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KPT-330, a potent and selective exportin-1 (XPO-1) inhibitor, shows antitumor effects modulating the expression of cyclin D1 and survivin [corrected] in prostate cancer models.

Gravina GL, Mancini A, Sanita P, Vitale F, Marampon F, Ventura L, Landesman Y, McCauley D, Kauffman M, Shacham S, Festuccia C - BMC Cancer (2015)

Bottom Line: SINE compounds inhibited proliferation and promoted apoptosis of tumor cells, but did not affect immortalized non-transformed prostate epithelial cells.Nuclei from SINE treated cells showed increased protein localization of XPO-1, survivin and cyclin D1 followed by degradation of these proteins leading to cell cycle arrest and apoptosis.Oral administration of KPT-251 and KPT-330 in PC3, DU145 and 22rv1 tumor-bearing nude mice reduced tumor cell proliferation, angiogenesis and induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnological and Applied Clinical Sciences, Laboratory of Radiobiology, University of L'Aquila, L'Aquila, Italy. giovanniluca.gravina@libero.it.

ABSTRACT

Background and aims: Increased expression of Chromosome Region Maintenance (CRM-1)/exportin-1 (XPO-1) has been correlated with poor prognosis in several aggressive tumors, making it an interesting therapeutic target. Selective Inhibitor of Nuclear Export (SINE) compounds bind to XPO-1 and block its ability to export cargo proteins. Here, we investigated the effects of a new class of SINE compounds in models of prostate cancer.

Material and methods: We evaluated the expression of XPO-1 in human prostate cancer tissues and cell lines. Next, six SINE (KPT-127, KPT-185, KPT-205, KPT-225, KPT-251 and KPT-330) compounds having different potency with broad-spectrum, tumor-selective cytotoxicity, tolerability and pharmacokinetic profiles were tested in a panel of prostate cancer cells representing distinct differentiation/progression states of disease and genotypes. Two SINE candidates for clinical trials (KPT-251 and KPT-330) were also tested in vivo in three cell models of aggressive prostate cancer engrafted in male nude mice.

Results and conclusions: XPO-1 is overexpressed in prostate cancer compared to normal or hyperplastic tissues. Increased XPO-1 expression, mainly in the nuclear compartment, was associated with increased Gleason score and bone metastatic potential supporting the use of SINEs in advanced prostate cancer. SINE compounds inhibited proliferation and promoted apoptosis of tumor cells, but did not affect immortalized non-transformed prostate epithelial cells. Nuclei from SINE treated cells showed increased protein localization of XPO-1, survivin and cyclin D1 followed by degradation of these proteins leading to cell cycle arrest and apoptosis. Oral administration of KPT-251 and KPT-330 in PC3, DU145 and 22rv1 tumor-bearing nude mice reduced tumor cell proliferation, angiogenesis and induced apoptosis. Our results provide supportive evidence for the therapeutic use of SINE compounds in advanced/castration resistant prostate cancers and warrants further clinical investigation.

No MeSH data available.


Related in: MedlinePlus

Molecular arrangements associated to CRM1 inhibition: a-c CRM1 and Cyclin D1 nuclear accumulation. a Western blots for the nuclear expression of cyclin D1 and CRM1 in PC3 cells treated with 100 nM KPT-330 for 4, 8, 12 and 24 hrs (early events); b Western blots for the cytoplasmic expression of cyclin D1 and CRM1 in PC3 cells treated with KPT-330 for 24, 48 or 72 hr and and immunocytochemical evaluation for CRM1 in the same conditions (late events) and c immunocytochemical evaluation for cyclin D1 expression in PC3 cells treated with 100 nM KPT-330 for 8 (T8), 24 (T24) and 72 (T72) hrs (late events). d Western blots for nuclear expression of Foxo proteins, p21, p27, cyclin E, cdk2, cyclin B1 and Cdk1 in PC3 cells treated with 100 nM KPT-330 for 2, 4, 8 and 12 hrs (early events). e Immunocytochemical evaluation for Foxo3a expression in 22rv1 cells treated with 100 nM KPT-330 for 2 (T2), 8 (T8) and 12 (T12) hrs (early events). f Western blots for nuclear expression of p53 and MDM2 in AR+/p53wt 22rv1 cells treated with 100 nM KPT-330 for 4, 8, 12 and 24 hrs (Early events). g Western blot for AR nuclear expression in 22rv1 cells treated with 100 nM KPT-330 for 2, 4, 8 and 12 hr (early events) and h western blot for AR nuclear expression in 22rv1 cells treated with 100 nM KPT-330 for 8 (T8), 24 (T24) and 72 (T72) hrs (late events). Nuclear expression was normalized to lamin whereas cytosolic expression was normalized to β-actin
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Fig3: Molecular arrangements associated to CRM1 inhibition: a-c CRM1 and Cyclin D1 nuclear accumulation. a Western blots for the nuclear expression of cyclin D1 and CRM1 in PC3 cells treated with 100 nM KPT-330 for 4, 8, 12 and 24 hrs (early events); b Western blots for the cytoplasmic expression of cyclin D1 and CRM1 in PC3 cells treated with KPT-330 for 24, 48 or 72 hr and and immunocytochemical evaluation for CRM1 in the same conditions (late events) and c immunocytochemical evaluation for cyclin D1 expression in PC3 cells treated with 100 nM KPT-330 for 8 (T8), 24 (T24) and 72 (T72) hrs (late events). d Western blots for nuclear expression of Foxo proteins, p21, p27, cyclin E, cdk2, cyclin B1 and Cdk1 in PC3 cells treated with 100 nM KPT-330 for 2, 4, 8 and 12 hrs (early events). e Immunocytochemical evaluation for Foxo3a expression in 22rv1 cells treated with 100 nM KPT-330 for 2 (T2), 8 (T8) and 12 (T12) hrs (early events). f Western blots for nuclear expression of p53 and MDM2 in AR+/p53wt 22rv1 cells treated with 100 nM KPT-330 for 4, 8, 12 and 24 hrs (Early events). g Western blot for AR nuclear expression in 22rv1 cells treated with 100 nM KPT-330 for 2, 4, 8 and 12 hr (early events) and h western blot for AR nuclear expression in 22rv1 cells treated with 100 nM KPT-330 for 8 (T8), 24 (T24) and 72 (T72) hrs (late events). Nuclear expression was normalized to lamin whereas cytosolic expression was normalized to β-actin

Mentions: The molecular pathways that are induced by XPO-1 inhibition were evaluated by immunoblots, ELISA and immunocytochemical analyses performed in PC3, DU145 and 22rv1 as models of CRPC using 100 nM KPT-330 as reference compound. The effects were monitored in early and chronic treatments. We found that KPT-330 induced an early XPO-1 nuclear accumulation. In parallel, cyclin D1 nuclear expression was increased whereas cytosolic levels were strongly reduced. However, prolonged treatments with KPT-330 dramatically reduced XPO-1 protein levels. In Fig. 3a, we show representative western blots for nuclear and cytoplasm expression of cyclin D1 and XPO-1 in PC3 cell extracts after 2–12 hr of treatment with KPT-330. In Fig. 3b, we demonstrate that total XPO-1 levels were also reduced after prolonged treatment of PC3 cells with KPT-330. Western blot and immune-cytochemistry showed similar results. In addition, prolonged treatment with KPT-330 determined a reduction of cyclin D1 protein expression in DU145 cell lines (Fig. 3c). Western analysis demonstrated a nuclear accumulation of FOXO, p21, p27, cyclin B1 as shown in Fig. 3d for PC3 cells. Increased nuclear expression of FOXO3a is visible also by IHC (Fig. 3e) in 22rv1 cells treated with KPT-330. In addition, we found that KPT-330 treatment increased nuclear expression of p53 and MDM2 (Fig. 3f) and AR (Fig. 3g) in AR+/p53wt 22rv1 cells. Also in this case, prolonged exposure to KPT-330 significantly reduced the total expression of AR, especially in the truncated androgen insensitive forms. Indeed, it has been demonstrated that AR expression is increased in CRPC and XPO-1 activity is necessary for a completely functionally active AR-mediated pathway. Therefore, the inhibition of XPO-1-mediated export machinery may enhance AR signaling (manuscript in preparation). In addition, the disrupted cyclin B1-XPO-1 interactions leading to a marked nuclear accumulation of cyclin B1 is necessary for cyclin B1-dependent apoptosis In Fig. 3h, we demonstrate that proteasome inhibition was able to block the degradation of XPO-1, Cyclin D1 and survivin reducing the loss of these proteins induced by KPT-330 treatment. Although proteasome inhibitors are considered for treatment of solid tumors, these compounds could reduce the effectiveness of selinexor, requiring further investigation. These molecular changes were associated with accumulation of cells in the G1 cell cycle phase with loss of cells in the S and G2/M phase (Fig. 4a).Fig. 3


KPT-330, a potent and selective exportin-1 (XPO-1) inhibitor, shows antitumor effects modulating the expression of cyclin D1 and survivin [corrected] in prostate cancer models.

Gravina GL, Mancini A, Sanita P, Vitale F, Marampon F, Ventura L, Landesman Y, McCauley D, Kauffman M, Shacham S, Festuccia C - BMC Cancer (2015)

Molecular arrangements associated to CRM1 inhibition: a-c CRM1 and Cyclin D1 nuclear accumulation. a Western blots for the nuclear expression of cyclin D1 and CRM1 in PC3 cells treated with 100 nM KPT-330 for 4, 8, 12 and 24 hrs (early events); b Western blots for the cytoplasmic expression of cyclin D1 and CRM1 in PC3 cells treated with KPT-330 for 24, 48 or 72 hr and and immunocytochemical evaluation for CRM1 in the same conditions (late events) and c immunocytochemical evaluation for cyclin D1 expression in PC3 cells treated with 100 nM KPT-330 for 8 (T8), 24 (T24) and 72 (T72) hrs (late events). d Western blots for nuclear expression of Foxo proteins, p21, p27, cyclin E, cdk2, cyclin B1 and Cdk1 in PC3 cells treated with 100 nM KPT-330 for 2, 4, 8 and 12 hrs (early events). e Immunocytochemical evaluation for Foxo3a expression in 22rv1 cells treated with 100 nM KPT-330 for 2 (T2), 8 (T8) and 12 (T12) hrs (early events). f Western blots for nuclear expression of p53 and MDM2 in AR+/p53wt 22rv1 cells treated with 100 nM KPT-330 for 4, 8, 12 and 24 hrs (Early events). g Western blot for AR nuclear expression in 22rv1 cells treated with 100 nM KPT-330 for 2, 4, 8 and 12 hr (early events) and h western blot for AR nuclear expression in 22rv1 cells treated with 100 nM KPT-330 for 8 (T8), 24 (T24) and 72 (T72) hrs (late events). Nuclear expression was normalized to lamin whereas cytosolic expression was normalized to β-actin
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Fig3: Molecular arrangements associated to CRM1 inhibition: a-c CRM1 and Cyclin D1 nuclear accumulation. a Western blots for the nuclear expression of cyclin D1 and CRM1 in PC3 cells treated with 100 nM KPT-330 for 4, 8, 12 and 24 hrs (early events); b Western blots for the cytoplasmic expression of cyclin D1 and CRM1 in PC3 cells treated with KPT-330 for 24, 48 or 72 hr and and immunocytochemical evaluation for CRM1 in the same conditions (late events) and c immunocytochemical evaluation for cyclin D1 expression in PC3 cells treated with 100 nM KPT-330 for 8 (T8), 24 (T24) and 72 (T72) hrs (late events). d Western blots for nuclear expression of Foxo proteins, p21, p27, cyclin E, cdk2, cyclin B1 and Cdk1 in PC3 cells treated with 100 nM KPT-330 for 2, 4, 8 and 12 hrs (early events). e Immunocytochemical evaluation for Foxo3a expression in 22rv1 cells treated with 100 nM KPT-330 for 2 (T2), 8 (T8) and 12 (T12) hrs (early events). f Western blots for nuclear expression of p53 and MDM2 in AR+/p53wt 22rv1 cells treated with 100 nM KPT-330 for 4, 8, 12 and 24 hrs (Early events). g Western blot for AR nuclear expression in 22rv1 cells treated with 100 nM KPT-330 for 2, 4, 8 and 12 hr (early events) and h western blot for AR nuclear expression in 22rv1 cells treated with 100 nM KPT-330 for 8 (T8), 24 (T24) and 72 (T72) hrs (late events). Nuclear expression was normalized to lamin whereas cytosolic expression was normalized to β-actin
Mentions: The molecular pathways that are induced by XPO-1 inhibition were evaluated by immunoblots, ELISA and immunocytochemical analyses performed in PC3, DU145 and 22rv1 as models of CRPC using 100 nM KPT-330 as reference compound. The effects were monitored in early and chronic treatments. We found that KPT-330 induced an early XPO-1 nuclear accumulation. In parallel, cyclin D1 nuclear expression was increased whereas cytosolic levels were strongly reduced. However, prolonged treatments with KPT-330 dramatically reduced XPO-1 protein levels. In Fig. 3a, we show representative western blots for nuclear and cytoplasm expression of cyclin D1 and XPO-1 in PC3 cell extracts after 2–12 hr of treatment with KPT-330. In Fig. 3b, we demonstrate that total XPO-1 levels were also reduced after prolonged treatment of PC3 cells with KPT-330. Western blot and immune-cytochemistry showed similar results. In addition, prolonged treatment with KPT-330 determined a reduction of cyclin D1 protein expression in DU145 cell lines (Fig. 3c). Western analysis demonstrated a nuclear accumulation of FOXO, p21, p27, cyclin B1 as shown in Fig. 3d for PC3 cells. Increased nuclear expression of FOXO3a is visible also by IHC (Fig. 3e) in 22rv1 cells treated with KPT-330. In addition, we found that KPT-330 treatment increased nuclear expression of p53 and MDM2 (Fig. 3f) and AR (Fig. 3g) in AR+/p53wt 22rv1 cells. Also in this case, prolonged exposure to KPT-330 significantly reduced the total expression of AR, especially in the truncated androgen insensitive forms. Indeed, it has been demonstrated that AR expression is increased in CRPC and XPO-1 activity is necessary for a completely functionally active AR-mediated pathway. Therefore, the inhibition of XPO-1-mediated export machinery may enhance AR signaling (manuscript in preparation). In addition, the disrupted cyclin B1-XPO-1 interactions leading to a marked nuclear accumulation of cyclin B1 is necessary for cyclin B1-dependent apoptosis In Fig. 3h, we demonstrate that proteasome inhibition was able to block the degradation of XPO-1, Cyclin D1 and survivin reducing the loss of these proteins induced by KPT-330 treatment. Although proteasome inhibitors are considered for treatment of solid tumors, these compounds could reduce the effectiveness of selinexor, requiring further investigation. These molecular changes were associated with accumulation of cells in the G1 cell cycle phase with loss of cells in the S and G2/M phase (Fig. 4a).Fig. 3

Bottom Line: SINE compounds inhibited proliferation and promoted apoptosis of tumor cells, but did not affect immortalized non-transformed prostate epithelial cells.Nuclei from SINE treated cells showed increased protein localization of XPO-1, survivin and cyclin D1 followed by degradation of these proteins leading to cell cycle arrest and apoptosis.Oral administration of KPT-251 and KPT-330 in PC3, DU145 and 22rv1 tumor-bearing nude mice reduced tumor cell proliferation, angiogenesis and induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnological and Applied Clinical Sciences, Laboratory of Radiobiology, University of L'Aquila, L'Aquila, Italy. giovanniluca.gravina@libero.it.

ABSTRACT

Background and aims: Increased expression of Chromosome Region Maintenance (CRM-1)/exportin-1 (XPO-1) has been correlated with poor prognosis in several aggressive tumors, making it an interesting therapeutic target. Selective Inhibitor of Nuclear Export (SINE) compounds bind to XPO-1 and block its ability to export cargo proteins. Here, we investigated the effects of a new class of SINE compounds in models of prostate cancer.

Material and methods: We evaluated the expression of XPO-1 in human prostate cancer tissues and cell lines. Next, six SINE (KPT-127, KPT-185, KPT-205, KPT-225, KPT-251 and KPT-330) compounds having different potency with broad-spectrum, tumor-selective cytotoxicity, tolerability and pharmacokinetic profiles were tested in a panel of prostate cancer cells representing distinct differentiation/progression states of disease and genotypes. Two SINE candidates for clinical trials (KPT-251 and KPT-330) were also tested in vivo in three cell models of aggressive prostate cancer engrafted in male nude mice.

Results and conclusions: XPO-1 is overexpressed in prostate cancer compared to normal or hyperplastic tissues. Increased XPO-1 expression, mainly in the nuclear compartment, was associated with increased Gleason score and bone metastatic potential supporting the use of SINEs in advanced prostate cancer. SINE compounds inhibited proliferation and promoted apoptosis of tumor cells, but did not affect immortalized non-transformed prostate epithelial cells. Nuclei from SINE treated cells showed increased protein localization of XPO-1, survivin and cyclin D1 followed by degradation of these proteins leading to cell cycle arrest and apoptosis. Oral administration of KPT-251 and KPT-330 in PC3, DU145 and 22rv1 tumor-bearing nude mice reduced tumor cell proliferation, angiogenesis and induced apoptosis. Our results provide supportive evidence for the therapeutic use of SINE compounds in advanced/castration resistant prostate cancers and warrants further clinical investigation.

No MeSH data available.


Related in: MedlinePlus