Naftopidil, a selective alpha-1 adrenoceptor antagonist, inhibits growth of human prostate cancer cells by G1 cell cycle arrest

Alpha-1 adrenoceptor antagonists are generally prescribed for benign prostate hyperplasia with lower urinary tract symptoms. Naftopidil, a selective alpha-1 adrenoceptor antagonist, is frequently used in Japan because it has fewer side effects. Here we demonstrate for the first time that naftopidil has growth inhibitory effect in androgen-sensitive and -insensitive human prostate cancer cell lines. The concentrations causing 50% inhibition (IC50) of cancer cell growth were 22.2 +/- 4.0 microM in androgen-sensitive LNCaP cells and 33.2 +/- 1.1 microM in androgen-insensitive PC-3 cells. FACS analysis revealed that cell growth inhibition by naftopidil was due to the arrest of the G1 cell cycle. Expressions of p27(kip1) and p21(cip1) were significantly increased in LNCaP cells treated with naftopidil. In PC-3 cells, naftopidil induced p21(cip1) but not p27(kip1). In vivo, oral administration of naftopidil to nude mice inhibited the growth of PC-3 tumors as compared to vehicle-treated controls. These results suggest that naftopidil may be useful in the chemoprevention of prostate cancer and the intervention of hormone refractory prostate cancer.     

Inhibition of gap junctional intercellular communication between primary human smooth muscle cells by tumor necrosis factor {alpha}

Tumor necrosis factor     

1'-Acetoxychavicol acetate suppresses angiogenesis-mediated human prostate tumor growth by targeting VEGF-mediated Src-FAK-Rho GTPase-signaling pathway

Cancer therapeutic agents that are safe, effective and affordable are urgently needed. We describe that 1'-acetoxychavicol acetate (ACA), a component of Siamese ginger (Languas galanga), can suppress prostate tumor growth by largely abrogating angiogenesis. ACA suppressed vascular endothelial growth factor (VEGF)-induced proliferation, migration, adhesion and tubulogenesis of primary cultured human umbilical vascular endothelial cells (HUVECs) in a dose-dependent manner. ACA also inhibited VEGF-induced microvessel sprouting from aortic rings ex vivo and suppressed new vasculature formation in Matrigel plugs in vivo. We further demonstrated that the mechanisms of this chavicol were to block the activation of VEGF-mediated Src kinase, focal adhesion kinase (FAK) and Rho family of small guanosine triphosphatases (GTPases) (Rac1 and Cdc42 but not RhoA) in HUVECs. Furthermore, treatment of human prostate cancer cells (PC-3) with ACA resulted in decreased cell viability and suppression of angiogenic factor production by interference with dual Src/FAK kinases. After subcutaneous administration to mice bearing human prostate cancer PC-3 xenografts, ACA (6 mg/kg/day) remarkably inhibited tumor volume and tumor weight and decreased levels of Src, CD31, VEGF and Ki-67. As indicated by immunohistochemistry and TUNEL analysis, microvessel density and cell proliferation were also dramatically suppressed in tumors from ACA-treated mice. Taken together, our findings suggest that ACA targets the Src-FAK-Rho GTPase pathway, leading to the suppression of prostate tumor angiogenesis and growth.     

Malignant transformatin of human bronchial epithelial cells by radon-simulated {alpha}-particles

Epidemiological studies have shown that inhalation of radonis associated with an increased risk for bronchogenic carcinomain uranium miners. These     

Suppression of human prostate cancer cell growth by alpha1-adrenoceptor antagonists doxazosin and terazosin via induction of apoptosis

Recent evidence from our laboratory has demonstrated that alpha1-adrenoceptor antagonists doxazosin and terazosin induced apoptosis in prostate epithelial and smooth muscle cells in patients with benign prostatic hypertrophy (BPH; J. Urol., 159: 1810-1815, 1998; J. Urol., 161: 2002-2007, 1999). In this study, we investigated the biological action of three alpha1-adrenoceptor antagonists, doxazosin, terazosin, and tamsulosin, against prostate cancer cell growth. The antigrowth effect of the three alpha1-adrenoceptor antagonists was examined in two human prostate cancer cell lines, PC-3 and DU-145, and a prostate smooth muscle cell primary culture, SMC-1, on the basis of: (a) cell viability assay; (b) rate of DNA synthesis; and (c) induction of apoptosis. Our results indicate that treatment of prostate cancer cells with doxazosin or terazosin results in a significant loss of cell viability, via induction of apoptosis in a dose-dependent manner, whereas tamsulosin had no effect on prostate cell growth. Neither doxazosin nor terazosin exerted a significant effect on the rate of cell proliferation in prostate cancer cells. Exposure to phenoxybenzamine, an irreversible inhibitor of alpha1-adrenoceptors, does not abrogate the apoptotic effect of doxazosin or terazosin against human prostate cancer or smooth muscle cells. This suggests that the apoptotic activity of doxazosin and terazosin against prostate cells is independent of their capacity to antagonize alpha1-adrenoceptors. Furthermore, an in vivo efficacy trial demonstrated that doxazosin administration (at tolerated pharmacologically relevant doses) in SCID mice bearing PC-3 prostate cancer xenografts resulted in a significant inhibition of tumor growth. These findings demonstrate the ability of doxazosin and terazosin (but not tamsulosin) to suppress prostate cancer cell growth in vitro and in vivo by inducing apoptosis without affecting cell proliferation. This evidence provides the rationale for targeting both drugs, already in clinical use and with established adverse-effect profiles, against prostatic tumors for the treatment of advanced prostate cancer.     

Cotargeting tumor and stroma in a novel chimeric tumor model involving the growth of both human prostate cancer and bone stromal cells

Stromal-epithelial interaction contributes to local prostate tumor growth, androgen-independent progression and distant metastasis. We have established in vitro coculture and in vivo chimeric tumor models to evaluate the roles of stromal cells isolated from either osteosarcoma or normal bone, a site where prostate cancer cells frequently metastasize, in contributing to the growth and survival of human prostate cancer cells. We have evaluated extensively the effects of toxic gene therapy using luciferase-tagged chimeric human prostate cancer models both in vitro and in vivo. In the in vitro cocultured cell model, we assessed cancer cell growth and residual cellular proteins after targeting either prostate cancer epithelial cells alone or both prostate cancer and bone stromal cells. In the in vivo animal model, we measured tumor volume and serum prostate-specific antigen (PSA) in mice bearing chimeric prostate tumors comprised of human prostate tumor cells and normal bone stromal cells. Our results demonstrated that: (1) The rate of human prostate cancer cell growth in vitro is accelerated by coculturing with human and rat osteosarcoma or normal mouse bone marrow stromal cell lines. No growth stimulation was noted when cocultured with a human prostate epithelial cell line. (2) Disabling the growth of normal bone stromal cells using transgenic targeting with a bystander gene, herpes simplex virus thymidine kinase (hsv-TK), plus the pro-drug ganciclovir (GCV) or acyclovir markedly depressed the growth of cocultured human prostate cancer cells in vitro and human prostate cancer-mouse normal bone stroma chimeric tumors in vivo. (3) By cotargeting both human prostate cancer and normal mouse bone stromal cells in vitro with an adenoviral construct, Ad-hOC-TK (a replication-defective Ad5 vector with the bystander transgene hsv-TK under the control of a human osteocalcin (hOC) promoter) plus GCV4, we observed greater inhibition of tumor cell growth than by targeting a single cell compartment with Ad-PSA-TK (a vector construct similar to Ad-hOC-TK except that the transgene expression is under regulation by a full-length human PSA promoter). These results, taken together, established a basic principle that cotargeting both tumor and its supporting stroma is more efficacious than targeting a single cell compartment in the treatment of human prostate cancer bone metastasis. This principle can be applied to other clinical conditions of cancer growth where stroma contribute to the overall growth and survival potential of the cancer.     

Human tumor cell strains both unable to repair O6-methylguanine and hypersensitive to killing by human {alpha} and {beta} interferons

Human brain tumor cell strains were previously found by othersto be sensitive to growth inhibition by human inter-feron-ß(HuIFN-ß). We noticed that the sensitive strains weresome that we had found deficient in the repair of O⁶-methyl-guanine(O⁶MeG), a characteristic of 20% of the human tumor cell strainswe have studied. We confirmed this sensitivity to HuIFN-ß,and have further shown that human brain tumor cells which repairO⁶MeG are resistant to the growth inhibitory effects of HuIFN-ß.In addition, treatment with HuIFN- or HuIFN-ß resultedin more killing (reproductive inactivation) of six human tumorcell strains deficient in repairing O⁶-methylguanine in DNAthan did such treatment of six strains of cells proficient insuch repair. Further, we found two human lines, altered to becomeO⁶MeG repair deficient after establishment of the primary tumorcell culture, that were resistant to interferon. IFN treatmentproduced no DNA damage detectable by either chemical or biologicalassays. It is suggested that the genes responsible for resistanceto IFN treatment and to agents that produce O⁶MeG are oftencoordinately shut down.     

WL-276, an antagonist against Bcl-2 proteins, overcomes drug resistance and suppresses prostate tumor growth

Patients with hormone-refractory prostate cancer (HRPC) have an estimated median survival of only 10 months because of acquired drug resistance, urging the need to develop therapies against the drug-resistant HRPC phenotype. Accumulating evidence suggests that overexpressing antiapoptotic Bcl-2 family proteins is at least partially responsible for the development of drug resistance among HRPC patients. Antagonizing the antiapoptotic Bcl-2 family proteins, therefore, is one potential approach to circumventing drug resistance in HRPC. WL-276 was developed as a small-molecule antagonist against antiapoptotic Bcl-2 family proteins, with binding potency comparable to (-)-gossypol. Overexpressing Bcl-2 or Bcl-X(L) failed to confer resistance to WL-276. WL-276 also effectively induced apoptosis in PC-3 cells. In addition, three PC-3 cell lines with acquired drug resistance against standard cancer chemotherapies were more sensitive to WL-276 than the parent PC-3 cell line. The increased cytotoxicity toward drug-resistant PC-3 cells shows the clinical potential of WL-276 against HRPC that is resistant to conventional therapies. The anticancer activity of WL-276 was manifested in its suppression of PC-3-induced prostate tumor growth in vivo. The selective toxicity of WL-276 against drug-resistant PC-3 cells and its in vivo suppression of PC-3 prostate tumor growth suggest that WL-276 is a promising lead candidate for the development of Bcl-2 antagonists against drug-resistant HRPC.     

Comparative genotoxicity of 2,3'-O-cyclocytidine, {beta}-xylocytidine and 1-{beta}-D-arabinofuranosylcytosine in human tumor cell lines

We have Investigated the genotoxicity of two 3'-derivativesof cytidine, 2,3'-O-cyclocytidine (3'-cycloC) and ß-xylocytidine(xyloC), in human leukemia and solid tumor cell lines. Bothderivatives were found to be cytotoxic at micromolar concentrations.For example, in the alveolar tumor cell line A549 which wasincluded in all experiments as a reference, drug concentrationsrequired to induce 50% inhibition of cell growth (DM values)equalled 55 (iMfor 3'-cycloC and 80 µM for xyloC. Comparedwith the response of this reference cell line, none of the solidtumor cell lines tested—representing five different malignancies—displayedsignificant hypersensitivity to these drugs, while the acutelymphoblastic leukemia cell lines proved to be hypersensitive(range of D₅₀ values, 5–13 (µM). To gain insightinto the modes of cytotoxic action of xyloC and 3'-cycloC, wecompared the effect on DNA metabolism of these compounds withthat of 1-p-D-arabinofuranosylcytosine (araC), a potent inhibitorof semi-conservative DNA replication and long-patch excisionrepair. As seen with araC, the xylo compound strongly inhibitedboth DNA replicative synthesis and the repair of DNA damageinduced by UV light and ⁶⁰Co -radiation. In -irradiated A549cells, the extent of repair inhibition by 1 mM xyloC was 40%of that inhibited by araC, and concomitant exposure of the irradiatedcultures to xyloC plus araC gave rise to a synergistic response.Since araC was employed at a concentration (0.1 mM) which produceda maximal effect on DNA repair when applied alone, the observedsynergistic response implies that the mode of action of xyloCon DNA repair is different from that of araC. In contrast tothat observed with xyloC, 3'-cycloC proved to be a very weakinhibitor of DNA replication and repair, strongly suggestingthat the genotoxic action of the latter analog may be througha mechanism other than inhibition of DNA synthesis.     

{alpha}-Difluoromethylornithine inhibits cell growth stimulated by a tumor-promoting rat urinary fraction

The growth stimulating activity of a tumor-promoting rat urinaryfraction (Fraction I), and its inhibition by -difluoromethylomithine(DFMO) were examined in vitro using a rat bladder carcinomacell line, 804G cells. Cell growth was markedly stimulated byFraction I when added to the basic medium containing 0.2% fetalcalf serum (FCS). The increased proliferative activity was associatedwith an increase in omithine decarboxylase (ODC) activity andintracellular polyamine content. DFMO effectively inhibitedthe growth of 804G cells stimulated by Fraction I or by 10%FCS, and the inhibition was associated with suppression of ODCactivity and partial depletion of intracellular putrescine andspermidine. Growth inhibition was reversed by exogenous putrescine.These results show that (i) urinary Fraction I, both a tumorpromoter in bladder carcinogenesis and an ODC inducer in 804Gcells, has potent mitogenicity in 804G cells, and (ii) the mitogenicityis inhibited by DMFO, an irreversible inhibitor of ODC.     

The alpha1-adrenoceptor antagonist terazosin induces prostate cancer cell death through a p53 and Rb independent pathway

Prostate cancer is the second leading cause of cancer-related death in men. Treatment failure in prostate cancer is usually due to the development of androgen independence and resistance to chemotherapeutic drugs at an advanced stage. Recently, it was reported that the alpha1-adrenoceptor antagonist terazosin was able to inhibit prostate cancer cell growth and indicated that it may have an implication in the treatment of prostate cancer. The aim of the present study was to investigate the mechanisms involved in terazosin-induced prostate cancer cell death using two androgen-independent cell lines, PC-3 and DU145. Our results showed that terazosin inhibited not only prostate cancer cell growth but also colony forming ability, which is the main target of chemotherapy. We also found that the sensitivity of these cells to terazosin was not affected by the presence of either functional p53 or Rb, suggesting that the terazosin-induced cell death was independent of p53 and Rb. However, the terazosin-induced cell death was associated with G1 phase cell cycle arrest and up-regulation of p27KIP1. In addition, up-regulation of Bax and down-regulation of Bcl-2 was also observed indicating that these two apoptotic regulators may play important roles in terazosin-mediated cell death pathway. Our results provide evidence for the first time that terazosin may have a therapeutic potential in the treatment of advanced prostate cancer.     

Cyr61 increases migration and MMP-13 expression via {alpha}v{beta}3 integrin, FAK, ERK and AP-1-dependent pathway in human chondrosarcoma cells

Cysteine-rich 61 (Cyr61), from the CCN gene family, is a secretedand matrix-associated protein, which is involved in many cellularactivities such as growth and differentiation. However, theeffect of Cyr61 on migration activity in human chondrosarcomacells is mostly unknown. Here, we found that Cyr61 increasedthe migration and expression of matrix metalloproteinase (MMP)-13in human chondrosarcoma cells (JJ012 cells). RGD peptide, vβ3monoclonal antibody and mitogen-activated protein kinase (MEK)inhibitors (PD98059 and U0126) but not RAD peptide inhibitedthe Cyr61-induced increase of the migration and MMP-13 upregulationof chondrosarcoma cells. Cyr61 stimulation increased the phosphorylationof focal adhesion kinase (FAK) and extracellular signal-regulatedkinase (ERK). In addition, activator protein-1 (AP-1) decoyoligodeoxynucleotide also suppressed the MMP-13 messenger RNAand enzyme activity enhanced by Cyr61. Moreover, Cyr61 increasedthe binding of c-Fos and c-Jun to the AP-1 element on the MMP-13promoter. Taken together, our results indicated that Cyr61 enhancesthe migration of chondrosarcoma cells by increasing MMP-13 expressionthrough the vβ3 integrin receptor, FAK, ERK, c-Fos/c-Junand AP-1 signal transduction pathway. Abbreviations: AP-1, activator protein-1; AS, antisense; Cyr61, cysteine-rich 61; ECM, extracellular matrix; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; mAb, monoclonal antibody; MEK, MAPK kinase; MMP, matrix metalloproteinase; mRNA, messenger RNA; MS, missense; NF-B, nuclear factor-kappa B; ODN, oligonucleotide; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; qPCR, quantitative real-time polymerase chain reaction; siRNA, small-interfering RNA     

Induction of genomic instability in normal human bronchial epithelial cells by 238Pu {alpha}-particles

Pulmonary deposition of     

Nitroxide tempo, a small molecule, induces apoptosis in prostate carcinoma cells and suppresses tumor growth in athymic mice

BACKGROUND: In previous studies, nitroxide tempo (2, 2, 6, 6-tetramethyl-piperidine-1-oxyl), a small molecule, induced cell death in cancer cells. The current study examined the antineoplastic properties of tempo in the human hormone-dependent/hormone-independent prostate carcinoma models (LNCaP, DU-145, and PC-3). METHODS: The apoptotic effects of tempo were examined by the flow cytometric analysis of cells labeled with fluorescein isothiocyanate-conjugated annexin-V, and by electron microscopy. Enzymatic assays were performed to measure the activities of 2 cysteine proteases, i.e., caspase-9 and caspase-3, in tempo-treated cells. The effects of tempo on cell proliferation and on cell cycle distribution profiles were measured by the flow cytometric assay using immunofluorescent staining of incorporated 5'-bromo-2'-deoxyuridine (BrdU) coupled with 7-amino-actinomycin D (7-AAD) staining of total DNA. The number of proliferating cells was also determined independently by enzyme-linked immunosorbent assay using chemiluminescent detection of incorporated BrdU. Subcutaneous growth of human prostate carcinoma in athymic mice was monitored after intratumoral administration of tempo into tumor-bearing mice. In addition, cell viability assays were performed to compare the cytotoxic effect of a combination of doxorubicin or mitoxantrone and tempo with single agents. RESULTS: Tempo treatment of prostate carcinoma cells caused a significant increase in the number of apoptotic cells compared with control groups (tempo, 2.5 mM, 24 hours: DU-145, approximately 3.4-fold; PC-3, approximately 6-7-fold; tempo 1 mM, 24 hours: LNCaP, approximately 12-fold). Tempo-induced loss of cell viability was blocked partially or completely after pretreatment of cells with actinomycin-D or cycloheximide, suggesting a de novo macromolecule synthesis-dependent mechanism of cell death. Electron microscopy revealed aggregation and marginalization of chromatin in the nuclei of a large number of tempo-treated LNCaP cells. Tempo treatment of LNCaP cells resulted in enhanced activities of caspase-9 (tempo, 5 mM, 15 hours: approximately 2-fold) and caspase-3 (tempo, 2.5 mM, 24 hours: approximately 12-fold). Tempo treatment also led to an enhanced number of cells in G2/M phase of the cell cycle (tempo, 5.0 mM, 24 hours: DU-145, approximately 1.6-fold; PC-3, approximately 1.5-fold; LNCaP, approximately 5.3-fold), and decreased BrdU incorporation indicative of a decline in the number of proliferating cells (tempo, 2.5 mM, 24 or 48 hours; DU-145, approximately 2-3-fold; PC-3, approximately 1.2-fold; LNCaP, approximately 5-10-fold). Administration of tempo into LNCaP tumor-bearing mice resulted in a significant inhibition of tumor growth (percent initial tumor volume [Day 30, n = 4]: vehicle, 845.35 +/- 272.83; tempo, 9.72 +/- 9.72; tempo vs. vehicle, P < 0.02). In hormone-refractory prostate carcinoma cells, a combination of relatively low doses of tempo and doxorubicin or mitoxantrone caused enhanced cytotoxicity as compared with single agents. CONCLUSIONS: These data demonstrated that nitroxide tempo induced apoptosis and activated a caspase-mediated signaling pathway in prostate carcinoma cells. Tempo treatment also caused cell cycle arrest in G2/M phase and decreased the number of proliferating cells (S phase). Tempo treatment of tumor-bearing mice led to inhibition of tumor growth, suggesting that tempo is a novel member of the small-molecule family of antineoplastic agents.     

Antisense suppression of the chloride intracellular channel family induces apoptosis, enhances tumor necrosis factor {alpha}-induced apoptosis, and inhibits tumor growth

mtCLIC/CLIC4 is a p53 and tumor necrosis factor alpha (TNFalpha) regulated intracellular chloride channel protein that localizes to cytoplasm and organelles and induces apoptosis when overexpressed in several cell types of mouse and human origin. CLIC4 is elevated during TNFalpha-induced apoptosis in human osteosarcoma cell lines. In contrast, inhibition of NFkappaB results in an increase in TNFalpha-mediated apoptosis with a decrease in CLIC4 protein levels. Cell lines expressing an inducible CLIC4-antisense construct that also reduces the expression of several other chloride intracellular channel (CLIC) family proteins were established in the human osteosarcoma lines SaOS and U2OS cells and a malignant derivative of the mouse squamous papilloma line SP1. Reduction of CLIC family proteins by antisense expression caused apoptosis in these cells. Moreover, CLIC4-antisense induction increased TNFalpha-mediated apoptosis in both the SaOS and U2OS derivative cell lines without altering TNFalpha-induced NFkappaB activity. Reducing CLIC proteins in tumor grafts of SP1 cells expressing a tetracycline-regulated CLIC4-antisense substantially inhibited tumor growth and induced tumor apoptosis. Administration of TNFalpha i.p. modestly enhanced the antitumor effect of CLIC reduction in vivo. These results suggest that CLIC proteins could serve as drug targets for cancer therapy, and reduction of CLIC proteins could enhance the activity of other anticancer drugs.