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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

PARP inhibition increases TMZ induced double stranded breaks, autophagy and apoptosis in GSCs. A) DSB induction is significantly enhanced by combination therapy. GSCs, MGMT(+) and MGMT(-) were scored for γH2Ax positive cells, as an indicator for DSB induction,16 hrs post-treatment. GS160 results are all significant as compared to non-treated controls. For the other cultures, significance is indicated as described in the following; * indicates p < 0,05 as compared to non-treated controls. # indicates p < 0.05 as compared to monotherapy. R = combination therapy responder, NR = non-responder. B) Apoptosis induction is significantly enhanced by combination therapy in responder GSCs but not in non-responders. Wells were scored for apoptosis positive cells by live imaging. Read out was performed at 48 hrs post-treatment. Apoptotic cells were significantly (p < 0.05) increased after combination therapy as compared to monontherapy in GSC79. For GS160 cells were not affected by TMZ/ABT-888 combination therapy while the positive control (etoposide 0.1 mM) was successfully inducing apoptosis. C) TMZ and ABT-888 induce autophagy in GS79, which is augmented by combination therapy. Autophagosomes were counted per well and compared to non-treated controls. Readout was performed 16 hrs post-treatment. Compared to controls, all treatment conditions were significantly increased p < 0.05. However, combination therapy vs. monotherapy with ABT-888 or TMZ was found not significantly increased (p = 0.3 and p = 0.4). D) Representative images acquired by the Incucyte FLR of GS79 stained for autophagosomes at indicated treatment conditions. Note the accumulation of speckled dots in treated cells, specifically localized in enlarged cytopathic cells. E) Combination therapy enhances autophagic flux as compared to monotherapy in GS79. Western blot 24 hrs post-treatment indicates the accumulation of LC3-II (lipidated isoform) that is indicative for increased autophagic flux. Actin blots serve as loading controls.
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Fig3: PARP inhibition increases TMZ induced double stranded breaks, autophagy and apoptosis in GSCs. A) DSB induction is significantly enhanced by combination therapy. GSCs, MGMT(+) and MGMT(-) were scored for γH2Ax positive cells, as an indicator for DSB induction,16 hrs post-treatment. GS160 results are all significant as compared to non-treated controls. For the other cultures, significance is indicated as described in the following; * indicates p < 0,05 as compared to non-treated controls. # indicates p < 0.05 as compared to monotherapy. R = combination therapy responder, NR = non-responder. B) Apoptosis induction is significantly enhanced by combination therapy in responder GSCs but not in non-responders. Wells were scored for apoptosis positive cells by live imaging. Read out was performed at 48 hrs post-treatment. Apoptotic cells were significantly (p < 0.05) increased after combination therapy as compared to monontherapy in GSC79. For GS160 cells were not affected by TMZ/ABT-888 combination therapy while the positive control (etoposide 0.1 mM) was successfully inducing apoptosis. C) TMZ and ABT-888 induce autophagy in GS79, which is augmented by combination therapy. Autophagosomes were counted per well and compared to non-treated controls. Readout was performed 16 hrs post-treatment. Compared to controls, all treatment conditions were significantly increased p < 0.05. However, combination therapy vs. monotherapy with ABT-888 or TMZ was found not significantly increased (p = 0.3 and p = 0.4). D) Representative images acquired by the Incucyte FLR of GS79 stained for autophagosomes at indicated treatment conditions. Note the accumulation of speckled dots in treated cells, specifically localized in enlarged cytopathic cells. E) Combination therapy enhances autophagic flux as compared to monotherapy in GS79. Western blot 24 hrs post-treatment indicates the accumulation of LC3-II (lipidated isoform) that is indicative for increased autophagic flux. Actin blots serve as loading controls.

Mentions: The progression of alkylation based single stranded breaks (SSBs) to double stranded breaks (DSBs) has been proposed to be fatal in cancer cells. While MGMT can facilitate repair of SSBs, the rationale behind the addition of ABT-888 to TMZ is to comprise the DNA-repair cascade needed to prevent the induction of DSBs after alkylation induced SSB formation. To address this proposed role of DSB induction as an outcome of therapeutic efficacy in our GSC panel, three MGMT(-) and two MGMT(+) GSCs were tested for DSB induction when treated with TMZ, ABT-888 or combination therapy. The induction of DSBs by TMZ monotherapy was significant (p < 0.01) in 2 out of 3 MGMT(-) GSCs (Figure 3A). Monotherapy with ABT-888 did not yield a significant increase in DSB induction. Interestingly, DSB induction was not apparent in one non-responder GS160 (MGMT(+)), suggestive of the premise that DSB induction is needed for combination therapy to potentiate TMZ-monotherapy.Figure 3


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)

PARP inhibition increases TMZ induced double stranded breaks, autophagy and apoptosis in GSCs. A) DSB induction is significantly enhanced by combination therapy. GSCs, MGMT(+) and MGMT(-) were scored for γH2Ax positive cells, as an indicator for DSB induction,16 hrs post-treatment. GS160 results are all significant as compared to non-treated controls. For the other cultures, significance is indicated as described in the following; * indicates p < 0,05 as compared to non-treated controls. # indicates p < 0.05 as compared to monotherapy. R = combination therapy responder, NR = non-responder. B) Apoptosis induction is significantly enhanced by combination therapy in responder GSCs but not in non-responders. Wells were scored for apoptosis positive cells by live imaging. Read out was performed at 48 hrs post-treatment. Apoptotic cells were significantly (p < 0.05) increased after combination therapy as compared to monontherapy in GSC79. For GS160 cells were not affected by TMZ/ABT-888 combination therapy while the positive control (etoposide 0.1 mM) was successfully inducing apoptosis. C) TMZ and ABT-888 induce autophagy in GS79, which is augmented by combination therapy. Autophagosomes were counted per well and compared to non-treated controls. Readout was performed 16 hrs post-treatment. Compared to controls, all treatment conditions were significantly increased p < 0.05. However, combination therapy vs. monotherapy with ABT-888 or TMZ was found not significantly increased (p = 0.3 and p = 0.4). D) Representative images acquired by the Incucyte FLR of GS79 stained for autophagosomes at indicated treatment conditions. Note the accumulation of speckled dots in treated cells, specifically localized in enlarged cytopathic cells. E) Combination therapy enhances autophagic flux as compared to monotherapy in GS79. Western blot 24 hrs post-treatment indicates the accumulation of LC3-II (lipidated isoform) that is indicative for increased autophagic flux. Actin blots serve as loading controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4359449&req=5

Fig3: PARP inhibition increases TMZ induced double stranded breaks, autophagy and apoptosis in GSCs. A) DSB induction is significantly enhanced by combination therapy. GSCs, MGMT(+) and MGMT(-) were scored for γH2Ax positive cells, as an indicator for DSB induction,16 hrs post-treatment. GS160 results are all significant as compared to non-treated controls. For the other cultures, significance is indicated as described in the following; * indicates p < 0,05 as compared to non-treated controls. # indicates p < 0.05 as compared to monotherapy. R = combination therapy responder, NR = non-responder. B) Apoptosis induction is significantly enhanced by combination therapy in responder GSCs but not in non-responders. Wells were scored for apoptosis positive cells by live imaging. Read out was performed at 48 hrs post-treatment. Apoptotic cells were significantly (p < 0.05) increased after combination therapy as compared to monontherapy in GSC79. For GS160 cells were not affected by TMZ/ABT-888 combination therapy while the positive control (etoposide 0.1 mM) was successfully inducing apoptosis. C) TMZ and ABT-888 induce autophagy in GS79, which is augmented by combination therapy. Autophagosomes were counted per well and compared to non-treated controls. Readout was performed 16 hrs post-treatment. Compared to controls, all treatment conditions were significantly increased p < 0.05. However, combination therapy vs. monotherapy with ABT-888 or TMZ was found not significantly increased (p = 0.3 and p = 0.4). D) Representative images acquired by the Incucyte FLR of GS79 stained for autophagosomes at indicated treatment conditions. Note the accumulation of speckled dots in treated cells, specifically localized in enlarged cytopathic cells. E) Combination therapy enhances autophagic flux as compared to monotherapy in GS79. Western blot 24 hrs post-treatment indicates the accumulation of LC3-II (lipidated isoform) that is indicative for increased autophagic flux. Actin blots serve as loading controls.
Mentions: The progression of alkylation based single stranded breaks (SSBs) to double stranded breaks (DSBs) has been proposed to be fatal in cancer cells. While MGMT can facilitate repair of SSBs, the rationale behind the addition of ABT-888 to TMZ is to comprise the DNA-repair cascade needed to prevent the induction of DSBs after alkylation induced SSB formation. To address this proposed role of DSB induction as an outcome of therapeutic efficacy in our GSC panel, three MGMT(-) and two MGMT(+) GSCs were tested for DSB induction when treated with TMZ, ABT-888 or combination therapy. The induction of DSBs by TMZ monotherapy was significant (p < 0.01) in 2 out of 3 MGMT(-) GSCs (Figure 3A). Monotherapy with ABT-888 did not yield a significant increase in DSB induction. Interestingly, DSB induction was not apparent in one non-responder GS160 (MGMT(+)), suggestive of the premise that DSB induction is needed for combination therapy to potentiate TMZ-monotherapy.Figure 3

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