Multiple readout assay for hormonal (androgenic and antiandrogenic) and cytotoxic activity of plant and fungal extracts based on differential prostate cancer cell line behavior

Ethnopharmacological relevance

Prostate cancer is one of the most diagnosed forms of cancer among men in western regions. Many traditional applications or phytotherapeutic concepts propose to inhibit the proliferation of prostate cancer cells. In order to detect influences of plant or fungal extracts and derived fractions on androgen receptor signaling pathways, a differentiating cell proliferation assay was established, which enables the simultaneous detection of hormonal and cytotoxic effects.

Material and methods

The well characterized prostate cancer cell lines LNCaP and PC-3 were used in a multiple readout assay. In all, 186 fractions of 23 traditionally used organisms were screened regarding their effects on proliferation of the two prostate cancer cell lines. The fractions were prepared by accelerated solvent extraction followed by gradient extrography. Extracts of the potential hormonally active plants Cibotium barometz, Heteropterys chrysophylla, and Sideroxylon obtusifolium (= Bumelia sartorum) were phytochemically investigated.

Results

Fractions from Cibotium barometz, Cortinarius rubellus, Cyrtomium falcatum, Heteropterys chrysophylla, Nephrolepis exaltata, Salvia miltiorrhiza, Sideroxylon obtusifolium, Trichilia emetica, and Trimeria grandifolia exhibited hormonal influences on prostate cancer cells. Cytotoxic activity towards human cell lines was detected for the first time for fractions from Aglaia spectabilis (A. gigantea), Nephrolepis exaltata and Cortinarius brunneus.

Conclusions

The differential behavior of the two prostate cancer cell lines allows the discrimination between potential androgenic or antiandrogenic activities and effects on the estrogen or glucocorticoid receptor as well as cytotoxic activities. The combined cell lines assay can help to assess the biological activities of material used in traditional medicine.     

Caveolin-1 secreting LNCaP cells induce tumor growth of caveolin-1 negative LNCaP cells in vivo

Caveolin-1 (Cav-1) was originally identified as a structural protein of caveolae, which is a plasma membrane domain that regulates a variety of signaling pathways involved in cell growth and migration. Here, we show that expression of Cav-1 in the Cav-1-deficient human prostate cancer cell line LNCaP both stimulates cell proliferation and promotes tumor growth in nude mice. Unexpectedly, Cav-1 expressing LNCaP (LNCaP(Cav-1)) cells injected into one side of a nude mouse promoted tumor growth of Cav-1 negative LNCaP cells injected on the contralateral side of the same animal. The LNCaP tumors were positive for Cav-1, however, this signal was not caused by migrated LNCaP(Cav-1) cells, but we show that this Cav-1 was secreted by the LNCaP(Cav-1) tumors. We demonstrate that conditioned media from LNCaP(Cav-1) cells contained Cav-1 that was associated with a lipoprotein particle ranging in size from 15 to 30 nm and a density similar to high density lipoprotein particle. These results suggest that LNCaP(Cav-1) cells secreting Cav-1 particle produce an endocrine factor that stimulates tumor growth.     

雄激素非依赖性前列腺癌细胞亚系模型LNCaP-AI的建立

目的:建立雄激素非依赖性前列腺癌细胞亚系模型LNCaP-AI.方法:长期在去雄激素的血清环境下培养雄激素依赖性LNCaP细胞,培养出能够适直无雄激素环境的LNCaP-AI细胞亚系.MTT、RT-PCR、免疫荧光方法观察LNCaP-AI细胞在无雄激素环境下的增殖能力和表达及分泌前列腺特异性抗原(PSA)的水平.结果:LNCaP前列腺癌细胞在无雄激素中培养3个月后逐渐适应了无雄激素的环境,成为雄激素非依赖的LNCaP-AI细胞亚系.LNCaP-AI细胞在无雄激素的环境下迅速增殖,能够分泌PSA,但其PSA mRNA的表达水平是正常生长LNCaP细胞的44%.结论:成功构建雄激素非依赖性前列腺癌细胞亚系模型LNCaP-AI,可以模拟前列腺癌由雄激素依赖发展为雄激素非依赖的过程.     

淫羊藿苷对前列腺癌原位移植瘤模型小鼠雄激素受体信号通路的影响

目的：探讨淫羊藿苷对前列腺癌原位移植瘤模型SCID小鼠雄激素受体信号通路的影响机制。方法：雄性SCID小鼠64只,均采用前列腺腺体背外侧包膜内注射人前列腺癌细胞株（LNCaP）悬液的方法构建前列腺癌原位移植瘤模型,然后分为移植瘤组、10 mg·kg-1组、40 mg·kg-1组和80 mg·kg-1组,除移植瘤组灌生理盐水对照外其它3组灌胃给予相应剂量的淫羊藿苷治疗5周。采用western blotting检测前列腺癌特异性抗原（PSA）和磷酸化AR（p-AR）表达,采用RT-PCR检测治疗前后前列腺瘤体雄激素受体（AR）和张力蛋白同源第10号染色体缺失的磷酸酶基因（PTEN）表达,采用流式细胞学方法检测前列腺体肿瘤瘤体LNCaP前列腺癌细胞在肿瘤瘤体增殖周期。结果：40 mg·kg-1组和80 mg·kg-1组治疗后AR mRNA为（0.25 ± 0.02,0.27 ± 0.03）、p-AR为（1.45 ± 0.22,1.64 ± 0.24）,PSA为（0.31 ± 0.02,0.38 ± 0.05）,两组PSA、p-AR和AR mRN治疗后相对表达量均低表达,而PTEN mRNA高表达（0.91 ± 0.07,0.95 ± 0.09）,与治疗前和移植瘤组比较差异均有统计学意义（P < 0.05）。两组治疗后抑瘤率分别为（41.59 ± 4.51）%和（42.76 ± 5.13）%,瘤质量为（86.34 ± 9.07,84.73 ±7.58）mg、瘤体积为（11.83 ± 0. 84,10.27 ± 1.14）mm2,均较同组治疗前和移植瘤组比较明显降低（P < 0.05）;两组治疗后G0/G1期比例降低[两组G0/G1分别为（34.97 ± 4.52,35.03 ± 3.97）%、且S期比例明显提高[两组S期比例分别为（39.59 ± 5.03,40.27 ± 4.82）%],与同组治疗前和移植瘤组比较差异均均有统计学意义（P < 0.05）。结论：淫羊藿苷抑制LNCaP增殖的机制应与其抑制雄激素受体信号通路中AR的磷酸化并增强PTEN表达而将癌细胞增阻滞于S期有关。     

Growth hormone (GH) receptors in prostate cancer: gene expression in human tissues and cell lines and characterization, GH signaling and androgen receptor regulation in LNCaP cells

Various hormones and growth factors have been implicated in progression of prostate cancer, but their role and the underlying molecular mechanism(s) involved remain poorly understood. In this study, we investigated the role of human growth hormone (GH) and its receptor (GHR) in human prostate cancer. We first demonstrated mRNA expression of GHR and of its exon 9-truncated isoform (GHR_tr) in benign prostate hyperplasia (BPH) and prostate adenocarcinoma patient tissues, as well as in LNCaP, PC3 and DU145 human prostate cancer cell lines. GHR mRNA levels were 80% higher and GHR_tr only 25% higher, in the carcinoma tissues than in BPH. Both isoforms were also expressed in LNCaP and PC3 cell lines and somewhat less so in DU145 cells. The LNCaP cell GHR protein was further characterized, on the basis of its M_r of 120 kDa, its binding to two different GHR monoclonal antibodies, its high affinity and purely somatogenic binding to ¹²⁵I-hGH and its ability to secrete GH binding protein, all characteristic of a functional GHR. Furthermore, GH induced rapid, time- and dose-dependent signaling events in LNCaP cells, including phosphorylation of JAK2 tyrosine kinase, of GHR itself and of STAT5A (JAK2-STAT5A pathway), of p42/p44 MAPK and of Akt/PKB. No effect of GH (72 h) could be shown on basal or androgen-induced LNCaP cell proliferation nor on PSA secretion. Interestingly, however, GH caused a rapid (2–12 h) though transient striking increase in immunoreactive androgen receptor (AR) levels (≤5-fold), followed by a slower (24–48 h) reduction (≤80%), with only modest parallel changes in serine-phosphorylated AR. In conclusion, the GH-induced activation of signaling pathways, its effects on AR protein in LNCaP cells and the isoform-specific regulation of GHR in prostate cancer patient tissues, suggest that GH, most likely in concert with other hormones and growth factors, may play an important role in progression of human prostate cancer.     

LNCaP衍生的雄激素非依赖型前列腺癌细胞系C4-2对辐射的应答反应

放射治疗是治疗局灶性前列腺癌相对有效的方法,然而,30％的病人会产生辐射治疗抵抗。放射协同雄激素撤除为临床提供了一种改善疗效的治疗方法。雄激素撤除诱导的信号通路相应地也会介导前列腺癌细胞的放射敏感。C4—2细胞是由雄激素非依赖的LNCaP母体细胞在雄激素撤除条件下衍生而来的,C4-2细胞获得了雄激素非依赖生长的特性。我们分析了LNCaP和WC4－2对辐射的应答反应,克隆形成、细胞存活和细胞周期分析结果显示,C4－2细胞经照射后较LNCaP细胞更容易存活,对辐射处理表现出更强的抵抗能力。基因表达分析表明,一整套与细胞周期阻滞和DNA损伤相关的基因经过辐射处理后在LNCaP和C4．2细胞中差异表达,其结果与C4—2细胞的辐射抵抗性质一致,这些结果明显提示,前列腺癌细胞对辐射的抵抗与向雄激素非依赖发展的过程可能同步进行。LNCaP和C4-2细胞模型不仅可应用于研究前列腺癌向雄激素非依赖发展的过程,而且也用于分析前列腺癌细胞的辐射抵抗机制。     

Cell-Surface labeling and internalization by a fluorescent inhibitor of prostate-specific membrane antigen

BACKGROUND: [corrected] Prostate-specific membrane antigen (PSMA) remains an attractive target for imaging and therapeutic applications for prostate cancer. Recent efforts have been made to conjugate inhibitors of PSMA with imaging agents. Compared to antibodies, small-molecule inhibitors of PSMA possess apparent advantages for in vivo applications. To date, there are no reports on the cellular fate of such constructs once bound the extracellular domain of PSMA. The present study was focused on precisely defining the binding specificity, time-dependent internalization, cellular localization, and retention of inhibitor conjugates targeted to PSMA on LNCaP cells. A novel fluorescent inhibitor was prepared as a model to examine these processes. METHODS: Fluorescence microscopy of LNCaP and PC-3 cell lines was used to monitor the specificity, time-dependent internalization, cellular localization, and retention of a fluorescent PSMA inhibitor. RESULTS: Fluorescent inhibitor 2 was found to be a potent inhibitor (IC50 = 0.35 nM) of purified PSMA. Its high affinity for PSMA on living cells was confirmed by antibody blocking and competitive binding experiments. Specificity for LNCaP cells was demonstrated as no labeling by 2 was observed for negative control PC-3 cells. Internalization of 2 by viable LNCaP cells was detected after 30 min incubation at 37 degrees C, followed by accumulation in the perinuclear endosomes. It was noted that internalized fluorescent inhibitor can be retained within endosomes for up to 150 min without loss of signal. CONCLUSIONS: Our results suggest that potent, small-molecule inhibitors of PSMA can be utilized as carriers for targeted delivery for prostate cancer for future imaging and therapeutic applications.     

Triiodothyronine modulates cell proliferation of human prostatic carcinoma cells by downregulation of the B-cell translocation gene 2

BACKGROUND: Studies suggest that triiodothyronine (T3) and cognate nuclear receptors (hTR) are involved in regulation of prostatic cell growth and differentiation. To probe mechanisms for T3 effects, we studied prostate carcinoma cells, investigating the effect of T3 on expression of the B-cell translocation gene 2 (BTG2), which regulates the G1/S transition of the cell cycle. METHODS: Effects of T3 on cell proliferation were determined by (3)H-thymidine incorporation. T3 modulation of BTG2 expression was investigated using immunoblots, Northern blots, and transient gene expression assays. The putative T3 response element was determined by electrophoretic mobility shift assay. RESULTS: T3 (0.1-1,000 nM) enhanced threefold the proliferation of prostate carcinoma cells and human androgen-dependent prostate carcinoma cells (LNCaP), but not PC-3 cells. T3 also inhibited BTG2 gene expression in LNCaP cells. Reporter assays showed that T3 downregulates by 50% promoter activity of the BTG2 gene in LNCaP cells but not PC-3 cells or thyroid-hormone receptor (TRbeta1)-overexpression PC-3 cells. Deleting the putative thyroid hormone response element (TRE; AGCGATGACCTCAGCG) blocked the inhibitory effect of T3 on BTG2 promoter activity. Electrophoretic mobility shift assays with purified TRbeta1 from in vitro translation, or with nuclear extracts from LNCaP cells and PC-3 cells, demonstrated the presence of T3 receptor binding sites in the TRE region. CONCLUSIONS: These results suggested that the T3 upregulates proliferation of LNCaP cells by downregulating BTG2 gene expression through the consensus TRE pathway.     

Hormonal regulation of beta2-adrenergic receptor level in prostate cancer

BACKGROUND: Androgen deprivation is the only effective systemic therapy available for patients with prostatic carcinoma, but is associated with a gradual transition to a hormone-refractory prostate cancer (HRCAP) in which ligand-independent activation of the androgen receptor has been implicated. The beta(2)-adrenergic receptor (beta(2)-AR) is a well-known activator of the androgen receptor. METHODS: Prostatic cell lines were analyzed using cDNA micro-array, real time RT-PCR, radioligand binding assay, cAMP measurements, transfection and thymidine incorporation assay. Clinical specimens were studied by immunohistochemistry and Affymetrix microarrays. RESULTS: Here, we show that beta(2)-AR was transiently down-regulated both at mRNA- and protein levels when hormone-sensitive prostate cancer cells, LNCaP, were cultured in steroid stripped medium (charcoal-stripped fetal calf serum) or when the cells were treated with the anti-androgen, bicalutamide (Casodex). The number of beta-adrenergic receptors was modestly up-regulated in androgen independent cell lines (LNCaP-C4, LNCaP-C4-2 and DU145) compared to LNCaP. Triiodothyronine (T3) increased the level of beta(2)-AR and the effect of T3 was inhibited by bicalutamide. Immunohistochemical staining of human prostate specimens showed high expression of beta(2)-AR in glandular, epithelial cells and increased expression in malignant cells compared to benign hyperplasia and normal tissue. Interestingly, beta(2)-AR mRNA was strongly down-regulated by androgen ablation therapy of prostate cancer patients. CONCLUSION: The level of beta(2)-AR was increased by T3 in prostatic adenocarcinoma cells and reduced in prostate cancer patients who had received androgen ablation therapy for 3 months.