Pharmacodynamics of Doxorubicin in Human Prostate Tumors1

The pharmacodynamics of doxorubicin in human prostate tumors were studied using histocultures of radical prostatectomy specimens. Drug treatment lasted 96 h. The antiproliferative effect was measured by the inhibition of DNA precursor ([3H]thymidine) incorporation, and the cytotoxic effect was measured by monitoring cells with fragmented DNA, as indicated by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. The average [3H]thymidine labeling index in 17 tumors was 39% (range, 20-56%). The antiproliferative and cytotoxic effects were concentration dependent and reached 100% at 6 and 17 LM doxorubicin. The cytotoxic concentrations were significantly higher than the antiproliferative concentrations, indicating that prostate tumors were more sensitive to the antiproliferative effect than they were to the cytotoxic effect of doxorubicin. The antiproliferative effect was inversely correlated with patient’s age (P < 0.02) and weakly correlated with LI and Gleason grade (P = 0.07 and 0.06, respectively), but it was not correlated with clinical stage, prostate-specific antigen secretion, or race of patients (P > 0.12). In contrast, the cytotoxic effect was positively correlated with Gleason grade (P < 005) and weakly correlated with stage (P < 0.08), but it was not correlated with the other parameters (P > 0.18). The opposite correlations between the two effects with tumor grade suggest that the two effects are not coupled. A comparison of the drug concentrations required to produce 50% antiproliferative (0.06 jLM) and cytotoxic (2 ELM) effects and the literature data on plasma drug concentrations derived from systemic treatment suggest that there are minimal drug effects at the clinically achievable drug concentrations and that regional delivery of doxorubicin to Received 5/2/97; revised 1 1/4/97; accepted 11/10/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. This study was supported in part by Research Grant RO1CA74I79 from the National Cancer Institute, Department of Health and Human Services. The Ohio State University Comprehensive Cancer Center Tissue Procurement Service was supported in part by Grant P30CA16058 from the National Cancer Institute, NIH, Department of Health and Human Services. 2 To whom requests for reprints should be addressed, at The Ohio State University, 410 West 12th Avenue, Columbus, OH 43210. Phone: (614) 292-4055; Fax: (614) 292-4055. the prostate may be necessary to provide adequate concentrations to produce antiproliferative and cytotoxic effects. INTRODUCTION Prostate cancer is the most common malignancy in man. About 60% of the 317,000 new cases will present with disease confined to the organ (1). Prostate cancer is a slowly progressing disease and is usually diagnosed in older men at a median age of 70.5 years, with fewer than 1% of patients being less than 50 years old. The survival rate is significantly and inversely influenced by the tumor stage at diagnosis. An emphasis on early detection and the availability of new screening techniques, in particular, the detection of PSA,3 have increased the rate of diagnosis and lowered the stage and age at the time of diagnosis (2). The current treatment options for locally confined disease include surgical radical prostatectomy, radiotherapy, and cryotherapy, with surgery being the most common treatment modality. Neoadjuvant hormone therapy prior to radical prostatectomy is under investigation to improve the control of localized prostate cancer. Some studies showed benefits of neoadjuvant hormone therapy (3, 4), although time to progression or overall survival did not improve in another study (5). Androgen ablation therapy and systemic chemotherapy are usually reserved for metastatic disease and are not used for local disease. Among the agents used to treat advanced prostate cancer, doxorubicin produces one of the highest combined partial and complete response rate of 29%, although complete responses are rare (6). However, doxorubicin, as a single agent or in combination with other chemotherapeutics, has not improved the survival rate (6, 7). Because chemotherapy is not commonly used in early prostate cancer, the activity of doxorubicin against early disease is not known. This study was designed to determine the pharmacological effects of doxorubicin in surgical specimens of early-stage human prostate tumors obtained by radical prostatectomy. The drug-induced antiproliferative and cytotoxic (cell kill) effects were quantified. The effective drug concentrations were compared with the literature data on clinically achievable drug concentrations in patients to infer potential therapeutic effectiveness of neoadjuvant or adjuvant systemic doxorubicin therapy. The study was performed using histocultures of surgical specimens of human prostate tumors obtained by radical prostatectomy. The histoculture system provides advantages over monolayer cell culture system. Histocultures maintain the threedimensional tissue structure and organization, and thus, the coexistence of tumor and stromal cells, cell-cell interaction, and interand intratumor heterogeneity are preserved (8). The maintenance of tissue architecture is critical because the interaction between the tumor and normal cells may be important for 3 The abbreviations used are: PSA, prostate-specific antigen; LI, [3H]thymidine labeling index; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Research. on December 31, 2017. © 1998 American Association for Cancer clincancerres.aacrjournals.org Downloaded from 278 Doxorubicin Pharmacodynamics in Human ProstateCancer prostatic epithelial growth and response to androgen stimulation. We previously reported that human prostate tumor histocultures maintained their characteristics for at least 8 weeks, as indicated by unchanged LI and secretion of PSA and prostatic acid phosphatase (9). The clinical relevance of the human tumor histoculture system was recently demonstrated by Hoffman and colleagues (10-12). These investigators show, in retrospective and semiprospective preclinical and clinical studies, that drug responses in human tumor histocultures, using inhibition of DNA precursor incorporation or inhibition of metabolic reduction of tetrazolium dye as end points, correlates with the sensitivity, resistance, and survival of head and neck, colorectal, and gastric cancer patients to treatment with mitomycin C, doxorubicin, 5-fluorouracil, or cisplatin. MATERIALS AND METHODS Chemicals and Supplies. Doxorubicin was a gift from Pharmacia (Albuquerque, NM). Sterile pigskin collagen (Spongostan standard) was purchased from Health Designs Industries (Rochester, NY); tissue culture supplies (i.e. , L-glutamine, sodium pyruvate, fetal bovine serum, gentamicin, DMEM, MEM, MEM nonessential amino acids solution, and MEM vitamin solution) were from Life Technologies, Inc. (Grand Island, NY); [3H]thymidine (specific activity, 65 Ci/mmol) was from Moravek Biochemicals Inc. (Brea, CA); NTB-2 nuclear track emulsion was from Eastman Chemicals (Rochester, NY); terminal deoxynucleotidyl transferase, digoxigenin-dUTP, and antidigoxigenin-peroxidase in ApopTag for in situ detection of dead and dying cells were from Oncor Inc. (Oaithersburg, MD); proteinase K and mouse normal IgO were from Sigma Chemical Co. (St. Louis, MO); the liquid 3,3’-diaminobenzidine substrate kit was from BioGenex (San Ramon, CA); the mouse monoclonal antibody ER-PR8 against human PSA and Labeled Streptavidin-Biotin universal detection kit were from DAKO Corp. (Carpinteria, CA); and the PSA detection kit was from Hybritech (San Diego, CA). All chemicals and reagents were used as received. Tumor Procurement Surgical specimens of human primary prostate tumors were obtained from the peripheral zone of the prostate gland in patients who had organ-confined prostatic adenocarcinoma and who underwent radical prostatectomy. Specimens were obtained via the Tumor Procurement Service at The Ohio State University Comprehensive Cancer Center and from the neighboring Doctor’s Hospital. Tumor specimens were placed in MEM within 10-30 mm after surgical excision, stored on ice, and prepared for culturing within 1 h after excision. The histopathology of tumor specimens was established using frozen sections of tumor fragments adjacent the histocultured specimens. Tumors were graded according to the Gleason grading system, with a score of 2 for well-differentiated tumors and a score of 10 for poorly differentiated tumors (13), by pathologists at the James Cancer Hospital (Columbus, OH). Histoculture. Tumor specimens were cultured as described previously (9). In brief, tumors were cut into 1-mm3 pieces under sterile conditions. Five to six tumor pieces were placed on a 1 -cm3 presoaked collagen gel and incubated at 37#{176}C in a humidified atmosphere of 95% air and 5% CO2. The culture medium consisted of a 1 : 1 mixture of MEM and DMEM, 10% fetal bovine serum, 2 nvvi L-glutamine, 1 ms sodium pyruvate, 40 p.g/ml gentamicin, 0. 1 ms’i MEM nonessential amino acids, and concentrated MEM vitamin solution (100-fold concentrated; 10 mllliter). The histocultures were fed every other day and used for doxorubicin pharmacodynamic studies on day 4. Detection of PSA in Media and in Histocultured Tissues. The secretion of PSA by human prostate tumor histocultures to the culture medium collected at the end of doxorubicin exposure was measured by a sandwich immunoassay using two antibodies against two different epitope sites on PSA, performed by technicians at the Immunology Laboratory of the James Cancer Hospital (Columbus, OH). PSA expression in histocultured tumors was measured by immunohistochemistry using a universal detection kit from DAKO Corp. After sequential dewaxing and rehydration in xylene, ethanol, and water, tissue sections were boiled in a 10 msi citrate buffer (pH 6.0) in a microwave oven for 15 mm, cooled at room temperature for 15 ruin, and washed once in PBS. Tissue sections were then incubated with the blocking solution for 10 mm and subsequently with the mouse antihumam PSA (1:20 dilution in PBS containing 5 mg/mI BSA) in a humidified chamber at room temperature for 2 h. Mouse normal IgO was used as the antibody for negative controls. After washing with PBS, the tissue sections were covered with the biotin-anti-IgO linker solution, and then with peroxidase-conjugated streptavidin solution. After washing twice with PBS, tissue sections were incubated for 3-7 mm with the chromogen 3,3’-diaminobenzidime and with the substrate hydrogen peroxide and then counterstained with hematoxylin, followed by dehydration and coverslipping for microscopic examination. Pharmacological Effects of Doxorubicin. The antiproliferative effect was measured by the inhibition of DNA precursor ([3H]thymidine) incorporation. The cytotoxic effect was measured by monitoring dead and dying cells using the TUNEL assay. Tumor histocultures were exposed to various concentrations of doxorubicin, ranging from 0.00017 to 17 p.M for 96 h. After drug treatments, the doxorubicin-containing medium was removed, and tumor histocultures were washed three times with drug-free medium. Tumors were incubated with 0.03 p.M [3H]thymidime for 96 h, washed 3 times with PBS, and them fixed in 10% neutralized formalin, dehydrated, and embedded in paraffin. The embedded tumor tissues were cut onto microscope slides at a 5-p.m thickness using a microtome. Two sets of the slides were collected for autoradiography and for TUNEL assay separately. Untreated controls were processed similarly. Autoradiography. Tumor sections on microscope slides were deparaffinized, rehydrated, and stained with hematoxylin. Slides were then coated with a thin layer of NTB-2 nuclear track emulsion and exposed for 7-10 days in the dark in a cold room. Slides were developed, dehydrated, and then coverslipped. The [3H]thymidine-labeled cells were scored, and the LI was calculated as (labeled cells)/(total cells). TUNEL Assay. Procedures were carried out as described in the manufacturer’s instructions for the ApopTag detection kit. Tissue sections on slides were deparaffinized, rehydrated, and incubated with proteimase K (20 p.g/ml) in PBS for 15 mm at room temperature, followed by 4 washes with distilled water for 2 mm each. This protease treatment is necessary to make DNA fragments accessible for reaction with enzyme and substrate. Endogenous peroxidase was quenched by incubating tissue Research. on December 31, 2017. © 1998 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Table 1 Patients and tumor characteristics and tumor sensitivity of tumors to 96-h doxorubicin treatments IC50 and IC90 are the concentrations that caused 50 and 90% inhibition of thymidine labeling, respectively. LC50 and LC ,) are the concentrations that caused 50 and 90% of cell death, respectively, as determined by the TUNEL assay.