ERK1/2 activity contributes to gemcitabine resistance in pancreatic cancer cells

Abstract

Objective

To test the hypothesis that chemoresistance in pancreatic cancer is mediated via extracellular signal-regulated protein kinase (ERK) 1/2 overactivity.

Methods

The human pancreatic cancer cell lines BxPC3, PANC-1 and a stably gemcitabine-resistant subline, PANC1^GemRes, were treated with combinations of gemcitabine and the ERK1/2 inhibitor, U0126. Phosphorylated (p)ERK1/2 was examined by Western blotting; cell proliferation and apoptosis were quantified. A nude mouse xenograft model was established with each cell line, and the therapeutic efficacy of gemcitabine and U0126 alone or in combination was examined.

Results

Gemcitabine treatment visibly increased pERK1/2 levels in BxPC-3 and PANC-1 cells. PANC-1^GemRes constitutively produced high levels of pERK1/2. U0126 treatment reversed the gemcitabine-associated increase in cell proliferation and reduction in apoptosis, in all three cell lines. Combination treatment with U0126 and gemcitabine inhibited tumour growth and promoted apoptosis in xenograft tumours derived from all three cell lines.

Conclusions

ERK1/2 activity may protect pancreatic cancer cells from chemotherapy-induced apoptosis. The combined use of an ERK1/2 inhibitor (such as U0126) together with gemcitabine may result in synergistic therapeutic effects at tolerable gemcitabine doses.

Introduction

Pancreatic cancer has the worst prognosis of all major cancers, with a 5-year overall survival rate of ∼5%. ¹ The current standard treatment for advanced pancreatic cancer is gemcitabine, which is a cytotoxic nucleoside analogue. The benefits of gemcitabine are modest, with a tumour response rate of 12% and a median survival time of 5 months. ² Data from a small number of studies suggest that a combination of targeted therapy and gemcitabine has statistically significant benefits. ³ The extracellular signal-regulated protein kinases (ERK) 1 and 2 are potential targets for combination therapy. ⁴

The ERK1/2 signalling pathway is involved in the control of diverse cellular processes such as proliferation, survival, differentiation and motility. ⁴ This pathway is often upregulated in human tumours^5,6 and represents an attractive target for the development of anticancer drugs.^7–10 The inhibitor of mitogen-activated protein kinase kinase 1/2 (MAP2K1/2), U0126, has been shown to inhibit ERK1/2 phosphorylation effectively, ¹¹ and to enhance radiosensitivity of rhabdomyosarcoma cells. ¹² ERK1/2 has been shown to be activated in pancreatic carcinoma, ⁵ but little is known regarding its role in chemoresistance.

This study investigated the effects of ERK1/2 inhibition by U0126 and gemcitabine, alone and in combination, on a pancreatic carcinoma model in vitro and in vivo.

Materials and methods

Cell culture

Human pancreatic adenocarcinoma PANC-1 and BxPC-3 cells were obtained from American Type Culture Collection (Shanghai, China) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (Life Technologies, Gaithersburg, MD). Cells were incubated in 75-cm² culture flasks in a humidified atmosphere at 37°C with 5% carbon dioxide in air, and passaged on reaching 60–80% confluence.

Development of PANC-1^GemRes

The pancreatic adenocarcinoma cell line PANC-1 was used to develop a gemcitabine-resistant cell line, PANC-1^GemRes, as described previously. ¹³ Briefly, PANC-1 cells were serially subcultured through incrementally increasing gemcitabine concentrations, starting at the 50% inhibitory drug concentration (IC₅₀), over 3 months. Persistence of stable resistance was confirmed by subculture in gemcitabine-free DMEM containing 10% fetal bovine serum; PANC-1^GemRes retained the capacity for proliferation when returned to medium containing 5 µM gemcitabine.

Determination of gemcitabine IC₅₀

The IC₅₀ of gemcitabine was determined in each cell line. BxPC-3, PANC-1 and PANC-1^GemRes cells were seeded at 1 × 10⁴ per well in 96-well plates, in 0.1 ml of culture medium and cultured overnight. The next day, the medium was supplemented with 0–100 µM gemcitabine (Sigma, St Louis, MO, USA) for 48 h at 37°C. The IC₅₀ was determined using 2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay (Roche Molecular Biochemicals, Indianapolis, IN, USA) according to the manufacturer’s instructions. Cell proliferation was quantified by measuring the absorbance at 450 nm, and the IC₅₀ for each of the cell lines was estimated from the semilogarithmic dose–response curves, by linear interpolation.

Cell proliferation and apoptosis analysis

Cells (BxPC-3, PANC-1 or PANC-1^GemRes) were seeded at 1 × 10⁴ per well in 96-well plates in 0.1 ml of culture medium and allowed to grow overnight. The next day, the medium was supplemented with IC₅₀ gemcitabine for 48 h at 37°C in the presence or absence of 25 µM U0126 (Sigma, St Louis, MO, USA) (added 1 h before gemcitabine), or treated with 25 µM U0126 alone. Cell proliferation was quantified by XTT assay as described above. Parallel cultures were immediately fixed in 37% formaldehyde at room temperature for 30 min and allowed to dry overnight. Apoptosis was determined using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) method (Roche in situ Apoptosis Detection System; Roche, Mannheim, Germany), according to the manufacturer's instructions. Cells were examined by light microscopy; apoptosis was quantified as the mean number of stained cells as a percentage of total cells in five random high-power fields (×100 magnification).

Western blotting

Cells (BxPC-3, PANC-1 or PANC-1^GemRes) were seeded at 1 × 10⁴ per well in 96-well plates, in 0.1 ml of culture medium, and allowed to grow overnight. The next day, the medium was supplemented with IC₅₀ gemcitabine for 48 h at 37°C. Cells were rinsed once with cold phosphate buffered saline (PBS; 20 mM, pH 7.6) and lysed in radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 8.0, 150 mM sodium chloride, 1% nonyl phenoxypolyethoxylethanol, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulphate [SDS]), containing 0.3 mg/l protease inhibitor (Roche), for 15 min on ice. Cell lysates were centrifuged at 28 340  g for 15 min, and 25 µg of protein for each sample was resolved by 10% SDS–polyacrylamide gel electrophoresis under reduced conditions. Proteins were transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA) and blocked with TBST (10 mM Tris-HCl, pH 7.4, 100 mM sodium chloride, 0.1% Tween-20) containing 5% skim milk for 30 min at 4°C. Membranes were incubated with mouse antihuman phosphorylated (p)ERK1/2 monoclonal antibody (1 : 200 dilution) or mouse antihuman β-actin monoclonal antibody (1 : 500 dilution; both Santa Cruz Biotechnology; Santa Cruz, CA, USA) overnight at 4°C. Membranes were washed four times with TBST at room temperature for 5 min each wash, then incubated with horseradish peroxidase-conjugated rabbit antimouse immunoglobulin G (1 : 5000 dilution; Zymed, San Francisco, CA, USA) for 60 min at room temperature. Membranes were washed four times with TBST at room temperature (5 min each wash) and immunoreactive protein bands were visualized using enhanced chemiluminescence (ECL detection system; Pierce, Rockford, IL, USA), followed by exposure to AGFA X-ray blue film (AGFA-Gevaert, Hortsel, Belgium).

Mouse tumour model

Male athymic nu/nu mice (aged 5 weeks; weight 18–20 g; specific pathogen free) were obtained from the Laboratory Centre of Shandong University. Mice were housed in microisolator cages, with autoclaved bedding in a specific pathogen-free facility, with a 12-h light/dark cycle. Animals received water and food ad libitum and were observed for signs of tumour growth, activity, feeding and pain, in accordance with the guidelines of the Standing Committee on Animals, Shandong University. The study protocol was approved by the Ethics Committee of Jinan Central Hospital of Shandong University.

Xenograft tumours were established by subcutaneous injection of 1 × 10⁶ BxPC-3, PANC-1 or PANC1^GemRes cells in 20 µl of PBS (pH 7.6). When tumours were 50–100 mm³ in diameter (after 4 weeks) the mice were divided into four groups (n = 8 per group), to receive the following treatments by intraperitoneal injection, three times per week for 4 weeks (all injections were of identical volume): 250 µg/kg U0126 (in dimethyl sulphoxide; DMSO); 100 mg/kg gemcitabine (in 1% DMSO); 250 µg/kg U0126 and 100 mg/kg gemcitabine (in DMSO); and control group (1% DMSO). Tumour growth was monitored with calipers three times per week during the treatment period; tumour volume was calculated according to the formula (length × width²)/2. On completion of treatment, mice were sacrificed and the tumours were excised.

Ttissue samples were immediately snap-frozen in liquid nitrogen and stored at −80°C until used for analysis. Apoptosis was quantified in frozen sections of tumour tissue (4-µm thick) using TUNEL assay (Roche in situ Apoptosis Detection System), according to the manufacturer’s instructions, as previously described. ¹⁴ Sections were examined by light microscopy and the apoptosis index was determined by counting ≥1000 cells in 5 randomly selected high-power fields (×200 magnification).

Statistical analyses

All experiments were performed a minimum of three times. Data were presented as mean ± SD and compared using Student’s t-test. A P-value <0.05 was considered to be statistically significant. Statistical analyses were performed using SPSS® software, version 11.0 (SPSS Inc., Chicago, IL, USA) for Windows®.

Results

Data regarding the gemcitabine IC₅₀ for PANC-1, BxPC-3 and PANC-1^GemRes cells are shown in Table 1. The gemcitabine IC₅₀ of PANC-1^GemRes cells was significantly greater than that of the parental cell line, PANC-1 (P < 0.05). Treatment with U0126 significantly reduced the gemcitabine IC₅₀ in PANC-1 and PANC-1^GemRes cells (P < 0.05; Table 1), but had no effect in BxPC-3 cells.

Table 1.

Effect of the extracellular signal-regulated protein kinase 1/2 inhibitor U0126 on the 50% inhibitory drug concentration (IC₅₀) of gemcitabine in the human pancreatic adenocarcinoma cell lines BxPC-3 and PANC-1, and in a gemcitabine-resistant PANC-1 derived cell line, PANC-1^GemRes.

Cell line	Gemcitabine	Gemcitabine  + U0126
BxPC-3	14.0 ± 2.1 nM	7.9 ± 1.2 nM
PANC-1	49.6 ± 5.4 nM	19.7 ± 4.3 nMb
PANC-1GemRes	12.4 ± 1.8 µM a	2.4 ± 0.5 µMb

Data presented as mean ± SD.

a

P < 0.05 vs PANC-1 cells, ^bP < 0.05 vs gemcitabine alone; Student’s t-test.

Western blot analysis of pERK1/2 in PANC-1, BxPC-3 and PANC-1^GemRes cells, after treatment with gemcitabine, is presented in Figure 1. BxPC-3 cells appeared to have no constitutive pERK1/2 (absence of protein at 0 h). Gemcitabine treatment resulted in visibly increased pERK1/2 levels in BxPC-3 and PANC-1 cells at 6 and 12 h after treatment, respectively. PANC-1^GemRes cells constitutively produced relatively high levels of pERK1/2 that were unchanged by gemcitabine treatment.

Figure 1.

Western blot analysis of phosphorylated signal regulated extracellular kinase (pERK)1/2 and β-actin in human pancreatic adenocarcinoma cell lines BxPC-3 and PANC-1, and a gemcitabine-resistant PANC-1 derived cell line, PANC-1^GemRes. Cells were treated with their 50% inhibitory concentration of gemcitabine (14 nM, 49.6 nM and 12.4 µM, respectively) for 48 h.

Treatment with U0126 alone had no effect on apoptosis in PANC-1, BxPC-3 or PANC-1^GemRes cells. Combined treatment with U0126 and gemcitabine resulted in significantly higher levels of apoptosis than gemcitabine alone, in all three cell lines (P < 0.01 for each cell line; Table 2)

Table 2.

Effect of treatment with U0126 and/or gemcitabine on apoptosis ^a in the human pancreatic adenocarcinoma cell lines BxPC-3 and PANC-1, and a gemcitabine-resistant PANC-1 derived cell line, PANC-1^GemRes.

Treatment	Cell line
	BxPC-3	PANC-1	PANC-1GemRes
Control	1.12 ± 0.02	1.04 ± 0.03	1.16 ± 0.02
U0126	1.14 ± 0.02	1.13 ± 0.03	1.18 ± 0.04
Gemcitabine	13.5 ± 1.46*	5.14 ± 1.05	1.23 ± 0.06
U0126 + gemcitabine	37.4 ± 6.3***	17.3 ± 2.54**	19.32 ± 2.46**

Data presented as mean ± SD.

a

mean percentage of apoptotic cells as a proportion of total cells in five random high power fields (× 100 magnification).

*

P < 0.05, **P < 0.01, ***P < 0.001 versus control; Student’s t-test.

All mice survived the treatment period. Tumours derived from all three cell lines were significantly smaller following combination U0126 and gemcitabine treatment compared with the tumours observed in control animals (P < 0.05 for each cell line; Table 3). Of the three cell lines, only BxPC-3-derived tumours were significantly smaller than controls, following gemcitabine treatment alone (P < 0.05; Table 3). U0126 treatment had no effect on the growth of tumours derived from any cell line.

Table 3.

Effect of treatment with U0126 and/or gemcitabine on tumour volume and apoptosis in mouse xenograft models of pancreatic carcinoma, derived from human pancreatic adenocarcinoma cell lines BxPC-3 and PANC-1, and a gemcitabine-resistant PANC-1 derived cell line, PANC-1^GemRes.

Parameter	Cell line
	BxPC-3	PANC-1	PANC-1GemRes
Tumour volume, mm 3
Control	2540 ± 250	2350 ± 280	2120 ± 320
U0126	2430 ± 235	2286 ± 315	2216 ± 280
Gemcitabine	1760 ± 85*	2032 ± 178	2080 ± 260
U0126 + gemcitabine	210 ± 35**	1100 ± 65*	1650 ± 120*
Apoptosis a
Control	1.02 ± 0.08	1.83 ± 0.12	1.14 ± 0.12
U0126	1.08 ± 0.07	1.92 ± 0.12	1.24 ± 0.13
Gemcitabine	4.46 ± 0.13	2.17 ± 0.15	2.15 ± 0.13
U0126 + gemcitabine	14.30 ± 1.16***	11.20 ± 2.30***	9.70 ± 2.54**

Data presented as mean ± SD.

a

mean percentage of apoptotic cells as a proportion of total cells in five random high power fields (×200 magnification).

*

P < 0.05, **P < 0.01, ***P < 0.001 versus control; Student’s t-test.

In vivo, combination treatment with U0126 and gemcitabine resulted in significantly increased apoptosis compared with controls, in tumours derived from all three cell lines (P < 0.001 for each comparison; Table 3). Neither U0126 nor gemcitabine treatment alone had any effect on apoptosis in vivo.

Discussion

Human pancreatic cancer is characterized by high intrinsic (de novo) and acquired (therapy induced) resistance to traditional chemo- and radiation therapy. ¹⁵ Understanding the underlying mechanisms of drug resistance in pancreatic cancer is critical in order for new and effective treatments to be developed for this deadly disease. Gemcitabine is the standard chemotherapeutic agent for pancreatic cancer patients, but its clinical impact is limited by the high degree of inherent and acquired resistance. ¹⁵

Studies have found ERK1/2 to be involved in tumour chemoresistance,^16,17 and increased ERK1/2 activity has been shown to correlate with inherent gemcitabine resistance in intrahepatic cholangiocarcinoma cells. ¹⁸ Levels of pERK1/2 appeared to be higher in PANC-1^GemRes cells than in the gemcitabine-sensitive PANC-1 parental cell line in the present study, suggesting that gemcitabine resistance may be mediated via pERK1/2.

Inhibition of ERK1/2 activity by U0126 promoted gemcitabine-induced apoptosis in human pancreatic adenocarcinoma cell lines and reversed acquired gemcitabine resistance in PANC1^GemRes in the present study. BxPC-3 cells, which were shown not to produce pERK1/2 constitutively in the present study, were sensitive to gemcitabine-induced apoptosis in vitro. These data suggest that inherent ERK1/2 activity may play an important role in gemcitabine resistance.

It has been suggested that the ERK1/2 pathway mediates apoptosis induced by various stimuli, including cisplatin. ¹⁹ This is supported by the finding that Nic-T24 cells (which have low ERK1/2 activity) tend to remain in G₀/G₁ phase rather than undergo apoptosis, in response to antitumour agents. ¹⁷

In conclusion, the findings of the present study suggest that high ERK1/2 activity protects tumour cells from chemotherapy-induced apoptosis, in pancreatic cancer. The combined use of an ERK1/2 inhibitor (such as U0126) and gemcitabine may result in synergistic therapeutic effects at tolerable gemcitabine doses. Further studies are required to elucidate the mechanism by which ERK1/2 inhibition enhances chemotherapeutic effects. The significance of pERK1/2 as a therapeutic marker in clinical specimens remains to be investigated.