Human Prostate Carcinoma PC-3 Cells Research Articles

Synthesis and cytotoxic analysis of some disodium 3β,6β-dihydroxysterol disulfates

Disodium 3β,6β-dihydroxy-5α-cholestane disulfate ( 1) was synthesized in 4 steps with a high overall yield from cholesterol. First, cholesterol ( 4a) was converted to cholest-4-en-3,6-dione ( 5a) via oxidation with pyridinium chlorochromate (PCC) and then 5a was reduced by NaBH 4 in the presence of NiCl 2 to produce cholest-3β,6β-diol ( 6a). The reaction of 6a with the triethylamine-sulfur trioxide complex generated diammonium 3β,6β-dihydroxy-5α-cholestane disulfate ( 7a) and the treatment of 7a by cation exchange resin 732 (sodium form)(Na +) yielded the target steroid 1. Disodium 24-ethyl-3β,6β-dihydroxycholest-22-ene disulfate ( 2) and disodium 24-ethyl-3β,6β-dihydroxycholestane disulfate ( 3) were synthesized using a similar method. The cytotoxicity of these compounds against Sk-Hep-1 (human liver carcinoma cell line), H-292 (human lung carcinoma cell line), PC-3 (human prostate carcinoma cell line) and Hey-1B (human ovarian carcinoma cell line) cells was investigated. Our results indicate that presence of a cholesterol-type side chain at position 17 is necessary for their biological activity.

Antiproliferative Activities of Five Chinese Medicinal Herbs and Active Compounds in Elephantopus scaber

Extracts and fractions prepared from five medicinal herbs used to treat cancer in Chinese folk medicine were tested for their antiproliferative activities on five cancer cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The MTT results revealed that the ethanolic extracts possessed stronger antiproliferative activities than the corresponding aqueous ones. The bioactive fractions of the ethanolic extracts were mostly found in n-hexane and/or ethyl acetate fractions, which by TLC analysis were shown to contain terpenes and/or phenolic compounds. The ethyl acetate fraction from the ethanolic extract of Elephantopus scaber showed the highest effect with IC50 values of less than 25 microg/mL on three susceptible cell lines, namely, PC-3 (human prostate carcinoma cell), CNE (human nasopharyngeal carcinoma epithelial cell) and HL-60 (human acute promyelocytic leukemia cell). Further isolation and analysis of this fraction led to the identification of three sesquiterpene lactones, two flavonoids and five triterpenes. MTT results showed that the sesquiterpene lactones were the most effective group with IC50 values ranging from 0.9 to 7.5 microM while the flavonoids possessed moderate to strong effect with IC50 values ranging from 17.3 to 42.6 microM. However, the triterpenes displayed weak effect with IC50 values larger than 50 microM. Among the active compounds, deoxyelephantopin, a sesquiterpene lactone, exhibited the strongest effect on the PC-3, CNE and HL-60 cells, with IC50 values of 4.6, 2.6 and 0.9 microM, respectively. Flow cytometric analysis showed that treatment with deoxyelephantopin caused subG-1 population augmentation in PC-3, CNE and HL-60 cells, suggesting apoptosis was induced in these cells.

Inositol hexaphosphate downregulates both constitutive and ligand-induced mitogenic and cell survival signaling, and causes caspase-mediated apoptotic death of human prostate carcinoma PC-3 cells

Constitutively active mitogenic and prosurvival signaling cascades due to aberrant expression and interaction of growth factors and their receptors are well documented in human prostate cancer (PCa). Epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1) are potent mitogens that regulate proliferation and survival of PCa cells via autocrine and paracrine loops involving both mitogen-activated protein kinase (MAPK)- and Akt-mediated signaling. Accordingly, here we assessed the effect of inositol hexaphosphate (IP6) on constitutive and ligand (EGF and IGF-1)-induced biological responses and associated signaling cascades in advanced and androgen-independent human PCa PC-3 cells. Treatment of PC-3 cells with 2 mM IP6 strongly inhibited both growth and proliferation and decreased cell viability; similar effects were also observed in other human PCa DU145 and LNCaP cells. IP6 also caused a strong apoptotic death of PC-3 cells together with caspase 3 and PARP cleavage. Mechanistic studies showed that biological effects of IP6 were associated with inhibition of both constitutive and ligand-induced Akt phosphorylation together with a decrease in total Akt levels, but a differential inhibitory effect on MAPKs extra cellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal protein kinase (JNK1/2), and p38 under constitutive and ligand-activated conditions. Under similar condition, IP6 also inhibited AP-1 DNA-binding activity and decreased nuclear levels of both phospho and total c-Fos and c-Jun. Together, these findings for the first time establish IP6 efficacy in inhibiting aberrant EGF receptor (EGFR) or IGF-1 receptor (IGF-1R) pathway-mediated sustained growth promoting and survival signaling cascades in advanced and androgen-independent human PCa PC-3 cells, which might have translational implications in advanced human PCa control and management.

A Novel Antitumor Prodrug Platform Designed to Be Cleaved by the Endoprotease Legumain

Chemotherapeutic treatment of neoplastic diseases is often restricted by adverse systemic toxicity, which limits the dose of drug that can be administered, or by the appearance of drug resistance. Therefore, novel targeted therapeutic approaches are being developed to improve current conventional therapy in order to increase specificity and biocompatibility, and decrease toxicity. Legumain represents a recently identified lysosomal protease that has been reported to be overexpressed in the majority of human solid tumors, to promote cell migration and is associated with enhanced tissue invasion and metastases. Therefore, it serves as a promising candidate for prodrug therapy. We synthesized a novel legumain-cleavable prodrug, carbobenzyloxy-alanine-alanine-asparagine-ethylenediamine-etoposide, which releases the chemotherapeutic agent, etoposide, as the active drug. The prodrug was characterized and analyzed by (1)H NMR and HPLC. 293 Human embryonic kidney (293 HEK) cells were stably transfected with human legumain, to achieve overexpression in vitro (293 HEK-Leg). 293 HEK-Leg cells expressed both active and inactive legumain and secreted it to the medium. Legumain expression was found to be elevated because of serum starvation in both 293 HEK cells and PC3 human prostate carcinoma cells. The commercial substrate of legumain, carbobenzyloxy-alanine-alanine-asparagine-amino-4-methyl coumarin (CBZ-Ala-Ala-Asn-AMC) and the synthesized prodrug were both cleaved by recombinant human legumain (rhlegumain) and legumain expressed in the 293 HEK-Leg cell lysate. Upon cleavage by rhlegumain, the prodrug showed an inhibitory effect on the proliferation of 293 HEK and 293 HEK-Leg cells. This study suggests a novel platform for prodrug therapy activated by legumain as a promising approach for cancer therapy.

Mechanism of Inhibition of Human Cytochrome P450 1A1 and 1B1 by Piceatannol

The resveratrol analogue piceatannol (3,5,3',4'-tetrahydroxy-trans-stilbene) is a polyphenol present in grapes and wine and reported to have anti-carcinogenic activities. To investigate the mechanism of anticarcinogenic activities of piceatannol, the effects on CYP 1 enzymes were determined in Escherichia coli membranes coexpressing recombinant human CYP1A1, CYP1A2 or CYP1B1 with human NADPH-P450 reductase. Piceatannol showed a strong inhibition of CYP1A1 and CYP1B1 in a concentration-dependent manner, and <TEX>$IC_{50}$</TEX> of human CYP1A1 and CYP1B1 was 5.8 <TEX>${\mu}M$</TEX> and 16.6 <TEX>${\mu}M$</TEX>, respectively. However, piceatannol did not inhibit CYP1A2 activity in the concentration of up to 100 <TEX>${\mu}M$</TEX>. Piceatannol exhibited 3-fold selectivity for CYP1B1 over CYP1A1. The mode of inhibition of piceatannol was non-competitive for CYP1A1 and CYP1B1. The result that piceatannol did not inhibit CYP1B1-mediated <TEX>$\alpha$</TEX>-naphthoflavone (<TEX>$\alpha$</TEX>-NF) metabolism suggests piceatannol may act as a non-competitive inhibitor as well. In human prostate carcinoma PC-3 cells, piceatannol induces apoptosis and prevents Aktmediated signal pathway. Taken together, abilities of piceatannol to induce apoptotic cell death as well as CYP1 enzyme inhibition make this compound a useful tool for cancer chemoprevention.

99mTc-Labeled Bombesin(7−14)NH2 with Favorable Properties for SPECT Imaging of Colon Cancer

In this report, we present the synthesis and evaluation of the (99m)Tc-labeled beta-Ala-BN(7-14)NH2 (ABN = beta-Ala-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2) as a new radiotracer for tumor imaging in the BALB/c nude mice bearing HT-29 human colon cancer xenografts. The gastrin releasing peptide receptor binding affinity of ABN and HYNIC-ABN (6-hydrazinonicotinamide) was assessed via a competitive displacement of (125)I-[Tyr4]BBN bound to the PC-3 human prostate carcinoma cells. The IC 50 values were calculated to be 24 +/- 2 nM and 38 +/- 1 nM for ABN and HYNIC-ABN, respectively. HYNIC is the bifunctional coupling agent for (99m)Tc-labeling, while tricine and TPPTS (trisodium triphenylphosphine-3,3',3''-trisulfonate) are used as coligands to prepare the ternary ligand complex [(99m)Tc(HYNIC-ABN)(tricine)(TPPTS)] in very high yield and high specific activity. Because of its high hydrophilicity (log P = -2.39 +/- 0.06), [(99m)Tc(HYNIC-ABN)(tricine)(TPPS)] was excreted mainly through the renal route with little radioactivity accumulation in the liver, lungs, stomach, and gastrointestinal tract. The tumor uptake at 30 min postinjection (p.i.) was 1.59 +/- 0.23%ID/g with a steady tumor washout over the 4 h study period. As a result, it had the best T/ B ratios in the blood (2.37 +/- 0.68), liver (1.69 +/- 0.41), and muscle (11.17 +/- 3.32) at 1 h p.i. Most of the injected radioactivity was found in the urine sample at 1 h p.i., and there was no intact [(99m)Tc(HYNIC-ABN)(tricine)(TPPTS)] detectable in the urine, kidney, and liver samples. Its metabolic instability may contribute to its rapid clearance from the liver, lungs, and stomach. Despite the steady radioactivity washout, the tumors could be clearly visualized in planar images of the BALB/c nude mice bearing the HT-29 human colon xenografts at 1 and 4 h p.i. The favorable excretion kinetics from the liver, lungs, stomach, and gastrointestinal tract makes [(99m)Tc(HYNIC-ABN)(tricine)(TPPTS)] a promising SPECT radiotracer for imaging colon cancer.

Vitamin E succinate induces NAG-1 expression in a p38 kinase-dependent mechanism

NAG-1 (nonsteroidal anti-inflammatory drug-activated gene), a member of the transforming growth factor-beta superfamily, is involved in many cellular processes, such as inflammation, apoptosis/survival, and tumorigenesis. Vitamin E succinate (VES) is the succinate derivative of alpha-tocopherol and has antitumorigenic activity in a variety of cell culture and animal models. In the current study, the regulation and role of NAG-1 expression in PC-3 human prostate carcinoma cells by VES was examined. VES treatment induced growth arrest and apoptosis as well as an increase in NAG-1 protein and mRNA levels in a time- and concentration-dependent manner. VES treatment induced nuclear translocation and activation of p38 kinase. Pretreatment with p38 kinase inhibitor blocked the VES-induced increase in NAG-1 protein and mRNA levels, whereas an inhibition of protein kinase C, Akt, c-Jun NH(2)-terminal kinase, or MEK activity had no effect on VES-induced NAG-1 levels. Forced expression of constitutively active MKK6, an upstream kinase for p38, induced an increase in NAG-1 promoter activity, whereas p38 kinase inhibitor blocked MKK6-induced increase in NAG-1 promoter activity. VES treatment resulted in >3-fold increase in the half-life of NAG-1 mRNA in a p38 kinase-dependent manner and transient transfection experiment showed that VES stabilizes NAG-1 mRNA through AU-rich elements in 3'-untranslated region of NAG-1 mRNA. The inhibition of NAG-1 expression by small interfering RNA significantly blocked VES-induced poly(ADP-ribose) polymerase cleavage, suggesting that NAG-1 may play an important role in VES-induced apoptosis. These results indicate that VES-induced expression of NAG-1 mRNA/protein is regulated by transcriptional/post-transcriptional mechanism in a p38 kinase-dependent manner and NAG-1 can be chemopreventive/therapeutic target in prostate cancer.

PC3 Human Prostate Carcinoma Cell Holoclones Contain Self-renewing Tumor-Initiating Cells

Primary keratinocytes exhibit three typical clonal morphologies represented by holoclones, meroclones, and paraclones, with holoclones containing self-renewing stem cells, and meroclones and paraclones containing more mature and differentiated cells. Interestingly, long-term-cultured human epithelial cancer cells in clonal cultures also form holoclones, meroclones, and paraclones, and tumor cell holoclones have been hypothesized to harbor stem-like cells or cancer stem cells. However, the key question of whether tumor cell holoclones genuinely contain tumor-initiating cells has not been directly addressed. Here, using PC3 human prostate carcinoma cells as a model, we provide direct experimental evidence that tumor cell holoclones contain stem-like cells that can initiate serially transplantable tumors. Importantly, holoclones derived from either cultured PC3 cells or holoclone-initiated tumors can be serially passaged and regenerate all three types of clones. In contrast, meroclones and paraclones cannot be continuously propagated and fail to initiate tumor development. Phenotypic characterizations reveal high levels of CD44, alpha(2)beta(1) integrin, and beta-catenin expression in holoclones, whereas meroclones and paraclones show markedly reduced expression of these stem cell markers. The present results have important implications in understanding morphologic heterogeneities and tumorigenic hierarchies in human epithelial cancer cells.

A facile and efficient synthesis of some (6 E)-hydroximino-4-en-3-one steroids, steroidal oximes from Cinachyrella spp. sponges

Using β-sitosterol as a starting material, (6 E)-hydroximino-24-ethylcholest-4-en-3-one ( 1), a natural steroidal oxime from Cinachyrella alloclada and C. apion, was synthesized in four steps with a high overall yield. First, β-sitosterol ( 5a) is transformed into the corresponding 24-ethylcholest-4-en-3,6-dione ( 6a) via oxidation with pyridinium chlorochromate (PCC). Selective reduction of 6a by NaBH 4 in the presence of CoCl 2 gives 24-ethylcholest- 4-en-3β-ol-6-one ( 7a). The reaction of 7a with hydroxylamine hydrochloride offers the oxime 8a and the oxidation of 8a by Jones reagent gives the target steroid 1. (6 E)-Hydroximinocholest-4-en-3-one ( 2) and (6 E)-hydroximino-24-ethylcholest-4,22-dien-3-one ( 4) were synthesized by a similar method. The cytotoxicity of the synthesized compounds against sk-Hep-1 (human liver carcinoma cell line), H-292 (human lung carcinoma cell line), PC-3 (human prostate carcinoma cell line) and Hey-1B (human ovarian carcinoma cell line) cells were investigated. The presence of a cholesterol-type side chain appears to be necessary for the biological activity.

RETRACTED: Theaflavins induce G2/M arrest by modulating expression of p21waf1/cip1, cdc25C and cyclin B in human prostate carcinoma PC-3 cells

Cancer of the prostate gland (PCA) is the most common invasive malignancy and is the second leading cause of cancer-related death in males. The polyphenolic constituents of black tea have gained considerable attention as chemopreventive agents. Many studies have shown that black tea reduces the risk of several cancer types. In the present study, we studied the effect of a black tea polyphenol, theaflavin (TF), on cellular proliferation and cell death in the human prostate cancer cell line, PC-3. We showed that TF inhibits cell proliferation in a dose- and time-dependent manner. Studies on cell cycle progression have shown that the anti-proliferative effect of TF is associated with an increase in the G2/M phase of PC-3 cells. Western blot results showed that TF-induced G2/M phase arrest was mediated through the inhibition of cyclin-regulated signaling pathways. TF induces cyclin kinase inhibitor p21(waf1/cip1) expression and inhibits cdc25C and cyclin B expression. Increased exposure time to TF caused apoptosis of PC-3 cells, which was associated with up-regulation of the pro-apoptotic proteins Bax, caspase-3 and caspase-9 and down-regulation of anti-apoptotic protein Bcl-2. The role of caspase-induced apoptosis was further confirmed by a reduction in mitochondria membrane potential and the appearance of a DNA laddering pattern. Thus, it can be concluded that TF acts as an effective anti-proliferative agent by modulating cell growth regulators in prostate cancer cells.

Prostasin induces protease-dependent and independent molecular changes in the human prostate carcinoma cell line PC-3

Expression of prostasin in the PC-3 human prostate carcinoma cells inhibited in vitro invasion, but the molecular mechanisms are unknown. Wild-type human prostasin or a serine active-site mutant prostasin was expressed in the PC-3 cells. Molecular changes were measured at the mRNA and the protein levels. Cell signaling changes were evaluated by measuring phosphorylation of the extracellular signal-regulated kinases (Erk1/2) following epidermal growth factor (EGF) treatment of the cells. Protein expression of the EGF receptor (EGFR) was differentially down-regulated by the wild-type and the active-site mutant prostasin. The mRNA expression of EGFR and the transcription repressor SLUG was reduced in cells expressing wild-type prostasin but not the active-site mutant. Phosphorylation of Erk1/2 in response to EGF was greatly reduced by the wild-type prostasin but not by the active-site mutant. The mRNA expression of the urokinase-type plasminogen activator (uPA), the uPA receptor (uPAR), cyclooxygenase-2 (COX-2), and the inducible nitric oxide synthase (iNOS) was decreased by the wild-type and the active-site mutant prostasin. The mRNA or protein expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), matriptase, and E-cadherin was greatly increased by the active-site mutant prostasin. In conclusion, prostasin expression elicits both protease-dependent and independent molecular changes in the PC-3 cells.

A novel anticancer agent, icaritin, induced cell growth inhibition, G1 arrest and mitochondrial transmembrane potential drop in human prostate carcinoma PC-3 cells

Icariin and icaritin with prenyl group have been demonstrated for their selective estrogen receptor modulating activities. We screened their effects on cell growth in human prostate carcinoma PC-3 cell line (estrogen receptor positive) in vitro. PC-3 cell line was used for the measurement of anti-carcinoma activities of 0–100 μmol/l icaritin and 30 μmol/l icariin. 1 μmol/l 17-β estradiol (E2) served as the estrogen positive control, and 1 μmol/l ICI 182,780 [7 α-[9 (4,4,5,5,5-pentafluoropentyl) sulfinyl] nonyl]-estra-1,3,5(10)-triene-3,17h-diol]] served as the specific estrogen receptor antagonist. Primary cultured rat prostate basal cells used as cell growth selective control. The growth-inhibitory effects were analyzed using MTT assay, and fluorochrome staining, flow cytometry, and immunoblotting were employed to illustrate the possible mechanisms. When treated with icaritin for 24 to 72 h, cell growth was strongly inhibited (at 48 h IC50 was 10.74±1.59 μmol/l, P<0.001) companied with a mitochondrial transmembrane potential (_Ψm) drop. Meanwhile, few changes in IC50 could be observed when co-incubated with ICI 182,780. Icaritin-induced growth inhibition was associated with G1 arrest (P<0.05), and G2-M arrest depending upon doses. Consistently with G1 arrest, icaritin increased protein expressions of pRb, p27Kip1 and p16Ink4a, while showed decrease in phosphorylated pRb, Cyclin D1 and CDK4. Comparatively, icariin has much lower effects on PC-3 cells and showed only weak G1 arrest, suggesting a possible structure–activity relationship. These findings suggested a novel anticancer efficacy of icaritin mediated selectively via induction of cell cycle arrest but not associated with estrogen receptors in PC-3 cells.

Atrial Natriuretic Peptide Attenuates Hypoxia Induced Chemoresistance in Prostate Cancer Cells

Low tumor oxygenation (hypoxia) correlates with resistance to chemotherapeutic agents. We recently reported that in vitro hypoxia induced resistance to various anti-cancer drugs can be attenuated by nitric oxide mimetic agents. Natriuretic peptides are molecules that mediate their cellular effects by activating a signaling pathway similar to that activated by nitric oxide. In the current study we determined whether atrial natriuretic peptide is able to inhibit hypoxia induced chemoresistance in prostate carcinoma cells. Reverse transcriptase-polymerase chain reaction and atrial natriuretic peptide binding studies were used to determine the presence and function of natriuretic peptide receptors on a panel of human cell lines as well as in tissue samples. Drug sensitivity assays of cell lines exposed to hypoxic or standard conditions were performed in the presence of various concentrations of atrial natriuretic peptide. These studies revealed the presence of the 3 known natriuretic peptide receptors A, B and C in PC-3 and DU-145 human prostate carcinoma cells (American Type Culture Collection, Manassas, Virginia) as well as in tissue samples of human prostate cancer. Atrial natriuretic peptide binding to these cells was unaffected by culture in 0.5% vs 20% O(2). Clonogenic assays revealed that incubation of these cells in 0.5% O(2) for 24 hours resulted in a subsequent 4 to 10-fold increase in their survival following 1-hour exposure to doxorubicin (Sigma) (12.5 microM) (p <0.001). While small concentrations of atrial natriuretic peptide (10(-7) to 10(-13) M) did not affect sensitivity to doxorubicin in cells incubated in 20% O(2), similar concentrations of atrial natriuretic peptide inhibited the survival of these cells incubated in 0.5% O(2) by up to 50% (p <0.006). Using the cyclic guanosine monophosphate dependent protein kinase G inhibitor KT5823 (15 microM) the chemosensitizing effect of atrial natriuretic peptide was abrogated. These results indicate the potential use of natriuretic peptides as adjuvants to chemotherapy for prostate cancer.

STAT3 is required but not sufficient for EGF receptor-mediated migration and invasion of human prostate carcinoma cell lines

Growth factor-induced migration is a rate-limiting step in tumour invasiveness. The molecules that regulate this cellular behaviour would represent novel targets for limiting tumour cell progression. Epidermal growth factor (EGF) receptor (EGFR)-mediated motility, present in both autocrine and paracrine modes in prostate carcinomas, requires de novo transcription to persist over times greater than a few hours. Therefore, we sought to define specific signalling pathways that directly alter cellular transcription. Signal transducer and activator of transcription 3 (STAT3) is activated, as determined by electrophoretic motility shift assays, by EGFR in DU145 and PC3 human prostate carcinoma cells in addition to the motility model NR6 fibroblast cell line. Inhibition of STAT3 activity by antisense or siRNA downregulation or expression of a dominant-negative construct limited cell motility as determined by an in vitro wound healing assay and invasiveness through a extracellular matrix barrier. The expression of constitutively activated STAT3 did not increase the migration, which indicates that STAT3 is necessary but not sufficient for EGFR-mediated migration. These findings suggest that STAT3 signalling may be a new target for limiting prostate tumour cell invasion. In a microarray gene analysis of what transcription units are altered by EGF in a STAT3-dependent manner we found that the expression of motility-limiting VASP protein and the apoptosis nexus caspase 3 were both downregulated upon EGF exposure. These findings suggest a molecular basis for the STAT3 dependence of EGFR-mediated prostate tumour progression.

Dual-Color-Coded Imaging of Viable Circulating Prostate Carcinoma Cells Reveals Genetic Exchange between Tumor Cells In Vivo, Contributing to Highly Metastatic Phenotypes

Color-coded imaging analysis revealed yellow fluorescent metastasis precursor cells that were readily recognized in the blood of tumor-bearing mice after mixtures of red fluorescent protein (RFP)- and green fluorescent protein (GFP)-expressing PC-3 human prostate carcinoma cells were implanted in the nude mouse prostate and metastasized. The yellow fluorescent cells were purified from the blood of nude mice to 99% homogeneity by FACS, expanded in culture, and re-implanted in the prostate of nude mice. The yellow fluorescent phenotype was heritable and stably maintained by tumor cells for many generations in vitro and in vivo. In the animals implanted with the yellow-fluorescing cells, 100% developed aggressive metastatic cancer. Lung metastases were demonstrated in 100% of the animals as early as four weeks after injection of the yellow-fluorescing cells in the mouse prostate. In contrast, when the GFP- and RFP-expressing parental cells were inoculated into the mouse prostate separately, none of the animals developed lung metastasis. All animals had almost exclusively yellow fluorescent cells in the blood and bone marrow. These results are consistent with the idea that spontaneous genetic exchange between tumor cells in vivo contributes to genomic instability and creation of highly metastatic cells.

900 PUBLICATION Synergistic interaction between ionizing radiation and 8-chloro-adenosine 3′, 5′-monophosphate in PC-3 human prostate carcinoma cells

900 PUBLICATION Synergistic interaction between ionizing radiation and 8-chloro-adenosine 3′, 5′-monophosphate in PC-3 human prostate carcinoma cells

A novel anticancer agent, decursin, induces G 1 arrest and apoptosis in human prostate carcinoma cells : Yim D, Singh RP, Agarwal C, Lee S, Chi H, Agarwal R, Department of Pharmacy, Sahm Yook University, Seoul, Korea

We isolated a coumarin compound decursin (C 19 H 20 O 5 ; molecular weight 328) from Korean angelica ( Angelica gigas ) root and characterized it by spectroscopy. Here, for the first time, we observed that decursin (25–100 μmol/L) treatment for 24 to 96 hours strongly inhibits growth and induces death in human prostate carcinoma DU145, PC-3, and LNCaP cells. Furthermore, we observed that decursinol [where (CH 3 ) 2 -C=CH-COO- side chain of decursin is substituted with -OH] has much lower effects compared with decursin, suggesting a possible structure-activity relationship. Decursin-induced growth inhibition was associated with a strong G 1 arrest ( P 1 , S as well as G 2 -M arrests depending upon doses and treatment times in PC-3 cells. Comparatively, decursin was nontoxic to human prostate epithelial PWR-1E cells and showed only moderate growth inhibition and G 1 arrest. Consistent with G 1 arrest in DU145 cells, decursin strongly increased protein levels of Cip1/p21 but showed a moderate increase in Kip1/p27 with a decrease in cyclin-dependent kinases (CDK); CDK2, CDK4, CDK6, and cyclin D1, and inhibited CDK2, CDK4, CDK6, cyclin D1, and cyclin E kinase activity, and increased binding of CDK inhibitor (CDKI) with CDK. Decursin-caused cell death was associated with an increase in apoptosis ( P

HIV-1 TAT protein transduction domain mediates enhancement of enzyme prodrug cancer gene therapy in vitro: a study with TAT-TK-GFP triple fusion construct

Protein transduction domain (PTD) from HIV-1 TAT protein has been reported to translocate across the mammalian cell membrane, also as a part of fusion proteins. However, the true nature of TAT-mediated intercellular spreading is still under debate because it has been claimed to be a fixation artifact. To study the spreading of TAT fusion proteins and their potency to enhance thymidine kinase/ ganciclovir (HSV-TK/GCV) cancer gene therapy, we constructed a novel triple fusion protein containing TAT PTD, HSV-TK and green fluorescent protein (TAT-TK-GFP). This fusion protein has three functional domains in the same polypeptide, allowing reliable determination of the relationship between transduction rate and cell killing efficiency. TAT-TK-GFP was cloned into a lentivirus vector and used for analyses of TAT-mediated protein translocation and enhancement of HSV-TK/GCV cytotoxicity. The triple fusion protein was expressed correctly in vitro, but cell-to-cell translocation was not observed in rat glioma cells (BT4C). However, TAT-TK-GFP made BT4C and SKOV3.ip1 (human ovarian carcinoma) cells significantly more sensitive to ganciclovir than TK-GFP, whereas the effect in PC-3 human prostate carcinoma cells was more subtle. It was also observed that growth in lower serum concentration (2.5-5%) abolished the enhancement in BT4C cells, suggesting that high proliferation rate is one of the factors that contribute to TAT PTD-mediated enhancement of cytotoxicity. In summary, our results indicate that TAT PTD fusion proteins do not translocate intercellularly at detectable levels, but enhancement of the HSV-TK/GCV cytotoxicity can be detected in rat and human tumor cell lines in vitro.

Milk Thistle and Prostate Cancer: Differential Effects of Pure Flavonolignans from Silybum marianum on Antiproliferative End Points in Human Prostate Carcinoma Cells

Extracts from the seeds of milk thistle, Silybum marianum, are known commonly as silibinin and silymarin and possess anticancer actions on human prostate carcinoma in vitro and in vivo. Seven distinct flavonolignan compounds and a flavonoid have been isolated from commercial silymarin extracts. Most notably, two pairs of diastereomers, silybin A and silybin B and isosilybin A and isosilybin B, are among these compounds. In contrast, silibinin is composed only of a 1:1 mixture of silybin A and silybin B. With these isomers now isolated in quantities sufficient for biological studies, each pure compound was assessed for antiproliferative activities against LNCaP, DU145, and PC3 human prostate carcinoma cell lines. Isosilybin B was the most consistently potent suppressor of cell growth relative to either the other pure constituents or the commercial extracts. Isosilybin A and isosilybin B were also the most effective suppressors of prostate-specific antigen secretion by androgen-dependent LNCaP cells. Silymarin and silibinin were shown for the first time to suppress the activity of the DNA topoisomerase IIalpha gene promoter in DU145 cells and, among the pure compounds, isosilybin B was again the most effective. These findings are significant in that isosilybin B composes no more than 5% of silymarin and is absent from silibinin. Whereas several other more abundant flavonolignans do ultimately influence the same end points at higher exposure concentrations, these findings are suggestive that extracts enriched for isosilybin B, or isosilybin B alone, might possess improved potency in prostate cancer prevention and treatment.

A Novel Anticancer Agent, Decursin, Induces G1 Arrest and Apoptosis in Human Prostate Carcinoma Cells

We isolated a coumarin compound decursin (C(19)H(20)O(5); molecular weight 328) from Korean angelica (Angelica gigas) root and characterized it by spectroscopy. Here, for the first time, we observed that decursin (25-100 micromol/L) treatment for 24 to 96 hours strongly inhibits growth and induces death in human prostate carcinoma DU145, PC-3, and LNCaP cells. Furthermore, we observed that decursinol [where (CH(3))(2)-C=CH-COO- side chain of decursin is substituted with -OH] has much lower effects compared with decursin, suggesting a possible structure-activity relationship. Decursin-induced growth inhibition was associated with a strong G(1) arrest (P < 0.001) in DU145 and LNCaP cells, and G(1), S as well as G(2)-M arrests depending upon doses and treatment times in PC-3 cells. Comparatively, decursin was nontoxic to human prostate epithelial PWR-1E cells and showed only moderate growth inhibition and G(1) arrest. Consistent with G(1) arrest in DU145 cells, decursin strongly increased protein levels of Cip1/p21 but showed a moderate increase in Kip1/p27 with a decrease in cyclin-dependent kinases (CDK); CDK2, CDK4, CDK6, and cyclin D1, and inhibited CDK2, CDK4, CDK6, cyclin D1, and cyclin E kinase activity, and increased binding of CDK inhibitor (CDKI) with CDK. Decursin-caused cell death was associated with an increase in apoptosis (P < 0.05-0.001) and cleaved caspase-9, caspase-3, and poly(ADP-ribose) polymerase; however, pretreatment with all-caspases inhibitor (z-VAD-fmk) only partially reversed decursin-induced apoptosis, suggesting the involvement of both caspase-dependent and caspase-independent pathways. These findings suggest the novel anticancer efficacy of decursin mediated via induction of cell cycle arrest and apoptosis selectively in human prostate carcinoma cells.