Phenolic fraction of tobacco smoke condensate potentiates benzo[a]pyerene diol epoxide-induced cell transformation: role of protein kinase C

Abstract

In this study we separated weakly acidic phenolic components from other neutral, acidic and basic components of tobacco smoke condensate (TSC) and observed that phenolic fraction of TSC significantly increased the number of colonies of promotion-sensitive JB6 Cl41 cells that showed anchorage-independent growth on soft agar in response to BPDE (an ultimate carcinogen produced by metabolic activation of the PAH benzo[a]pyrene). Anchorage-independent cell growth is indicative of cell transformation resulting in acquisition of tumorigenic potential. In order to understand the underlying mechanism by which TSC phenolic fraction potentiates BPDE-induced tumorigenicity, we examined its effect on the activation of two transcription factors AP-1 and NF-kappaB which are known to be influenced by established tumor promoter TPA. BPDE treatment caused induction of both AP-1 and NF-kappaB activity as determined by luciferase reporter assay and only NF-kappaB induction in response to BPDE was significantly attenuated by TSC phenolic fraction whereas AP-1 induction remains unaltered. Attenuation of NF-kappaB activation by TSC phenolic fraction was associated with significant decrease of intracellular PKC substrate phosphorylation in BPDE treated cells. Non-specific PKC inhibitors staurosporine and bisindolylmaleimide II as well as inhibitors specific to conventional PKCs (Go6976) and PKC-delta (rottlerin) attenuated NF-kappaB activation in BPDE treated cells to a varying degree indicating a possible link between PKC down-regulation and the attenuation of NF-kappaB activity by TSC phenolic fraction. Treatment of cells with PKC inhibitors also potentiated anchorage-independent growth of BPDE treated cells on soft agar. Our data suggest a possible role of PKC down-regulation in potentiation of BPDE-induced tumorogenicity by TSC phenolic fraction.

Figure 1

Effect of TSC phenolic fraction on anchorage-independent growth of BPDE treated JB6 Cl41 cells. Experimental procedure is described in the text. Each bar shows the average of three independent experiments and the error bars indicate SD. (*) indicates statistical analysis of significance with paired t-Test (p<0.05).

Figure 2

Effect of TSC phenolic fraction on activation of AP-1 and NF-κB in JB6 (P⁺) Cl 41 cells harboring AP1-luciferase and NF-κB-luciferase reporter plasmids. Activation of AP-1 (A) and NF-κB (B) is measured as described in “Methods”. Cells were either untreated; treated with TSC phenolic fraction (10 µg/ml); with BPDE (0.2 µM, 0.5 µM); with 0.5 µM BPDE followed by TSC phenolic fraction (10 µg/ml or 30 µg/ml). Each bar indicates the mean ± SD of three parallel experiments. (*) indicates statistical analysis of significance with paired t-Test (p<0.05).

Figure 3

Inhibition of PKC substrate phosphorylation by TSC phenolic fraction. Cells were either untreated or treated with 0.5 µM BPDE for 1 hr. followed by noncytotoxic concentration of TSC phenolic fraction for 6 hours and PKC substrate phosphorylation in the cell extract was determined by Western immunoblotting using phospho-(Ser) PKC Substrate antibody which is specific for PKC substrates phosphorylated at Ser residues within a particular motif.

Figure 4

Effect of PKC inhibitors on BPDE-induced NF-κB activation in JB6 Cl 41 cells harboring NF-κB-luciferase reporter plasmid. Bar graph shows NF-κB activation in cells that were untreated (A); treated with 0.5 µM BPDE (B); treated with 0.5 µM BPDE plus respective PKC inhibitors staurosporine, 7 nM (C); bisindolylmaleimide, 100 nM (D); Go6976, 0.5 µM (E) and rottlerin, 30 µM (F). Each bar indicates the mean ± SD of three parallel experiments. (*) indicates statistical analysis of significance with paired t-Test (p<0.05).

Figure 5

Effect of PKC inhibitors on anchorage-independent growth of JB6 cl41 cells. Experimental procedure is described in the text. Each bar shows the average of three independent experiments and the error bar indicates SD.

Chart 1

Preparation of weakly acidic phenolic fraction from tobacco smoke condensate