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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, not cathepsin B, mediates chemotherapy drug toxicity in A2780 cells. (A) A2780 cells and OVCAR cells plated on Petri dishes were exposed or not for 24 hrs to VP16 or taxol (Tax) in the absence or the presence of the CD inhibitor Pst or the CB inhibitor CA074Me (CA). At the end, cells were harvested and counted. The experiment was repeated four times in triplicate. Data are given as mean ± S.D. In A2780 cells (top panel) both VP16 and taxol induced cell loss, which was prevented by Pst not by Ca074Me (compare final density at day 1 in treated versus initial density at d0 in cultures; note that Pst abrogated cell death by the chemotherapy drugs though it not completely rescued the cells from a growth block). Inhibition of CB and CL, on the other hand, did not protect from drug toxicity and, on itself, caused some cell loss in A2780 culture. In OVCAR cells (bottom panel) VP16 and taxol were not toxic and no effect were exerted by the cathepsin inhibitors either alone or in combination with the chemotherapy drugs. (B) A2780 cells plated on Petri dishes were transfected with a control duplex RNA (sham) or a CD specific siRNA and 24 hrs later incubated in fresh medium for further 24 hrs in the absence or in the presence of VP16 or taxol (Tax). Prior to start the treatment, CD activity was assayed in parallel homogenates to ascertain down-regulation of CD in siRNA-transfected cultures (upper panel). At the end of the incubation, cell survival was estimated counting the adherent trypan-blue excluding cells (lower panel). Data (mean ± S.D.) are given as percentage of controls and arise from three independent experiments.
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fig03: Cathepsin D, not cathepsin B, mediates chemotherapy drug toxicity in A2780 cells. (A) A2780 cells and OVCAR cells plated on Petri dishes were exposed or not for 24 hrs to VP16 or taxol (Tax) in the absence or the presence of the CD inhibitor Pst or the CB inhibitor CA074Me (CA). At the end, cells were harvested and counted. The experiment was repeated four times in triplicate. Data are given as mean ± S.D. In A2780 cells (top panel) both VP16 and taxol induced cell loss, which was prevented by Pst not by Ca074Me (compare final density at day 1 in treated versus initial density at d0 in cultures; note that Pst abrogated cell death by the chemotherapy drugs though it not completely rescued the cells from a growth block). Inhibition of CB and CL, on the other hand, did not protect from drug toxicity and, on itself, caused some cell loss in A2780 culture. In OVCAR cells (bottom panel) VP16 and taxol were not toxic and no effect were exerted by the cathepsin inhibitors either alone or in combination with the chemotherapy drugs. (B) A2780 cells plated on Petri dishes were transfected with a control duplex RNA (sham) or a CD specific siRNA and 24 hrs later incubated in fresh medium for further 24 hrs in the absence or in the presence of VP16 or taxol (Tax). Prior to start the treatment, CD activity was assayed in parallel homogenates to ascertain down-regulation of CD in siRNA-transfected cultures (upper panel). At the end of the incubation, cell survival was estimated counting the adherent trypan-blue excluding cells (lower panel). Data (mean ± S.D.) are given as percentage of controls and arise from three independent experiments.

Mentions: Preliminary experiments in which the cells were exposed for 24 to 72 hrs to increasing doses of cis-platinum (0.5–5.0 μM), resveratrol (10–200 μM), VP16 (1–20 μM) or taxol (10–100 nM) revealed that A2780 cells are sensitive whereas OVCAR cells are resistant towards all tested chemotherapy drugs. These drugs exert their cytotoxicity through the activation of the intrinsic apoptosis pathway [12, 22, 28, 31, 32]. To identify the molecular basis for the different sensitivity to chemotherapy drugs, we first explored the functionality of this pathway in the two cell lines. VP16, a drug widely used in the therapy of ovarian cancers that show resistance to cis-Platinum [25], was employed as a paradigm of anticancer drug. In some experiments we also included taxol, which also is employed in the second line therapy of drug resistant ovarian cancers [4]. For the cytotoxic treatments, we chose the lowest dose of VP16 and of taxol that revealed to be most effective in a 24-hr incubation, i.e. 10 μM VP16 and 50 nM taxol, in the chemosensitive A2780 cell line. Both the treatments elicited an increased expression of p53 in the two cell lines, although this effect was prominent in A2780 cells (Fig. 1A). Counting of viable cells (data not shown and Fig. 3) and flow cytometry quantification of cell surface phos-phatidylserine (which mirrors apoptosis) indicated that after such a treatment a large proportion (∼50%) of A2780 cells underwent apoptotic cell death, whereas the cytotoxic effect of both drugs was negligible in OVCAR cells (Fig. 1B). In a separate experiment, the co-treatment of A2780 cells with the antiblastic drugs and the pan-caspase inhibitor ZVAD-fmk resulted in absence of cytotoxicity proving that cell death occurred via true apoptosis (not shown; see below). Parallel cultures in coverslips were double-stained with DAPI and mitotracker red, which allowed assessing the occurrence of mitochondrial membrane permeabilization in apoptotic cells. The latter were identified on the basis of the typical chromatin alterations (i.e. condensation and fragmentation) demonstrated by the fluorescent dye DAPI. The images in Fig. 1B show nuclei with condensation and fragmentation of the chromatin (arrows) in treated A2780, not in OVCAR cells. Of note, apoptosis in A2780 cells was associated with mitochondrial permeabilization, as demonstrated by loss of mitotracker staining (Fig. 1C). We then tested whether Bax, a bcl-2 family member involved in mitochondrial permeabilization, was differently targeted upon chemotherapy drug treatment in the two cell lines. The images shown in Fig. 1D clearly indicate that in A2780, not in OVCAR, Bax localizes onto mitochondrial membranes (and this coincides with permeabilization of mitochondria, as demonstrated by loss of mitotracker staining) in response to VP16.


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, not cathepsin B, mediates chemotherapy drug toxicity in A2780 cells. (A) A2780 cells and OVCAR cells plated on Petri dishes were exposed or not for 24 hrs to VP16 or taxol (Tax) in the absence or the presence of the CD inhibitor Pst or the CB inhibitor CA074Me (CA). At the end, cells were harvested and counted. The experiment was repeated four times in triplicate. Data are given as mean ± S.D. In A2780 cells (top panel) both VP16 and taxol induced cell loss, which was prevented by Pst not by Ca074Me (compare final density at day 1 in treated versus initial density at d0 in cultures; note that Pst abrogated cell death by the chemotherapy drugs though it not completely rescued the cells from a growth block). Inhibition of CB and CL, on the other hand, did not protect from drug toxicity and, on itself, caused some cell loss in A2780 culture. In OVCAR cells (bottom panel) VP16 and taxol were not toxic and no effect were exerted by the cathepsin inhibitors either alone or in combination with the chemotherapy drugs. (B) A2780 cells plated on Petri dishes were transfected with a control duplex RNA (sham) or a CD specific siRNA and 24 hrs later incubated in fresh medium for further 24 hrs in the absence or in the presence of VP16 or taxol (Tax). Prior to start the treatment, CD activity was assayed in parallel homogenates to ascertain down-regulation of CD in siRNA-transfected cultures (upper panel). At the end of the incubation, cell survival was estimated counting the adherent trypan-blue excluding cells (lower panel). Data (mean ± S.D.) are given as percentage of controls and arise from three independent experiments.
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Related In: Results  -  Collection

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fig03: Cathepsin D, not cathepsin B, mediates chemotherapy drug toxicity in A2780 cells. (A) A2780 cells and OVCAR cells plated on Petri dishes were exposed or not for 24 hrs to VP16 or taxol (Tax) in the absence or the presence of the CD inhibitor Pst or the CB inhibitor CA074Me (CA). At the end, cells were harvested and counted. The experiment was repeated four times in triplicate. Data are given as mean ± S.D. In A2780 cells (top panel) both VP16 and taxol induced cell loss, which was prevented by Pst not by Ca074Me (compare final density at day 1 in treated versus initial density at d0 in cultures; note that Pst abrogated cell death by the chemotherapy drugs though it not completely rescued the cells from a growth block). Inhibition of CB and CL, on the other hand, did not protect from drug toxicity and, on itself, caused some cell loss in A2780 culture. In OVCAR cells (bottom panel) VP16 and taxol were not toxic and no effect were exerted by the cathepsin inhibitors either alone or in combination with the chemotherapy drugs. (B) A2780 cells plated on Petri dishes were transfected with a control duplex RNA (sham) or a CD specific siRNA and 24 hrs later incubated in fresh medium for further 24 hrs in the absence or in the presence of VP16 or taxol (Tax). Prior to start the treatment, CD activity was assayed in parallel homogenates to ascertain down-regulation of CD in siRNA-transfected cultures (upper panel). At the end of the incubation, cell survival was estimated counting the adherent trypan-blue excluding cells (lower panel). Data (mean ± S.D.) are given as percentage of controls and arise from three independent experiments.
Mentions: Preliminary experiments in which the cells were exposed for 24 to 72 hrs to increasing doses of cis-platinum (0.5–5.0 μM), resveratrol (10–200 μM), VP16 (1–20 μM) or taxol (10–100 nM) revealed that A2780 cells are sensitive whereas OVCAR cells are resistant towards all tested chemotherapy drugs. These drugs exert their cytotoxicity through the activation of the intrinsic apoptosis pathway [12, 22, 28, 31, 32]. To identify the molecular basis for the different sensitivity to chemotherapy drugs, we first explored the functionality of this pathway in the two cell lines. VP16, a drug widely used in the therapy of ovarian cancers that show resistance to cis-Platinum [25], was employed as a paradigm of anticancer drug. In some experiments we also included taxol, which also is employed in the second line therapy of drug resistant ovarian cancers [4]. For the cytotoxic treatments, we chose the lowest dose of VP16 and of taxol that revealed to be most effective in a 24-hr incubation, i.e. 10 μM VP16 and 50 nM taxol, in the chemosensitive A2780 cell line. Both the treatments elicited an increased expression of p53 in the two cell lines, although this effect was prominent in A2780 cells (Fig. 1A). Counting of viable cells (data not shown and Fig. 3) and flow cytometry quantification of cell surface phos-phatidylserine (which mirrors apoptosis) indicated that after such a treatment a large proportion (∼50%) of A2780 cells underwent apoptotic cell death, whereas the cytotoxic effect of both drugs was negligible in OVCAR cells (Fig. 1B). In a separate experiment, the co-treatment of A2780 cells with the antiblastic drugs and the pan-caspase inhibitor ZVAD-fmk resulted in absence of cytotoxicity proving that cell death occurred via true apoptosis (not shown; see below). Parallel cultures in coverslips were double-stained with DAPI and mitotracker red, which allowed assessing the occurrence of mitochondrial membrane permeabilization in apoptotic cells. The latter were identified on the basis of the typical chromatin alterations (i.e. condensation and fragmentation) demonstrated by the fluorescent dye DAPI. The images in Fig. 1B show nuclei with condensation and fragmentation of the chromatin (arrows) in treated A2780, not in OVCAR cells. Of note, apoptosis in A2780 cells was associated with mitochondrial permeabilization, as demonstrated by loss of mitotracker staining (Fig. 1C). We then tested whether Bax, a bcl-2 family member involved in mitochondrial permeabilization, was differently targeted upon chemotherapy drug treatment in the two cell lines. The images shown in Fig. 1D clearly indicate that in A2780, not in OVCAR, Bax localizes onto mitochondrial membranes (and this coincides with permeabilization of mitochondria, as demonstrated by loss of mitotracker staining) in response to VP16.

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