Human Cancer Cell Line LNCaP Research Articles

Bioactive secondary metabolites from a soybean-derived fungus Aspergillus versicolor IMBC-NMTP02

• Isolation of 10 compounds from Aspergillus versicolor IMBC-NMTP02. • Structural identification of 3 new, 2 new natural, and 5 known compounds. • Cytotoxicity toward HepG2, KB, HL-60, MCF-7, SK-Mel-2, and LNCaP cancer cells. • Nitric oxide inhibitory effects of the isolated compounds. • Antimicrobial effects of the isolated compounds. In the present study, 10 secondary metabolites, including three new asperversilactones A–C ( 2 , 4 , and 5 ) and two new natural metabolites (4 Z ,6 S ,7 R )-6,7-dihydroxyocta-2,4-dien-4-lactone ( 1 ) and (4 E ,6 S ,7 R )-6,7-dihydroxyocta-2,4-dien-4-lactone ( 3 ) were isolated from a culture fermentation of the soybean-derived fungus Aspergillus versicolor IMBC-NMTP02. Their structures were elucidated by comprehensive analyses of spectroscopic methods, including NMR, mass, and ECD spectra. Compounds 5 , 6 , and 9 were shown to have cytotoxic effects toward the HepG2, KB, HL-60, MCF-7, SK-Mel-2, and LNCaP human cancer cell lines (IC 50 = 31.7–81.5 μM), whereas all of the compounds inhibited NO overproduction in LPS-stimulated BV2 cells (IC 50 = 19.0–41.9 μM). Furthermore, all of the compounds showed antimicrobial effects against Enterococcus faecalis and Candida albican s, with MIC values in the range of 32–256 μM. Our investigation contributes to clarification of the chemical constituents of the soybean-derived A. versicolor and warrants further studies of some bioactive metabolites for therapeutic uses.

Abstract 2101: Comparison of transcriptional response to phorbol ester, bryostatin 1, and bryostatin analogue in LNCaP and U937 cells provides insight into their differential mechanism.

Abstract Bryostatin 1 (bryo 1) is a promising cancer chemotherapeutic agent with a unique mechanism of action. Whereas bryo 1 binds to and activates protein kinase C (PKC) like the phorbol esters, it paradoxically antagonizes most phorbol ester responses. Previously, we compared the changes in transcription in response to the phorbol ester phorbol 12-myristate 13-acetate (PMA), to bryo 1, and to the synthetic bryo 1 derivative Merle 23 in the human cancer cell lines LNCaP and U937. Bryo1 failed to induce a typical phorbol ester biological response in either cell line, whereas the bryo 1 analog Merle 23 resembled PMA in the U937 cells and bryo 1 in the LNCaP cells. In both cell lines, bryo 1 differed from PMA in inducing a shorter duration of the transcriptional responses for a panel of PKC-regulated genes as quantitated by qPCR. Merle 23, in contrast, induced a pattern very similar to that of PMA. Here, we analyzed by microarray the genome wide transcriptional changes in LNCaP and U937 cells treated with maximally effective doses of PMA, bryo 1 and Merle 23 for 6 and 8 hours, respectively. Several thousand genes were significantly up- or down-regulated by PMA (3131 and 3206), bryo 1 (1569 and 2454) and Merle 23 (3533 and 3207) compared to control in LNCaP and U937 cells, respectively. As seen previously for selected genes, the magnitude of the changes was much greater for LNCaP cells than for U937 cells. The great majority of the genes followed a pattern in which bryo 1 induced changes similar to PMA but to a significantly smaller extent (3096 and 1610 genes significantly different in LNCaP and U937 cells), whereas Merle 23 was very similar to PMA (only 230 and 30 genes different between them). A detailed qPCR analysis (dose and time courses) of representative outlier genes revealed that the shorter duration of bryo 1 induced response was responsible for the differences between bryo 1 and PMA effect in all cases. The dose response experiments did not reveal a difference in bryo 1 potency linked to any specific pattern of response. Analysis of transcription factors revealed overrepresentation of the same families of transcription factors (13 in the LNCaP cells; 12 in the U937 cells) among the genes differentially regulated by PMA and bryo 1. These included AP2F, E2F, EGRF, KLF, NRF1, SP1F. However, the target genes of the transcription factor families were largely different between the two cell lines. The analysis of 45 signaling pathways using reporter assays identified significant changes in 7 pathways induced both by PMA and bryo 1 at 6 hours. While the analysis of the microarray data is ongoing, we conclude that early turn-off of the induced responses by bryo 1 is the central feature accounting for its difference from PMA in its transcriptional response. Understanding the basis for this transiency of response may reveal a more general strategy for inhibition of PKC pathways. Citation Format: Noemi Kedei, Aleksandra M. Michalowski, Mark E. Petersen, Gary E. Keck, Peter M. Blumberg. Comparison of transcriptional response to phorbol ester, bryostatin 1, and bryostatin analogue in LNCaP and U937 cells provides insight into their differential mechanism. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2101. doi:10.1158/1538-7445.AM2013-2101

Biologically active alkylated coumarins from Kayea assamica

Four coumarin derivatives, theraphins A ( 1), B ( 2), C ( 3), and D ( 4), along with three known xanthones, 2-hydroxyxanthone, 1,7-dihydroxyxanthone, and 5-hydroxy-1-methoxyxanthone, were isolated from the bark of Kayea assamica (Clusiaceae) native to Myanmar. Their structures were determined using spectroscopic and chemical techniques. The absolute configuration of 1 was established by the modified Mosher ester method. Theraphins A ( 1), B ( 2), and C ( 3) exhibited good cytotoxicity against Col2, KB, and LNCaP human cancer cell lines. Theraphin D ( 4) showed mild activity only against the KB cell line. The coumarins also exhibited mild antimalarial activities.

Specificity of glucuronosyltransferase activity in the human cancer cell line LNCaP, evidence for the presence of at least two glucuronosyltransferase enzymes

Recent findings obtained by our group showed that incubation of LNCaP cells with labeled steroids leads to the formation of 3- and 17-hydroxysteroid glucuronides. In this study, the specificity and the kinetic properties of 3-hydroxy-C 19 steroid uridine diphospho-glucuronosyltransferase (3-OH-UGT) and 17-hydroxy-C 19 steroid UGT (17-OH-UGT) activities in LNCaP cells were investigated. Results indicate that the UGT has a high affinity for testosterone, dihydrotestosterone (DHT), androsterone (ADT) and androstane-3α,17β-diol (3α-DIOL), with K mvalues ranging from 0.25 to 0.68 μM. The K mvalues are approx. 10-fold higher for androst-5-ene-3β,17β-diol (5-ene-DIOL) and androstane-3β,17β-diol (3β-DIOL). The relative specificities ( V max/ K m) also showed higher turnover rates for testosterone, DHT, ADT and 3α-DIOL with values ranging from 2.93 to 5.71, than for 3β-DIOL and 5-ene-DIOL with ratios of 0.41 and 1.10, respectively. Dixon plot and Cornish-Bowden analysis demonstrate that testosterone, DHT, ADT, and 3α-DIOL inhibit the glucuronidation of DHT and ADT in a competitive fashion. In contrast, when the studies are performed with 3β-diol and 5-ene-DIOL the inhibition of ADT glucuronidation is uncompetitive while the glucuronidation of DHT is inhibited competitively, suggesting the presence of two UGT enzymes, one for glucuronidation of the 17β-OH group and a second for the 3α-OH group. Further evidence for the presence of two UGTs in LNCaP cells was obtained by incubation with a variety of 3β-OH-C 19 steroids which caused a marked inhibition of DHT-G formation but had no effect on the glucuronidation of ADT. In summary, our data demonstrate the presence of at least two UGTs in the human prostate cancer cell line LNCaP. The relative specificity of the 17-OH-UGT in LNCaP cells is 3α-DIOL > DHT > testosterone, while ADT is glucuronidated by the 3-OH-UGT.

Glucuronosyltransferase activity in human cancer cell line LNCaP

LNCaP, a human prostate cancer cell line, metabolizes testosterone into a variety of 5α-reduced C19 steroids, such as dihydrotestosterone (DHT), androstane-3α,17β-diol (3α-DIOL), androstane-3β,17β-diol (3β-DIOL), and androsterone (ADT). Recent reports also suggest that 5α-reduced C19 steroid glucuronides can be detected in the medium. The purpose of this work was to characterize by liquid chromatograph ion spray mass spectroscopy (LCMS) the metabolites formed by LNCaP during incubation with testosterone and its 5α-reduced C19 steroids. Time course studies using 10 nM labeled testosterone, 3α-DIOL, 3β-DIOL, or ADT showed that a large proportion of polar steroids were produced by LNCaP. Identification of metabolites produced by LNCaP was carried out by LCMS using 1 μM substrates. Analysis of testosterone metabolism indicated that testosterone glucuronide was formed at 77 ± 2% after 96 h of incubation. Using DHT as substrate, 3α-DIOL-G and DHT-G were the major metabolites, accounting for 46 ± 4% and 38 ± 3%, respectively, of the total radioactivity in the medium; ADT-G accounted for 8 ± 1%. Further analysis by LCMS also indicated that the glucuronide group in 3α-DIOL-G was at position 17-carbon. 3α-DIOL-G (86 ± 3%) was the prominent metabolite formed from 3α-DIOL, a minor product was detected at 7 ± 1% and identified by mass spectrometry to correspond to a trihydroxylated C19 steroid. After a 96-h incubation period, 3β-DIOL was transformed into 3β-DIOL-G, DHT-G, and a trihydroxylated metabolite of C19 steroid at 48 ± 3, 26 ±1, and 24 ± 2%, respectively. When ADT was incubated, ADT-G (89 ± 1%) and two minor products, 3α-DIOL and 3α-DIOL-G, at less than 10% of total metabolites were detected. It was also shown that glucuronosyltransferase activity in homogenized cells was about 8% of that in intact cells, whereas the addition of the cofactor (2 mM uridine 5′-diphosphoglucuronic acid (UDPGA)) partially restored the activity to 25%. Our data clearly indicate that extensive glucuronidation of testosterone and its metabolites, namely DHT, 3α-DIOL, 3β-DIOL and ADT, occurs in LNCaP cells. Because these polar metabolites are not active and cannot be reconverted into active androgens, it is suggested that glucuronosyltransferase may be a major factor in the regulation of androgen action.