PROTEASOME INHIBITION AND NOT NF-ΚB INHIBITION INDUCES APOPTOSIS TO RESISTANT CELLS IN...

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PROTEASOME INHIBITION AND NOT NF- PROTEASOME INHIBITION AND NOT NF- Κ Κ B INHIBITION INDUCES APOPTOSIS B INHIBITION INDUCES APOPTOSIS TO RESISTANT CELLS IN GLUCOCORTICOID-INDUCED APOPTOSIS TO RESISTANT CELLS IN GLUCOCORTICOID-INDUCED APOPTOSIS George I Lambrou George I Lambrou 1 1 , , Apostolos Zaravinos Apostolos Zaravinos 2 2 , Maria , Maria Adamaki Adamaki 1 1 , Dimitrios Spandidos , Dimitrios Spandidos 2 2 and Spiros and Spiros Vlahopoulos Vlahopoulos 1 1 1 1 1 st st Department of Pediatrics, University of Athens, Choremeio Research Laboratory, Department of Pediatrics, University of Athens, Choremeio Research Laboratory, 11527 Goudi, Athens. 11527 Goudi, Athens. 2 2 Department of Clinical Virology, University of Crete, School of Medicine, 71110 Department of Clinical Virology, University of Crete, School of Medicine, 71110 Heraklio, Crete, Greece. Heraklio, Crete, Greece. INTRODUCTION AIM OF STUDY MATERIALS AND METHODS The cell system used was the CCRF-CEM cell line. Cells were The cell system used was the CCRF-CEM cell line. Cells were cultured in RPMI-1640 medium with 15% FBS supplement. Cells were cultured in RPMI-1640 medium with 15% FBS supplement. Cells were treated with various concentrations of prednisolone, a treated with various concentrations of prednisolone, a glucocorticoid, MG132 (Sigma-Aldrich) a potent proteasome glucocorticoid, MG132 (Sigma-Aldrich) a potent proteasome inhibitor and Bay11 (Sigma-Aldrich), a known IKK kinase inhibitor and Bay11 (Sigma-Aldrich), a known IKK kinase inhibitor. Cells were counted every 24h with a NIHON-KOHDEN inhibitor. Cells were counted every 24h with a NIHON-KOHDEN CellTaq- CellTaq- a a cytometer. Samples were taken daily and apoptosis, as cytometer. Samples were taken daily and apoptosis, as defined by DNA fragmentation and cell cycle distribution were defined by DNA fragmentation and cell cycle distribution were estimated using a FC500 flow cytometer (Beckman Coulter). estimated using a FC500 flow cytometer (Beckman Coulter). RESULTS DISCUSSION Elucidating the mechanisms of resistance to GC-induced apoptosis is of crucial importance to the therapy of leukemia. We Elucidating the mechanisms of resistance to GC-induced apoptosis is of crucial importance to the therapy of leukemia. We have identified two potent molecules that would possibly play a role in eradicating resistance mechanisms in childhood have identified two potent molecules that would possibly play a role in eradicating resistance mechanisms in childhood leukemia. In particular, it appeared that both compounds exhibit an inhibitory effect on cell proliferation at least at the leukemia. In particular, it appeared that both compounds exhibit an inhibitory effect on cell proliferation at least at the highest concentrations used, indicating that highest concentrations used, indicating that they they both act on the cell cycle machinery. Yet, not both have the same effect both act on the cell cycle machinery. Yet, not both have the same effect on apoptosis. The MG132 compound appears to be more effective than the Bay11 compound. This is a strong indication that on apoptosis. The MG132 compound appears to be more effective than the Bay11 compound. This is a strong indication that sensitization to apoptosis is probably regulated through the proteasome pathway and not through the NFkB pathway. It is sensitization to apoptosis is probably regulated through the proteasome pathway and not through the NFkB pathway. It is however, possible that resistance to GC-induced apoptosis is induced through NFkB activation, yet sensitization does not however, possible that resistance to GC-induced apoptosis is induced through NFkB activation, yet sensitization does not pass through the same pathway regulatory mechanism. This makes the phenomenon more complicate and interesting at the same pass through the same pathway regulatory mechanism. This makes the phenomenon more complicate and interesting at the same time. Further investigations are required to elucidate the mechanisms of resistance to glucocorticoids. time. Further investigations are required to elucidate the mechanisms of resistance to glucocorticoids. The CCRF-CEM cell line it is known to exhibit resistance to GC-induced apoptosis. It is also known that the NF- The CCRF-CEM cell line it is known to exhibit resistance to GC-induced apoptosis. It is also known that the NF- κ κ B, B, transcription factor is an immediate target of the GR. Also, the proteasome mechanism is closely connected to the GR transcription factor is an immediate target of the GR. Also, the proteasome mechanism is closely connected to the GR pathway. It appeared that the present cell system appeared to be sensitive to concentrations of MG132 starting already from pathway. It appeared that the present cell system appeared to be sensitive to concentrations of MG132 starting already from 1uM, which 1uM, which is is a concentration that approaches the expected drug levels if administered to an organism. Cellular apoptosis, a concentration that approaches the expected drug levels if administered to an organism. Cellular apoptosis, as defined by DNA fragmentation reached 90%, while this effect was already evident from the very first hours of incubation. as defined by DNA fragmentation reached 90%, while this effect was already evident from the very first hours of incubation. On the other hand, inhibiting the IKK kinase, and subsequently the NF- On the other hand, inhibiting the IKK kinase, and subsequently the NF- κ κ B pathway, by inhibiting the transcription factor to B pathway, by inhibiting the transcription factor to enter the nucleus did not have the same result as, cells reached an apoptosis level of ~70%. Co-incubation of the inhibitors enter the nucleus did not have the same result as, cells reached an apoptosis level of ~70%. Co-incubation of the inhibitors with the GC did not affect the levels of observed cell death for the MG132 factor nor for the Bay11 inhibitor. with the GC did not affect the levels of observed cell death for the MG132 factor nor for the Bay11 inhibitor. Interestingly, both compounds manifested an inhibitory effect on cell proliferation, yet the MG132 compound induces Interestingly, both compounds manifested an inhibitory effect on cell proliferation, yet the MG132 compound induces apoptosis while Bay11 not in the same extent. apoptosis while Bay11 not in the same extent. Figure 1 Figure 1 . Proliferation assay of the . Proliferation assay of the CCRF-CEM cell line under Bay11 and MG132 CCRF-CEM cell line under Bay11 and MG132 treatments respectively treatments respectively Figure 2 Figure 2 . Apoptosis, as measured by . Apoptosis, as measured by DNA fragmentation, of the CCRF-CEM DNA fragmentation, of the CCRF-CEM cell line under Bay11 and MG132 cell line under Bay11 and MG132 treatments respectively. treatments respectively. Figure 4 Figure 4 . Apoptosis, as measured by . Apoptosis, as measured by DNA fragmentation, of the CCRF-CEM DNA fragmentation, of the CCRF-CEM cell line under MG132 and prednisolone cell line under MG132 and prednisolone incubation. incubation. Figure 3 Figure 3 . Comparative apoptosis, as . Comparative apoptosis, as measured by DNA fragmentation and trypan measured by DNA fragmentation and trypan blue, of the CCRF-CEM cell line under blue, of the CCRF-CEM cell line under MG132 treatments. MG132 treatments.

Transcript of PROTEASOME INHIBITION AND NOT NF-ΚB INHIBITION INDUCES APOPTOSIS TO RESISTANT CELLS IN...

Page 1: PROTEASOME INHIBITION AND NOT NF-ΚB INHIBITION INDUCES APOPTOSIS TO RESISTANT CELLS IN GLUCOCORTICOID-INDUCED APOPTOSIS George I Lambrou 1, Apostolos Zaravinos.

PROTEASOME INHIBITION AND NOT NF-PROTEASOME INHIBITION AND NOT NF-ΚΚB INHIBITION INDUCES APOPTOSIS TO B INHIBITION INDUCES APOPTOSIS TO RESISTANT CELLS IN GLUCOCORTICOID-INDUCED APOPTOSIS RESISTANT CELLS IN GLUCOCORTICOID-INDUCED APOPTOSIS

George I LambrouGeorge I Lambrou11, , Apostolos ZaravinosApostolos Zaravinos22, Maria Adamaki, Maria Adamaki11 , Dimitrios , Dimitrios SpandidosSpandidos22 and Spiros Vlahopoulos and Spiros Vlahopoulos11

11 1 1stst Department of Pediatrics, University of Athens, Choremeio Research Laboratory, 11527 Goudi, Athens. Department of Pediatrics, University of Athens, Choremeio Research Laboratory, 11527 Goudi, Athens.2 2 Department of Clinical Virology, University of Crete, School of Medicine, 71110 Heraklio, Crete, Greece.Department of Clinical Virology, University of Crete, School of Medicine, 71110 Heraklio, Crete, Greece.

INTRODUCTION

AIM OF STUDY MATERIALS AND METHODSThe cell system used was the CCRF-CEM cell line. Cells were cultured in RPMI-1640 medium The cell system used was the CCRF-CEM cell line. Cells were cultured in RPMI-1640 medium with 15% FBS supplement. Cells were treated with various concentrations of prednisolone, a with 15% FBS supplement. Cells were treated with various concentrations of prednisolone, a glucocorticoid, MG132 (Sigma-Aldrich) a potent proteasome inhibitor and Bay11 (Sigma-glucocorticoid, MG132 (Sigma-Aldrich) a potent proteasome inhibitor and Bay11 (Sigma-Aldrich), a known IKK kinase inhibitor. Cells were counted every 24h with a NIHON-KOHDEN Aldrich), a known IKK kinase inhibitor. Cells were counted every 24h with a NIHON-KOHDEN CellTaq-CellTaq-aa cytometer. Samples were taken daily and apoptosis, as defined by DNA fragmentation cytometer. Samples were taken daily and apoptosis, as defined by DNA fragmentation and cell cycle distribution were estimated using a FC500 flow cytometer (Beckman Coulter).and cell cycle distribution were estimated using a FC500 flow cytometer (Beckman Coulter).

RESULTS

DISCUSSION

Elucidating the mechanisms of resistance to GC-induced apoptosis is of crucial importance to the therapy of leukemia. We have identified two potent molecules that would possibly Elucidating the mechanisms of resistance to GC-induced apoptosis is of crucial importance to the therapy of leukemia. We have identified two potent molecules that would possibly play a role in eradicating resistance mechanisms in childhood leukemia. In particular, it appeared that both compounds exhibit an inhibitory effect on cell proliferation at least at the play a role in eradicating resistance mechanisms in childhood leukemia. In particular, it appeared that both compounds exhibit an inhibitory effect on cell proliferation at least at the highest concentrations used, indicating that highest concentrations used, indicating that theythey both act on the cell cycle machinery. Yet, not both have the same effect on apoptosis. The MG132 compound appears to be more both act on the cell cycle machinery. Yet, not both have the same effect on apoptosis. The MG132 compound appears to be more effective than the Bay11 compound. This is a strong indication that sensitization to apoptosis is probably regulated through the proteasome pathway and not through the NFkB pathway. effective than the Bay11 compound. This is a strong indication that sensitization to apoptosis is probably regulated through the proteasome pathway and not through the NFkB pathway. It is however, possible that resistance to GC-induced apoptosis is induced through NFkB activation, yet sensitization does not pass through the same pathway regulatory mechanism. It is however, possible that resistance to GC-induced apoptosis is induced through NFkB activation, yet sensitization does not pass through the same pathway regulatory mechanism. This makes the phenomenon more complicate and interesting at the same time. Further investigations are required to elucidate the mechanisms of resistance to glucocorticoids.This makes the phenomenon more complicate and interesting at the same time. Further investigations are required to elucidate the mechanisms of resistance to glucocorticoids.

The CCRF-CEM cell line it is known to exhibit resistance to GC-induced apoptosis. It is also known that the NF-The CCRF-CEM cell line it is known to exhibit resistance to GC-induced apoptosis. It is also known that the NF-κκB, transcription factor is an immediate target of the GR. Also, the B, transcription factor is an immediate target of the GR. Also, the proteasome mechanism is closely connected to the GR pathway. It appeared that the present cell system appeared to be sensitive to concentrations of MG132 starting already from 1uM, proteasome mechanism is closely connected to the GR pathway. It appeared that the present cell system appeared to be sensitive to concentrations of MG132 starting already from 1uM, which which isis a concentration that approaches the expected drug levels if administered to an organism. Cellular apoptosis, as defined by DNA fragmentation reached 90%, while this effect a concentration that approaches the expected drug levels if administered to an organism. Cellular apoptosis, as defined by DNA fragmentation reached 90%, while this effect was already evident from the very first hours of incubation. On the other hand, inhibiting the IKK kinase, and subsequently the NF-was already evident from the very first hours of incubation. On the other hand, inhibiting the IKK kinase, and subsequently the NF-κκB pathway, by inhibiting the transcription factor to B pathway, by inhibiting the transcription factor to enter the nucleus did not have the same result as, cells reached an apoptosis level of ~70%. Co-incubation of the inhibitors with the GC did not affect the levels of observed cell death for enter the nucleus did not have the same result as, cells reached an apoptosis level of ~70%. Co-incubation of the inhibitors with the GC did not affect the levels of observed cell death for the MG132 factor nor for the Bay11 inhibitor. Interestingly, both compounds manifested an inhibitory effect on cell proliferation, yet the MG132 compound induces apoptosis while the MG132 factor nor for the Bay11 inhibitor. Interestingly, both compounds manifested an inhibitory effect on cell proliferation, yet the MG132 compound induces apoptosis while Bay11 not in the same extent. Bay11 not in the same extent.

Figure 1Figure 1. Proliferation assay of the CCRF-CEM cell line . Proliferation assay of the CCRF-CEM cell line under Bay11 and MG132 treatments respectively under Bay11 and MG132 treatments respectively

Figure 2Figure 2. Apoptosis, as measured by DNA . Apoptosis, as measured by DNA fragmentation, of the CCRF-CEM cell line under fragmentation, of the CCRF-CEM cell line under Bay11 and MG132 treatments respectively. Bay11 and MG132 treatments respectively.

Figure 4Figure 4. Apoptosis, as measured by DNA . Apoptosis, as measured by DNA fragmentation, of the CCRF-CEM cell line under fragmentation, of the CCRF-CEM cell line under MG132 and prednisolone incubation.MG132 and prednisolone incubation.

Figure 3Figure 3. Comparative apoptosis, as measured by DNA . Comparative apoptosis, as measured by DNA fragmentation and trypan blue, of the CCRF-CEM cell line fragmentation and trypan blue, of the CCRF-CEM cell line under MG132 treatments. under MG132 treatments.