Limits...
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 potentiates TMZ therapy in conventional GBM cell lines on serum-supplemented medium. A) Viability of T98 and U373 cell lines treated at the indicated doses with TMZ and PARPi. Viability is indicated as a measure of ATP conversion into luminescence. Lines above columns indicate significance between two conditions with a p-value <0.05. If monotherapy with TMZ is significant when compared to non-treated controls, the significance of combination therapy when compared to non-treated controls is related and therefore not defined as such in the figure. B) Viability as a measure of confluence per well as measured by microscopy. Read out is performed at 5 days post treatment. C) Microscopic images of U373 and T98. From left to right the PARPi dosage increases from 0 to 10 uM. Lower panel depicts combination therapy with TMZ at indicated dosages. Note the differential effect of PARPi monotherapy between T98 and U373. D) Combination Index (CI) plotted against Fraction affected (Fa) for T98 and U373. PARPi with TMZ combination therapy is synergistic in T98 but not in U373. CI < 1 demonstrates synergistic interaction of combination therapy with TMZ and PARPi at the indicated Fa. CI > 1 demonstrates antagonistic effect of combination therapy at the indicated Fa.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: PARPi potentiates TMZ therapy in conventional GBM cell lines on serum-supplemented medium. A) Viability of T98 and U373 cell lines treated at the indicated doses with TMZ and PARPi. Viability is indicated as a measure of ATP conversion into luminescence. Lines above columns indicate significance between two conditions with a p-value <0.05. If monotherapy with TMZ is significant when compared to non-treated controls, the significance of combination therapy when compared to non-treated controls is related and therefore not defined as such in the figure. B) Viability as a measure of confluence per well as measured by microscopy. Read out is performed at 5 days post treatment. C) Microscopic images of U373 and T98. From left to right the PARPi dosage increases from 0 to 10 uM. Lower panel depicts combination therapy with TMZ at indicated dosages. Note the differential effect of PARPi monotherapy between T98 and U373. D) Combination Index (CI) plotted against Fraction affected (Fa) for T98 and U373. PARPi with TMZ combination therapy is synergistic in T98 but not in U373. CI < 1 demonstrates synergistic interaction of combination therapy with TMZ and PARPi at the indicated Fa. CI > 1 demonstrates antagonistic effect of combination therapy at the indicated Fa.

Mentions: The addition of PARPi to TMZ treatment was tested in two conventionally used GBM cell lines, T98 and U373 respectively, for which the MGMT gene methylation and protein status have been previously reported [17]. Quantification of cytotoxicity was assessed by cell viability as well as monolayer confluency (Figure 1A and B). In accordance with previous reports [18], U373 was sensitive to TMZ monotherapy, whereas T98 was resistant (p>0.05 for both dosages compared to non-treated control). Conversely, T98 was sensitive to PARPi monotherapy whereas U373 was not. Comparative analysis of confluency and ATP-based viability generally correlated well. However, T98 susceptibility for ABT-888 monotherapy appeared more pronounced when assessed for viability than the confluency results suggest. Microscopic examination (Figure 1C) revealed fewer viable cells and drifting debris suggestive of cytotoxic effect of ABT-888 at 10 uM in T98 cells, which was not apparent in U373.Figure 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 potentiates TMZ therapy in conventional GBM cell lines on serum-supplemented medium. A) Viability of T98 and U373 cell lines treated at the indicated doses with TMZ and PARPi. Viability is indicated as a measure of ATP conversion into luminescence. Lines above columns indicate significance between two conditions with a p-value <0.05. If monotherapy with TMZ is significant when compared to non-treated controls, the significance of combination therapy when compared to non-treated controls is related and therefore not defined as such in the figure. B) Viability as a measure of confluence per well as measured by microscopy. Read out is performed at 5 days post treatment. C) Microscopic images of U373 and T98. From left to right the PARPi dosage increases from 0 to 10 uM. Lower panel depicts combination therapy with TMZ at indicated dosages. Note the differential effect of PARPi monotherapy between T98 and U373. D) Combination Index (CI) plotted against Fraction affected (Fa) for T98 and U373. PARPi with TMZ combination therapy is synergistic in T98 but not in U373. CI < 1 demonstrates synergistic interaction of combination therapy with TMZ and PARPi at the indicated Fa. CI > 1 demonstrates antagonistic effect of combination therapy at the indicated Fa.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: PARPi potentiates TMZ therapy in conventional GBM cell lines on serum-supplemented medium. A) Viability of T98 and U373 cell lines treated at the indicated doses with TMZ and PARPi. Viability is indicated as a measure of ATP conversion into luminescence. Lines above columns indicate significance between two conditions with a p-value <0.05. If monotherapy with TMZ is significant when compared to non-treated controls, the significance of combination therapy when compared to non-treated controls is related and therefore not defined as such in the figure. B) Viability as a measure of confluence per well as measured by microscopy. Read out is performed at 5 days post treatment. C) Microscopic images of U373 and T98. From left to right the PARPi dosage increases from 0 to 10 uM. Lower panel depicts combination therapy with TMZ at indicated dosages. Note the differential effect of PARPi monotherapy between T98 and U373. D) Combination Index (CI) plotted against Fraction affected (Fa) for T98 and U373. PARPi with TMZ combination therapy is synergistic in T98 but not in U373. CI < 1 demonstrates synergistic interaction of combination therapy with TMZ and PARPi at the indicated Fa. CI > 1 demonstrates antagonistic effect of combination therapy at the indicated Fa.
Mentions: The addition of PARPi to TMZ treatment was tested in two conventionally used GBM cell lines, T98 and U373 respectively, for which the MGMT gene methylation and protein status have been previously reported [17]. Quantification of cytotoxicity was assessed by cell viability as well as monolayer confluency (Figure 1A and B). In accordance with previous reports [18], U373 was sensitive to TMZ monotherapy, whereas T98 was resistant (p>0.05 for both dosages compared to non-treated control). Conversely, T98 was sensitive to PARPi monotherapy whereas U373 was not. Comparative analysis of confluency and ATP-based viability generally correlated well. However, T98 susceptibility for ABT-888 monotherapy appeared more pronounced when assessed for viability than the confluency results suggest. Microscopic examination (Figure 1C) revealed fewer viable cells and drifting debris suggestive of cytotoxic effect of ABT-888 at 10 uM in T98 cells, which was not apparent in U373.Figure 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