Protein Kinase C-β Gene Variants, Pathway Activation, and Enzastaurin Activity in Lung Cancer

Sang-Haak Lee; Tingan Chen; Jun Zhou; Jennifer Hofmann; Gerold Bepler


Clin Lung Cancer. 2010;11(3) 

In This Article


PRKCB1 Gene Sequencing

Within the coding regions of PKCβ1/2 (the last 173 nucleotides of exon 1, all 1690 nucleotides of exons 2–16, the first 153 nucleotides of exon 17 for PKCβ2, and the first 159 nucleotides of exon 18 for PKCβ1), we observed six single-nucleotide variations and no deletions or insertions in 28 lung-cancer cell lines. Five variants were silent alterations (C79A in 13 cases, C606T in one case, G702A in one case, T1191C in 12 cases, and C1770T in nine cases). Only one variant (C119T in exon 1), as found in two SCLC cell lines (H69 and SW210.5), resulted in an amino-acid substitution (threonine 40 to isoleucine; Table 2). No apparent sequence variations led to splice variants.

Effect of T40I on Enzastaurin's in Vitro Activity

We studied the effect of enzastaurin on the level of phosphorylation of PKCβ2 and the downstream molecules GSK3β and S6RP, and on proliferation in SCLC cell lines with (H69 and SW210.5) and without (H211 and DMS79) the T40I amino-acid substitution. In all four cell lines, maximal inhibition of PKCβ2 phosphorylation was achieved after 2 hours of exposure to 1 μM enzastaurin. The maximal inhibition of GSK3β and S6RP phosphorylation was achieved after 4 hours of exposure, and no clear differences among these four cell lines were evident. The 50% inhibitory concentration (IC50) for the inhibition of proliferation was 0.2 μM for H69, 0.07 μM for SW210.5, 0.05 μM for H211, and 0.08 μM for DMS79. These results suggest that the T40I substitution has no effect on enzastaurin's in vitro efficacy.

Effect of Enzastaurin on NSCLC and SCLC Cell Lines

We assessed whether enzastaurin would have differential activity in NSCLC versus SCLC cell lines. In all four NSCLC cell lines studied, the maximal inhibition of PKCβ2, GSK3β, and S6RP phosphorylation was achieved after 2–4 hours of exposure to 1 μM enzastaurin. The IC50 concentrations for the inhibition of proliferation were 0.09 μM for H23, 0.08 μM for H125, 0.08 μM H322, and 0.07 μM for A549. These concentrations were similar to those observed in SCLC cell lines, suggesting that enzastaurin is equally effective in NSCLC and SCLC cell lines (Figure 1).

Figure 1.

Phosphorylation (A and C) and Proliferation (B and D) Inhibition by Enzastaurin in 2 Small-Cell Lung Cancer Cell Lines
Gene sequencing of PRKCB1 revealed the presence of a T40I variant in exon 1 in SW210.5; H211 presented no evidence of sequence variations.

Effect of Enzastaurin Concentration on Phosphorylation and Proliferation Inhibition

We assessed the relationship between inhibition of protein phosphorylation and proliferation, using different concentrations of enzastaurin. A clear concentration-dependent inhibition of PKCβ2, GSK3β, S6RP, Akt (Thr308), and FKHR phosphorylation occurred in cell line H23, with an IC50 of approximately 0.08 μM for PKCβ2. This concentration was similar to that required for a 50% inhibition of proliferation and suggests that the inhibition of PKCβ2 phosphorylation may be a useful surrogate marker of proliferation inhibition in lung cancer (Figure 2).

Figure 2.

Concentration-Dependent Phosphorylation Inhibition of PKCβ2 (A and B) and Downstream Targets GSK3β (C, Red), S6RP (C, Blue), Akt (D, Red), and FKHR (D, Blue) After 1 Hour of Exposure to Enzastaurin in Non–Small-Cell Lung Cancer Cell Line H23