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Cisplatin-induced apoptosis proceeds by caspase-3-dependent and -independent pathways in cisplatin-resistant and -sensitive human ovarian cancer cell lines.
Cancer Res. 1999 Jul 01; 59(13):3077-83.CR

Abstract

We have assessed in detail the effect of cisplatin-activated programmed cell death in the cisplatin-sensitive human ovarian cancer cell line A2780 and two drug-resistant subclones, CP70 and C30. To determine whether the differential extent of apoptosis observed between the sensitive and resistant ovarian cancer cell lines was the result of dissimilar upstream signaling events, we assessed the execution of apoptotic events that precede target protein proteolysis and subsequent chromosomal DNA degradation. Proteolytic degradation of procaspase-3 was observed in both the CP70 and C30 cells following IC50 cisplatin treatment, whereas no proteolyzed caspase-3 subunits were detected in the A2780 cells. However, using a direct enzymatic assay measuring cleavage of the synthetic peptide substrate (N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide), activity was detected in extracts prepared from A2780 cells treated at the IC90 level of cisplatin and was 2-3-fold less than that of extracts prepared from CP70 and C30 cells. Because the activation of procaspase-3 by caspase-9 requires the release of cytochrome c into the cytoplasm, we determined the level of cytoplasmic cytochrome c in each cell line in response to cisplatin treatment. Consistent with the caspase-3 activation data, a very small increase in cytoplasmic cytochrome c was observed in A2780 cells following cisplatin treatment, whereas dramatic increases were evident in both the CP70 and C30 cell lines. The expression of the mitochondrial factors Bcl-2, Bcl-x, and Bax was determined because each has been implicated in the regulation or release of cytochrome c at the level of the mitochondria. Bcl-2 and Bcl-xL proteins remained relatively unchanged in expression for over 48 h after exposure to cisplatin in the A2780 cell lines. However, within the same time period, expression of Bcl-2 decreased in the CP70- and C30-resistant cell lines, whereas an increase in Bcl-xL expression was observed. Expression of the proapoptotic Bcl-xS protein was observed in only the resistant CP70 and C30 cell lines independent of cisplatin treatment. A change in the expression of Mr 24,000 Bax to a Mr 21,000 isoform was evidenced in the A2780 cells within 48 h of cisplatin treatment and, to a greater extent, in the CP70 and C30 cells, which also expressed a Mr 16,000 Bax variant. Evidence for an alternative apoptotic pathway in A2780 cells was obtained by demonstrating increased FADD expression in response to cisplatin treatment. These results support a model in which cisplatin-induced programmed cell death in the cisplatin-sensitive A2780 and -resistant CP70 and C30 cells proceeds via caspase-3-independent and -dependent pathways, respectively.

Authors+Show Affiliations

Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, Ohio 45435, USA.No affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

10397248

Citation

Henkels, K M., and J J. Turchi. "Cisplatin-induced Apoptosis Proceeds By Caspase-3-dependent and -independent Pathways in Cisplatin-resistant and -sensitive Human Ovarian Cancer Cell Lines." Cancer Research, vol. 59, no. 13, 1999, pp. 3077-83.
Henkels KM, Turchi JJ. Cisplatin-induced apoptosis proceeds by caspase-3-dependent and -independent pathways in cisplatin-resistant and -sensitive human ovarian cancer cell lines. Cancer Res. 1999;59(13):3077-83.
Henkels, K. M., & Turchi, J. J. (1999). Cisplatin-induced apoptosis proceeds by caspase-3-dependent and -independent pathways in cisplatin-resistant and -sensitive human ovarian cancer cell lines. Cancer Research, 59(13), 3077-83.
Henkels KM, Turchi JJ. Cisplatin-induced Apoptosis Proceeds By Caspase-3-dependent and -independent Pathways in Cisplatin-resistant and -sensitive Human Ovarian Cancer Cell Lines. Cancer Res. 1999 Jul 1;59(13):3077-83. PubMed PMID: 10397248.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Cisplatin-induced apoptosis proceeds by caspase-3-dependent and -independent pathways in cisplatin-resistant and -sensitive human ovarian cancer cell lines. AU - Henkels,K M, AU - Turchi,J J, PY - 1999/7/9/pubmed PY - 2000/4/15/medline PY - 1999/7/9/entrez SP - 3077 EP - 83 JF - Cancer research JO - Cancer Res VL - 59 IS - 13 N2 - We have assessed in detail the effect of cisplatin-activated programmed cell death in the cisplatin-sensitive human ovarian cancer cell line A2780 and two drug-resistant subclones, CP70 and C30. To determine whether the differential extent of apoptosis observed between the sensitive and resistant ovarian cancer cell lines was the result of dissimilar upstream signaling events, we assessed the execution of apoptotic events that precede target protein proteolysis and subsequent chromosomal DNA degradation. Proteolytic degradation of procaspase-3 was observed in both the CP70 and C30 cells following IC50 cisplatin treatment, whereas no proteolyzed caspase-3 subunits were detected in the A2780 cells. However, using a direct enzymatic assay measuring cleavage of the synthetic peptide substrate (N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide), activity was detected in extracts prepared from A2780 cells treated at the IC90 level of cisplatin and was 2-3-fold less than that of extracts prepared from CP70 and C30 cells. Because the activation of procaspase-3 by caspase-9 requires the release of cytochrome c into the cytoplasm, we determined the level of cytoplasmic cytochrome c in each cell line in response to cisplatin treatment. Consistent with the caspase-3 activation data, a very small increase in cytoplasmic cytochrome c was observed in A2780 cells following cisplatin treatment, whereas dramatic increases were evident in both the CP70 and C30 cell lines. The expression of the mitochondrial factors Bcl-2, Bcl-x, and Bax was determined because each has been implicated in the regulation or release of cytochrome c at the level of the mitochondria. Bcl-2 and Bcl-xL proteins remained relatively unchanged in expression for over 48 h after exposure to cisplatin in the A2780 cell lines. However, within the same time period, expression of Bcl-2 decreased in the CP70- and C30-resistant cell lines, whereas an increase in Bcl-xL expression was observed. Expression of the proapoptotic Bcl-xS protein was observed in only the resistant CP70 and C30 cell lines independent of cisplatin treatment. A change in the expression of Mr 24,000 Bax to a Mr 21,000 isoform was evidenced in the A2780 cells within 48 h of cisplatin treatment and, to a greater extent, in the CP70 and C30 cells, which also expressed a Mr 16,000 Bax variant. Evidence for an alternative apoptotic pathway in A2780 cells was obtained by demonstrating increased FADD expression in response to cisplatin treatment. These results support a model in which cisplatin-induced programmed cell death in the cisplatin-sensitive A2780 and -resistant CP70 and C30 cells proceeds via caspase-3-independent and -dependent pathways, respectively. SN - 0008-5472 UR - https://www.unboundmedicine.com/medline/citation/10397248/Cisplatin_induced_apoptosis_proceeds_by_caspase_3_dependent_and__independent_pathways_in_cisplatin_resistant_and__sensitive_human_ovarian_cancer_cell_lines_ L2 - http://cancerres.aacrjournals.org/cgi/pmidlookup?view=long&pmid=10397248 DB - PRIME DP - Unbound Medicine ER -