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An antimitotic and antivascular agent BPR0L075 overcomes multidrug resistance and induces mitotic catastrophe in paclitaxel-resistant ovarian cancer cells.

Wang X, Wu E, Wu J, Wang TL, Hsieh HP, Liu X - PLoS ONE (2013)

Bottom Line: Thus, new therapy that can overcome paclitaxel resistance will be of significant clinical importance.BPR0L075 blocks cell cycle at the G2/M phase in paclitaxel-resistant cells while equal concentration of paclitaxel treatment was ineffective.Immunoblotting analysis shows that BPR0L075 treatment induced up-regulation of cyclin B1, BubR1, MPM-2, and survivin protein levels and Bcl-XL phosphorylation in parental cells; however, in resistant cells, the endogenous expressions of BubR1 and survivin were depleted, BPR0L075 treatment failed to induce MPM-2 expression and phosphorylation of Bcl-XL.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas, United States of America.

ABSTRACT
Paclitaxel plays a major role in the treatment of ovarian cancer; however, resistance to paclitaxel is frequently observed. Thus, new therapy that can overcome paclitaxel resistance will be of significant clinical importance. We evaluated antiproliferative effects of an antimitotic and antivascular agent BPR0L075 in paclitaxel-resistant ovarian cancer cells. BPR0L075 displays potent and broad-spectrum cytotoxicity at low nanomolar concentrations (IC50 = 2-7 nM) against both parental ovarian cancer cells (OVCAR-3, SKOV-3, and A2780-1A9) and paclitaxel-resistant sublines (OVCAR-3-TR, SKOV-3-TR, 1A9-PTX10), regardless of the expression levels of the multidrug resistance transporter P-gp and class III β-tubulin or mutation of β-tubulin. BPR0L075 blocks cell cycle at the G2/M phase in paclitaxel-resistant cells while equal concentration of paclitaxel treatment was ineffective. BPR0L075 induces cell death by a dual mechanism in parental and paclitaxel-resistant ovarian cancer cells. In the parental cells (OVCAR-3 and SKOV-3), BPR0L075 induced apoptosis, evidenced by poly(ADP-ribose) polymerase (PARP) cleavage and DNA ladder formation. BPR0L075 induced cell death in paclitaxel-resistant ovarian cancer cells (OVCAR-3-TR and SKOV-3-TR) is primarily due to mitotic catastrophe, evidenced by formation of giant, multinucleated cells and absence of PARP cleavage. Immunoblotting analysis shows that BPR0L075 treatment induced up-regulation of cyclin B1, BubR1, MPM-2, and survivin protein levels and Bcl-XL phosphorylation in parental cells; however, in resistant cells, the endogenous expressions of BubR1 and survivin were depleted, BPR0L075 treatment failed to induce MPM-2 expression and phosphorylation of Bcl-XL. BPR0L075 induced cell death in both parental and paclitaxel-resistant ovarian cancer cells proceed through caspase-3 independent mechanisms. In conclusion, BPR0L075 displays potent cytotoxic effects in ovarian cancer cells with a potential to overcome paclitaxel resistance by bypassing efflux transporters and inducing mitotic catastrophe. BPR0L075 represents a novel microtubule therapeutic to overcome multidrug resistance and trigger alternative cell death by mitotic catastrophe in ovarian cancer cells that are apoptosis-resistant.

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BPR0L075 is not a substrate for efflux transporters.(A) Western blot analysis of the P-gp and βIII-tubulin expressions in the paired ovarian cancer cell lines. Pan β-tubulin and β-actin were loading controls. (B) LC/MS/MS analysis of intracellular BPR0L075 levels, with LC chromatogram showing the retention time of internal standard [1-(1H-indol-3-ylcarbonyl)-1H-imidazole] and BPR0L075 as 2.2 and 2.8 min, respectively. The mass transitions of m/z 212→144 for internal standard and m/z 342→195 for BPR0L075 were monitored for quantification. (C) The intracellular BPR0L075 levels in the paired ovarian cancer cell lines. Cells were incubated with 20 nM BPR0L075 for 6 hours, then cells were washed, harvested, and lysed for LC/MS/MS measurement. Bars represent mean ± SD of three independent tests. No statistically significant difference was observed (P>0.05, student t-test).
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pone-0065686-g002: BPR0L075 is not a substrate for efflux transporters.(A) Western blot analysis of the P-gp and βIII-tubulin expressions in the paired ovarian cancer cell lines. Pan β-tubulin and β-actin were loading controls. (B) LC/MS/MS analysis of intracellular BPR0L075 levels, with LC chromatogram showing the retention time of internal standard [1-(1H-indol-3-ylcarbonyl)-1H-imidazole] and BPR0L075 as 2.2 and 2.8 min, respectively. The mass transitions of m/z 212→144 for internal standard and m/z 342→195 for BPR0L075 were monitored for quantification. (C) The intracellular BPR0L075 levels in the paired ovarian cancer cell lines. Cells were incubated with 20 nM BPR0L075 for 6 hours, then cells were washed, harvested, and lysed for LC/MS/MS measurement. Bars represent mean ± SD of three independent tests. No statistically significant difference was observed (P>0.05, student t-test).

Mentions: Immunoblotting analysis showed that the resistant cells OVCAR-3-TR and SKOV-3-TR have high expression of P-gp protein while the parental OVCAR-3 and SKOV-3 cells have no detectable P-gp expression (Figure 2A). To confirm that BPR0L075 is not a substrate for efflux pumps, we measured the intracellular BPR0L075 levels in the two pairs of cell lines OVCAR-3/OVCAR-3-TR and SKOV-3/SKOV-3-TR using a sensitive LC/MS/MS method (Figure 2B). Both pairs of cells obtained similar intracellular BPR0L075 concentrations after BPR0L075 treatment (20 nM for 6 hours, Figure 2C) with no statistical difference (P>0.05). BPR0L075 is not a substrate for another ATP-binding cassette efflux transporter breast cancer resistance protein (BCRP), evidenced by the comparable cytotoxicity in human breast cancer MCF7 cells (IC50 = 3.5 nM) and mitoxantrone-resistant MCF7/MX cells (IC50 = 4.0 nM), MCF7/MX cells are known to overexpress BCRP [23], [24], [25]. Together, the results show that efflux transporter P-gp contributed to the selected paclitaxel resistance in SKOV-3-TR and OVCAR-3-TR cells, but BPR0L075 is not a substrate for efflux pumps, which contributes to its ability to overcome paclitaxel resistance.


An antimitotic and antivascular agent BPR0L075 overcomes multidrug resistance and induces mitotic catastrophe in paclitaxel-resistant ovarian cancer cells.

Wang X, Wu E, Wu J, Wang TL, Hsieh HP, Liu X - PLoS ONE (2013)

BPR0L075 is not a substrate for efflux transporters.(A) Western blot analysis of the P-gp and βIII-tubulin expressions in the paired ovarian cancer cell lines. Pan β-tubulin and β-actin were loading controls. (B) LC/MS/MS analysis of intracellular BPR0L075 levels, with LC chromatogram showing the retention time of internal standard [1-(1H-indol-3-ylcarbonyl)-1H-imidazole] and BPR0L075 as 2.2 and 2.8 min, respectively. The mass transitions of m/z 212→144 for internal standard and m/z 342→195 for BPR0L075 were monitored for quantification. (C) The intracellular BPR0L075 levels in the paired ovarian cancer cell lines. Cells were incubated with 20 nM BPR0L075 for 6 hours, then cells were washed, harvested, and lysed for LC/MS/MS measurement. Bars represent mean ± SD of three independent tests. No statistically significant difference was observed (P>0.05, student t-test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675084&req=5

pone-0065686-g002: BPR0L075 is not a substrate for efflux transporters.(A) Western blot analysis of the P-gp and βIII-tubulin expressions in the paired ovarian cancer cell lines. Pan β-tubulin and β-actin were loading controls. (B) LC/MS/MS analysis of intracellular BPR0L075 levels, with LC chromatogram showing the retention time of internal standard [1-(1H-indol-3-ylcarbonyl)-1H-imidazole] and BPR0L075 as 2.2 and 2.8 min, respectively. The mass transitions of m/z 212→144 for internal standard and m/z 342→195 for BPR0L075 were monitored for quantification. (C) The intracellular BPR0L075 levels in the paired ovarian cancer cell lines. Cells were incubated with 20 nM BPR0L075 for 6 hours, then cells were washed, harvested, and lysed for LC/MS/MS measurement. Bars represent mean ± SD of three independent tests. No statistically significant difference was observed (P>0.05, student t-test).
Mentions: Immunoblotting analysis showed that the resistant cells OVCAR-3-TR and SKOV-3-TR have high expression of P-gp protein while the parental OVCAR-3 and SKOV-3 cells have no detectable P-gp expression (Figure 2A). To confirm that BPR0L075 is not a substrate for efflux pumps, we measured the intracellular BPR0L075 levels in the two pairs of cell lines OVCAR-3/OVCAR-3-TR and SKOV-3/SKOV-3-TR using a sensitive LC/MS/MS method (Figure 2B). Both pairs of cells obtained similar intracellular BPR0L075 concentrations after BPR0L075 treatment (20 nM for 6 hours, Figure 2C) with no statistical difference (P>0.05). BPR0L075 is not a substrate for another ATP-binding cassette efflux transporter breast cancer resistance protein (BCRP), evidenced by the comparable cytotoxicity in human breast cancer MCF7 cells (IC50 = 3.5 nM) and mitoxantrone-resistant MCF7/MX cells (IC50 = 4.0 nM), MCF7/MX cells are known to overexpress BCRP [23], [24], [25]. Together, the results show that efflux transporter P-gp contributed to the selected paclitaxel resistance in SKOV-3-TR and OVCAR-3-TR cells, but BPR0L075 is not a substrate for efflux pumps, which contributes to its ability to overcome paclitaxel resistance.

Bottom Line: Thus, new therapy that can overcome paclitaxel resistance will be of significant clinical importance.BPR0L075 blocks cell cycle at the G2/M phase in paclitaxel-resistant cells while equal concentration of paclitaxel treatment was ineffective.Immunoblotting analysis shows that BPR0L075 treatment induced up-regulation of cyclin B1, BubR1, MPM-2, and survivin protein levels and Bcl-XL phosphorylation in parental cells; however, in resistant cells, the endogenous expressions of BubR1 and survivin were depleted, BPR0L075 treatment failed to induce MPM-2 expression and phosphorylation of Bcl-XL.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas, United States of America.

ABSTRACT
Paclitaxel plays a major role in the treatment of ovarian cancer; however, resistance to paclitaxel is frequently observed. Thus, new therapy that can overcome paclitaxel resistance will be of significant clinical importance. We evaluated antiproliferative effects of an antimitotic and antivascular agent BPR0L075 in paclitaxel-resistant ovarian cancer cells. BPR0L075 displays potent and broad-spectrum cytotoxicity at low nanomolar concentrations (IC50 = 2-7 nM) against both parental ovarian cancer cells (OVCAR-3, SKOV-3, and A2780-1A9) and paclitaxel-resistant sublines (OVCAR-3-TR, SKOV-3-TR, 1A9-PTX10), regardless of the expression levels of the multidrug resistance transporter P-gp and class III β-tubulin or mutation of β-tubulin. BPR0L075 blocks cell cycle at the G2/M phase in paclitaxel-resistant cells while equal concentration of paclitaxel treatment was ineffective. BPR0L075 induces cell death by a dual mechanism in parental and paclitaxel-resistant ovarian cancer cells. In the parental cells (OVCAR-3 and SKOV-3), BPR0L075 induced apoptosis, evidenced by poly(ADP-ribose) polymerase (PARP) cleavage and DNA ladder formation. BPR0L075 induced cell death in paclitaxel-resistant ovarian cancer cells (OVCAR-3-TR and SKOV-3-TR) is primarily due to mitotic catastrophe, evidenced by formation of giant, multinucleated cells and absence of PARP cleavage. Immunoblotting analysis shows that BPR0L075 treatment induced up-regulation of cyclin B1, BubR1, MPM-2, and survivin protein levels and Bcl-XL phosphorylation in parental cells; however, in resistant cells, the endogenous expressions of BubR1 and survivin were depleted, BPR0L075 treatment failed to induce MPM-2 expression and phosphorylation of Bcl-XL. BPR0L075 induced cell death in both parental and paclitaxel-resistant ovarian cancer cells proceed through caspase-3 independent mechanisms. In conclusion, BPR0L075 displays potent cytotoxic effects in ovarian cancer cells with a potential to overcome paclitaxel resistance by bypassing efflux transporters and inducing mitotic catastrophe. BPR0L075 represents a novel microtubule therapeutic to overcome multidrug resistance and trigger alternative cell death by mitotic catastrophe in ovarian cancer cells that are apoptosis-resistant.

Show MeSH
Related in: MedlinePlus