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Agonist activation of estrogen receptor beta (ERβ) sensitizes malignant pleural mesothelioma cells to cisplatin cytotoxicity.

Pinton G, Manente AG, Daga A, Cilli M, Rinaldi M, Nilsson S, Moro L - Mol. Cancer (2014)

Bottom Line: KB9520 showed significant anti-proliferative effect in ERβ positive human malignant pleural mesothelioma cells in vitro.Furthermore, in CD1 nude mice mesothelioma tumor growth was significantly inhibited when KB9520 was added on top of the standard of care chemo combination cisplatin/pemetrexed, as compared to the cisplatin/pemetrexed alone group.Importantly, KB9520 exerted a protective effect to cisplatin toxicity in the non-malignant mesothelium derived MET5A cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, University of Piemonte Orientale "A, Avogadro", Lgo Donegani 2, 28100 Novara, Italy. moro@pharm.unipmn.it.

ABSTRACT

Background: Estrogen receptor (ER) β acts as a tumor suppressor in malignant mesotheliomas.

Methods: Here we explored the anti-proliferative and anti-tumorigenic efficacy of the selective ERβ agonist, KB9520, in human mesothelioma cell lines in vitro and in a mesothelioma mouse model in vivo.

Results: KB9520 showed significant anti-proliferative effect in ERβ positive human malignant pleural mesothelioma cells in vitro. Selective activation of ERβ with KB9520 sensitized the cells to treatment with cisplatin, resulting in enhanced growth inhibition and increased apoptosis. Furthermore, in CD1 nude mice mesothelioma tumor growth was significantly inhibited when KB9520 was added on top of the standard of care chemo combination cisplatin/pemetrexed, as compared to the cisplatin/pemetrexed alone group. Importantly, KB9520 exerted a protective effect to cisplatin toxicity in the non-malignant mesothelium derived MET5A cells.

Conclusions: Together, the data presented suggest that selective targeting of ERβ may be an efficacious stand-alone treatment option and/or become an important add-on to existing malignant mesothelioma therapy.

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Related in: MedlinePlus

Mechanism of KB9520 sensitization to cisplatin cytotoxicity. A) Western blot analysis and relative densitometry of PARP1 cleavage and AKT phosphorylation in REN cells treated for 24 hours with cisplatin (25, 50 and 100 μM) or pre-treated 2 hours with KB9520 (10 nM) followed by wash-off and continued growth in normal medium ± cisplatin (25, 50 and 100 μM), for additional 24 hours. Total AKT and Tubulin staining were used for normalization. B) Effect on MET5A cell viability after 24 hours treatment with cisplatin (25, 50 and 100 μM) or 2 hours pre-treatment with KB9520 (10 nM) followed by wash-off and continued growth in normal medium ± different concentrations of cisplatin (25, 50 and 100 μM), for additional 24 hours. Each point represents mean ± s.d. *p ≤ 0.05. C) Western blot analysis and relative densitometry of PARP1 cleavage and AKT phosphorylation in MET5A cells treated with cisplatin (25, 50 and 100 μM) for 24 hours or pre-treated for 2 hours with KB9520 (10 nM) followed by wash-off and continued growth in normal medium ± different concentrations of cisplatin (25, 50 and 100 μM), for additional 24 hours. Total AKT and Tubulin staining were used for normalization. Data are representative of three separate experiments.
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Fig6: Mechanism of KB9520 sensitization to cisplatin cytotoxicity. A) Western blot analysis and relative densitometry of PARP1 cleavage and AKT phosphorylation in REN cells treated for 24 hours with cisplatin (25, 50 and 100 μM) or pre-treated 2 hours with KB9520 (10 nM) followed by wash-off and continued growth in normal medium ± cisplatin (25, 50 and 100 μM), for additional 24 hours. Total AKT and Tubulin staining were used for normalization. B) Effect on MET5A cell viability after 24 hours treatment with cisplatin (25, 50 and 100 μM) or 2 hours pre-treatment with KB9520 (10 nM) followed by wash-off and continued growth in normal medium ± different concentrations of cisplatin (25, 50 and 100 μM), for additional 24 hours. Each point represents mean ± s.d. *p ≤ 0.05. C) Western blot analysis and relative densitometry of PARP1 cleavage and AKT phosphorylation in MET5A cells treated with cisplatin (25, 50 and 100 μM) for 24 hours or pre-treated for 2 hours with KB9520 (10 nM) followed by wash-off and continued growth in normal medium ± different concentrations of cisplatin (25, 50 and 100 μM), for additional 24 hours. Total AKT and Tubulin staining were used for normalization. Data are representative of three separate experiments.

Mentions: REN cells were treated for 24 hours with 100 μM cisplatin or pre-treated 2 hours with 10 nM KB9520 followed by wash-off and continued growth in normal medium ± 100 μM cisplatin, for additional 24 hours. After treatments, cells were stained with propidium iodide and analyzed for cellular DNA content by flow cytometry. Pre-treatment with KB9520 for 2 hours followed by 24 hours cisplatin treatment resulted in significant and efficient block in the G0/G1 phase and inhibition of cells entering the S-phase of the cell cycle compared to any other treatment (data reported in Table 2 represent mean ± s.d. (n = 3) of the percentage of cells in each phase of the cell cycle). Moreover, a significant higher percentage of dead cells were found in wells pre-treated with KB9520 followed by cisplatin compared to other treatment regimens. A plausible explanation for the higher number of dead cells in the KB9520/cisplatin treated cells was induction of apoptosis. As expected from the cell cycle analysis and percentage of dead cells, 2 hours KB9520 treatment, prior to addition of cisplatin, had the greatest effect on the appearance of cleaved PARP1 (Figure 6A). Interestingly, neither cisplatin nor KB9520 alone resulted in significant PARP1 cleavage. As increased AKT activity has been implicated in the control of proliferation, apoptosis and cisplatin resistance [44], we analyzed its activation status following different treatments. As shown in Figure 6A, KB9520 treatment significantly reduced AKT phosphorylation both in the absence and in the presence of cisplatin. The mechanism for the combined effect of KB9520 and cisplatin on AKT pathway modulation, PARP1 cleavage and increased cell death needs further studies.Table 2


Agonist activation of estrogen receptor beta (ERβ) sensitizes malignant pleural mesothelioma cells to cisplatin cytotoxicity.

Pinton G, Manente AG, Daga A, Cilli M, Rinaldi M, Nilsson S, Moro L - Mol. Cancer (2014)

Mechanism of KB9520 sensitization to cisplatin cytotoxicity. A) Western blot analysis and relative densitometry of PARP1 cleavage and AKT phosphorylation in REN cells treated for 24 hours with cisplatin (25, 50 and 100 μM) or pre-treated 2 hours with KB9520 (10 nM) followed by wash-off and continued growth in normal medium ± cisplatin (25, 50 and 100 μM), for additional 24 hours. Total AKT and Tubulin staining were used for normalization. B) Effect on MET5A cell viability after 24 hours treatment with cisplatin (25, 50 and 100 μM) or 2 hours pre-treatment with KB9520 (10 nM) followed by wash-off and continued growth in normal medium ± different concentrations of cisplatin (25, 50 and 100 μM), for additional 24 hours. Each point represents mean ± s.d. *p ≤ 0.05. C) Western blot analysis and relative densitometry of PARP1 cleavage and AKT phosphorylation in MET5A cells treated with cisplatin (25, 50 and 100 μM) for 24 hours or pre-treated for 2 hours with KB9520 (10 nM) followed by wash-off and continued growth in normal medium ± different concentrations of cisplatin (25, 50 and 100 μM), for additional 24 hours. Total AKT and Tubulin staining were used for normalization. Data are representative of three separate experiments.
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Fig6: Mechanism of KB9520 sensitization to cisplatin cytotoxicity. A) Western blot analysis and relative densitometry of PARP1 cleavage and AKT phosphorylation in REN cells treated for 24 hours with cisplatin (25, 50 and 100 μM) or pre-treated 2 hours with KB9520 (10 nM) followed by wash-off and continued growth in normal medium ± cisplatin (25, 50 and 100 μM), for additional 24 hours. Total AKT and Tubulin staining were used for normalization. B) Effect on MET5A cell viability after 24 hours treatment with cisplatin (25, 50 and 100 μM) or 2 hours pre-treatment with KB9520 (10 nM) followed by wash-off and continued growth in normal medium ± different concentrations of cisplatin (25, 50 and 100 μM), for additional 24 hours. Each point represents mean ± s.d. *p ≤ 0.05. C) Western blot analysis and relative densitometry of PARP1 cleavage and AKT phosphorylation in MET5A cells treated with cisplatin (25, 50 and 100 μM) for 24 hours or pre-treated for 2 hours with KB9520 (10 nM) followed by wash-off and continued growth in normal medium ± different concentrations of cisplatin (25, 50 and 100 μM), for additional 24 hours. Total AKT and Tubulin staining were used for normalization. Data are representative of three separate experiments.
Mentions: REN cells were treated for 24 hours with 100 μM cisplatin or pre-treated 2 hours with 10 nM KB9520 followed by wash-off and continued growth in normal medium ± 100 μM cisplatin, for additional 24 hours. After treatments, cells were stained with propidium iodide and analyzed for cellular DNA content by flow cytometry. Pre-treatment with KB9520 for 2 hours followed by 24 hours cisplatin treatment resulted in significant and efficient block in the G0/G1 phase and inhibition of cells entering the S-phase of the cell cycle compared to any other treatment (data reported in Table 2 represent mean ± s.d. (n = 3) of the percentage of cells in each phase of the cell cycle). Moreover, a significant higher percentage of dead cells were found in wells pre-treated with KB9520 followed by cisplatin compared to other treatment regimens. A plausible explanation for the higher number of dead cells in the KB9520/cisplatin treated cells was induction of apoptosis. As expected from the cell cycle analysis and percentage of dead cells, 2 hours KB9520 treatment, prior to addition of cisplatin, had the greatest effect on the appearance of cleaved PARP1 (Figure 6A). Interestingly, neither cisplatin nor KB9520 alone resulted in significant PARP1 cleavage. As increased AKT activity has been implicated in the control of proliferation, apoptosis and cisplatin resistance [44], we analyzed its activation status following different treatments. As shown in Figure 6A, KB9520 treatment significantly reduced AKT phosphorylation both in the absence and in the presence of cisplatin. The mechanism for the combined effect of KB9520 and cisplatin on AKT pathway modulation, PARP1 cleavage and increased cell death needs further studies.Table 2

Bottom Line: KB9520 showed significant anti-proliferative effect in ERβ positive human malignant pleural mesothelioma cells in vitro.Furthermore, in CD1 nude mice mesothelioma tumor growth was significantly inhibited when KB9520 was added on top of the standard of care chemo combination cisplatin/pemetrexed, as compared to the cisplatin/pemetrexed alone group.Importantly, KB9520 exerted a protective effect to cisplatin toxicity in the non-malignant mesothelium derived MET5A cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, University of Piemonte Orientale "A, Avogadro", Lgo Donegani 2, 28100 Novara, Italy. moro@pharm.unipmn.it.

ABSTRACT

Background: Estrogen receptor (ER) β acts as a tumor suppressor in malignant mesotheliomas.

Methods: Here we explored the anti-proliferative and anti-tumorigenic efficacy of the selective ERβ agonist, KB9520, in human mesothelioma cell lines in vitro and in a mesothelioma mouse model in vivo.

Results: KB9520 showed significant anti-proliferative effect in ERβ positive human malignant pleural mesothelioma cells in vitro. Selective activation of ERβ with KB9520 sensitized the cells to treatment with cisplatin, resulting in enhanced growth inhibition and increased apoptosis. Furthermore, in CD1 nude mice mesothelioma tumor growth was significantly inhibited when KB9520 was added on top of the standard of care chemo combination cisplatin/pemetrexed, as compared to the cisplatin/pemetrexed alone group. Importantly, KB9520 exerted a protective effect to cisplatin toxicity in the non-malignant mesothelium derived MET5A cells.

Conclusions: Together, the data presented suggest that selective targeting of ERβ may be an efficacious stand-alone treatment option and/or become an important add-on to existing malignant mesothelioma therapy.

Show MeSH
Related in: MedlinePlus