Serum-free complete medium, an alternative medium to mimic androgen deprivation in human prostate cancer cell line models.

BACKGROUND: Almost all men who present with advanced prostate cancer (CaP) and some men who fail therapy for clinically localized CaP are treated with androgen deprivation therapy (ADT). CaP cell lines are used to identify and characterize new agents for ADT or investigate mechanisms of ADT resistance. CaP cell lines are maintained in culture medium that contains fetal bovine serum, which contains testosterone (T). Androgen deprivation experiments are performed using media supplemented with androgen-free serum, such as charcoal stripped fetal bovine serum (CS-FBS). However, CS-FBS composition varies from batch-to-batch and variations may impact experimental reproducibility. Serum free media (SFM) may provide a better defined alternative to media supplemented with CS-FBS (CSM). METHODS: Cell growth of six human CaP cell lines was assessed using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Androgen levels were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: MTT assays showed 5 of 6 CaP cell lines grew after 6 days of culture in androgen- deprived SFM or CSM. LNCaP and VCaP growth was stimulated when cells were cultured in SFM or CSM supplemented with T. LNCaP, C4-2, LAPC-4, and VCaP cell growth was inhibited when cultured in SFM or CSM with T and bicalutamide. LC-MS/MS data showed LAPC-4 cells produced similar DHT levels when cultured in T-supplemented SFM or CSM. Dutasteride impaired T to DHT metabolism in LAPC-4. CONCLUSIONS: Media composition contributed to growth differences observed between CaP cells cultured in SFM or CSM. However, the differences in media composition did not impair CaP cell response to T-stimulated growth, bicalutamide growth inhibition, metabolism of T, or dutasteride efficiency. SFM can be used as a better defined alternative to CSM for androgen deprivation experiments.

Thioredoxin Reductase 1 Expression and Castration-recurrent Growth of Prostate Cancer.

INTRODUCTION: Many genes are differentially expressed between androgen-dependent and androgen-independent prostate cancer (CaP). Differential expression analysis and subtractive hybridization previously identified nine genes expressed in intact mice bearing CWR22 tumors and castrated mice bearing recurrent CWR22 tumors but not in regressed tumors. The objectives of this study were to develop an immunostaining method to dual-label foci of proliferating tumor cells [the origin of castration-recurrent CaP (CR-CaP)], to determine which of the nine candidate proteins were differentially expressed in proliferating versus nonproliferating cells at the onset of growth after castration, and to test preclinical findings using clinical specimens of androgen-stimulated benign prostate (AS-BP) and CaP (AS-CaP) and CR-CaP. METHODS: Paraffin-embedded, bromodeoxyuridine-injected CWR22 tumors were hydrated, antigen-retrieved using high heat and high pressure, labeled for each of the nine antigens of interest, visualized using peroxidase, and counterstained with hematoxylin. Mean optical density was calculated for proliferating and nonproliferating areas using automated (nuclear staining) or manual (cytoplasmic staining) image analysis. Prostate tissue microarray sections were immunostained and visually scored. RESULTS: Immunohistochemistry revealed higher nuclear expression of thioredoxin reductase 1 (TrxR1) in proliferating cells than nonproliferating cells (P < .005). There were no statistical differences between cell types in the expression of other proteins. TrxR1 expression was higher (P < .01) in CR-CaP compared with AS-BP or AS-CaP. CONCLUSIONS: Increased TrxR1 expression in CR-CaP was consistent with increased TrxR1 and BrdU expression at the onset of growth in the CWR22 model. Thioredoxin reductase 1 should be targeted in an attempt to delay or prevent CaP recurrence after castration.

A new nonlinear mixture response surface paradigm for the study of synergism: a three drug example.

A flexible approach to response surface modeling for the study of the joint action of three active anticancer agents is used to model a complex pattern of synergism, additivity and antagonism in an in vitro cell growth assay. The method for determining a useful nonlinear response surface model depends upon a series of steps using appropriate scaling of drug concentrations and effects, raw data modeling, and hierarchical parameter modeling. The method is applied to a very large in vitro study of the combined effect of Trimetrexate (TMQ), LY309887 (LY), and Tomudex (TDX) on inhibition of cancer cell growth. The base model employed for modeling dose-response effect is the four parameter Hill equation [1]. In the hierarchical aspect of the final model, the base Hill model is treated as a function of the total amount of the three drug mixture and the Hill parameters, background B, dose for 50% effect D50, and slope m, are understood as functions of the three drug fractions. The parameters are modeled using the canonical mixture polynomials from the mixture experiment methodologies introduced by Scheff [2]. We label the model generated a Nonlinear Mixture Amount model with control observations, or zero amounts, an "NLMAZ" model. This modeling paradigm provides for the first time an effective statistical approach to modeling complex patterns of local synergism, additivity, and antagonism in the same data set, the possibility of including additional experimental components beyond those in the mixture, and the capability of modeling three or more drugs.

Chk1 signaling pathways that mediated G(2)M checkpoint in relation to the cellular resistance to the novel topoisomerase I poison BNP1350.

A novel karenitecin, BNP1350, is a topoisomerase I-targeting anticancer agent with significant antitumor activity in vitro and in vivo. A BNP1350-resistant human head and neck carcinoma A253 cell line, denoted A253/BNPR, was developed. The A253/BNPR cell line was approximately 9-fold resistant to BNP1350 and 4-fold cross-resistant to another topoisomerase I inhibitor SN-38, the active metabolite of irinotecan. After drug treatment with equimolar concentrations of BNP1350 (0.7 microM) for 2h, activation of the DNA double-strand break repair protein complexes was similar in the two cell lines, suggesting that DNA dsb repair is not attributable to resistance to BNP1350 in the A253/BNPR cells. Cell cycle analysis indicates that the A253 cell line accumulated primarily in S phase, but G(2) phase accumulation was observed in the A253/BNPR cell line at 48 h after drug removal. Elevated chk1 phosphorylation at Ser(345) following DNA damage induced by BNP1350 was accompanied by G(2) accumulation in the A253/BNPR cell line, while exposure to equimolar concentrations of BNP1350 (0.7 microM) induced S-phase arrest and no increased phosphorylation of chk1 at Ser(345) in the A253 cell line. Under the same conditions, increased chk1 activity was observed in the A253/BNPR cell line, but not in the A253 cell line. Moreover, stimulated binding of 14-3-3 proteins to chk1 was observed in BNP1350-treated A253/BNPR cells. To confirm relationship between chk1 expression/phosphorylation and drug resistance to topo I poisons, we examined the effects of chk1 or chk2 antisense oligonucleotides on the cellular growth inhibition. Chk1 antisense oligonucleotide can sensitize the A253/BNPR cells to killing by topo I inhibitor BNP1350, but no significant sensitization of BNP1350-induced growth inhibition was observed in the drug-sensitive cell line. Chk2 antisense oligonucleotide has only a small sensitization effect on BNP1350-induced growth inhibition in both cell lines. The data indicate that the chk1 signaling pathways that mediate cell cycle checkpoint are associated with cellular resistance to BNP1350 in the A253/BNPR cell line.