LY294002

LY294002 Enhances Inhibitory Effect of Gemcitabine on Proliferation of Human Pancreatic Carcinoma PANC-1 Cells

Summary: Phosphatidylinositide 3-kinase (PI3K)/protein kinase B (PKB, Akt) pathway plays a major role in proliferation and survival of many types of cells. The inhibitory effect of LY294002, widely ap- plied as an inhibitor of PI3K, in combination with gemcitabine on proliferation of PANC-1 cells was investigated. The expression of PI3K, phosphorylated Akt (p-Akt) and multidrug-resistance like protein (MRP) in normal pancreas tissues, chronic pancreatitis tissues and pancreatic carcinoma tissues was de- tected. The effects of LY294002 combined with gemcitabine on proliferation of PANC-1 cells and pro- tein levels of p-Akt and MRP were detected. The results showed that the positive expression rate of PI3K, p-Akt and MRP in pancreatic carcinoma tissues was significantly higher than that in normal pan- creas tissues and chronic pancreatitis tissues (P<0.01 and P<0.05 respectively). LY294002 could effec- tively enhance the inhibitory effect of gemcitabine on proliferation of PANC-1 cells. Furthermore, Western blotting revealed that LY294002 combined with gemcitabine reduced the protein levels of p-Akt and MRP, which contributed to the inhibition of proliferation. It is concluded that LY294002 in combination with gemcitabine may represent an alternative therapy for pancreatic carcinoma. Key words: pancreatic carcinoma; phosphatidylinositide 3-kinase; phosphorylated protein kinase B; multidrug-resistance like protein; LY294002 Pancreatic carcinoma lacks effective treatment and has an ominous prognosis. Presently, gemcitabine is one of the most common chemotherapy drugs for pancreatic carcinoma patients[1, 2]. However, the objective response rate of gemcitabine treatment is less than 20% in clinic[3, 4]. Therefore, alternative strategy is in urgent need, to increase the efficacy of gemcitabine as chemotherapeutic agent[5]. It is well documented that certain genetic abnor- malities occur at very high frequencies in pancreatic car- cinoma, which may potentially up-regulate the activity of the phosphatidylinositide 3-kinase (PI3K)/protein kinase B (PKB, Akt) pathway[6]. PI3K, a heterodimer consisting of a p85 regulatory subunit and a p110 catalytic subunit, has been demonstrated to phosphorylate the ser- ine/threonine kinase PKB/Akt. Akt is an important downstream target of PI3K and functions as regulating cell survival, proliferation, and protein synthesis[7]. Inac- tivation of PI3K/Akt-dependent pathway may improve sensitivity of gemcitabine in pancreatic carcinoma cells such as PANC-1 and BxPC-3[8, 9]. The PI3K-Akt pathway plays an important role in proliferation in human pancre- atic carcinoma cells. The PI3K inhibitor LY294002 might inactivate Akt, consequently inhibit cell proliferation in human pancreatic carcinoma cell lines PSN-1, PANC-1, and KP-4[10, 11]. LY294002, as a dominant in- hibitor of PI3K signaling pathway, is a promising drug candidate in clinic[12, 13]. The expression of drug efflux pumps including multidrug-resistance like protein (MRP) family is com- mon and detected frequently in pancreatic carcinoma[14]. MRP1- and p-glycoprotein-overexpressing cells are more sensitive to gemcitabine than their parental cells in hu- man cancer cell lines[15]. PI3K/Akt pathway may modu- late the expression of the MRP-1 gene and be necessary for maximal expression of MRP-1[16]. Inhibition of PI3K/Akt signaling pathways combined with cytotoxic drugs is recently gaining popularity in pancreatic carci- noma researching[17]. Likewise, PI3K inhibition com- bined with gemcitabine may be regarded as an alterna- tive strategy that could be effectively applied in patients with pancreatic carcinoma. Based on the above facts, we investigated the inhibitory effects of LY294002 (a PI3K specific inhibitor) combined with gemcitabine on prolif- eration of human pancreatic carcinoma PANC-1 cells, and observed the change of Akt, phosphorylated Akt (p-Akt) and MRP in the cells. 1 MATERIALS AND METHODS 1.1 Clinical Data Tumor samples were obtained from 43 pancreatic carcinoma patients (29 males and 14 females; of them 12 aged ≤45 years and 31 aged >45 years) surgically treated at Tongji Hospital of Tongji Medical College, Wuhan, China, from Year 2000 to 2006. All of the tumors were confirmed as pancreatic carcinoma by the Clinic Patho- logical Department of Tongji Hospital. Lymph node staging was made according to cancer staging manual of the American Joint Committee on Cancer (AJCC)[18]. Another 9 chronic pancreatitis tissues and 8 normal pan- creas tissues for control were obtained from the same hospital.

1.2 Reagents and Cell Culture

Gemcitabine was purchased from Lily Pharmaceu- tical Co., France, and LY294002 from Sigma Pharma- ceutical Co., USA. The polyclonal antibody including PI3K, Akt, p-Akt and the monoclonal antibody MRP were bought from Abcam Biotechnology Inc. UK. The polyclonal antibody β-actin was procured from Sant Cruz Biotechnology Inc., USA. Human pancreatic car- cinoma cell line PANC-1 was obtained from the Re- search Institute of Liver Diseases of Tongji Hospital.

Gemcitabine and LY294002 were dissolved in 0.9% NaCl and DMSO, respectively. PANC-1 cells were cul- tured in DMEM high glucose medium supplemented with 10% fetal bovine serum at 37°C and in a humidified atmosphere with 5% CO2. There were 2 experiments performed as below. The first one: cells grown were classified into five groups according to different LY294002 concentrations of 0, 10, 25, 50, 100 μmol/L for 24-h incubation. The second one: cells were exposed to 20 μmol/L gemcitabine or 25 μmol/L LY294002 for 24 h, and combination studies involved treatment with an initial 24-h exposure to 20 μmol/L gemcitabine, followed by additional 24-h treatment with 25 μmol/L LY294002. The final concentration of DMSO was 0.1% or less. To- tal proteins were isolated from PANC-1 cells and ana- lyzed by Western blotting.

1.3 Immunohistochemistry

The 5-μm thick histological sections were prepared from each patient. A standard streptavidin-biotin peroxi- dase method (SP Kit, ZhongShan Goldenbridge Bio- technology Co. LTD, China) was used to detect the pro- teins of PI3K, p-Akt and MRP. Briefly, the sections were dewaxed by using xylene, rehydrated with alcohol, im- mersed in a solution of 3% H2O2, then transferred to cit- rate buffer and boiled for 15 min in a microwave oven. Afterwards, the sections were washed 3 times in phos- phate buffered saline (PBS) and incubated in goat serum for 30 min, to reduce nonspecific antibody binding. After further washing with PBS, the sections were re-incubated for 15 h at 4°C with the following antibodies: rabbit polyclonal antibody against PI3K, rabbit polyclonal an- tibody against p-Akt, mouse monoclonal antibody against MRP. Omission of the primary antibody was used as a negative control. Finally, the reaction was visualized with a chromogen, diaminobenzidine in 3% hydrogen peroxidase. Sections were then counterstained with he- matoxylin, dehydrated and mounted.

1.4 Determination of Proliferation of PANC-1 Cells

The cells were collected and transferred to a 96-well plate for 24-h normoxic incubation. Then, 3 different experiments were carried out as follows: (1) Gemcitabine alone was added to the culture medium at final concentrations of 10, 20, 40, 60 and 80 μmol/L; (2) LY294002 alone was added to the culture medium at final concen- trations of 10, 25, 50, 80 and 120 μmol/L; (3) The cells were incubated with LY294002 at final concentrations of 10, 25, 50, 80 and 120 μmol/L, combined with gemcit- abine at a final concentration of 20 μmol/L. And the cells cultured under normoxic condition without gemcitabine or LY294002 addition were used as control. After con- tinuous incubation for 24 h, 20 μL 3-[4,5-dimethylth- iazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT, 5 mg/mL) was added into each well, and the plate was continuously incubated for another 4 h. After the entire medium was discarded, 150 μL DMSO was added into each well, and then gently mixed for 10 min. The viable cell number was indirectly reflected by the absorbance (A) value at 492 nm on the microplate reader.

1.5 Western Blotting

Cells were washed thrice with PBS, and lysed with lysis buffer [50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 5 mmol/L EDTA, and 1% Triton X-100] and pro- tease inhibitor mixture (Roche Diagnostics, Germany) on ice for 30 min. Whole cell lysates were centrifuged at 12 000 g for 30 min at 4°C. Protein concentration of each sample was determined by using the Bradford protein assay (Bio-Rad, USA). Cell lysates (30 μg/lane) were separated by 8% SDS-polyacrylamide gel electrophoresis. After electrophoresis, proteins were transferred to nitro- cellulose membranes (Millipore, USA). After 1-h incu- bation in a blocking solution (5% non-fat milk in PBS-1% Tween 20), the membrane was incubated with the following antibodies overnight: rabbit polyclonal antibody against Akt, rabbit polyclonal antibody against p-Akt, mouse monoclonal antibody against MRP and mouse polyclonal antibody against β-actin, followed by incubation with the second antibodies, which were horseradish peroxidase-conjugated goat anti-rabbit an- tiserum and horseradish peroxidase-conjugated goat anti-mouse anti-serum (ZhongShan Biotechnology Co., China). After washing with PBST, the complex was visualized by Enhanced Chemiluminescent Detection Kit (Pierce Biotechnology, USA). β-actin was used to semi-quantify the expression of Akt, p-Akt and MRP.

1.6 Statistical Analysis

All data were presented as mean±SD of at least three independent experiments, and analyzed by SPSS13.0 software. Fisher’s exact test or Pearson’s χ2 test of equality was used to compare expression rate in clinic pathological cases. Two tailed paired Student’s t test and One-way ANOVA correction were used to iden- tify differences between the control and treatment groups. Differences between groups were considered statistically significant at P<0.05. 2 RESULTS 2.1 Expression of PI3K, p-Akt, MRP and Their Cor- relation with Clinicopathological Features The staining pattern of PI3K, p-Akt and MRP ex- pression was predominantly observed in cytoplasm (fig. 1). The positive expression rate of PI3K, p-Akt and MRP in pancreatic carcinoma tissues was 46.51%, 55.81% and 39.53% respectively, which was significantly higher than that in the normal pancreas tissues and chronic pancreatitis tissues (P<0.01 and P<0.05, respectively) (table 1). The aberrant expression of PI3K and p-Akt was associated with lymph node metastasis and TNM stages (P<0.05, table 2). Fig. 1 Immunohistochemical staining of PI3K, p-Akt and MRP proteins (×400) A: negative staining of PI3K protein in normal pancreas tissues; B, C and D: positive staining of PI3K, p-Akt and MRP protein in pancreatic carcinoma tissues respectively. 2.2 LY294002 Enhanced Inhibitory Effect of Gem- citabine on Proliferation of PANC-1 Cells After the cells were incubated with gemcitabine (10, 20, 40, 60 and 80 μmol/L) alone for 24 h, the inhibitory rate was (15.52±2.74)%, (22.63±2.35)%, (26.82±2.50)%, (34.21±3.01)%, (39.73±2.98)%, respectively. As for cells treated with LY294002 (10, 25, 50, 80 and 120 μmol/L) alone for 24-h incubation, the inhibitory rate was (21.47±3.01)%,(25.93±2.53)%,(31.62±2.18)%, (37.81±3.56)%, (45.82±2.38)%, respectively. For cells treated with combination of LY294002 (10, 25, 50, 80 and 120 μmol/L) and gemcitabine (20 μmol/L) for 24-h incubation, the inhibitory rate was (33.92±2.92)%, (45.36±2.27)%, (58.23±2.64)%, (66.32±3.18)%, (78.51±2.63)%, respectively which was significantly higher than that of the cells only treated with gemitabine or LY294002 respectively (P<0.05). Hereby, gemcitabine and LY294002 could inhibit proliferation of PANC-cells in a dose-dependent manner. With the increasing concentration of gemcitabine or LY294002, the proliferation rate of PANC-1 cells was reduced gradually. LY294002 could enhance the inhibitory effect of gem- citabine on proliferation of PANC-1 cells in a concentra- tion-dependent manner (fig. 2). Fig. 2 Effects of LY294002 (A), gemcitabine (B), LY294002 combined with gemcitabine (20 μmol/L, C) on proliferation of PANC-1 cells The cells were incubated in normoxic condition for 24 h after adding the drugs. The inhibitory rate in different groups was in- directly reflected by A492 nm value of viable cells. 2.3 Effect of LY294002 Combined With Gemcitabine on Protein Levels of Akt, p-Akt and MRP In order to determine whether the antiproliferative effect of LY294002 combined with gemcitabine was correlated to the inhibition of p-Akt and MRP, the activa- tion status of p-Akt and MRP was analyzed by Western blotting. LY294002 significantly reduced the levels of p-Akt and MRP in PANC-1 cells in a concentration-dependent manner (F=191.34, P<0.05 for p-Akt; F=389.03, P<0.05 for MRP) (fig. 3A). Treatment of cells with gemcitabine alone for 24 h did not alter p-Akt and MRP levels, but treatment with gemcitabine for 24 h followed by LY294002 for 24 h reduced p-Akt and MRP to a greater degree than did LY294002 alone (F=419.16, P<0.05 for p-Akt; F=493.61, P<0.05 for MRP) (fig. 3B). The level of total Akt was not changed by any drug. Fig. 3 Expression of Akt, p-Akt and MRP in PANC-1 cells determined by Western blotting A: Protein levels of Akt, p-Akt and MRP in PANC-1 cells after treatment with LY294002 alone for 24 h. LY294002 signifi- cantly reduced the levels of p-Akt and MRP in PANC-1 cells in a concentration-dependent manner (F=191.34, P<0.05 for p-Akt; F=389.03, P<0.05 for MRP); B: Protein levels of Akt, p-Akt and MRP in PANC-1 cells under treatment of LY294002 combined with gemcitabine. Treatment with gemcitabine for 24 h followed by LY294002 (G+LY) for 24 h reduced p-Akt and MRP to a greater degree than did LY294002 (LY) alone (F=419.16, P<0.05 for p-Akt; F=493.61, P<0.05 for MRP). Con: con- trol; G: gemcitabine; LY: LY294002. 3 DISCUSSION Pancreatic carcinoma is a lethal disease with rapid progression and generally considered to be incurable[3]. One of the main reasons for its low survival rate is that pancreatic carcinoma exhibits extensive local invasion and frequent regional lymph node metastasis in stage Ⅰ and Ⅱ[1]. Till recently, most patients are not candidates for curative surgical resection, and there has been no effective chemotherapeutic drug for this disease[4]. Some studies have reported that gemcitabine-based therapies are the best possible option available currently for pancreatic carcinoma patients. Unfortunately, pancreatic carcinoma is a deadly cancer with limited sensitivity to gemcitabine[19]. Hence, how to increase the efficacy of gemcitabine becomes very critical for pancreatic carci- noma patients. PI3K signaling pathway is well established as an important signaling in cancer. Several studies have dem- onstrated that the PI3K-Akt pathway plays an important role in proliferation and survival of pancreatic carcinoma cells[20, 21]. In some instances, activation of the PI3K-Akt pathway suggests the increase of Akt phosphorylation in human pancreatic carcinoma cells. Inhibition of the PI3K-Akt pathway induces apoptosis in pancreatic carcinoma cells in vitro and in vivo[22]. PI3K/Akt pathway may modulate the expression of MRP-1 gene and be necessary for maximal expression of MRP-1[8, 23]. In ad- dition, inhibition of the PI3K signaling pathway in- creases the responsiveness of pancreatic carcinoma cells to chemotherapeutic agents[24, 25]. We hypothesize LY294002, which is valuable as early and still frequently employed as PI3K inhibitor[26], may enhance the inhibitory effect of gemcitabine on pro- liferation of pancreatic carcimoma cells. Firstly, our re- sults indicated the positive expression of PI3K, p-Akt and MRP was predominantly observed in cytoplasm. In addition, the positive rate of PI3K, Akt and MRP expres- sion in the pancreatic carcinoma tissues was significantly higher than that in the normal pancreas tissues and chronic pancreatitis tissues. We confirmed that the ex- pression of PI3K, p-Akt and MRP was up-regulated in pancreatic carcinoma tissues, and the aberrant expression of PI3K and Akt was associated with lymph node metas- tasis and TNM stages. Then, we examined the inhibitory effect of LY294002, gemcitabine, and combination of them respectively on proliferation of PANC-1 cells. The inhibition rate of proliferation for cells treated with gem- citabine and/or LY294002 was detected by a colorimetric proliferation assay and cell counts. Gemcitabine or LY294002 inhibited proliferation of PANC-1 cells in a dose-dependent manner. As the concentration of gemcit- abine or LY294002 increased, the proliferation rate of PANC-1 cells was reduced gradually. Different concen- trations of LY294002 combined with low-dose gemcit- abine (20 μmol/L) enhanced inhibitory effect of gemcit- abine on proliferation of PANC-1 cells. Furthermore, we examined the effect of PI3K specific inhibitor LY294002 combined with gemcitabine on protein levels of p-Akt and MRP in PANC-1 cells. Our data showed that differ- ent concentrations of LY294002 significantly inhibited the protein levels of p-Akt and MRP. Along with the raising dose of LY294002, the levels of p-Akt and MRP proteins were reduced gradually, and reached the highest inhibitory effect at the dose of 100 μmol/L. When gem- citabine was followed by LY294002, the protein levels of p-Akt and MRP were reduced, and lower than LY294002 used alone. These findings indicated that the inhibitory effect of LY294002 combined with gemcitabine on pro- liferation of PANC-1 cells was correlated with the reduc- tion of p-Akt and MRP protein levels in the cells. In an- other words, LY294002 might enhance the inhibitory effect of gemcitabine on proliferation of PANC-1 cells via reducing the levels of p-Akt and MRP in the cells. To sum up, LY294002, as PI3K inhibitor, enhances the inhibitory effect of gemcitabine on proliferation of pancreatic carcinoma PANC-1 cells, which is associated with the reduction of p-Akt/MPR protein levels. The results presented herein imply that PI3K inhibitor com- bined with chemotherapy may represent a potential therapeutic alternative that could be effectively used in patients with pancreatic carcinoma.