The effects of a novel MEK inhibitor PD184161 on MEK-ERK signaling and growth in human liver cancer

Abstract

The MEK-ERK growth signaling pathway is important in human hepatocellular carcinoma (HCC). To evaluate the targeting of this pathway in HCC, we characterized a novel, orally-active MEK inhibitor, PD184161, using human HCC cells (HepG2, Hep3B, PLC, and SKHep) and in vivo human tumor xenografts. PD184161 inhibited MEK activity (IC50 = 10-100 nM) in a time- and concentration-dependent manner more effectively than PD098059 or U0126. PD184161 inhibited cell proliferation and induced apoptosis at concentrations of > or = 1.0 microM in a time- and concentration-dependent manner. In vivo, tumor xenograft P-ERK levels were significantly reduced 3 to 12 hours after an oral dose of PD184161 (P < .05). Contrarily, tumor xenograft P-ERK levels following long-term (24 days) daily dosing of PD184161 were refractory to this signaling effect. PD184161 significantly suppressed tumor engraftment and initial growth (P < .0001); however, established tumors were not significantly affected. In conclusion, PD184161 has antitumor effects in HCC in vitro and in vivo that appear to correlate with suppression of MEK activity. These studies demonstrate that PD184161 is unable to suppress MEK activity in HCC xenografts in the long term. Thus, we speculate that the degree of success of MEK targeted treatment in HCC and other cancers may, in part, depend on the discovery of mechanisms governing MEK inhibitor signaling resistance.

Figure 1

Chemical structure of PD184161 and PD184352.

Figure 2

Representative Western immunoblots of phosphorylated ERK1,2 and total ERK1,2 proteins from lysates of human HCC cell lines following treatment with PD098059, U0126, and PD184161. Actively dividing cells (Hep3B, HepG2, PLC, and SKHep) were treated for 1 hour with one of the following mitogen-activated protein kinase kinase (MEK) inhibitors: PD098059, U0126, or PD184161 (0.1 and 1.0 µM). Following treatment, the cells were lysed, and Western immunoblots were performed using antibodies to phosphorylated ERK1,2 (A) and total ERK1,2 (B). Similar results were obtained in at least two independent experiments.

Figure 3

The effect of the MEK inhibitor PD184161 on the proliferation of four human HCC cell lines (Hep3B, HepG2, PLC, and SKHep). Actively dividing cells were treated for 24, 48, or 72 hours with concentrations of PD184161 varying from 1.0 to 20 µM. A colorimetric proliferation assay was used to determine proliferation relative to control dimethyl sulfoxide-treated cells. Data are expressed as percentage of control. Each point on the graph represents the mean of triplicate samples, and error bars represent SEM. Similar results were obtained in at least two independent experiments.

Figure 4

The effect of the MEK inhibitor PD184161 on apoptosis in four human HCC cell lines (Hep3B, HepG2, PLC and SKHep). Actively dividing cells were treated for 48 hours with concentrations of PD184161 varying from 1.0 to 20 µM. A DNA fragmentation ELISA assay was used to determine apoptosis relative to control dimethyl sulfoxide-treated cells. Data are expressed as the percentage of control. Each point on the graph represents the mean of triplicate samples, and error bars represent SD. Similar results were obtained in at least two independent experiments.

Figure 5

The effect of twice-daily oral dosing with the MEK inhibitor PD184161 on the growth of newly inoculated Hep3B tumor xenografts in nude mice (n = 7). Mice were treated with PD184161 (300 mg/kg) beginning 12 hours after Hep3B inoculation and continued for 38 days. Tumors were measured every 3 days, and volumes are represented in the figure (▲ vehicle; ■ PD184161). Tumor measurements in PD184161-treated animals were found to be statistically significant from control at all time points shown (t-test; P < .05).

Figure 6

The effect of twice-daily oral dosing with the MEK inhibitor PD184161 on the growth of established Hep3B tumor xenografts in nude mice (n = 7). Nude mice were inoculated in the flank with 3 to 5 million Hep3B cells. Once the tumors had reached 100 mm³, the mice were dosed orally with 300 mg/kg twice daily for 24 days. Tumor measurements were taken every 3 days, and tumor volume is represented in the figure (▲ vehicle; ■ PD184161). There was no statistically significant difference between treatment groups (P = .17).

Figure 7

The effect of single and multiple oral dosing with the MEK inhibitor PD184161 on the expression of phosphorylated ERK1,2 (A) and total ERK1,2 (B) in nude mouse Hep3B tumor xenografts and lung. Homogenates from Hep3B tumors were prepared from treated and control mice and assessed by Western blot analysis for phosphorylated ERK1,2 and total ERK1,2 levels. Representative Western blot analyses are shown here. Results were reproducible in multiple assays. Densitometric analysis was performed, and results were analyzed by t-test (P < .05).