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Targeted inhibition of FAK, PYK2 and BCL-XL synergistically enhances apoptosis in ovarian clear cell carcinoma cell lines.

Yoon H, Choi YL, Song JY, Do I, Kang SY, Ko YH, Song S, Kim BG - PLoS ONE (2014)

Bottom Line: We then searched for single agents that exhibited a synergistic effect on cell death in combination with PF271.We found that co-treatment of PF271 with ABT-737, a BCL-2/BCL-XL antagonist, was profoundly effective at inducing apoptosis.Mechanistically, PF271 treatment resulted in the transient down-regulation of the anti-apoptotic protein MCL1 via the PI3K/AKT pathway.

View Article: PubMed Central - PubMed

Affiliation: Institute for Refractory Cancer Research, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.

ABSTRACT
Ovarian clear cell carcinoma (OCCC) displays a higher resistance to first line chemotherapy, requiring the development of new therapeutics. We previously identified a frequent chromosomal gain at 8q24 that harbors the focal-adhesion kinase (FAK) gene; the potential of this gene as a therapeutic target remains to be evaluated in OCCCs. We first examined the dependence of OCCCs on FAK and the PI3K/AKT signaling pathway. FAK was overexpressed in 20% of 67 OCCC samples, and this overexpression was correlated with its copy number gain. FAK copy number gains and mutations in PIK3CA accounted for about 40% of OCCC samples, suggesting that the FAK/PI3K/AKT axis is an attractive candidate for targeted therapeutics. We, therefore, treated ovarian cancer cell lines, including OCCC subtypes, with the FAK inhibitors PF-562,271 (PF271), and PF-573,228 (PF228). Ovarian cancer cells were more sensitive to PF271 than PF228. We then searched for single agents that exhibited a synergistic effect on cell death in combination with PF271. We found that co-treatment of PF271 with ABT-737, a BCL-2/BCL-XL antagonist, was profoundly effective at inducing apoptosis. RMGI and OVISE cells were more sensitive to ABT-737 than OVMANA and SKOV3 cells, which have PIK3CA mutations. Mechanistically, PF271 treatment resulted in the transient down-regulation of the anti-apoptotic protein MCL1 via the PI3K/AKT pathway. Therefore, PF271/ABT-737 treatment led to the inhibition of the anti-apoptotic proteins MCL1 and BCL-XL/BCL-2. We suggest that pharmacological inhibition of BCL-XL and FAK/PYK2 can be a potential therapeutic strategy for the treatment of OCCC.

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FAK and BCL2/BCL-XL inhibitors induced apoptosis.A, OCCC cell lines were treated with 1 µM ABT-737 (A), 1 µM BEZ235 (B), or 5 µM PF271 (P), individually or in combinations (AB indicates ABT-737/BEZ235; AP, ABT-737/PF271; BP, BEZ235/PF271; ABP, ABT-737/BEZ235/PF271) at the same drug concentrations for 6 hr. Cell lysates were prepared and subjected to Western blot analysis using the indicated antibodies. Cleavage of caspase-3 (CASP3), caspase-8 (CASP8), PARP, and FAK indicated that apoptosis had occurred. BCL-2 was not expressed in OVMANA and OVISE. B, TOV21G, RMGI and OVISE cells were treated individually or in combination with 1 µM ABT-737, 1 µM BEZ235 and/or 5 µM PF271 for 6 hr. The cells were fixed with 70% ethanol, were stained with propidium iodide (PI) and analyzed using a FACS Calibur flow cytometer.
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pone-0088587-g005: FAK and BCL2/BCL-XL inhibitors induced apoptosis.A, OCCC cell lines were treated with 1 µM ABT-737 (A), 1 µM BEZ235 (B), or 5 µM PF271 (P), individually or in combinations (AB indicates ABT-737/BEZ235; AP, ABT-737/PF271; BP, BEZ235/PF271; ABP, ABT-737/BEZ235/PF271) at the same drug concentrations for 6 hr. Cell lysates were prepared and subjected to Western blot analysis using the indicated antibodies. Cleavage of caspase-3 (CASP3), caspase-8 (CASP8), PARP, and FAK indicated that apoptosis had occurred. BCL-2 was not expressed in OVMANA and OVISE. B, TOV21G, RMGI and OVISE cells were treated individually or in combination with 1 µM ABT-737, 1 µM BEZ235 and/or 5 µM PF271 for 6 hr. The cells were fixed with 70% ethanol, were stained with propidium iodide (PI) and analyzed using a FACS Calibur flow cytometer.

Mentions: To investigate the synergistic mechanism by which ABT-737 and PF271 induce apoptosis, we treated cells with ABT-737, PF271, and BEZ235 alone or in combinations for 6 hr (Fig. 5) and analyzed the cell lysates and cell cycle. Dramatic increases in the levels of cleaved caspase-8, caspase-3, PARP, and FAK, which are markers of the activation of apoptosis, were observed in the cells co-treated with ABT-737 and either PF271 or BEZ235, but not with PF271 and BEZ235 alone (Fig. 5A), which is in agreement with the result of the cytotoxicity test (Fig. 4B). ABT-737 alone induced to a minor extent cleavage of caspase-3 and caspase-8, PARP, and FAK in RMGI and OVISE cells, which seemed to be sensitive to ABT-737, but not in TOV21G and OVMANA cells (Fig. 5A). Interestingly, the levels of cleaved caspase-8 and PARP were correlated with the differential cytotoxicity (Figs. 4 and 5A), but the levels of p-AKT were not.


Targeted inhibition of FAK, PYK2 and BCL-XL synergistically enhances apoptosis in ovarian clear cell carcinoma cell lines.

Yoon H, Choi YL, Song JY, Do I, Kang SY, Ko YH, Song S, Kim BG - PLoS ONE (2014)

FAK and BCL2/BCL-XL inhibitors induced apoptosis.A, OCCC cell lines were treated with 1 µM ABT-737 (A), 1 µM BEZ235 (B), or 5 µM PF271 (P), individually or in combinations (AB indicates ABT-737/BEZ235; AP, ABT-737/PF271; BP, BEZ235/PF271; ABP, ABT-737/BEZ235/PF271) at the same drug concentrations for 6 hr. Cell lysates were prepared and subjected to Western blot analysis using the indicated antibodies. Cleavage of caspase-3 (CASP3), caspase-8 (CASP8), PARP, and FAK indicated that apoptosis had occurred. BCL-2 was not expressed in OVMANA and OVISE. B, TOV21G, RMGI and OVISE cells were treated individually or in combination with 1 µM ABT-737, 1 µM BEZ235 and/or 5 µM PF271 for 6 hr. The cells were fixed with 70% ethanol, were stained with propidium iodide (PI) and analyzed using a FACS Calibur flow cytometer.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3921183&req=5

pone-0088587-g005: FAK and BCL2/BCL-XL inhibitors induced apoptosis.A, OCCC cell lines were treated with 1 µM ABT-737 (A), 1 µM BEZ235 (B), or 5 µM PF271 (P), individually or in combinations (AB indicates ABT-737/BEZ235; AP, ABT-737/PF271; BP, BEZ235/PF271; ABP, ABT-737/BEZ235/PF271) at the same drug concentrations for 6 hr. Cell lysates were prepared and subjected to Western blot analysis using the indicated antibodies. Cleavage of caspase-3 (CASP3), caspase-8 (CASP8), PARP, and FAK indicated that apoptosis had occurred. BCL-2 was not expressed in OVMANA and OVISE. B, TOV21G, RMGI and OVISE cells were treated individually or in combination with 1 µM ABT-737, 1 µM BEZ235 and/or 5 µM PF271 for 6 hr. The cells were fixed with 70% ethanol, were stained with propidium iodide (PI) and analyzed using a FACS Calibur flow cytometer.
Mentions: To investigate the synergistic mechanism by which ABT-737 and PF271 induce apoptosis, we treated cells with ABT-737, PF271, and BEZ235 alone or in combinations for 6 hr (Fig. 5) and analyzed the cell lysates and cell cycle. Dramatic increases in the levels of cleaved caspase-8, caspase-3, PARP, and FAK, which are markers of the activation of apoptosis, were observed in the cells co-treated with ABT-737 and either PF271 or BEZ235, but not with PF271 and BEZ235 alone (Fig. 5A), which is in agreement with the result of the cytotoxicity test (Fig. 4B). ABT-737 alone induced to a minor extent cleavage of caspase-3 and caspase-8, PARP, and FAK in RMGI and OVISE cells, which seemed to be sensitive to ABT-737, but not in TOV21G and OVMANA cells (Fig. 5A). Interestingly, the levels of cleaved caspase-8 and PARP were correlated with the differential cytotoxicity (Figs. 4 and 5A), but the levels of p-AKT were not.

Bottom Line: We then searched for single agents that exhibited a synergistic effect on cell death in combination with PF271.We found that co-treatment of PF271 with ABT-737, a BCL-2/BCL-XL antagonist, was profoundly effective at inducing apoptosis.Mechanistically, PF271 treatment resulted in the transient down-regulation of the anti-apoptotic protein MCL1 via the PI3K/AKT pathway.

View Article: PubMed Central - PubMed

Affiliation: Institute for Refractory Cancer Research, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.

ABSTRACT
Ovarian clear cell carcinoma (OCCC) displays a higher resistance to first line chemotherapy, requiring the development of new therapeutics. We previously identified a frequent chromosomal gain at 8q24 that harbors the focal-adhesion kinase (FAK) gene; the potential of this gene as a therapeutic target remains to be evaluated in OCCCs. We first examined the dependence of OCCCs on FAK and the PI3K/AKT signaling pathway. FAK was overexpressed in 20% of 67 OCCC samples, and this overexpression was correlated with its copy number gain. FAK copy number gains and mutations in PIK3CA accounted for about 40% of OCCC samples, suggesting that the FAK/PI3K/AKT axis is an attractive candidate for targeted therapeutics. We, therefore, treated ovarian cancer cell lines, including OCCC subtypes, with the FAK inhibitors PF-562,271 (PF271), and PF-573,228 (PF228). Ovarian cancer cells were more sensitive to PF271 than PF228. We then searched for single agents that exhibited a synergistic effect on cell death in combination with PF271. We found that co-treatment of PF271 with ABT-737, a BCL-2/BCL-XL antagonist, was profoundly effective at inducing apoptosis. RMGI and OVISE cells were more sensitive to ABT-737 than OVMANA and SKOV3 cells, which have PIK3CA mutations. Mechanistically, PF271 treatment resulted in the transient down-regulation of the anti-apoptotic protein MCL1 via the PI3K/AKT pathway. Therefore, PF271/ABT-737 treatment led to the inhibition of the anti-apoptotic proteins MCL1 and BCL-XL/BCL-2. We suggest that pharmacological inhibition of BCL-XL and FAK/PYK2 can be a potential therapeutic strategy for the treatment of OCCC.

Show MeSH
Related in: MedlinePlus