This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tang, Y. Articles by Hussain, A. Search for Related Content PubMed PubMed Citation Articles by Tang, Y. Articles by Hussain, A.

Docetaxel Followed by Castration Improves Outcomes in LNCaP Prostate Cancer–Bearing Severe Combined Immunodeficient Mice

Authors' Affiliations: ¹ Department of Medicine, ² Division of Biostatistics, ³ Greenebaum Cancer Center, and ⁴ Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine and ⁵ Baltimore Veterans Affairs Medical Center, Baltimore, Maryland

Requests for reprints: Arif Hussain, Department of Medicine, University of Maryland School of Medicine, BRB 9-041, 655 West Baltimore Street, Baltimore, MD 21201. Phone: 410-328-3911; Fax: 410-328-6559; E-mail: ytang{at}som.umaryland.edu.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Purpose: Androgen ablation is the standard initial treatment for advanced prostate cancer; however, tumors eventually develop androgen independence and become incurable. Chemotherapy is commonly used after hormone treatment fails but has not shown significant survival benefit. Studies suggest that androgen ablation can select for a population of hormone-independent cells that are also relatively chemotherapy resistant. Thus, it may be therapeutically advantageous to target prostate cancer with chemotherapy before hormone ablation. This study was undertaken to determine the relative efficacy of such an approach in a preclinical model of prostate cancer.

Experimental Design: Severe combined immunodeficient mice bearing human LNCaP prostate tumors were treated with docetaxel and/or surgical castration applied singly, concurrently, or in different sequences. Treatment efficacy was determined by tumor volume and growth delay measurements. The extent of apoptosis in tumors in response to treatments was assessed via terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL) assays. In addition, Western blots were done to study the relative expression of Bcl-2 and Bax in the tumors.

Results: Docetaxel followed by castration showed the most potent antitumor effects. In contrast, with the exception of castration alone, castration followed by docetaxel produced the least antitumor activity. TUNEL assays confirmed that the density of apoptotic tumor cells was significantly greater for docetaxel followed by castration than for any other treatment. In tumors of mice treated with single modality therapies, Bax to Bcl-2 ratios decreased significantly after castration, whereas this ratio remained high after docetaxel treatment.

Conclusion: A treatment sequence of docetaxel followed by hormone ablation may be more effective in treating prostate cancer than concurrent docetaxel/hormone therapy or hormone ablation followed by docetaxel.

Preclinical and clinical studies suggest that androgen ablation can select for a population of androgen-independent prostate cancer cells that may also be relatively resistant to chemotherapy (5, 6). This might account, in part, for the apparent ineffectiveness of chemotherapy in HRPC. It is likely that cytotoxic agents, such as docetaxel, that have shown activity in HRPC may also be active, and possibly more so, in hormone-sensitive prostate cancer. Thus, one potential way to improve the outcome of the traditional treatment modalities, like hormone ablation and chemotherapy, might be to use chemotherapy relatively early in the disease process rather than in the HRPC stage.

Here, we present our recent studies on severe combined immunodeficient mice bearing human LNCaP prostate cancer xenografts; the results show that of the numerous treatment methods tested, sequential therapy with docetaxel followed by castration provided the best antitumor effects.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Animals and tumor inoculation. Male severe combined immunodeficient mice (National Cancer Institute, Frederick, MD), 4 to 6 weeks old, were inoculated with human LNCaP prostate cancer cells (American Type Culture Collection, Rockville, MD) at 2 x 10⁶ per flank, resulting in two tumors per mouse. Three to 4 weeks after inoculation, mice were assigned to different treatment groups such that the average tumor volume in each group was 300 mm³. There was no difference in mean tumor volumes across all groups (P = 0.78). At this time point (designated week 0), treatments were initiated. The average body weight of the mice at week 0 was 19.36 ± 2.46 g; there was no significant difference in mean body weight across all groups (P = 0.10). All animal studies were done under a protocol approved by the Institutional Animal Care and Use Committee, University of Maryland, Baltimore, MD.

Treatments. The different treatments are listed in Table 1. Most of the treatments were tested in three to four separate groups of mice (four to five mice per group) in independent experiments.

Tumors from 30 additional treated mice were used for terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL) assays and Western blot analysis. Tumors were harvested at different time points posttreatment (two to three mice for each time point). For the TUNEL assays, the time points were 1 or 2 weeks after castration (C-1 or C-2), 1 or 2 weeks of docetaxel (D-1 or D-2), or 2 weeks of treatment consisting of D-1 followed by C-1 (D-1//C-1) or castration first followed by D-1 (C//D-1). For the Western blots, the time points were 2 or 4 weeks after castration (C-2 and C-4) or 2 or 4 weeks of docetaxel treatment (D-2 and D-4). Tumors from mice treated with vehicle only served as controls.

Tumor measurements. Tumors were measured weekly during the first 4 weeks after treatment started and twice a week thereafter. Tumor volumes were calculated using the following formula: 4/3a²b, where a = 0.5 width; b = 0.5 length (a < b). Efficacy of the different treatment methods was assessed using two metrics: (a) average tumor volumes per group after 4 weeks of treatment (i.e., week 4 time point) and (b) growth delay, measured as the time from the start of treatment for either tumor in a mouse to reach a volume of 800 mm³.

TUNEL assay. Tumor tissues were fixed in 10% formaldehyde for routine paraffin sections and H&E staining. TUNEL assays for apoptosis were done using the ApopTag kit (Intergen, Norcross, GA) as per the manufacturer's instructions. After counterstaining with hematoxylin (Sigma, St. Louis, MO), slides were observed under a Nikon TE200 microscope. Based on the morphology of H&E stains, areas of tumors free of necrosis and with similar cellular densities were selected. Within these areas, 10 random fields (x400) per slide were captured with Nikon Digital Still Camera DXM1200 and saved as image files with Act-1 software (Nikon Corp., Tokyo, Japan). The apoptotic index in each field was calculated using the formula: apoptotic index (%) = A x 100 / (A + C), where A = apoptotic cells, C = unlabeled cells. For each tumor, the mean of the apoptotic index and the SE were calculated using SigmaPlot.

Western blot analysis. Tumor tissues were homogenized in Tissue Protein Extraction Reagent (Pierce, Rockford, IL) with brief ultrasound sonication. Equal amounts of protein were loaded onto 12% Tris-glycine polyacrylamide gels (Bio-Rad, Hercules, CA), separated, and then transferred onto Hybond-P polyvinylidene difluoride membrane (Amersham, Piscataway, NJ). Nonspecific signals were blocked by incubating the membranes in 10% nonfat dry milk in TBST (TBS plus 0.05% Tween 20) solution for 1 hour. The membranes were briefly rinsed with 1% bovine serum albumin in TBST and incubated overnight in 1:200 diluted anti-human Bcl-2 monoclonal antibody (Oncogene, San Diego, CA), anti-human Bax monoclonal antibody (Upstate, Lake Placid, NY), or anti-human p-Bcl-2 polyclonal antibodies (which recognize phosphorylated serine residues 70 and 87; Santa Cruz Biotechnology, Santa Cruz, CA). Anti-human ß-actin antibody (Sigma), diluted 1:1,000, was used to assess housekeeping protein expression. Signals were detected with an enhanced chemiluminescence–linked detection kit (Amersham) after incubating the membranes with horseradish peroxidase–coupled secondary antibodies. Signal intensity was measured via a Molecular Dynamics Image Quant Densitometer and was normalized to ß-actin expression.

Statistical analyses. The experiment/cage effect was tested in a statistical model (ANOVA). No sufficient evidence was found that experiment/cage effect was present (P = 0.28-0.51 for the treatment groups). Therefore, groups that were identical with respect to the treatments given were pooled for the statistical modeling.

Linear mixed-effects models were used to estimate average animal body weight, tumor volume, and growth rate across the treatment groups (7). The treatment groups were compared with one another at 0.05 level of statistical significance. All statistical tests were two sided. No adjustments for multiple comparisons were made due to the exploratory nature of the analysis. The results of the comparisons across treatment groups are presented as the difference in group means, along with the corresponding SE or 95% confidence interval. Tumor growth delay was modeled using the time-to-event approach (i.e., the time required for a tumor to grow to the prespecified volume of 800 mm³). The Kaplan-Meier estimates of growth delay function were compared using the log-rank test for right-censored data.

The morphologic data were compared between the different treatment groups using standard or Welch modified two-sample t test.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Tumor size at week 4. The mean tumor volumes at week 4 of treatment are shown in Table 1. As expected, the average tumor volume in the castration-only group (C-only; 602 mm³) was significantly less than in the control group (819 mm³), with P = 0.009 (Table 1). However, compared with the C-only group, the mean tumor volumes were at least 50% smaller for all groups whose treatment included docetaxel. The group with the smallest tumor size at week 4 was the one that received docetaxel for 2 weeks followed by castration (D-2//C). The opposite treatment sequence, C//D-2 (castration followed 2 weeks later by 2 weeks of docetaxel), resulted in tumors about thrice as large. No significant difference was found between the groups treated with concurrent docetaxel and castration (D-2+C) versus docetaxel only (D-2).

Growth delay in tumors after treatment. The median time for tumors to reach 800 mm³ in the control group was about 3.5 weeks; castration delayed tumor growth by only 7 to 10 days relative to the controls (data not shown). Growth delay results for other treatment groups are shown in Fig. 1A and B. Among the four groups that received docetaxel for 2 weeks (Fig. 1A), D-2//C was again the most effective. It produced the longest growth delay, whereas C//D-2 had the shortest. Among the groups treated with docetaxel for 4 weeks (Fig. 1B), chemotherapy followed by castration (D-4//C) also resulted in the best outcome.

View larger version (22K): [in this window] [in a new window]   Fig. 1. Kaplan-Meier estimates of growth delay functions. A, groups that received 2 weeks of docetaxel. B, groups that received 4 weeks of docetaxel.

View larger version (56K): [in this window] [in a new window]   Fig. 2. Apoptosis in tumor tissues determined by TUNEL assay. A, typical morphologic changes in selected fields on H&E stains (x200) and TUNEL assays (x400). B, average number of apoptotic cells per field for different treatments and time points. D-1, 1 week of docetaxel; D-2, 2 weeks of docetaxel; C-1, 1 week after castration; C-2, 2 weeks after castration; C-1//D-1, 1 week after castration followed by docetaxel given for 1 week; and D-1//C-1, 1 week of docetaxel followed by castration, with tumors harvested a week after the castration. Controls received vehicle only for 2 weeks.

View larger version (22K): [in this window] [in a new window]   Fig. 3. Bax and Bcl-2 expression in tumors treated with castration or docetaxel. C-2, 2 weeks after castration; D-2, 2 weeks of docetaxel; C-4, 4 weeks after castration; D-4, 4 weeks of docetaxel.

	Discussion

Top Abstract Materials and Methods Results Discussion References

The LNCaP cell line (10) has been widely used for both in vitro and in vivo prostate cancer studies due primarily to its androgen-sensitive characteristics. Surgical castration in mice bearing LNCaP tumors arrests tumor growth (11). However, this antitumor effect of androgen ablation is temporary. In our experiments, tumors resumed growth between 2 and 3 weeks after castration, suggesting that they had evolved to be hormone independent by this time.

All groups that received docetaxel for 2 weeks showed significantly longer growth delays (over 6 weeks) than did the castration-only group (7-10 days). Extending docetaxel treatment provided an even greater antitumor effect; the median growth delay time in D-2 mice was 9 weeks, whereas in the D-4 group it was 17 weeks (Table 2). However, significant loss of body weight was also associated with the extended chemotherapy (data not shown).

It is noteworthy that the antitumor effects of docetaxel were reduced if the docetaxel treatments were begun after the mice had been castrated compared with being given alone. This is evidenced by the significant difference in growth delay between the D-2 and C//D-2 treatment groups (P = 0.009; Fig. 1A; Table 2). A similar difference is also seen between the D-4 and C//D-4 groups (Fig. 1B), although the statistical significance is reduced (P = 0.12); this might be influenced by the smaller sample size of the C//D-4 group (n = 5). TUNEL assays confirmed that apoptosis in tumors was significantly reduced if docetaxel followed castration rather than being used alone (Fig. 2). Taken together, these observations suggest that after castration therapy, LNCaP tumors become less sensitive to chemotherapy.

The sequential treatment consisting of docetaxel followed by castration (D-2//C and D-4//C) provided the most potent antitumor effects among all the treatments tested, as shown by both metrics (i.e., tumor volumes at week 4 and growth delay; Tables 1 and 2; Fig. 1). The TUNEL assay also showed that castration after docetaxel treatment results in a further increase in apoptotic cells over either docetaxel or castration alone.

An intricate network of cellular mediators modulates apoptosis. Mechanistically, altered expression in some of these mediators could contribute, at least in part, to the observed outcomes in the above xenograft model. As shown in Fig. 3 and Table 3, enhanced Bcl-2 expression occurs in the LNCaP tumors after hormone ablation, consistent with several prior preclinical studies (12–14). Other reports have also shown that with the development of clinical hormone resistance, Bcl-2 expression in men with prostate cancer tends to increase (15–18). In our studies, we noted that the tumors start to regrow around 2 to 3 weeks after castration (data not shown); this growth reflects resistance to the hormone ablation. In parallel, higher Bcl-2 levels are found at both weeks 2 and 4 after castration compared with control and docetaxel-treated tumors (Fig. 3; Table 3).

Perhaps a more critical event in regulating apoptosis is the Bax to Bcl-2 ratio (19). An excess of Bax relative to Bcl-2 favors formation of proapoptotic Bax/Bax homodimers. As this ratio decreases, enhanced Bax/Bcl-2 heterodimerization and Bcl-2/Bcl-2 homodimerization occur; these events have antiapoptotic effects on cells (19). In the LNCaP model, untreated tumors are relatively sensitive, at least initially, to either hormone ablation or docetaxel, in keeping with the significant excess of Bax over Bcl-2 at baseline (Table 4). The decrease in the Bax to Bcl-2 ratio after castration favors formation of Bax/Bcl-2 heterodimers and Bcl-2/Bcl-2 homodimers, resulting in a relatively apoptosis-resistant state. In contrast, after 2 weeks of docetaxel treatment, >5-fold excess of Bax over Bcl-2 is observed in the tumors, and this ratio increases further after 4 weeks of chemotherapy (Table 4). Thus, initial hormone treatment can lead to the emergence of tumors that are relatively resistant to subsequent chemotherapy. With the use of docetaxel first, on the other hand, tumors retain the ability to respond to additional apoptotic stimuli, such as castration. That is, LNCaP tumors retain hormone sensitivity after chemotherapy. In addition to producing the smallest tumors at week 4, the D-2//C treatment resulted in tumors that were nearly half the size of those treated with D-2 only (Table 1). Furthermore, both D-2//C and D-4//C provided the longest growth delays among all 2-week and 4-week treatment groups, respectively (Table 2). These data show that castration after chemotherapy contributes to a further reduction in tumor size and a longer delay in tumor growth. A recent clinical trial evaluating the sequential use of docetaxel followed by hormone therapy in men with prostate cancer experiencing prostate-specific antigen failure after their definitive primary local treatments also showed that patients responded to hormone therapy after the initial docetaxel treatment (20).

In addition to the possible selection of cells with altered Bax/Bcl-2 ratios during hormone ablation, a subpopulation of hormone-sensitive prostate cancer cells could also potentially enter the G₀ phase of the cell cycle (which is a relatively chemotherapy-resistant state) rather than undergo apoptosis. Thus, theoretically, concurrent docetaxel plus hormone therapy may also be less effective than sequential docetaxel hormone therapy. Consistent with this notion, tumor volumes at week 4 (Table 1) as well as growth delay measurements (Table 2) reveal the greater antitumor activity of D-2//C versus D-2+C and D-4//C versus D-4+C.

In summary, the above studies suggest that the earlier use of docetaxel (i.e., before the development of hormone resistance) is therapeutically advantageous. The relative insensitivity of prostate cancer to chemotherapy in the clinical setting could be due, in part, to the fact that patients generally receive chemotherapy after androgen ablation; the latter may be instrumental in desensitizing tumors to chemotherapy. More work is needed to fully understand the molecular mechanisms underlying the therapeutic outcomes of our studies, and to verify that these results can be translated to clinical practice. Nevertheless, our data suggest that a treatment sequence of docetaxel before hormone ablation might provide significant therapeutic benefits in androgen-sensitive prostate cancer.

	Footnotes

 
Grant support: NIH grant R01-CA27440 (A.M. Brodie), Medical Research Service Department of Veterans Affairs Merit Review Award (A. Hussain), and Sanofi-Aventis Pharmaceuticals (A. Hussain).

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.

Received 6/27/05; revised 10/ 7/05; accepted 10/13/05.

	References

Top Abstract Materials and Methods Results Discussion References

 

1. Denmeade SR, Isaacs JT. A history of prostate cancer treatment. Nat Rev 2002;2:389–96.
2. Beer TM, Garzotto M, Henner WD, Eilers KM, Wersinger EM. Multiple cycles of intermittent chemotherapy in metastatic androgen-independent prostate cancer. Br J Cancer 2004;91:1425–7.[Medline]
3. Tannock IF, Wit RD, Berry WR, et al. Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer. N Engl J Med 2004;351:1502–12.[Abstract/Free Full Text]
4. Petrylak DP, Tangen CM, Hussain MHA, et al. Docetaxel and estramustine compared with mitoxantrone and prednisone for advanced refractory prostate cancer. N Engl J Med 2004;351:1513–20.[Abstract/Free Full Text]
5. Tso CL, McBride WH, Sun J, et al. Androgen deprivation induces selective outgrowth of aggressive hormone refractory prostate cancer clones expressing distinct cellular and molecular properties not present in parental androgen-dependent cancer cells. Cancer J 2000;6:213–4.[Medline]
6. Craft N, Chhor C, Tran C, et al. Evidence for clonal outgrowth of androgen-independent prostate cancer cells from androgen-dependent tumors through a two-step process. Cancer Res 1999;59:5030–6.[Abstract/Free Full Text]
7. Pinheiro JC, Bates DM. Mixed-effects models in S and D-PLUS. New York: Springer; 2001.
8. Haldar S, Jena N, Croce CM. Inactivation of bcl-2 by phosphorylation. Proc Natl Acad Sci U S A 1995;92:4507–11.[Abstract/Free Full Text]
9. Basu A, Haldar S. Microtubule-damaging drugs triggered bcl-2 phosphorylation: requirement of phosphorylation on both serine-70 and serine-87 residues of bcl-2 protein. Int J Oncol 1998;13:659–64.[Medline]
10. Horoszewicz JS, Leong SS, Kawinski E, et al. LNCaP model of human prostatic carcinoma. Cancer Res 1983;43:1809–18.[Abstract/Free Full Text]
11. Nicholson B, Gulding K, Conaway M, Wedge GR, Theodorescu D. Combination antiangiogenic and androgen deprivation therapy for prostate cancer: a promising therapeutic approach. Clin Cancer Res 2004;10:8728–34.[Abstract/Free Full Text]
12. Furuya Y, Krajewski S, Epstein JI, Reed JC, Isaacs JT. Expression of bcl-2 and the progression of human and rodent prostatic cancer. Clin Cancer Res 1996;2:389–98.[Abstract/Free Full Text]
13. Lu S, Tsai SY, Tsai MJ. Molecular mechanisms of androgen-independent growth of human prostate cancer LNCaP-AI cells. Endocrinology 1999;140:5054–9.[Abstract/Free Full Text]
14. Gleave M, Tolcher A, Miyake H, et al. Progression to androgen independence is delayed by adjuvant treatment with antisense Bcl-2 oligodeoxynucleotides after castration in the LNCaP prostate tumor model. Clin Cancer Res 1999;5:2891–8.[Abstract/Free Full Text]
15. McDonnell TJ, Troncoso P, Brisbay SM, et al. Expression of the protooncogene bcl-2 in the prostate and its association with emergence of androgen-independent prostate cancer. Cancer Res 1992;52:6940–4.[Abstract/Free Full Text]
16. Colombel M, Symmans F, Gil S, et al. Detection of the apoptosis-suppressing oncoprotein bcl-2 in hormone-refractory human prostate cancers. Am J Pathol 1993;143:390–400.[Abstract]
17. Apakama I, Robinson MC, Walter NM, et al. bcl-2 overexpression combined with p53 protein accumulation correlates with hormone-refractory prostate cancer. Br J Cancer 1996;74:1258–62.[Medline]
18. McDonnell TJ, Navone NM, Troncoso P, et al. Expression of bcl-2 oncoprotein and p53 protein accumulation in bone marrow metastases of androgen independent prostate cancer. J Urol 1997;157:569–74.[CrossRef][Medline]
19. Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 1993;74:609–19.[CrossRef][Medline]
20. Hussain A, Dawson N, Amin P, et al. Docetaxel followed by hormone therapy in men experiencing increasing prostate-specific antigen after primary local treatments for prostate cancer. J Clin Oncol 2005;23:2789–96.[Abstract/Free Full Text]

This article has been cited by other articles:

				Y. Tang, L. Wang, O. Goloubeva, M. Afnan Khan, B. Zhang, and A. Hussain Divergent Effects of Castration on Prostate Cancer in TRAMP Mice: Possible Implications for Therapy Clin. Cancer Res., May 15, 2008; 14(10): 2936 - 2943. [Abstract] [Full Text] [PDF]

				J. K. Hess-Wilson, H. K. Daly, W. A. Zagorski, C. P. Montville, and K. E. Knudsen Mitogenic Action of the Androgen Receptor Sensitizes Prostate Cancer Cells to Taxane-Based Cytotoxic Insult Cancer Res., December 15, 2006; 66(24): 11998 - 12008. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tang, Y. Articles by Hussain, A. Search for Related Content PubMed PubMed Citation Articles by Tang, Y. Articles by Hussain, A.