Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Trizol was purchased from Invitrogen (Carlsbad, CA, USA). Urapidil hydrochloride, yohimbine, streptomycin, penicillin, and 3,3-dimethyl-2-thionotetrahydro-1,3,5-thiadiazine (MTT) were purchased from Sigma Chemicals (St. Louis, MO, USA).

PC-2 and PC-3 were obtained from the Pathology Department of China Beijing Union Medical University (PC-3 cell line established from human primary exocrine pancreatic head-cancer, PC-2 cell line from human exocrine pancreatic cancer metastasis). Cells were cultured in a high glucose-DMEM medium supplemented with 10% heat-inactivated fetal calf serum, penicillin (100 U/mL), and streptomycin (100 mg/mL) at 37°C in an atmosphere containing 50 mL/L CO₂ and 950 mL/L with a relative humidity.

RNA was isolated from PC-2 and PC-3 cells lysed with Trizol. Dense RNA was quantified by UV spectrophotometry (at 260/280 nm) and stored at -70°C. For reverse transcriptase reaction, 0.5 &mgr;g of 18 oligo (dT) primers in diethyl pyrocarbonate (DEPC) water was heated to 70°C for 5 min to allow annealing of the primer to RNA followed by chilling on ice. A total of 10 mmol/L dNTPs, 20 units of RiboLock ribonuclease inhibitor, 5 × reaction buffer [250 mmol/L Tris-HCl (pH 8.3), 250 mmol/L KCl, 20 mmol/L MgCl₂, and 50 mmol/L dithiothreitol (DTT)] were added and the reaction mixture was incubated at 37°C for 5 min. Then, 200 units of M-MuLV reverse transcriptase was added and the mixture was incubated at 42°C for 1 h, followed by heat inactivation for 10 min at 70°C.

PCR was performed with 5 &mgr;L RT reaction mixture containing 1 &mgr;L of 10 mmol/L dNTPs, 5 &mgr;L of 10× PCR buffer [100 mmol/L Tris-HCl (pH 8.3), 500 mmol/L KCl, and 15 mmol/L MgCl₂, 1.5 &mgr;L of Taq polymerase (Perkin Elmer, Branchburg, NJ, USA), 1 &mgr;L primers for human α_1A-, α_1B-, α_1D-, α_2A-, α_2C-, α_2B-, β₁-, and β₂-adrenoceptors, and DEPC water in a final volume of 50 &mgr;L. Adrenoceptor primers were designed according to Genbank, NCBI. The α_1A adrenoceptor primers (sense: 5’-CTCTGCGTCTGGGCACTCT-3’ and antisense: 5’-GAAGCAGCCGACCACGAT-3’) were used to amplify a 402-bp fragment. The α_1B primers (sense: 5’-AAGAACTTTCACGAGGACACCC-3’ and antisense: 5’-CAGAACACCACCTTGAACACG-3’) were used to amplify a 233-bp fragment. The α_1D primers (sense: 5’-TCACTCAAGTACCCAGCCATCA-3’ and antisense: 5’-GGAACCAGCAGAGCACGAAG-3’) were used to amplify a 487-bp fragment. The α_2A primers (sense: 5’-ATCGGAGTGTTCGTGG-3’ and antisense: 5’-AGGAAGCAATAGTGATTAGGG-3’) were used to amplify a 489-bp fragment. The α_2C primers (sense: 5’-TGTTCGTGCTCTGCTGGTTC-3’ and antisense: 5’-GGGGAAGGCAAAGGGGTC-3’) were used to amplify a 427-bp fragment. The α_2B primers (sense: 5’-AAATCGGGGCGACAATAG-3’ and antisense: 5’-GAGACCCAAGCCACTAAAA-3’) were used to amplify a 367-bp fragment. The β₁ primers (sense: 5’-GGGAGAAGCATTAGGG-3’ and antisense: 5’-CAAGGAAAGCAAGGTGGG-3’) were used to amplify a 270-bp fragment. The β₂ primers (sense: 5’-CAGCAAAGGGACGAGGTG-3’ and antisense: 5’-AAGTAATGGCAAAGTAGCG-3’) were used to amplify a 334 bp fragment. The PCR conditions for the receptors of the above-mentioned primers were as follows: 1 cycle at 94°C for 2 min; 35 cycles at 94°C for 30 s, at 62°C for 60 s, and at 70°C for 2 min; and a final extension at 70°C for 7 min. Reactions were run on a MJ Research PTC-200 thermal cycler. The PCR products and a 100-bp DNA ladder were run on a 1.5% agarose gel for 2 h at 90 V. The gel was imaged using ethidium bromide staining with a UVP GDS 7500 or an Ultra Lum TUI-5000 gel documentation system.

The effects of yohimbine and urapidil hydrochloride on cell proliferation were detected by colorimetric MTT assay based on the NADH-dependent enzymatic reduction of the tetrazolium salt MTT in metabolically active cells but not in dead cells. Cells were seeded in a basal medium at the concentration of 1 × 10⁴ cells/well in 96-well plates, and allowed to grow and adhere in complete media at 37°C with 50 mL/L CO₂ for 20-24 h. The cells were then transferred to basal media with 0.05% FBS and incubated for 24 h with control (no treatment), yohimbine (25-200 &mgr;mol/L), and urapidil hydrochloride (25-200 &mgr;mol/L). Following incubation, 20 &mgr;L MTT (5 mg/mL) was added to each well, and the cells grew in complete media at 37°C with 50 mL/L CO₂ for 4 h. The supernatant was removed, then 150 &mgr;L DMSO was added to each well of the 96-well plate and swung for 10 min. The optical density at 490 nm was determined with an enzyme linked immunosorbent assay (ELISA) reader.

TUNEL assay was performed with the in situ cell apoptosis detection kit following its manufacturer’s instructions. Briefly, the cells were replaced on cover slips after exposure to urapidil and yohimbine for 24 h and fixed with 4% paraformaldehyde for 30 min and adhered to slides with balsam. Nonspecific chromogen reaction, induced by endogenous peroxidase, was inhibited with 3% H₂O₂ for 10 min at room temperature. Terminal deoxynucleotidyl transferase (TdT) and biotin-11-dUTP reactions were performed for 1 h at 37°C in a humidified box, and blocking reagent was applied for 30 min at room temperature, followed by Avidin-HRP for 1 h at 37°C in a humidified box. For biochemical controls, positive control slides were treated with DNase and negative control slides were treated with PBS instead of TdT. DNA fragments were stained using DAB as a substrate for peroxidase, and hematoxylin was used as a counter stain. Apoptotic index was calculated as a ratio of the number of apoptotic cells to the total number of tumor cells in each slide.

The apoptosis rate was measured by FCM according to the instructions supplied with the annexin V-FITC kit. In brief, after treatment with urapidil and yohimbine, PC-2 and PC-3 cells were harvested by centrifugation, washed once with ice-cold PBS and resuspended in binding buffer at a concentration of 1 × 10⁶ cells/mL, in which 100 mL of cell suspension was added in a 5 mL FCM tube. A total of 5 mL of annexin V-FITC and 10 mL of 20 mg/mL PI were added and incubated for 15 min in the dark prior to a further addition of 400 mL PBS. Quantitative analysis of apoptotic level was performed using a flow cytometer (BD Biosystems, CA, USA). The apoptotic percentage of 10 000 cells was determined and all the experiments reported in this study were performed in triplicate.

In vitro data were analyzed by one-way ANOVA and nonparametric Kruskal-Wallis test by using SPSS version 13.0. Values were expressed as mean ± SD, and P < 0.05 was considered statistically significant.

RT-PCR results demonstrated that PC-2 expressed mRNA in α_1D, α_1A, α_2A, β₁ and β₂-adrenoceptors and PC-3 expressed mRNA in α_2B, β₁, and β₂-adrenoceptors (Figure 1).

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Figure 1 Expression of mRNA in adrenoceptors of human ductal pancreatic adenocarcinoma cell lines PC-2 and PC-3 by RT-PCR. Lanes 1-8 represent the α_1D-, α_1A-, α_2A-, α_2B-, α_2C-, α_1B-, β₁-, and β₂-adrenoceptor subtypes, respectively. Lane maker = 100 bp DNA ladder. PC-2 expressed mRNA in α_1D-, α_1A-, α_2A-, β₁-, and β₂-adrenoceptors and PC-3 expressed mRNA in α_2B-, β₁-, and β₂-adrenoceptors.

MTT assays showed that urapidil hydrochloride had no effect on PC-3 cell lines, whereas exposure to urapidil hydrochloride increased DNA synthesis in the PC-2 cell line as compared to the control group. PC-2 was sensitive to both drugs. The proliferation of 2 cell lines was inhibited by yohimbine. However, the rate of suppression by yohimbine was weaker than that by 5-FU used as a positive control (Figure 2).

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Figure 2 MTT assay shows that PC-2 cells treated with urapidil hydrochloride for 24 h could increase cellular proliferation in a concentration-dependent manner. Yohimbine inhibited the proliferation of PC-2 cells (A) and urapidil hydrochloride had no effect on PC-3 cell lines while the proliferation of PC-3 cell lines was inhibited by yohimbine (B).

Nuclei of apoptotic cells were condense and stained brown whereas the negative cells were stained blue in the TUNEL assay. The results were in accordance with the MTT assay and revealed that yohimbine could induce apoptosis of both cell lines whereas urapidil hydrochloride had no effect on the 2 cell lines (Figure 3 and Table 1).

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Figure 3 TUNEL assay showing apoptotic PC-2 and PC-3 cells after treatment with 100 &mgr;mol/L urapidil hydrochloride and yohimbine for PC-2 cells and 100 &mgr;mol/L yohimibine and 100 &mgr;mol/L urapidil for PC-3 cells compared with control group.

PC-2 and PC-3 cells were treated with the α-adrenoreceptor antagonists, urapidil hydrochloride (200 &mgr;mol/L) and yohimbine (200 &mgr;mol/L) for 24 h. The cells treated with urapidil hydrochloride and yohimbine were double stained with annexin V and PI and analyzed by FCM (Figure 4).

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Figure 4 Apoptotic PC-2 and PC-3 cells after treatment with 200 &mgr;mol/L urapidil hydrochloride (A) and 200 &mgr;mol/L yohimbine (B) for PC-2 cells and 200 &mgr;mol/L yohimibine for PC-3 cells (C) detected by FCM. Significant apoptosis was induced by yohimbine on both PC-2 and PC-3 cells, whereas urapidil hydrochloride had no apoptotic effect on PC-2 cell.

It is supposed that neurobiology would play a key role in development of malignant tumors, by its direct effect on malignant tumors or on tumor micro-environment. Autonomic nerves are closely related with various activities of the body. A few reports are available on the beta-adrenergic receptors in cancer of colon, breast, and prostate, etc. However, little is known about alpha-adrenergic receptors in malignant tumor, we, therefore, studied the distribution and function of alpha-adrenergic receptors of malignant tumour cells.

Pancreas is a complex organ, including blood, nerve and lymph. Pancreatic cancer is generally characterized by less blood supply, innervation complex, and lymphatic irregularity, which have been brought difficulty to diagnosis and treatment of pancreatic cancer.

In recent years, basic researches of malignant tumor concentrated on their biological characteristics, such as invasion and metastasis. This study explored the development, metastasis and infiltration of pancreatic cancer by neurobiology.

The study is only a part of our research. The study would screen the drugs suppressing malignant tumor, from cardiovascular drugs.

The study investigated the effects of α-adrenoreceptor antagonists on proliferation and apoptosis of pancreatic cancer cells. The subject is important because very little is known about the role of adrenergic stimulation in the regulation of proliferation and death of pancreatic cancer cells. The study was well designed.