title: Actions of the selective protein kinase C inhibitor PKC412 on B-chronic lymphocytic leukemia cells in vitro.
abstract: OBJECTIVE
  The staurosporine derivative PKC412 (CGP41251) is a more selective inhibitor of the conventional isoforms of protein kinase C (PKC) than is the parent compound. In addition to its growth inhibitory properties, PKC412 reverses the efflux function of the multidrug resistance (MDR)-1 gene product, P-glycoprotein (P-gp).
  
  
  METHODS
  The in vitro actions of PKC412 were investigated in peripheral blood lymphocytes (PBL) from 4 normal volunteers, B-cell isolates from 3 normal tonsils and 31 patients with B-cell chronic lymphocytic leukemia (B-CLL). Following incubation with PKC412 for 2 days, the viability of B-CLL cells was decreased relative to that of controls (63+/-23% at 1 micromole/L; 52+/-30% at 10 micromole/L; n=20). Normal PBL were significantly more resistant to the drug (91+/-5% viable cells at 1 micromole/L; 73+/-18% at 10 micromole/L; n=4). Thirteen of the B-CLL patients were treated with oral PKC412 in a phase II trial.
  
  
  RESULTS
  PKC activity in malignant cells from these patients showed a reduction post-treatment of 25-96% of their respective pre-treatment levels. Morphologic analysis, as well as in situ assay for DNA strand breaks (TUNEL assay) showed that B-CLL cells were killed by an apoptotic mechanism. In B-CLL cells the mean IC50, for PKC412, as measured by the reduction of 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), was 2.1 micromol/L in 16 samples in which the IC50 were below the maximum concentration of PKC412 used for the assay. In tonsillar B-cells, the mean IC50 was 11 micromol/L whereas PBL cells were resistant. Four of eight and 1/3 B-CLL samples that were resistant to chlorambucil and fludarabine, respectively, were sensitive to PKC412. In 15/31 B-CLL samples a dose-dependent reversal of P-gp-mediated drug efflux by PKC412 was observed. A statistically significant correlation (p<0.001) was observed between P-gp protein expression as measured by FACScan analysis and the reversal of efflux activity by either PKC412 or verapamil. PKC412 increased the sensitivity of B-CLL cells to 2'-chlorodeoxyadenosine and chlorambucil.
  
  
  CONCLUSIONS
  This study establishes the in vitro cytotoxic and multidrug resistance (MDR) modulatory properties of PKC412 towards malignant cells from B-CLL patients. The direct antitumor activity combined with the potential for P-gp modulation make PKC412 an attractive drug for the treatment of malignancies expressing the MDR phenotype, or in combination with conventional drugs.
authors.affiliation: Department of Hematology, Royal Free & University College Medical School, Royal Free Campus, Rowland Hill Street, London NW3 2PF, UK. k.ganeshaguru@rfc.ucl.ac.uk
authors.forename: Kanagasabai
authors.initials: K
authors.lastname: Ganeshaguru
authors.forename: Ranmohan G
authors.initials: RG
authors.lastname: Wickremasinghe
authors.forename: Dylan T
authors.initials: DT
authors.lastname: Jones
authors.forename: Mathew
authors.initials: M
authors.lastname: Gordon
authors.forename: Steve M
authors.initials: SM
authors.lastname: Hart
authors.forename: Andres E
authors.initials: AE
authors.lastname: Virchis
authors.forename: H Grant
authors.initials: HG
authors.lastname: Prentice
authors.forename: A Victor
authors.initials: AV
authors.lastname: Hoffbrand
authors.forename: Anthony
authors.initials: A
authors.lastname: Man
authors.forename: Kate
authors.initials: K
authors.lastname: Champain
authors.forename: Katalin
authors.initials: K
authors.lastname: Csermak
authors.forename: Atul B
authors.initials: AB
authors.lastname: Mehta
mesh_terms: D015762:2-Chloroadenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000368:Aged; D000369:Aged, 80 and over; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001402:B-Lymphocytes / Q000187:drug effects / Q000201:enzymology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002699:Chlorambucil / Q000494:pharmacology; D003839:Deoxyadenosines / Q000494:pharmacology; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D004354:Drug Screening Assays, Antitumor; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D006801:Humans; D020287:In Situ Nick-End Labeling; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors*; D014066:Palatine Tonsil / Q000166:cytology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D019311:Staurosporine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000201:enzymology; D014700:Verapamil / Q000494:pharmacology; D014740:Vidarabine / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D002121:Calcium Channel Blockers; D003839:Deoxyadenosines; D004791:Enzyme Inhibitors; D009363:Neoplasm Proteins; D015762:2-Chloroadenosine; D002699:Chlorambucil; C036854:2'-chloro-2'-deoxyadenosine; D014700:Verapamil; D011493:Protein Kinase C; D014740:Vidarabine; D019311:Staurosporine; C059539:midostaurin; C024352:fludarabine