Ribozyme-mediated inhibition of PKCα sensitizes androgen-independent human prostate cancer cells to cisplatin-induced apoptosis

Linda Orlandi, Mara Binda, Marco Folini, Alessandra Bearzatto, Raffaella Villa, Maria Grazia Daidone, Nadia Zaffaroni

Research output: Contribution to journalArticle

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Abstract

BACKGROUND. Therapeutic strategies to target the molecular basis of hormone and drug resistance of prostate cancer cells are needed. Since protein kinase Cα (PKCα) is thought to have a role in the development of the androgen-independent phenotype of prostate cancer cells and in apoptosis suppression, the objective of the present study was to test whether specific inhibition of PKCα by a hammerhead ribozyme was able to sensitize androgen-independent prostate cancer cells the effects of apoptosis-inducing anticancer drugs. METHODS. An active ribozyme (PKCαRZ) targeting codon 4 in human PKCα mRNA was synthesized by in vitro transcription. A mutant ribozyme (PKCαmutRZ) was also made by deleting G12 from the catalytic core of the active ribozyme and used as a control throughout the study. The double-stranded, ribozyme-encoding sequences were then inserted into an expression vector under the control of the cytomegalovirus promoter and delivered to growing prostate cancer cells (DU145 and PC-3) by a DOTAP-mediated transfer. A neomycin resistance gene on the vector was used to select ribozyme-expressing clones. The clones were analyzed for PKCα expression, sensitivity to anticancer drugs and ability to undergo drug-induced apoptosis. RESULTS. Two DU145-derived cell clones expressing the active ribozyme (DURZ 2 and DURZ 12) and one clone expressing the catalytically inactive ribozyme (DUmutRZ) were selected for the study. DURZ 2 and DURZ 12 were characterized by a markedly (about 40-50%) lower PKCα protein level than parental DU145 cells, whereas no reduction in PKCα expression was observed in DUmutRZ cells. Results of cytotoxicity experiments indicated that DURZ 2 and DURZ 12 but not DUmutRZ cells were significantly more sensitive than parental DU145 cells to a 1 hr exposure to the mononuclear platinum compounds (cisplatin and oxaliplatin) and showed an increased susceptibility to undergo cisplatin-induced apoptosis. A significantly enhanced apoptotic response to cisplatin was also observed in a PC-3-derived polyclonal cell population endogenously expressing the active ribozyme. CONCLUSIONS. Results of the study highlight the importance of PKCα in the response of prostate cancer cells to mononuclear platinum compounds and indicate specific inhibition of the enzyme as a potential therapeutic strategy to sensitize androgen-independent prostate cancer cells to these drugs.

Original languageEnglish
Pages (from-to)133-143
Number of pages11
JournalProstate
Volume54
Issue number2
DOIs
Publication statusPublished - Feb 1 2003

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Catalytic RNA
Protein Kinase C
Cisplatin
Androgens
Prostatic Neoplasms
Apoptosis
Clone Cells
Platinum Compounds
oxaliplatin
Pharmaceutical Preparations
Neomycin
Cytomegalovirus
Drug Resistance
Codon
Catalytic Domain
Hormones

Keywords

  • Apoptosis
  • Cisplatin
  • Oxaliplatin
  • Protein kinase Cα
  • Ribozyme

ASJC Scopus subject areas

  • Urology

Cite this

Ribozyme-mediated inhibition of PKCα sensitizes androgen-independent human prostate cancer cells to cisplatin-induced apoptosis. / Orlandi, Linda; Binda, Mara; Folini, Marco; Bearzatto, Alessandra; Villa, Raffaella; Daidone, Maria Grazia; Zaffaroni, Nadia.

In: Prostate, Vol. 54, No. 2, 01.02.2003, p. 133-143.

Research output: Contribution to journalArticle

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title = "Ribozyme-mediated inhibition of PKCα sensitizes androgen-independent human prostate cancer cells to cisplatin-induced apoptosis",
abstract = "BACKGROUND. Therapeutic strategies to target the molecular basis of hormone and drug resistance of prostate cancer cells are needed. Since protein kinase Cα (PKCα) is thought to have a role in the development of the androgen-independent phenotype of prostate cancer cells and in apoptosis suppression, the objective of the present study was to test whether specific inhibition of PKCα by a hammerhead ribozyme was able to sensitize androgen-independent prostate cancer cells the effects of apoptosis-inducing anticancer drugs. METHODS. An active ribozyme (PKCαRZ) targeting codon 4 in human PKCα mRNA was synthesized by in vitro transcription. A mutant ribozyme (PKCαmutRZ) was also made by deleting G12 from the catalytic core of the active ribozyme and used as a control throughout the study. The double-stranded, ribozyme-encoding sequences were then inserted into an expression vector under the control of the cytomegalovirus promoter and delivered to growing prostate cancer cells (DU145 and PC-3) by a DOTAP-mediated transfer. A neomycin resistance gene on the vector was used to select ribozyme-expressing clones. The clones were analyzed for PKCα expression, sensitivity to anticancer drugs and ability to undergo drug-induced apoptosis. RESULTS. Two DU145-derived cell clones expressing the active ribozyme (DURZ 2 and DURZ 12) and one clone expressing the catalytically inactive ribozyme (DUmutRZ) were selected for the study. DURZ 2 and DURZ 12 were characterized by a markedly (about 40-50{\%}) lower PKCα protein level than parental DU145 cells, whereas no reduction in PKCα expression was observed in DUmutRZ cells. Results of cytotoxicity experiments indicated that DURZ 2 and DURZ 12 but not DUmutRZ cells were significantly more sensitive than parental DU145 cells to a 1 hr exposure to the mononuclear platinum compounds (cisplatin and oxaliplatin) and showed an increased susceptibility to undergo cisplatin-induced apoptosis. A significantly enhanced apoptotic response to cisplatin was also observed in a PC-3-derived polyclonal cell population endogenously expressing the active ribozyme. CONCLUSIONS. Results of the study highlight the importance of PKCα in the response of prostate cancer cells to mononuclear platinum compounds and indicate specific inhibition of the enzyme as a potential therapeutic strategy to sensitize androgen-independent prostate cancer cells to these drugs.",
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T1 - Ribozyme-mediated inhibition of PKCα sensitizes androgen-independent human prostate cancer cells to cisplatin-induced apoptosis

AU - Orlandi, Linda

AU - Binda, Mara

AU - Folini, Marco

AU - Bearzatto, Alessandra

AU - Villa, Raffaella

AU - Daidone, Maria Grazia

AU - Zaffaroni, Nadia

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N2 - BACKGROUND. Therapeutic strategies to target the molecular basis of hormone and drug resistance of prostate cancer cells are needed. Since protein kinase Cα (PKCα) is thought to have a role in the development of the androgen-independent phenotype of prostate cancer cells and in apoptosis suppression, the objective of the present study was to test whether specific inhibition of PKCα by a hammerhead ribozyme was able to sensitize androgen-independent prostate cancer cells the effects of apoptosis-inducing anticancer drugs. METHODS. An active ribozyme (PKCαRZ) targeting codon 4 in human PKCα mRNA was synthesized by in vitro transcription. A mutant ribozyme (PKCαmutRZ) was also made by deleting G12 from the catalytic core of the active ribozyme and used as a control throughout the study. The double-stranded, ribozyme-encoding sequences were then inserted into an expression vector under the control of the cytomegalovirus promoter and delivered to growing prostate cancer cells (DU145 and PC-3) by a DOTAP-mediated transfer. A neomycin resistance gene on the vector was used to select ribozyme-expressing clones. The clones were analyzed for PKCα expression, sensitivity to anticancer drugs and ability to undergo drug-induced apoptosis. RESULTS. Two DU145-derived cell clones expressing the active ribozyme (DURZ 2 and DURZ 12) and one clone expressing the catalytically inactive ribozyme (DUmutRZ) were selected for the study. DURZ 2 and DURZ 12 were characterized by a markedly (about 40-50%) lower PKCα protein level than parental DU145 cells, whereas no reduction in PKCα expression was observed in DUmutRZ cells. Results of cytotoxicity experiments indicated that DURZ 2 and DURZ 12 but not DUmutRZ cells were significantly more sensitive than parental DU145 cells to a 1 hr exposure to the mononuclear platinum compounds (cisplatin and oxaliplatin) and showed an increased susceptibility to undergo cisplatin-induced apoptosis. A significantly enhanced apoptotic response to cisplatin was also observed in a PC-3-derived polyclonal cell population endogenously expressing the active ribozyme. CONCLUSIONS. Results of the study highlight the importance of PKCα in the response of prostate cancer cells to mononuclear platinum compounds and indicate specific inhibition of the enzyme as a potential therapeutic strategy to sensitize androgen-independent prostate cancer cells to these drugs.

AB - BACKGROUND. Therapeutic strategies to target the molecular basis of hormone and drug resistance of prostate cancer cells are needed. Since protein kinase Cα (PKCα) is thought to have a role in the development of the androgen-independent phenotype of prostate cancer cells and in apoptosis suppression, the objective of the present study was to test whether specific inhibition of PKCα by a hammerhead ribozyme was able to sensitize androgen-independent prostate cancer cells the effects of apoptosis-inducing anticancer drugs. METHODS. An active ribozyme (PKCαRZ) targeting codon 4 in human PKCα mRNA was synthesized by in vitro transcription. A mutant ribozyme (PKCαmutRZ) was also made by deleting G12 from the catalytic core of the active ribozyme and used as a control throughout the study. The double-stranded, ribozyme-encoding sequences were then inserted into an expression vector under the control of the cytomegalovirus promoter and delivered to growing prostate cancer cells (DU145 and PC-3) by a DOTAP-mediated transfer. A neomycin resistance gene on the vector was used to select ribozyme-expressing clones. The clones were analyzed for PKCα expression, sensitivity to anticancer drugs and ability to undergo drug-induced apoptosis. RESULTS. Two DU145-derived cell clones expressing the active ribozyme (DURZ 2 and DURZ 12) and one clone expressing the catalytically inactive ribozyme (DUmutRZ) were selected for the study. DURZ 2 and DURZ 12 were characterized by a markedly (about 40-50%) lower PKCα protein level than parental DU145 cells, whereas no reduction in PKCα expression was observed in DUmutRZ cells. Results of cytotoxicity experiments indicated that DURZ 2 and DURZ 12 but not DUmutRZ cells were significantly more sensitive than parental DU145 cells to a 1 hr exposure to the mononuclear platinum compounds (cisplatin and oxaliplatin) and showed an increased susceptibility to undergo cisplatin-induced apoptosis. A significantly enhanced apoptotic response to cisplatin was also observed in a PC-3-derived polyclonal cell population endogenously expressing the active ribozyme. CONCLUSIONS. Results of the study highlight the importance of PKCα in the response of prostate cancer cells to mononuclear platinum compounds and indicate specific inhibition of the enzyme as a potential therapeutic strategy to sensitize androgen-independent prostate cancer cells to these drugs.

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KW - Cisplatin

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