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Mitochondrial Malfunctioning, Proteasome Arrest and Apoptosis in Cancer Cells by Focused Intracellular Generation of Oxygen Radicals.

Postiglione I, Chiaviello A, Barra F, Roscetto E, Soriano AA, Catania MR, Palumbo G, Pierantoni GM - Int J Mol Sci (2015)

Bottom Line: A similar result is obtained by treating cells with Antimycin and Rotenone, indicating, as a common denominator of this effect, the ATP decrease.Both inhibitors, however, were more toxic to cells as the recovery of proteasomal activity was incomplete.The evaluation of the mitochondrial membrane depolarization, residual proteasome and mitochondrial enzymatic activities, colony-forming capabilities, and changes in protein expression profiles in A549 and H1299 cells under a combined therapeutic regimen gave results consistent with our hypothesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples 80131, Italy. ilariapostiglione@gmail.com.

ABSTRACT
Photofrin/photodynamic therapy (PDT) at sub-lethal doses induced a transient stall in proteasome activity in surviving A549 (p53(+/+)) and H1299 (p53(-/-)) cells as indicated by the time-dependent decline/recovery of chymotrypsin-like activity. Indeed, within 3 h of incubation, Photofrin invaded the cytoplasm and localized preferentially within the mitochondria. Its light activation determined a decrease in mitochondrial membrane potential and a reversible arrest in proteasomal activity. A similar result is obtained by treating cells with Antimycin and Rotenone, indicating, as a common denominator of this effect, the ATP decrease. Both inhibitors, however, were more toxic to cells as the recovery of proteasomal activity was incomplete. We evaluated whether combining PDT (which is a treatment for killing tumor cells, per se, and inducing proteasome arrest in the surviving ones) with Bortezomib doses capable of sustaining the stall would protract the arrest with sufficient time to induce apoptosis in remaining cells. The evaluation of the mitochondrial membrane depolarization, residual proteasome and mitochondrial enzymatic activities, colony-forming capabilities, and changes in protein expression profiles in A549 and H1299 cells under a combined therapeutic regimen gave results consistent with our hypothesis.

No MeSH data available.


Related in: MedlinePlus

(A) Expression profiles of p27 and IκBα in H1299 and A549 cells incubated with Bortezomib (BZT) at the indicated concentrations (lanes 2–4) and after 24 h of release in fresh medium (lanes 5–7). Tubulin was used as a loading control; (B) Reversible effects of Bortezomib at various concentrations (3 h incubation) on cell viability of H1299 and A549 cells (XTT assay); the analyses were performed after 24 h of release in fresh medium; (C) Expression profile of PARP (poly ADP-ribose polymerase) in H1299 (lanes 1–4) and A549 cells (lanes 5–8) incubated with BZT at the indicated concentrations. Tubulin was used as gel loading control.
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ijms-16-20375-f004: (A) Expression profiles of p27 and IκBα in H1299 and A549 cells incubated with Bortezomib (BZT) at the indicated concentrations (lanes 2–4) and after 24 h of release in fresh medium (lanes 5–7). Tubulin was used as a loading control; (B) Reversible effects of Bortezomib at various concentrations (3 h incubation) on cell viability of H1299 and A549 cells (XTT assay); the analyses were performed after 24 h of release in fresh medium; (C) Expression profile of PARP (poly ADP-ribose polymerase) in H1299 (lanes 1–4) and A549 cells (lanes 5–8) incubated with BZT at the indicated concentrations. Tubulin was used as gel loading control.

Mentions: Among the numerous in vitro studies that concern the effect of Bortezomib on cell lines, the screening performed in the 1990s on a panel of 60 cell lines by the National Cancer Institute is particularly important. It showed that Bortezomib potently inhibited the growth of all cells at a concentration around 7–10 nM [32,35,36]. Keeping in mind the use of Bortezomib in combination with PDT, we first decided to analyze the effects of the drug alone in H1299 and A549 lung adenocarcinoma cell lines. For this purpose, we exposed these cells to Bortezomib (2.5, 5.0, and 10 nM) for 3 h and estimated p27, a cyclin dependent kinase (Cdk) inhibitor protein, and IκBα expression levels in both cell lines by Western blot. As shown in Figure 4A (lanes 2–4), the expression of these proteins increased in both H1299 and A549 cells within the first 3 h at all employed concentrations, suggesting a significant inhibition of proteasome activity. The hypothesized arrest of proteasome in these conditions has been confirmed by direct chymotrypsin-like activity quantitative assay (not shown). In these conditions, however, we could not demonstrate any sign of cytotoxicity as assessed by XTT assay and, more importantly, we could not demonstrate any sign of apoptosis as suggested by the absence of PARP (poly ADP-ribose polymerase) cleavage (Figure 4B,C).


Mitochondrial Malfunctioning, Proteasome Arrest and Apoptosis in Cancer Cells by Focused Intracellular Generation of Oxygen Radicals.

Postiglione I, Chiaviello A, Barra F, Roscetto E, Soriano AA, Catania MR, Palumbo G, Pierantoni GM - Int J Mol Sci (2015)

(A) Expression profiles of p27 and IκBα in H1299 and A549 cells incubated with Bortezomib (BZT) at the indicated concentrations (lanes 2–4) and after 24 h of release in fresh medium (lanes 5–7). Tubulin was used as a loading control; (B) Reversible effects of Bortezomib at various concentrations (3 h incubation) on cell viability of H1299 and A549 cells (XTT assay); the analyses were performed after 24 h of release in fresh medium; (C) Expression profile of PARP (poly ADP-ribose polymerase) in H1299 (lanes 1–4) and A549 cells (lanes 5–8) incubated with BZT at the indicated concentrations. Tubulin was used as gel loading control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4613209&req=5

ijms-16-20375-f004: (A) Expression profiles of p27 and IκBα in H1299 and A549 cells incubated with Bortezomib (BZT) at the indicated concentrations (lanes 2–4) and after 24 h of release in fresh medium (lanes 5–7). Tubulin was used as a loading control; (B) Reversible effects of Bortezomib at various concentrations (3 h incubation) on cell viability of H1299 and A549 cells (XTT assay); the analyses were performed after 24 h of release in fresh medium; (C) Expression profile of PARP (poly ADP-ribose polymerase) in H1299 (lanes 1–4) and A549 cells (lanes 5–8) incubated with BZT at the indicated concentrations. Tubulin was used as gel loading control.
Mentions: Among the numerous in vitro studies that concern the effect of Bortezomib on cell lines, the screening performed in the 1990s on a panel of 60 cell lines by the National Cancer Institute is particularly important. It showed that Bortezomib potently inhibited the growth of all cells at a concentration around 7–10 nM [32,35,36]. Keeping in mind the use of Bortezomib in combination with PDT, we first decided to analyze the effects of the drug alone in H1299 and A549 lung adenocarcinoma cell lines. For this purpose, we exposed these cells to Bortezomib (2.5, 5.0, and 10 nM) for 3 h and estimated p27, a cyclin dependent kinase (Cdk) inhibitor protein, and IκBα expression levels in both cell lines by Western blot. As shown in Figure 4A (lanes 2–4), the expression of these proteins increased in both H1299 and A549 cells within the first 3 h at all employed concentrations, suggesting a significant inhibition of proteasome activity. The hypothesized arrest of proteasome in these conditions has been confirmed by direct chymotrypsin-like activity quantitative assay (not shown). In these conditions, however, we could not demonstrate any sign of cytotoxicity as assessed by XTT assay and, more importantly, we could not demonstrate any sign of apoptosis as suggested by the absence of PARP (poly ADP-ribose polymerase) cleavage (Figure 4B,C).

Bottom Line: A similar result is obtained by treating cells with Antimycin and Rotenone, indicating, as a common denominator of this effect, the ATP decrease.Both inhibitors, however, were more toxic to cells as the recovery of proteasomal activity was incomplete.The evaluation of the mitochondrial membrane depolarization, residual proteasome and mitochondrial enzymatic activities, colony-forming capabilities, and changes in protein expression profiles in A549 and H1299 cells under a combined therapeutic regimen gave results consistent with our hypothesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples 80131, Italy. ilariapostiglione@gmail.com.

ABSTRACT
Photofrin/photodynamic therapy (PDT) at sub-lethal doses induced a transient stall in proteasome activity in surviving A549 (p53(+/+)) and H1299 (p53(-/-)) cells as indicated by the time-dependent decline/recovery of chymotrypsin-like activity. Indeed, within 3 h of incubation, Photofrin invaded the cytoplasm and localized preferentially within the mitochondria. Its light activation determined a decrease in mitochondrial membrane potential and a reversible arrest in proteasomal activity. A similar result is obtained by treating cells with Antimycin and Rotenone, indicating, as a common denominator of this effect, the ATP decrease. Both inhibitors, however, were more toxic to cells as the recovery of proteasomal activity was incomplete. We evaluated whether combining PDT (which is a treatment for killing tumor cells, per se, and inducing proteasome arrest in the surviving ones) with Bortezomib doses capable of sustaining the stall would protract the arrest with sufficient time to induce apoptosis in remaining cells. The evaluation of the mitochondrial membrane depolarization, residual proteasome and mitochondrial enzymatic activities, colony-forming capabilities, and changes in protein expression profiles in A549 and H1299 cells under a combined therapeutic regimen gave results consistent with our hypothesis.

No MeSH data available.


Related in: MedlinePlus