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MDM2 antagonists boost antitumor effect of androgen withdrawal: implications for therapy of prostate cancer.

Tovar C, Higgins B, Kolinsky K, Xia M, Packman K, Heimbrook DC, Vassilev LT - Mol. Cancer (2011)

Bottom Line: Using charcoal-stripped serum as a cellular model of androgen deprivation, we show an increased apoptotic effect of p53 activation by nutlin-3a in the androgen-dependent LNCaP cells and to a lesser extent in androgen-independent but responsive 22Rv1 cell line.This effect is due, at least in part, to an enhanced downregulation of AR expression by activated p53.Since majority of prostate tumors express wild-type p53, its activation by MDM2 antagonists in combination with androgen depletion may offer an efficacious new approach to prostate cancer therapy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Discovery Oncology, Roche Research Center, Hoffmann-La Roche Inc., Nutley, NJ 07110, USA.

ABSTRACT

Background: Hormone therapy is the standard of care for newly diagnosed or recurrent prostate cancers. It uses anti-androgen agents, castration, or both to eliminate cancer promoting effect of testicular androgen. The p53 tumor suppressor controls a major pathway that can block cell proliferation or induce apoptosis in response to diverse forms of oncogenic stress. Activation of the p53 pathway in cancer cells expressing wild-type p53 has been proposed as a novel therapeutic strategy and recently developed MDM2 antagonists, the nutlins, have validated this in preclinical models of cancer. The crosstalk between p53 and androgen receptor (AR) signaling suggest that p53 activation could augment antitumor outcome of androgen ablation in prostate cancer. Here, we test this hypothesis in vitro and in vivo using the MDM2 antagonist, nutlin-3 and the p53 wild-type prostate cancer cell line, LNCaP.

Results: Using charcoal-stripped serum as a cellular model of androgen deprivation, we show an increased apoptotic effect of p53 activation by nutlin-3a in the androgen-dependent LNCaP cells and to a lesser extent in androgen-independent but responsive 22Rv1 cell line. This effect is due, at least in part, to an enhanced downregulation of AR expression by activated p53. In vivo, androgen deprivation followed by two weeks of nutlin administration in LNCaP-bearing nude mice led to a greater tumor regression and dramatically increased survival.

Conclusions: Since majority of prostate tumors express wild-type p53, its activation by MDM2 antagonists in combination with androgen depletion may offer an efficacious new approach to prostate cancer therapy.

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Related in: MedlinePlus

Nutlin-3a activates p53 signaling in LNCaP cells. A) Antitumor activity of Nutlin-3a. Cells were incubated with nutlin-3a for 5 days and cell growth/viability measured by the MTT assay. B) Changes in key proteins. Exponentially proliferating LnCAP cells were treated with 10 μM Nutlin-3a, or DMSO for 20 h and cell lysates were analyzed by Western blotting. C) Nutlin inhibits cell cycle progression. LNCaP cells were treated with 10 μM nutlin-3a for 20 h and analyzed by BrdU labeling and flow cytometry. Box indicates the S phase compartments. D) Nutlin induces dose and time-dependent apoptosis in LNCaP cells. Cells were incubated with nutlin-3a at the indicated concentrations and the percentage of the Annexin V-positive cells was determined. Time-dependant apoptosis was determined at 10 μM concentration.
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Figure 1: Nutlin-3a activates p53 signaling in LNCaP cells. A) Antitumor activity of Nutlin-3a. Cells were incubated with nutlin-3a for 5 days and cell growth/viability measured by the MTT assay. B) Changes in key proteins. Exponentially proliferating LnCAP cells were treated with 10 μM Nutlin-3a, or DMSO for 20 h and cell lysates were analyzed by Western blotting. C) Nutlin inhibits cell cycle progression. LNCaP cells were treated with 10 μM nutlin-3a for 20 h and analyzed by BrdU labeling and flow cytometry. Box indicates the S phase compartments. D) Nutlin induces dose and time-dependent apoptosis in LNCaP cells. Cells were incubated with nutlin-3a at the indicated concentrations and the percentage of the Annexin V-positive cells was determined. Time-dependant apoptosis was determined at 10 μM concentration.

Mentions: To study the combined effect of p53 activation and androgen deprivation, we chose the androgen-depenedent prostate cancer cell line LNCaP as a cellular model. We have shown previously that nutlin-3a activates p53 and inhibits the growth of LNCaP cells and xenograft tumors in nude mice [27]. Incubation of proliferating LNCaP cells with nutlin-3a for 5 days showed a dose-dependent effect with EC50 of 0.5 μM (Figure 1A). Western blot analysis following 24 h nutlin treatment revealed p53 accumulation and concurrent increases in its transcriptional targets MDM2 and p21Waf1/CIP1 (Figure 1B). Additionally, BrdU cell cycle analysis showed a loss of S-phase and increase of G1 and G2/M phase fractions consistent with p53-mediated cell cycle block in G1 and G2 phases (Figure 1C). To assess the apoptotic activity of nutlin-3a we used the Annexin V assay. Nutlin showed both a dose-dependant and time-dependant increase in the apoptotic fraction (Figure 1D). These results confirm that MDM2 antagonists stabilize p53 protein in LNCaP cells and effectively activate p53 signaling and the main p53 functions, cell cycle arrest and apoptosis.


MDM2 antagonists boost antitumor effect of androgen withdrawal: implications for therapy of prostate cancer.

Tovar C, Higgins B, Kolinsky K, Xia M, Packman K, Heimbrook DC, Vassilev LT - Mol. Cancer (2011)

Nutlin-3a activates p53 signaling in LNCaP cells. A) Antitumor activity of Nutlin-3a. Cells were incubated with nutlin-3a for 5 days and cell growth/viability measured by the MTT assay. B) Changes in key proteins. Exponentially proliferating LnCAP cells were treated with 10 μM Nutlin-3a, or DMSO for 20 h and cell lysates were analyzed by Western blotting. C) Nutlin inhibits cell cycle progression. LNCaP cells were treated with 10 μM nutlin-3a for 20 h and analyzed by BrdU labeling and flow cytometry. Box indicates the S phase compartments. D) Nutlin induces dose and time-dependent apoptosis in LNCaP cells. Cells were incubated with nutlin-3a at the indicated concentrations and the percentage of the Annexin V-positive cells was determined. Time-dependant apoptosis was determined at 10 μM concentration.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3094321&req=5

Figure 1: Nutlin-3a activates p53 signaling in LNCaP cells. A) Antitumor activity of Nutlin-3a. Cells were incubated with nutlin-3a for 5 days and cell growth/viability measured by the MTT assay. B) Changes in key proteins. Exponentially proliferating LnCAP cells were treated with 10 μM Nutlin-3a, or DMSO for 20 h and cell lysates were analyzed by Western blotting. C) Nutlin inhibits cell cycle progression. LNCaP cells were treated with 10 μM nutlin-3a for 20 h and analyzed by BrdU labeling and flow cytometry. Box indicates the S phase compartments. D) Nutlin induces dose and time-dependent apoptosis in LNCaP cells. Cells were incubated with nutlin-3a at the indicated concentrations and the percentage of the Annexin V-positive cells was determined. Time-dependant apoptosis was determined at 10 μM concentration.
Mentions: To study the combined effect of p53 activation and androgen deprivation, we chose the androgen-depenedent prostate cancer cell line LNCaP as a cellular model. We have shown previously that nutlin-3a activates p53 and inhibits the growth of LNCaP cells and xenograft tumors in nude mice [27]. Incubation of proliferating LNCaP cells with nutlin-3a for 5 days showed a dose-dependent effect with EC50 of 0.5 μM (Figure 1A). Western blot analysis following 24 h nutlin treatment revealed p53 accumulation and concurrent increases in its transcriptional targets MDM2 and p21Waf1/CIP1 (Figure 1B). Additionally, BrdU cell cycle analysis showed a loss of S-phase and increase of G1 and G2/M phase fractions consistent with p53-mediated cell cycle block in G1 and G2 phases (Figure 1C). To assess the apoptotic activity of nutlin-3a we used the Annexin V assay. Nutlin showed both a dose-dependant and time-dependant increase in the apoptotic fraction (Figure 1D). These results confirm that MDM2 antagonists stabilize p53 protein in LNCaP cells and effectively activate p53 signaling and the main p53 functions, cell cycle arrest and apoptosis.

Bottom Line: Using charcoal-stripped serum as a cellular model of androgen deprivation, we show an increased apoptotic effect of p53 activation by nutlin-3a in the androgen-dependent LNCaP cells and to a lesser extent in androgen-independent but responsive 22Rv1 cell line.This effect is due, at least in part, to an enhanced downregulation of AR expression by activated p53.Since majority of prostate tumors express wild-type p53, its activation by MDM2 antagonists in combination with androgen depletion may offer an efficacious new approach to prostate cancer therapy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Discovery Oncology, Roche Research Center, Hoffmann-La Roche Inc., Nutley, NJ 07110, USA.

ABSTRACT

Background: Hormone therapy is the standard of care for newly diagnosed or recurrent prostate cancers. It uses anti-androgen agents, castration, or both to eliminate cancer promoting effect of testicular androgen. The p53 tumor suppressor controls a major pathway that can block cell proliferation or induce apoptosis in response to diverse forms of oncogenic stress. Activation of the p53 pathway in cancer cells expressing wild-type p53 has been proposed as a novel therapeutic strategy and recently developed MDM2 antagonists, the nutlins, have validated this in preclinical models of cancer. The crosstalk between p53 and androgen receptor (AR) signaling suggest that p53 activation could augment antitumor outcome of androgen ablation in prostate cancer. Here, we test this hypothesis in vitro and in vivo using the MDM2 antagonist, nutlin-3 and the p53 wild-type prostate cancer cell line, LNCaP.

Results: Using charcoal-stripped serum as a cellular model of androgen deprivation, we show an increased apoptotic effect of p53 activation by nutlin-3a in the androgen-dependent LNCaP cells and to a lesser extent in androgen-independent but responsive 22Rv1 cell line. This effect is due, at least in part, to an enhanced downregulation of AR expression by activated p53. In vivo, androgen deprivation followed by two weeks of nutlin administration in LNCaP-bearing nude mice led to a greater tumor regression and dramatically increased survival.

Conclusions: Since majority of prostate tumors express wild-type p53, its activation by MDM2 antagonists in combination with androgen depletion may offer an efficacious new approach to prostate cancer therapy.

Show MeSH
Related in: MedlinePlus