Lines DU145 Research Articles

Radiation-induced effects in PC-3 and DU-145 human prostate cancer cells

Background: Prostate cancer is the first as an incidence and the second as a cause of the oncologic mortality in the male population. There is a broad range of possibilities in the prostate cancer therapy. However, there is also much controversy on the most appropriate treatment in the various stages of the disease. Advanced disease is mostly treated by radiation therapy, sometimes in combination with hormone or chemotherapy. Irradiation induces damage of cell biomolecules, which can lead to the arrest in cell division, or to apoptotic or necrotic cell death. The aim of this study was to determine the dose dependence of radiation-induced cell death in two human prostate cancer cell lines, and to define the form of death of these cells. Methods: Human prostate cancer cell lines PC-3 and DU-145 were irradiated with 2 - 30 Gy from 60 Co g-source, at the dose rate of 20 Gy/h. The effect of irradiation on cell viability, morphology and DNA structure were followed 24 - 72 hours after treatment. Cells were analyzed by trypan blue exclusion assay, flow cytometry and DNA gel electrophoresis. Simultaneous staining of cells with Annexin V-FITC and propidium iodide enabled distinction of early apoptosis from late apoptosis and/or necrosis. Results: The results of trypan blue staining indicated that radiation-induced cell death was both time and dose dependent process. According to flow-cytometry and DNA fragmentation assay, necrosis was the prevailing form of the radiation-induced cell death in both PC-3 and DU-145 cells. The apoptosis occurred in insignificant number of cells, probably due to the mutant p53 gene present in both cell lines. The cell necrosis was dose dependent and was most pronounced 72 hours post treatment. Conclusion The prevailing form of radiation-induced PC-3 and DU-145 cell death is necrosis. Both PC-3 and DU-145 are rather radioresistant cell lines, as the dose necessary to induce 50% decrease in viable cell number is about 10 Gy.

The effect of EGFR-related tyrosine kinase activity inhibition on the growth and invasion mechanisms of prostate carcinoma cell lines

Increased urokinase plasminogen activator (uPA) levels and epidermal growth factor receptor (EGFR)-related tyrosine kinase activity are associated with poor prognosis in several cancers. We studied the effect of epidermal growth factor (EGF) and a specific inhibitor of EGFR, ZM252868, on the growth and invasiveness of the prostate cancer cell lines PC3 and DU145. PC3 cell growth was stimulated by exogenous EGF but DU145 cell growth was not. EGFR-specific tyrosine kinase inhibitor significantly inhibited the growth of both cell types. EGF increased uPA protein level and uPA activity in both cell types. EGF stimulation also resulted in increased uPAR transcript in both cell lines. uPA production and activity were suppressed by the inhibitor to well below the levels in control cells. Matrigel invasion of PC3 cells was increased by EGF. ZM252868 also reversed the EGF-stimulated matrigel invasion by PC3 cells. Our results indicate that EGF is a potent stimulative agent for both growth and invasion in prostate cancer cells, and that targeting the EGFR function inhibits not only tumor growth but also invasiveness.

Contributions of mitogen-activated protein kinase and nuclear factor kappa B to N-(4-hydroxyphenyl)retinamide-induced apoptosis in prostate cancer cells.

The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) has been shown to induce apoptosis in various types of tumors, including prostate cancer. We sought to examine the key mechanisms affecting the resistance to 4-HPR-induced apoptosis in three human prostate cancer cell lines, PC-3, DU145, and LNCaP. Concentrations of more than 40 microM 4-HPR produced apoptosis to almost the same extent in all cell lines; however, only the LNCaP line remained highly sensitive to concentrations less than 10 microM. These differing sensitivities at low concentrations correlated well with the level of constitutive activation of nuclear factor kappa B (NFkappaB) in the individual cell lines. We found that NFkappaB activation inhibited c-jun NH(2)-terminal kinase and caspase 3 activation induced by 4-HPR and that NFkappaB inhibition by the I kappa B alpha phosphorylation inhibitor compound Bay 117082 resulted in increasing sensitization of both PC-3 and DU145 lines to apoptosis induced by 4-HPR at low concentrations. Furthermore, we found that inhibition of extracellular signal-regulated kinase (ERK) enhanced the suppression of NFkappaB by 4-HPR and also resulted in sensitization to apoptosis in the DU145 cell line, in which ERK is activated constitutively. It thus appears that mitogen-activated protein kinase associated with the activity of NFkappaB plays an important role in the degree of resistance to 4-HPR-induced apoptosis in human prostate cancer cells.

Liprin-α2 gene, protein tyrosine phosphatase LAR interacting protein related gene, is downregulated by androgens in the human prostate cancer cell line LNCaP

Prostate cancer is one of the most common neoplasms in the USA and Europe. We used differential display PCR (DD-PCR) to identify androgen-regulated genes in prostate cancer. The RNA of LNCaP cells treated with dihydrotestosterone (DHT) was analyzed for differentially expressed genes. Using DD-PCR, we identified a down-regulated cDNA fragment by DHT in LNCaP cells. This fragment was cloned and expressions of this fragment in prostate cancer cell lines were analyzed by RT-PCR. Sequence analysis revealed that a cDNA fragment is identical to protein tyrosine phosphatase LAR related gene, liprin-alpha2. liprin-alpha2 was downregulated by dihydrotestosterone (DHT) in LNCaP cells in a time- and androgen concentration-dependent manner. Downregulation by DHT was not inhibited by the protein synthesis inhibitor cycloheximide. This liprin-alpha2 gene was not expressed in androgen independent prostate cancer cell lines PC-3 and DU-145 at the mRNA level. And also, we first revealed here that liprin-alpha2 mRNA is expressed in LNCaP cells as well as human prostate cancer tissues and normal prostate tissues. These data suggest that liprin-alpha2 might play a role in androgen responsive human prostate cancer cell line as well as human prostate cells, and the loss of this gene expression might be associated with the androgen independent characteristics of prostate cancer.

Altered response to thyroid hormones by prostate and breast cancer cells.

Transferrin, an abundant bone marrow constituent, has been shown to be a potent mitogen in vitro in the prostate cancer cell line PC3. T4 (L-thyroxine) and T3 (3',3,5-tri-iodo-L-thyronine) are regulators of cell metabolism. In this study, the effects of nonphysiological concentrations (about two orders of magnitude higher) of T4, T3, T2 (3,5-di-iodo-L-thyronine), RT3 (reverse T3, 3',5', 3-tri-iodo-L-thyronine) and transferrin (about three orders of magnitude lower) were tested on the prostate cancer cell lines PC3, DU145 and LNCaP, and the breast cancer cell line MCF-7. In PC3 cells, increased proliferation by transferrin could be reversed by the addition of T3 or T4. T4 decreased proliferation in all cell lines tested, while transferrin increased proliferation in PC3 cells only. T3 decreased proliferation in PC3, LNCaP and MCF-7 cells but had no effect on DU145 cells. T4 and T3 gave two-state behavior in LNCaP cells. These results were combined to determine the essential iodines which produced the observed proliferative effects. Cell lines responded differently to T4, T3, T2, RT3 and transferrin suggesting a specific interaction among the compounds tested and the different cell lines. Finally, regulation of gene expression was demonstrated using DU145 cells. Upregulation of c-fos mRNA was observed in cultures at early time-points in the presence of T4, transferrin or both. Decreased expression was observed at later time-points with no expression at 4 h. An explanation for these results may be a change in thyroid hormone receptor/ligand affinity. Thus, the interactions between thyroid hormones and cancer cells may be different from those between thyroid hormones and normal cells.

Expression of receptor protein tyrosine phosphatase alpha mRNA in human prostate cancer cell lines.

Receptor protein tyrosine phosphatase alpha (RPTPalpha) is a transmembrane protein phosphatase, and has been proposed to be involved in the differentiation of the neuronal system. In the present study, we demonstrated the expression of RPTPalpha mRNA in several normal human tissues. We further investigated the regulation of expression of RPTPalpha mRNA in epithelial cells utilizing three commercially available human prostate cancer cell lines LNCaP, PC-3 and DU145. This is because these cells exhibit different levels of differentiation, defined by the expression of a tissue-specific differentiation antigen, prostatic acid phosphatase (PAcP), and their androgen sensitivity. LNCaP cells express PAcP and are androgen-sensitive cells, while PC-3 and DU145 cells do not express PAcP and are androgen-insensitive cells. Northern blot analyses revealed that, in LNCaP cells, fetal bovine serum (FBS) and 5alpha-dihydrotestosterone (DHT) down-regulates RPTPalpha mRNA expression, similar to the effect on PAcP. Contrarily, FBS up-regulated the RPTPalpha mRNA level in PC-3 and DU145 cells. In LNCaP cells, sodium butyrate inhibited cell growth and up-regulated RPTPalpha as well as PAcP mRNA expression. Although, sodium butyrate also inhibited the growth of PC-3 and DU145 cells, the level of RPTPalpha mRNA was decreased in PC-3, while increased in DU145 cells. Thus, data taken together indicate that the expression of RPTPalpha is apparently regulated by a similar mechanism to that of PAcP in LNCaP cells.

Characterization of insulin-like growth factor-binding protein-related protein-1 in prostate cells.

Insulin-like growth factor-binding protein-related protein-1 (IGFBP-rP1; also known as Mac25, TAF, and PSF) is a member of the IGFBP superfamily. It is a cysteine-rich protein that shares structural and functional similarities with the conventional IGFBPs. In situ hybridization of prostate tissue sections show intense IGFBP-rP1 messenger ribonucleic acid (mRNA) expression in normal stroma and glandular epithelium. There was a significant loss of detectable IGFBP-rP1 mRNA in metastatic prostate tissue. IGFBP-rP1 mRNA (Northern blots) and protein (immunoblots) were detectable in primary cultures ofprostatic stromal and epithelial cells as well as in the immortalized nonmalignant prostatic human epithelial cells, P69, and in the P69 metastatic subline, M12. IGFBP-rP1 expression was not detectable in the prostatic cancer cell lines PC-3, DU145, and LNCaP. IGFBP-rP1 expression was regulated in P69 cells but not in M12 cells. Protein and mRNA expression was up-regulated by IGF-I, transforming growth factor-beta, and retinoic acid. The observations that IGFBP-rP1 expression is significantly diminished in prostate tumorigenesis and is regulated in nonmalignant prostate cells suggest IGFBP-rP1 is important in normal prostatic cell growth.

Involvement of prostate specific antigen in the stimulation of LNCaP cell growth

Since its identification in 1979, prostatic specific antigen (PSA) has been used extensively as a serum marker for diagnosis and prognosis of prostate cancer. In addition, PSA is an immunohistochemical marker for the identification of prostatic tissues and cells in histological specimens. PSA is found in normal prostate, benign prostatic hypertrophy (BPH) tissue, in cancer of the prostate, and its metastases as well as in other hormone dependent cancers, such as breast and ovarian carcinoma. However, the importance of PSA as a regulator of cell growth generally has not been appreciated. The role of PSA in the development of prostate or other hormone-dependent cancers has remained unclear. We therefore examined the role of PSA in the control of cell growth using both the PSA positive cell line, LNCaP cells and the PSA negative cell line PC-3 and DU145. LNCaP cell growth was stimulated by the conditioned medium (CM) from LNCaP cells, but not by CM from PC-3 or DU145 cells. No such stimulation was observed when PC-3 or DU145 cells were exposed to CM from LNCaP cells nor from CM produced by their own lines. The stimulation of LNCaP cell growth by its own CM could not be attributed to the high level of insulin-like growth factor binding protein-2 (IGFBP-2) present in the CM since even higher level of IGFBP-2 was also found to be present in CM from both PC-3 and DU145 CMs. High level of PSA and 66 kDa epidermal growth factor (EGF) were present in LNCaP CM as measured by Western blotting. The stimulation of LNCaP cell growth by its own CM was eliminated partially by PSA or EGF antibody. Stimulation of DNA biosynthesis in LNCaP cells by LNCaP CM or pure PSA was also observed. These data indicate that PSA and EGF are involved in the growth regulation of PSA positive LNCaP cell line.

Expression of fibroblast growth factor-8 in adult rat tissues and human prostate carcinoma cells

Androgens are essential for normal prostatic and testicular function. However, paracrine and/or autocrine actions of a number of growth factors have been implicated in the function of these tissues. A recent addition to the fibroblast growth factor family, the so called androgen-induced growth factor (AIGF) or fibroblast growth factor-8 (FGF-8), has been proposed to be under strict androgen regulation and induction in the mouse mammary carcinoma cell line SC3. FGF-8, therefore, may have a local role in the prostate, which is known to be an androgen-responsive organ. This study reports, for the first time, the presence of FGF-8 mRNA in normal adult rat tissues (heart, brain, lung, kidney, testis, prostate and ovary), using an optimised reverse transcription and nested polymerase chain reaction (RT-PCR) procedure, although androgen-dependent FGF-8 expression was not demonstrated in these adult tissues. Consistent with the oncogenic characteristics of FGF-8, the corresponding mRNA was detected in the human prostate tumour cell lines LNCaP and DU145. Because the DU145 cell line is known to be androgen-independent, and the expression of FGF-8 mRNA in cultured LNCaP cells also occurred in the absence of exogenous androgens, it can be concluded that the expression of FGF-8 mRNA in these human cell lines, in the rat prostate and in other rat tissues is not under the regulation of androgens as hitherto proposed.

Characterization of an early growth response gene, which encodes a zinc finger transcription factor, potentially involved in cell cycle regulation.

Using differential display polymerase chain reaction, early growth response gene alpha (EGR alpha) was first isolated as a 291-base pair 3'-cDNA clone, which was highly expressed in the androgen-independent prostate carcinoma cell lines PC3 and DU145, as compared with the androgen-responsive prostate carcinoma cell line LNCaP. Full length cloning of the EGR alpha coding region revealed that EGR alpha was a new member of an important subfamily of nuclear zinc finger transcription factors (others members e.g. Sp1, EGR-2, and Wilms' tumor gene). Moreover, it was observed that EGR alpha, as with most Sp1 subfamily members, was conserved between mammalian species ranging from human to rabbit. Two hormones important for prostate development and differentiation were found to be potent regulators of EGR alpha mRNA expression. Androgens were observed to induce a down-regulation of EGR alpha mRNA expression (70% in 72 h), while epidermal growth factor induced a rapid transient up-regulation (6-fold in 100 min). The up-regulation was controlled at the transcriptional level and effectively blocked by staurosporine (which suggests the involvement of the protein kinase C pathway). Functional analysis demonstrated that EGR alpha could bind to, and stimulate transcription from, a basic transcription element (BTE) consensus sequence on DNA (BTE is a transcription-modulating sequence in the promoter region of some cytochrome P450 family members). Furthermore, in stage-synchronized prostate cells, EGR alpha mRNA was highly expressed in the early G1 phase of the cell cycle, similar to c-fos mRNA expression. These results indicated that the zinc finger transcription factor EGR alpha seems to play a role in cell cycle regulation.

Diversity and modulation of plasminogen activator activity in human prostate carcinoma cell lines.

Baseline cellular plasminogen activator (PA) activity, and the cellular proteins responsible for variations in PA activity were evaluated in three human prostate carcinoma cell lines. Net PA activity in the cell lines PC-3, DU-145, and LNCaP was measured using a plasminogen-dependent fibrin lysis assay. These three cell lines were then analyzed to determine the specific protein(s) responsible for differences in PA activity. mRNA and protein levels of cellular urinary PA (uPA), tissue PA (tPA), PA inhibitor 1 (PAI1), PA inhibitor 2 (PAI2), and uPA receptor (uPAr) were measured using Northern analysis and ELISA assays. Net cellular PA activity in the three cell lines varied over a 3-fold range (PC3 > DU145 >> LNCaP). Net PA activity in the fibrinolysis assay demonstrated a direct correlation with mRNA transcript levels of uPA, tPA, PAI1, and uPAr (PC-3 > DU-145 > LNCaP). uPA protein was identified in both the PC-3 and the DU-145 lines. tPA, PAI1, and PAI2 proteins were identified only in PC-3 cells. In general, cellular protein levels correlated with mRNA levels. These findings demonstrate that prostate carcinoma cell lines vary in their net PA activity. This variability results from both qualitative and quantitative differences in the cellular expression of PA regulatory proteins.

PRECLINICAL STUDIES OF GOSSYPOL IN PROSTATE CARCINOMA

Hormone refractory prostate cancer remains an incurable disease and the discovery of newer agents with higher cytotoxic activity is required. Gossypol is a phenolic compound isolated from cottonseed oil which has been shown to have anti-spermatogenic effects. In in vitro studies, gossypol appears to inhibit the growth of rat prostate cancer cell line MAT-LyLu and human prostate adenocarcinoma cell lines PC-3, LNCaP and DU-145. In vive, gossypol appeared to inhibit tumor growth of subcutaneously implanted MAT-LyLu cells in Copenhagen rats. Gossypol may be an active agent for the treatment of hormone refractory metastatic prostate cancer.

CHARACTERIZATION OF PROTEASE EXPRESSION IN HUMAN PROSTATE-CANCER CELL-LINES

To better understand the biochemical mechanisms necessary for prostate cancer invasion and metastases, we studied the expression and interaction of proteolytic enzymes cathepsin D, cathepsin B, urokinase and collagenase IV in human prostate cell lines LNCAP (hormone sensitive) and DU145 (hormone refractory). Cellular fractionation and immunoblotting revealed that both cell lines expressed similar amounts of the 34 kD form of cathepsin D, 72 kD form of collagenase IV and the 55 kD form of urokinase. However, DU145 expressed an increased amount of the 28 kD form of cathepsin B. When E64, a cysteine protease inhibitor was added, a decreased amount of the active cathepsin D was expressed. Furthermore, when cathepsin B was added to concentrated plasma membrane homogenates, urokinase was processed to its active form at 33 kD. E64 inhibited the ability of both cell lines to degrade fibronectin. An in vitro Boyden chamber demonstrated that DU145 was more motile than LNCAP and that preincubation with E64 could decrease motility of both cell lines. We suggest that cathepsin B may promote tumor invasion not only by proteolysis of basement membrane components, but also by activating other proteases.

Radiobiological studies of PC-3 and DU-145 human prostate cancer cells: X-ray sensitivity in vitro and hypoxic fractions of xenografted tumors in vivo

Purpose : To determine the quantitative responses to x-irradiation of exponentially growing human prostatic cancer cell lines PC-3 and DU-145 in vitro, and to determine the hypoxic percentages of these two cell lines when grown in vivo as xenografted solid tumors in nude mice. Methods and Materials : Radiation survival in vitro was quantitated using both single-hit, multitarget and linearquadratic formalisms. Hypoxic fractions in vivo were determined from tumors of average sizes of about 750 mm 3 using clonogenic assay. Results : In vitro, the average single-hit, multitarget survival values for 7 replicate experiments for the DU-145 line were n = 1.92 (1.39 – 2.65), D q(Gy) = 1.25, and D o(Gy) = 1.91 (1.88 – 1.94) (all values in parentheses indicate 95% confidence limits). For the PC-3 line (10 replicate experiments), these values were n = 2.84 (2.11 – 2.79), D q(Gy) = 1.02, and D o(Gy) = 1.06 (0.87 – 1.25). For the linear-quadratic formalism, values of α(Gy −1 × 10 1) and β(Gy −2 × 10 2) for the DU-145 and PC-3 lines were, respectively, 1.55 (0.42) and 5.21 (1.09); and 4.87 (1.11) and 5.50 (1.88). The mean percentage survival of the DU-145 and PC-3 lines at a dose of 2 Gy were, respectively, 59.8 (53.3 – 67.0) and 32.0 (25.8 – 38.2). In vivo, the hypoxic fractions for the DU-145 and PC-3 tumors were, respectively, 7.20 (4.30 – 11.5), and 52.3 (42.8 – 63.9). Results : The data from the in vitro experiments show that the DU-145 cell line is significantly more radioresistant than the PC-3 cell line. In vivo, the DU-145 tumors exhibit a significantly lower hypoxic percentage than do PC-3 neoplasms. Conclusions : Results indicate that significantly variability exists within human prostate tumors in regard to both intrinsic radiosensitivity in vitro and levels of hypoxia in vivo. Because these data appear to be the first published information on the intrinsic radiosensitivity and intratumor hypoxia characteristics of human prostate cancer, additional studies are needed to define the distributional aspects of these clinically relevant endpoints.