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ABT-888 enhances cytotoxic effects of temozolomide independent of MGMT status in serum free cultured glioma cells.

Balvers RK, Lamfers ML, Kloezeman JJ, Kleijn A, Berghauser Pont LM, Dirven CM, Leenstra S - J Transl Med (2015)

Bottom Line: PARPi monotherapy was found to decrease viability by more than 25% in 4 of the 20 GSCs (20%) at 10 μM.TMZ resistance to 100 μM was found in 7 of 8 MGMT protein positive cultures.Potentiation of TMZ therapy through PARPi was found in 90% (n = 20) of GSCs, of which 6 were initially resistant and 7 were sensitive to TMZ monotherapy.

View Article: PubMed Central - PubMed

Affiliation: Brain Tumor Center; Department of Neurosurgery, Erasmus MC, Molewaterplein 50, Ee2236, 3015GE, Rotterdam, The Netherlands. r.balvers@erasmusmc.nl.

ABSTRACT

Background: The current standard of care for Glioblastoma Multiforme (GBM) consists of fractionated focal irradiation with concomitant temozolomide (TMZ) chemotherapy. A promising strategy to increase the efficacy of TMZ is through interference with the DNA damage repair machinery, by poly(ADP-ribose) polymerase protein inhibition(PARPi). The objective of the present study was to investigate the therapeutic benefit of combination therapy in patient-derived glioma stem-like cells (GSC).

Methods: Combination therapy feasibility was tested on established GBM cell lines U373 and T98. We developed an in vitro drug-screening assay based on GSC cultures derived from a panel of primary patient tissue samples (n = 20) to evaluate the effect of PARPi (ABT-888) monotherapy and combination therapy with TMZ. Therapeutic effect was assessed by viability, double stranded breaks, apoptosis and autophagy assays and longitudinal microscopic cell monitoring was performed. O-6-methylguanine-DNA methyltransferase (MGMT) status was determined by methylation assay and protein expression by western blots.

Results: PARPi monotherapy was found to decrease viability by more than 25% in 4 of the 20 GSCs (20%) at 10 μM. TMZ monotherapy at 50 μM and 100 μM was effective in 12 and 14 of the 20 GSCs, respectively. TMZ resistance to 100 μM was found in 7 of 8 MGMT protein positive cultures. Potentiation of TMZ therapy through PARPi was found in 90% (n = 20) of GSCs, of which 6 were initially resistant and 7 were sensitive to TMZ monotherapy. Increased induction of double stranded breaks and apoptosis were noted in responsive GSCs. There was a trend noted, albeit statistically insignificant, of increased autophagy both in western blots and accumulation of autophagosomes.

Conclusion: PARPi mediated potentiation of TMZ is independent of TMZ sensitivity and can override MGMT(-) mediated resistance when administered simultaneously. Response to combination therapy was associated with increased double strand breaks induction, and coincided by increased apoptosis and autophagy. PARPi addition potentiates TMZ treatment in primary GSCs. PARPi could potentially enhance the therapeutic efficacy of the standard of care in GBM.

No MeSH data available.


Related in: MedlinePlus

PARPi and TMZ monotherapy efficacy in GS cultures. A) Panel of GS cultures tested for sensitivity to PARPi at 10 uM. Readout of viability was performed at day 5 and is depicted as a percentage when compared to non-treated controls. * Illustrates a p-value <0.05. B) GS culture panel tested against indicated concentrations in uM of TMZ monotherapy. Viability at day 5 is indicated as percentages when compared to paired non-treated controls. * indicates p < 0.05 as compared to non treated controls. C) Overview of western blotting results derived from GS cultures. MGMT protein expression is indicated with actin protein expression used as a loading control. TMZ sensitivity is indicated with an S (sensitive), R (resistant) and ND (TMZ sensitivity not determined in monotherapy screen). For several GSCs protein isolates were loaded in separate runs as technical controls.
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Fig2: PARPi and TMZ monotherapy efficacy in GS cultures. A) Panel of GS cultures tested for sensitivity to PARPi at 10 uM. Readout of viability was performed at day 5 and is depicted as a percentage when compared to non-treated controls. * Illustrates a p-value <0.05. B) GS culture panel tested against indicated concentrations in uM of TMZ monotherapy. Viability at day 5 is indicated as percentages when compared to paired non-treated controls. * indicates p < 0.05 as compared to non treated controls. C) Overview of western blotting results derived from GS cultures. MGMT protein expression is indicated with actin protein expression used as a loading control. TMZ sensitivity is indicated with an S (sensitive), R (resistant) and ND (TMZ sensitivity not determined in monotherapy screen). For several GSCs protein isolates were loaded in separate runs as technical controls.

Mentions: Molecular characteristics as found in primary GBM tissue, are inferiorly recapitulated in conventional cell lines such as T98 and U373, when compared to early passages of serum-free patient-derived cultures [10,19]. Therefore, we tested 20 GSC cultures from high-grade malignant glioma for the PARPi ABT-888 and TMZ sensitivity (overview of clinically relevant information in Table 1). GSC cultures were labeled sensitive if more than 25% reduction in viability was measured as compared to non-treated controls. For PARPi monotherapy at 10μM, this was found in 4/20 cultures (20%), (Figure 2A).Table 1


ABT-888 enhances cytotoxic effects of temozolomide independent of MGMT status in serum free cultured glioma cells.

Balvers RK, Lamfers ML, Kloezeman JJ, Kleijn A, Berghauser Pont LM, Dirven CM, Leenstra S - J Transl Med (2015)

PARPi and TMZ monotherapy efficacy in GS cultures. A) Panel of GS cultures tested for sensitivity to PARPi at 10 uM. Readout of viability was performed at day 5 and is depicted as a percentage when compared to non-treated controls. * Illustrates a p-value <0.05. B) GS culture panel tested against indicated concentrations in uM of TMZ monotherapy. Viability at day 5 is indicated as percentages when compared to paired non-treated controls. * indicates p < 0.05 as compared to non treated controls. C) Overview of western blotting results derived from GS cultures. MGMT protein expression is indicated with actin protein expression used as a loading control. TMZ sensitivity is indicated with an S (sensitive), R (resistant) and ND (TMZ sensitivity not determined in monotherapy screen). For several GSCs protein isolates were loaded in separate runs as technical controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4359449&req=5

Fig2: PARPi and TMZ monotherapy efficacy in GS cultures. A) Panel of GS cultures tested for sensitivity to PARPi at 10 uM. Readout of viability was performed at day 5 and is depicted as a percentage when compared to non-treated controls. * Illustrates a p-value <0.05. B) GS culture panel tested against indicated concentrations in uM of TMZ monotherapy. Viability at day 5 is indicated as percentages when compared to paired non-treated controls. * indicates p < 0.05 as compared to non treated controls. C) Overview of western blotting results derived from GS cultures. MGMT protein expression is indicated with actin protein expression used as a loading control. TMZ sensitivity is indicated with an S (sensitive), R (resistant) and ND (TMZ sensitivity not determined in monotherapy screen). For several GSCs protein isolates were loaded in separate runs as technical controls.
Mentions: Molecular characteristics as found in primary GBM tissue, are inferiorly recapitulated in conventional cell lines such as T98 and U373, when compared to early passages of serum-free patient-derived cultures [10,19]. Therefore, we tested 20 GSC cultures from high-grade malignant glioma for the PARPi ABT-888 and TMZ sensitivity (overview of clinically relevant information in Table 1). GSC cultures were labeled sensitive if more than 25% reduction in viability was measured as compared to non-treated controls. For PARPi monotherapy at 10μM, this was found in 4/20 cultures (20%), (Figure 2A).Table 1

Bottom Line: PARPi monotherapy was found to decrease viability by more than 25% in 4 of the 20 GSCs (20%) at 10 μM.TMZ resistance to 100 μM was found in 7 of 8 MGMT protein positive cultures.Potentiation of TMZ therapy through PARPi was found in 90% (n = 20) of GSCs, of which 6 were initially resistant and 7 were sensitive to TMZ monotherapy.

View Article: PubMed Central - PubMed

Affiliation: Brain Tumor Center; Department of Neurosurgery, Erasmus MC, Molewaterplein 50, Ee2236, 3015GE, Rotterdam, The Netherlands. r.balvers@erasmusmc.nl.

ABSTRACT

Background: The current standard of care for Glioblastoma Multiforme (GBM) consists of fractionated focal irradiation with concomitant temozolomide (TMZ) chemotherapy. A promising strategy to increase the efficacy of TMZ is through interference with the DNA damage repair machinery, by poly(ADP-ribose) polymerase protein inhibition(PARPi). The objective of the present study was to investigate the therapeutic benefit of combination therapy in patient-derived glioma stem-like cells (GSC).

Methods: Combination therapy feasibility was tested on established GBM cell lines U373 and T98. We developed an in vitro drug-screening assay based on GSC cultures derived from a panel of primary patient tissue samples (n = 20) to evaluate the effect of PARPi (ABT-888) monotherapy and combination therapy with TMZ. Therapeutic effect was assessed by viability, double stranded breaks, apoptosis and autophagy assays and longitudinal microscopic cell monitoring was performed. O-6-methylguanine-DNA methyltransferase (MGMT) status was determined by methylation assay and protein expression by western blots.

Results: PARPi monotherapy was found to decrease viability by more than 25% in 4 of the 20 GSCs (20%) at 10 μM. TMZ monotherapy at 50 μM and 100 μM was effective in 12 and 14 of the 20 GSCs, respectively. TMZ resistance to 100 μM was found in 7 of 8 MGMT protein positive cultures. Potentiation of TMZ therapy through PARPi was found in 90% (n = 20) of GSCs, of which 6 were initially resistant and 7 were sensitive to TMZ monotherapy. Increased induction of double stranded breaks and apoptosis were noted in responsive GSCs. There was a trend noted, albeit statistically insignificant, of increased autophagy both in western blots and accumulation of autophagosomes.

Conclusion: PARPi mediated potentiation of TMZ is independent of TMZ sensitivity and can override MGMT(-) mediated resistance when administered simultaneously. Response to combination therapy was associated with increased double strand breaks induction, and coincided by increased apoptosis and autophagy. PARPi addition potentiates TMZ treatment in primary GSCs. PARPi could potentially enhance the therapeutic efficacy of the standard of care in GBM.

No MeSH data available.


Related in: MedlinePlus