcitriol. The analysis was powered to detect a 1.23-fold change between the baseline and day 8 serum PSA. CONCLUSIONS: At clinically achievable concentrations, calcitriol inhibits growth and induces AR and PSA expression in LNCaP cells. We did not detect similar changes in serum PSA or free PSA in patients exposed to similar concentrations of calcitriol. Thus, a PSA flare, predicted by preclinical systems, is unlikely to occur in patients and therefore unlikely to complicate interpretation of clinical trial outcomes.     

Differentiated prostatic antigen expression in LNCaP cells following treatment with bispecific antisense oligonucleotides directed against BCL-2 and EGFR

Antisense oligonucleotides (oligos) have been administered against in vivo and in vitro prostate cancer models employing LNCaP and PC-3 cell lines. While most oligos consist of a single mRNA binding site targeting a single gene product or those with sequence homology, our lab has developed bispecific oligos directed toward two unrelated proteins. In LNCaP cells, we initially identified bispecifics that increased the expression of prostate-specific membrane antigen (PSMA) while not affecting secreted prostate-specific antigen (PSA). We postulated that surface antigen expression is increased by bispecifics able to form double-stranded regions, acting as interferon (IFN-γ) inducers. In other systems, when induced, IFN-γ promotes cell surface antigen expression, including HLA and receptors for tumor necrosis factor. To test this hypothesis, we measured the effect of oligo treatment on both IFN-γ induction and the expression of another secreted product of differentiated prostate cells, prostatic acid phosphatase (PAP). This study initially evaluated the inhibition of in vitro propagating LNCaP cells employing mono- and bispecific oligos directed against bcl-2 (the second bispecific binding site was against the epidermal growth factor receptor). Employing RT-PCR, the expression of non-targeted proteins encoded by mRNA for PSMA, PSA, PAP, and IFN-γ was subsequently valuated. When LNCaP prostate tumor cells were incubated with oligos and compared to lipofectin-containing controls significant growth inhibition resulted. Employing RT-PCR, the levels of mRNA encoding PSMA were unexpectedly found to be elevated following treatment with the bispecific oligos but not with a monospecific directed solely against bcl-2. No differences were detected in mRNA levels encoding PSA following treatment with either oligo type. IFN-γ was significantly induced only by bispecific oligos, and PAP expression was similar to PSA. These data support the hypothesis that double strand-forming bispecific oligos induce IFN-γ that enhances cell surface PSMA expression. Expression of tumor-associated surface antigens could increase their recognition and targeting by immunologic defense mechanisms and increase the effectiveness of tumor vaccines.