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Prolonged pemetrexed pretreatment augments persistence of cisplatin-induced DNA damage and eliminates resistant lung cancer stem-like cells associated with EMT.

Tièche CC, Peng RW, Dorn P, Froment L, Schmid RA, Marti TM - BMC Cancer (2016)

Bottom Line: Prolonged pemetrexed pretreatment for 48 h prior to cisplatin treatment maximally delayed long-term cell growth and significantly reduced the number of recovering clones.Therefore, this study warrants further investigations to elucidate whether such an adaptation could enhance the effectiveness of the standard clinical treatment regimen.In addition, a subpopulation of therapy resistant cells with EMT and cancer stem cell features was identified that was resistant to the standard treatment regimen but sensitive to pemetrexed pretreatment combined with cisplatin.

View Article: PubMed Central - PubMed

Affiliation: Division of General Thoracic Surgery, Inselspital, Bern University Hospital, Department of Clinical Research, University of Bern, Murtenstrasse 50, 3008, Bern, Switzerland. colin.tieche@insel.ch.

ABSTRACT

Background: Lung cancer is the leading cause of cancer-related mortality, and new therapeutic options are urgently needed. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers, with the current standard regimen of care for NSCLC including chemotherapy with pemetrexed as a single agent or in combination with platinum-based agents, e.g. cisplatin. Pemetrexed is a folic acid antagonist that inhibits the synthesis of precursor nucleotides, whereas cisplatin directly induces DNA adducts, the repair of which is dependent on sufficiently high nucleotide levels. In the clinical setting, the pemetrexed-cisplatin combination therapy is administered concomitantly. We hypothesized that prolonged pretreatment with pemetrexed could be beneficial, as prior depletion of nucleotide pools could sensitize cancer cells to subsequent treatment with cisplatin.

Methods: NSCLC A549 and H460 cells were treated with pemetrexed for 72 h. In addition, 24 h of cisplatin treatment was initiated at day 1, 2 or 3 resulting in either simultaneous pemetrexed application or pemetrexed pretreatment for 24 or 48 h, respectively. Cell growth and colony formation as well as senescence induction were quantified after treatment. Cell cycle distribution and phosphorylation of histone variant H2AX as a surrogate marker for DNA damage was quantified by flow cytometry. Relative changes in gene expression were determined by quantitative real time PCR.

Results: Prolonged pemetrexed pretreatment for 48 h prior to cisplatin treatment maximally delayed long-term cell growth and significantly reduced the number of recovering clones. Moreover, apoptosis and senescence were augmented and recovery from treatment-induced DNA damage was delayed. Interestingly, a cell population was identified that displayed an epithelial-to-mesenchymal transition (EMT) and which had a stem cell phenotype. This population was highly resistant to concomitant pemetrexed-cisplatin treatment but was sensitized by pemetrexed pretreatment.

Conclusions: Adaptation of the standard treatment schedule to include pretreatment with pemetrexed optimizes the anticancer efficiency of pemetrexed-cisplatin combination therapy, which correlates with a persistence of treatment-induced DNA damage. Therefore, this study warrants further investigations to elucidate whether such an adaptation could enhance the effectiveness of the standard clinical treatment regimen. In addition, a subpopulation of therapy resistant cells with EMT and cancer stem cell features was identified that was resistant to the standard treatment regimen but sensitive to pemetrexed pretreatment combined with cisplatin.

No MeSH data available.


Related in: MedlinePlus

Prolonged MTA pretreatment augments cisplatin-induced senescence in A549 cells. a Forward and side scatter analysis by flow cytometry (without reseeding) at day 10 (10d rec) and day 14 (14d rec) of the recovery phase. b Representative images of cells acquired by phase contrast-based microscopy at day 17 of the recovery phase (reseeded at day 10). Arrows indicate cells which stain positive for senescence associated β-galactosidase activity. c Quantification of senescent cells based on increased β-galactosidase activity b. Represented are three independent experiments and bars indicate means and standard deviations. ***, P < 0.001, **, P < 0.01 and *, P < 0.05
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Fig2: Prolonged MTA pretreatment augments cisplatin-induced senescence in A549 cells. a Forward and side scatter analysis by flow cytometry (without reseeding) at day 10 (10d rec) and day 14 (14d rec) of the recovery phase. b Representative images of cells acquired by phase contrast-based microscopy at day 17 of the recovery phase (reseeded at day 10). Arrows indicate cells which stain positive for senescence associated β-galactosidase activity. c Quantification of senescent cells based on increased β-galactosidase activity b. Represented are three independent experiments and bars indicate means and standard deviations. ***, P < 0.001, **, P < 0.01 and *, P < 0.05

Mentions: As mentioned above, visual examination revealed that the cells surrounding the recovering clones displayed morphologic changes that are associated with senescence, namely increased cell size and flattened shape (Fig. 1c; reviewed in [17]). Detectable by flow cytometry, increased forward (cell size) and side (cellular granularity) scatter intensity (F/S-high; Fig. 2a and Additional file 2: Figure S2) is an additional characteristic associated with senescence (reviewed in [18]). Flow cytometric analysis at day 10 and 14 of the recovery phase (without reseeding) revealed that the highest ratio of F/S-high versus F/S-low cells was observed after treatment #3. In detail, the ratios of FS-high versus FS-low cells were 12, 14 and 15.5 after day 10 and 2.3, 7.3 and 24 after day 14 for treatment #1/2/3, respectively. In other words, at day 14 of the recovery phase (without reseeding), a significant fraction of cells after treatment #1 restored normal forward and side scatter intensity (F/S-low), which was less pronounced after treatment #3 (31 % versus 7 %, respectively, p > 0.01). To quantify senescence induction, cells from recovery day 10 were reseeded at low density. At day 17 of the recovery phase, quantification of senescence-associated β-galactosidase (SA-β-Gal) activity revealed that the fraction of SA-β-Gal-positive cells (indicated by black arrows in Fig. 2b) was 3.5-fold higher after treatment #3 compared to treatment #1 (Fig. 2c). However, visual examination revealed that a fraction of the cells was clearly proliferating (see also Fig. 1d) giving rise to distinct colonies as described below.Fig. 2


Prolonged pemetrexed pretreatment augments persistence of cisplatin-induced DNA damage and eliminates resistant lung cancer stem-like cells associated with EMT.

Tièche CC, Peng RW, Dorn P, Froment L, Schmid RA, Marti TM - BMC Cancer (2016)

Prolonged MTA pretreatment augments cisplatin-induced senescence in A549 cells. a Forward and side scatter analysis by flow cytometry (without reseeding) at day 10 (10d rec) and day 14 (14d rec) of the recovery phase. b Representative images of cells acquired by phase contrast-based microscopy at day 17 of the recovery phase (reseeded at day 10). Arrows indicate cells which stain positive for senescence associated β-galactosidase activity. c Quantification of senescent cells based on increased β-galactosidase activity b. Represented are three independent experiments and bars indicate means and standard deviations. ***, P < 0.001, **, P < 0.01 and *, P < 0.05
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4759918&req=5

Fig2: Prolonged MTA pretreatment augments cisplatin-induced senescence in A549 cells. a Forward and side scatter analysis by flow cytometry (without reseeding) at day 10 (10d rec) and day 14 (14d rec) of the recovery phase. b Representative images of cells acquired by phase contrast-based microscopy at day 17 of the recovery phase (reseeded at day 10). Arrows indicate cells which stain positive for senescence associated β-galactosidase activity. c Quantification of senescent cells based on increased β-galactosidase activity b. Represented are three independent experiments and bars indicate means and standard deviations. ***, P < 0.001, **, P < 0.01 and *, P < 0.05
Mentions: As mentioned above, visual examination revealed that the cells surrounding the recovering clones displayed morphologic changes that are associated with senescence, namely increased cell size and flattened shape (Fig. 1c; reviewed in [17]). Detectable by flow cytometry, increased forward (cell size) and side (cellular granularity) scatter intensity (F/S-high; Fig. 2a and Additional file 2: Figure S2) is an additional characteristic associated with senescence (reviewed in [18]). Flow cytometric analysis at day 10 and 14 of the recovery phase (without reseeding) revealed that the highest ratio of F/S-high versus F/S-low cells was observed after treatment #3. In detail, the ratios of FS-high versus FS-low cells were 12, 14 and 15.5 after day 10 and 2.3, 7.3 and 24 after day 14 for treatment #1/2/3, respectively. In other words, at day 14 of the recovery phase (without reseeding), a significant fraction of cells after treatment #1 restored normal forward and side scatter intensity (F/S-low), which was less pronounced after treatment #3 (31 % versus 7 %, respectively, p > 0.01). To quantify senescence induction, cells from recovery day 10 were reseeded at low density. At day 17 of the recovery phase, quantification of senescence-associated β-galactosidase (SA-β-Gal) activity revealed that the fraction of SA-β-Gal-positive cells (indicated by black arrows in Fig. 2b) was 3.5-fold higher after treatment #3 compared to treatment #1 (Fig. 2c). However, visual examination revealed that a fraction of the cells was clearly proliferating (see also Fig. 1d) giving rise to distinct colonies as described below.Fig. 2

Bottom Line: Prolonged pemetrexed pretreatment for 48 h prior to cisplatin treatment maximally delayed long-term cell growth and significantly reduced the number of recovering clones.Therefore, this study warrants further investigations to elucidate whether such an adaptation could enhance the effectiveness of the standard clinical treatment regimen.In addition, a subpopulation of therapy resistant cells with EMT and cancer stem cell features was identified that was resistant to the standard treatment regimen but sensitive to pemetrexed pretreatment combined with cisplatin.

View Article: PubMed Central - PubMed

Affiliation: Division of General Thoracic Surgery, Inselspital, Bern University Hospital, Department of Clinical Research, University of Bern, Murtenstrasse 50, 3008, Bern, Switzerland. colin.tieche@insel.ch.

ABSTRACT

Background: Lung cancer is the leading cause of cancer-related mortality, and new therapeutic options are urgently needed. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers, with the current standard regimen of care for NSCLC including chemotherapy with pemetrexed as a single agent or in combination with platinum-based agents, e.g. cisplatin. Pemetrexed is a folic acid antagonist that inhibits the synthesis of precursor nucleotides, whereas cisplatin directly induces DNA adducts, the repair of which is dependent on sufficiently high nucleotide levels. In the clinical setting, the pemetrexed-cisplatin combination therapy is administered concomitantly. We hypothesized that prolonged pretreatment with pemetrexed could be beneficial, as prior depletion of nucleotide pools could sensitize cancer cells to subsequent treatment with cisplatin.

Methods: NSCLC A549 and H460 cells were treated with pemetrexed for 72 h. In addition, 24 h of cisplatin treatment was initiated at day 1, 2 or 3 resulting in either simultaneous pemetrexed application or pemetrexed pretreatment for 24 or 48 h, respectively. Cell growth and colony formation as well as senescence induction were quantified after treatment. Cell cycle distribution and phosphorylation of histone variant H2AX as a surrogate marker for DNA damage was quantified by flow cytometry. Relative changes in gene expression were determined by quantitative real time PCR.

Results: Prolonged pemetrexed pretreatment for 48 h prior to cisplatin treatment maximally delayed long-term cell growth and significantly reduced the number of recovering clones. Moreover, apoptosis and senescence were augmented and recovery from treatment-induced DNA damage was delayed. Interestingly, a cell population was identified that displayed an epithelial-to-mesenchymal transition (EMT) and which had a stem cell phenotype. This population was highly resistant to concomitant pemetrexed-cisplatin treatment but was sensitized by pemetrexed pretreatment.

Conclusions: Adaptation of the standard treatment schedule to include pretreatment with pemetrexed optimizes the anticancer efficiency of pemetrexed-cisplatin combination therapy, which correlates with a persistence of treatment-induced DNA damage. Therefore, this study warrants further investigations to elucidate whether such an adaptation could enhance the effectiveness of the standard clinical treatment regimen. In addition, a subpopulation of therapy resistant cells with EMT and cancer stem cell features was identified that was resistant to the standard treatment regimen but sensitive to pemetrexed pretreatment combined with cisplatin.

No MeSH data available.


Related in: MedlinePlus