Suppression of ErbB-2 in androgen-independent human prostate cancer cells enhances cytotoxic effect by gemcitabine in an androgen-reduced environment

Abstract

We examined the efficacy of combination treatments utilizing cytotoxic drugs plus inhibitors to members of the ErbB-ERK signal pathway in human prostate cancer (PCa) LNCaP C-81 cells. Under an androgen-reduced condition, 50nM gemcitabine caused about 40% growth suppression on C-81 cells. Simultaneous treatment of gemcitabine plus 10microM AG825 produced 60% suppression (p<0.03); while, 85% growth inhibition (p<0.02) was seen if AG825 was added to gemcitabine-treated cells after a 24h-interval. Our data thus showed that in androgen-reduced conditions, inhibition of ErbB-2 increases the cytotoxic efficacy of gemcitabine in PCa cells. This finding has significant implications in the choice of drugs for combination therapy as well as the order of administration for treating cancer patients.

Fig. 1

Chemosensitivity of LNCaP C-81 cells to gemcitabine or docetaxel in a steroid-reduced condition. (A) Upper panel: LNCaP C-81 cells were cultured in androgen-reduced conditions for 48 hrs and then were treated with docetaxel or gemcitabine alone for 72 hours. Cells were harvested, and total cell number was counted. Bar represents SEs of triplicates in 2 sets of independent experiments. Lower panel: TUNEL Assay for control cells treated with vehicle alone (Control), cells treated with 50 nM gemcitabine (Gem) or 1.2 nM Docetaxel (Doc). (B) An equal amount of total cell lysate proteins was separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blotted with antibodies against of Bcl-2 or Bcl-X_L. Hybridization with anti-β-actin Ab was used as a loading control for each lane. Similar results were obtained from at least 2 sets of independent experiments.

Fig. 2

Growth suppression of LNCaP C-81 cells by combination treatments of gemcitabine, Docetaxel and ErbB-2 inhibitor in steroid-reduced conditions. C-81 cells in androgen-reduced conditions were treated with gemcitabine (Gem, 50 nM), docetaxel (Doc, 2.4 nM), 10µM AG 825, 10µM PD98059 or different combinations as indicated in the figure for 3 days. Control cells received solvent alone. Cell growth was measured by cell counting. The treatment in each lane is as follows: lane 1- solvent alone; lane 2–50 nM gemcitabine alone; lane 3- 2.4 nM docetaxel alone; lane 4– 10 µM AG825 alone; lane 5–10 µM PD98059 alone; lane 6-Simultaneous treatment of 50 nM gemcitabine plus 2.4 nM docetaxel; lane 7- Simultaneous treatment of 50 nM gemcitabine plus 10 µM PD98059; lane 8-Simultaneous treatment of 50 nM gemcitabine plus 10 µM AG825; lane 9–10 µM AG825 given 24h after treatment with gemcitabine. Bars represent SEs of triplicates in 3 sets of independent experiments. *p<0.03, growth inhibition by 50 µM gemcitabine plus 10 µM AG825 vs. 50 µM gemcitabine alone; **p<0.02, growth inhibition by 50 µM gemcitabine plus 10 µM AG825 in which AG825 was added after 24 hours treatment with gemcitabine vs. 50 µM gemcitabine alone.

Fig. 3

Molecular analyses on growth-suppressed LNCaP C-81 cells by combined treatments with gemcitabine, docetaxel and ErbB-2 inhibitor. C-81 cells in androgen-reduced conditions were treated with gemcitabine (Gem, 50 nM), docetaxel (Doc, 2.4 nM), 10 µM AG 825, 10 µM PD98059 or different combinations as indicated in the figure for 3 days. Control cells received solvent alone. The treatment in each lane is as follows: lane 1- solvent alone; lane 2–50 nM gemcitabine alone; lane 3- 2.4 nM docetaxel alone; lane 4– 10 µM AG825 alone; lane 5–10 µM PD98059 alone; lane 6- Simultaneous treatment of 50 nM gemcitabine plus 2.4 nM docetaxel; lane 7- Simultaneous treatment of 50 nM gemcitabine plus 10 µM PD98059; lane 8-Simultaneous treatment of 50 nM gemcitabine plus 10 µM AG825; lane 9–10 µM AG825 given 24h after treatment with gemcitabine. An aliquot of total cell lysates containing an equal amount of proteins was separated by SDS-PAGE and then transferred to nitrocellulose membranes. The membranes were blotted with antibodies against Bcl-2, Bcl-X_L or Bax. Hybridization of the same membrane with anti-β-actin Ab was used as a loading control for each lane. Similar results were obtained from at least 3 sets of independent experiments.

Fig. 4

Growth suppression of LNCaP C-81 cells by combination treatment of gemcitabine and ErbB inhibitors. The C-81 cells in androgen-reduced conditions were treated with 50 nM gemcitabine (Gem) in the presence or absence of 10 µM AG 825 or 10 µM AG1478 for 3 days. Control cells received solvent alone. (A) Cell growth was determined by cell counting. Bars represent SEs of triplicates in 2 sets of independent experiments. *p<0.03, growth inhibition by gemcitabine plus 10 µM AG825 (lane #5) vs. gemcitabine alone (lane #4). (B) An equal amount of total cell lysate proteins was separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blotted with antibodies against of Bcl-2 or Bax. Hybridization of the same membrane with anti-β-actin Ab was used as a loading control for each lane. Similar results were obtained from at least 2 sets of independent experiments.