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Chemotherapy drug response in ovarian cancer cells strictly depends on a cathepsin D-Bax activation loop.

Castino R, Peracchio C, Salini A, Nicotra G, Trincheri NF, Démoz M, Valente G, Isidoro C - J. Cell. Mol. Med. (2008)

Bottom Line: We hypothesized that the two cell lines differ in their ability to activate the intrinsic death pathway and have, therefore, dissected the lysosome-mitochondrion signalling pathway by pharmacological inhibition or genetic manipulation of key regulators and executioners.The present data are consistent with a model in which on treatment with a cytotoxic drug the activation of a CD-Bax loop leads to the generalized permeabilization of lysosomes and eventually of mitochondria, thus reaching the point of no return, and culminates with the activation of the caspase cascade.Our data also imply that dysfunctional permeabilization of lysosomes contributes to the development of chemoresistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Sciences, "A. Avogadro" University, Novara, Italy.

ABSTRACT
The ovarian cancer cell lines A2780 (wild-type p53) and NIHOVCAR3 (mutated p53) showed, respectively, sensitivity and resistance towards several chemotherapy drugs. We hypothesized that the two cell lines differ in their ability to activate the intrinsic death pathway and have, therefore, dissected the lysosome-mitochondrion signalling pathway by pharmacological inhibition or genetic manipulation of key regulators and executioners. Biochemical and morphological confocal fluorescence studies showed that: (1) In A2780 cells bcl-2 is expressed at an undetectable level, whereas Bax is expressed at a rather high level; by contrast, bcl-2 is highly expressed and Bax is expressed at extremely low levels in NIHOVCAR3 cells; (2) Chemotherapy treatment reduced the expression of bcl-2 in NIHOVCAR3 cells, yet these cells resisted to drug toxicity; (3) Cathepsin D (CD), not cathepsin B or L, mediates the activation of the mitochondrial intrinsic death pathway in A2780 cells; (4) Lysosome leakage and cytosolic relocation of CD occurs in the chemosensitive A2780 cells, not in the chemoresistant NIHOVCAR3 cells; (5) Bax is essential for the permeabilization of both lysosomes and mitochondria in A2780 cells exposed to chemotherapy drugs; (6) CD activity is mandatory for the oligomerization of Bax on both mitochondrial and lysosomal membranes; (7) Bax activation did not occur in the resistant NIHOVCAR3 cells despite their high content in CD. The present data are consistent with a model in which on treatment with a cytotoxic drug the activation of a CD-Bax loop leads to the generalized permeabilization of lysosomes and eventually of mitochondria, thus reaching the point of no return, and culminates with the activation of the caspase cascade. Our data also imply that dysfunctional permeabilization of lysosomes contributes to the development of chemoresistance.

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Cathepsin D triggers Bax-mediated permeabilization of mitochondria and TUNEL-positive cell death in response to VP16. A2780 cells were let adhere on coverslips and pre-incubated for 16 hrs with Pst (A) or sham-transfected or transfected with a CD-specific siRNA (B). The cells were then treated or not with VP16 for 24 hrs and thereafter processed for mitotracker staining and Bax immunofluorescence. Both genetic down-regulation and pharmacological inhibition of CD prevented the activation and oligomerization of Bax onto mitochondria induced by VP16. (C) A2780 cells adherent on coverslips were exposed for 24 hrs to VP16 and then stained with the TUNEL technique that demonstrated the presence of nicked DNA in apoptotic cells. Images are representative of three separate experiments. Selected fields are shown. Note that only cells still adherent on coverslips were labelled (detached cells were discarded).
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fig04: Cathepsin D triggers Bax-mediated permeabilization of mitochondria and TUNEL-positive cell death in response to VP16. A2780 cells were let adhere on coverslips and pre-incubated for 16 hrs with Pst (A) or sham-transfected or transfected with a CD-specific siRNA (B). The cells were then treated or not with VP16 for 24 hrs and thereafter processed for mitotracker staining and Bax immunofluorescence. Both genetic down-regulation and pharmacological inhibition of CD prevented the activation and oligomerization of Bax onto mitochondria induced by VP16. (C) A2780 cells adherent on coverslips were exposed for 24 hrs to VP16 and then stained with the TUNEL technique that demonstrated the presence of nicked DNA in apoptotic cells. Images are representative of three separate experiments. Selected fields are shown. Note that only cells still adherent on coverslips were labelled (detached cells were discarded).

Mentions: Bax has been shown to promote mitochondrial permeabilization and relocation of pro-apoptotic proteins from mitochondria into the cytosol [46]. In various paradigms of cytotoxic treatments conformational changes and oligomerization on mitochondrial membrane of Bax were shown to depend on CD [15, 28, 29]. We thus tested whether CD was implicated in Bax-dependent activation of the intrinsic death pathway in A2780 cells. Both Pst-mediated inhibition of CD activity and siRNA-mediated silencing of CD expression prevented the oligomerization of Bax and the mitochondria permeabilization induced by VP16 in A2780 (Fig. 4A and B). Caspase 3-mediated hydrolysis of poly(ADP-ribose) polymerase, an enzyme involved in DNA repair, results in the accumulation of nicked DNA in nuclei of apoptotic cells that can be demonstrated with the TUNEL technique. Thus, to definitely prove the involvement of CD in caspase 3 activation we checked for the presence of TUNEL-positive cells in A2780 cultures exposed to VP16 along with Pst. As expected in case of true caspase-dependent apoptosis, A2780 cells exposed to VP16 showed TUNEL positive (Fig. 4C). However, when the cytotoxic treatment was performed in the presence of Pst the cell monolayer appeared well preserved and nuclei did not stain for TUNEL (Fig. 4C), indicating that in these cells activation of caspase 3 was precluded. These data indicate that in chemotherapy drug-sensitive cells activation of the CD-mediated proteolytic pathway precedes that of the caspase cascade.


Chemotherapy drug response in ovarian cancer cells strictly depends on a cathepsin D-Bax activation loop.

Castino R, Peracchio C, Salini A, Nicotra G, Trincheri NF, Démoz M, Valente G, Isidoro C - J. Cell. Mol. Med. (2008)

Cathepsin D triggers Bax-mediated permeabilization of mitochondria and TUNEL-positive cell death in response to VP16. A2780 cells were let adhere on coverslips and pre-incubated for 16 hrs with Pst (A) or sham-transfected or transfected with a CD-specific siRNA (B). The cells were then treated or not with VP16 for 24 hrs and thereafter processed for mitotracker staining and Bax immunofluorescence. Both genetic down-regulation and pharmacological inhibition of CD prevented the activation and oligomerization of Bax onto mitochondria induced by VP16. (C) A2780 cells adherent on coverslips were exposed for 24 hrs to VP16 and then stained with the TUNEL technique that demonstrated the presence of nicked DNA in apoptotic cells. Images are representative of three separate experiments. Selected fields are shown. Note that only cells still adherent on coverslips were labelled (detached cells were discarded).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4496106&req=5

fig04: Cathepsin D triggers Bax-mediated permeabilization of mitochondria and TUNEL-positive cell death in response to VP16. A2780 cells were let adhere on coverslips and pre-incubated for 16 hrs with Pst (A) or sham-transfected or transfected with a CD-specific siRNA (B). The cells were then treated or not with VP16 for 24 hrs and thereafter processed for mitotracker staining and Bax immunofluorescence. Both genetic down-regulation and pharmacological inhibition of CD prevented the activation and oligomerization of Bax onto mitochondria induced by VP16. (C) A2780 cells adherent on coverslips were exposed for 24 hrs to VP16 and then stained with the TUNEL technique that demonstrated the presence of nicked DNA in apoptotic cells. Images are representative of three separate experiments. Selected fields are shown. Note that only cells still adherent on coverslips were labelled (detached cells were discarded).
Mentions: Bax has been shown to promote mitochondrial permeabilization and relocation of pro-apoptotic proteins from mitochondria into the cytosol [46]. In various paradigms of cytotoxic treatments conformational changes and oligomerization on mitochondrial membrane of Bax were shown to depend on CD [15, 28, 29]. We thus tested whether CD was implicated in Bax-dependent activation of the intrinsic death pathway in A2780 cells. Both Pst-mediated inhibition of CD activity and siRNA-mediated silencing of CD expression prevented the oligomerization of Bax and the mitochondria permeabilization induced by VP16 in A2780 (Fig. 4A and B). Caspase 3-mediated hydrolysis of poly(ADP-ribose) polymerase, an enzyme involved in DNA repair, results in the accumulation of nicked DNA in nuclei of apoptotic cells that can be demonstrated with the TUNEL technique. Thus, to definitely prove the involvement of CD in caspase 3 activation we checked for the presence of TUNEL-positive cells in A2780 cultures exposed to VP16 along with Pst. As expected in case of true caspase-dependent apoptosis, A2780 cells exposed to VP16 showed TUNEL positive (Fig. 4C). However, when the cytotoxic treatment was performed in the presence of Pst the cell monolayer appeared well preserved and nuclei did not stain for TUNEL (Fig. 4C), indicating that in these cells activation of caspase 3 was precluded. These data indicate that in chemotherapy drug-sensitive cells activation of the CD-mediated proteolytic pathway precedes that of the caspase cascade.

Bottom Line: We hypothesized that the two cell lines differ in their ability to activate the intrinsic death pathway and have, therefore, dissected the lysosome-mitochondrion signalling pathway by pharmacological inhibition or genetic manipulation of key regulators and executioners.The present data are consistent with a model in which on treatment with a cytotoxic drug the activation of a CD-Bax loop leads to the generalized permeabilization of lysosomes and eventually of mitochondria, thus reaching the point of no return, and culminates with the activation of the caspase cascade.Our data also imply that dysfunctional permeabilization of lysosomes contributes to the development of chemoresistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Sciences, "A. Avogadro" University, Novara, Italy.

ABSTRACT
The ovarian cancer cell lines A2780 (wild-type p53) and NIHOVCAR3 (mutated p53) showed, respectively, sensitivity and resistance towards several chemotherapy drugs. We hypothesized that the two cell lines differ in their ability to activate the intrinsic death pathway and have, therefore, dissected the lysosome-mitochondrion signalling pathway by pharmacological inhibition or genetic manipulation of key regulators and executioners. Biochemical and morphological confocal fluorescence studies showed that: (1) In A2780 cells bcl-2 is expressed at an undetectable level, whereas Bax is expressed at a rather high level; by contrast, bcl-2 is highly expressed and Bax is expressed at extremely low levels in NIHOVCAR3 cells; (2) Chemotherapy treatment reduced the expression of bcl-2 in NIHOVCAR3 cells, yet these cells resisted to drug toxicity; (3) Cathepsin D (CD), not cathepsin B or L, mediates the activation of the mitochondrial intrinsic death pathway in A2780 cells; (4) Lysosome leakage and cytosolic relocation of CD occurs in the chemosensitive A2780 cells, not in the chemoresistant NIHOVCAR3 cells; (5) Bax is essential for the permeabilization of both lysosomes and mitochondria in A2780 cells exposed to chemotherapy drugs; (6) CD activity is mandatory for the oligomerization of Bax on both mitochondrial and lysosomal membranes; (7) Bax activation did not occur in the resistant NIHOVCAR3 cells despite their high content in CD. The present data are consistent with a model in which on treatment with a cytotoxic drug the activation of a CD-Bax loop leads to the generalized permeabilization of lysosomes and eventually of mitochondria, thus reaching the point of no return, and culminates with the activation of the caspase cascade. Our data also imply that dysfunctional permeabilization of lysosomes contributes to the development of chemoresistance.

Show MeSH
Related in: MedlinePlus