The authors have declared that no competing interests exist.
Conceived and designed the experiments: KG PW. Performed the experiments: PW XW TL XN. Analyzed the data: PW NZ TL XN. Contributed reagents/materials/analysis tools: KG NZ CZ. Wrote the paper: PW.
Co-expression of erythropoietin (Epo) and erythropoietin receptor (EpoR) has been found in various non-hematopoietic cancers including hereditary and sporadic renal cell carcinomas (RCC), but the Epo/EpoR autocrine and paracrine mechanisms in tumor progression have not yet been identified. In this study, we used RNA interference method to down-regulate EpoR to investigate the function of Epo/EpoR pathway in human RCC cells. Epo and EpoR co-expressed in primary renal cancer cells and 6 human RCC cell lines. EpoR signaling was constitutionally phosphorylated in primary renal cancer cells, 786-0 and Caki-1 cells, and recombinant human Epo (rhEpo) stimulation had no significant effects on further phosphorylation of EpoR pathway, proliferation, and invasiveness of the cells. Down-regulation of EpoR expression in 786-0 cells by lentivirus-introduced siRNA resulted in inhibition of growth and invasiveness
Von Hippel-Lindau (VHL) disease is an autosomal dominant inheritable disorder characterized by the inactivation of VHL tumor suppressor gene due to mutations in
It becomes clear now that the co-expression of Epo and erythropoietin receptor (EpoR) is not restricted to hematopoietic cells and can be detected in several tumors including RCC
To evaluate the function of Epo/EpoR pathway in RCC cells, we used RNA interference method to down-regulate EpoR expression in 786-0 cells and then observed the changes in growth, invasiveness, apoptosis, and sensitivity to the clinically used multi-kinases inhibitor Sunitinib. Our data provided the evidences that Epo/EpoR system in RCC may be involved in tumor growth, invasion, survival and sensitivity to Sunitinib.
RCC cell lines, primary renal tumor cells and the normal renal proximal tubular epithelial cell line HK-2 expressed EpoR mRNA and EpoR protein. RCC cell lines and primary renal tumor cells expressed EpoR higher than HK-2 cells, but much lower than the Epo-dependent UT-7 leukemia cells (
Real-time RT-PCR was used for quantification of (A) EpoR mRNA and (C) Epo mRNA, western blot for (B) EpoR protein detection. Error bar indicates mean ± SD from 3 independent experiments.
To assess the effect of exogenous Epo on Epo/EpoR pathway in RCC cells, we used the method similar to that described by Paragh et al.
(A) RCC cells were treated with 0–100 U/ml Epo for 5 min after serum starvation for 24 hours. Compared with Epo-dependent UT-7 cells, the phosphorylation of EpoR, STAT5, Akt and Erk1/2 in PRTC, 786-0 and Caki-1 cells increased insignificantly, if any, after rhEpo stimulations. Moreover, RCC cells expressed higher levels of baseline phosphorylation of all examined EpoR signaling proteins than UT-7 cells in the absence of exogenous Epo. In UT-7 cells, p-EpoR, p-STAT5, p-Erk1/2 and p-Akt were at lower levels before Epo stimulation, and increased significantly at 1 U/ml Epo. Experiments were done in triplicate with similar results. (B) PRTC, 786-0 cells and Caki-1 cells were cultured in media containing 0, 1, 10 and 100 U/ml Epo after serum starvation for 24 hours. Viable cells were evaluated after various incubation periods by modified MTT assay. Exogenous Epo had little effect on proliferation of RCC cells (6 wells for one sample, and experiments in triplicate;
In the cells stably expressing EpoR siRNA, EpoR and p-EpoR reduced by more than 90%, p-STAT5 decreased by more than 60%, whereas p-Erk1/2 increased by more than 36%, as compared with those in stably expressing negative siRNA and the parental cells (
(A) Signaling molecules were assayed in 786-0 expressing EpoR siRNA (EpoR siRNA) by western blot, using parental 786-0 cells (control) and 786-0 expressing negative siRNA (Neg siRNA) as controls. In 786-0 cells expressing EpoR siRNA, EpoR and p-EpoR decreased by >90%, p-STAT5 decreased by >60%, but p-Erk1/2 increased by >36%, as compared with those in the two controls. (B) Proliferation rate reduced significantly in 786-0 expressing EpoR siRNA by modified MTT method (experiments in triplicate;
Prior studies reported that exogenous Epo influenced the effects of chemoradiotherapy on cancer cells
(A) 786-0 cells stably expressing EpoR siRNA, 786-0 cells stably expressing negative siRNA, and parental 786-0 cells were incubated in the medium containing various concentrations of Sunitinib for 24 hours, and the viable cells were measured by modified MTT method. Error bar represents mean ± SD from 6 wells for each time point and triplicate experiments for each sample. 786-0 cells stably expressing EpoR siRNA were slightly more sensitive to Sunitinib than the two controls. (B) 786-0 cells stably expressing EpoR siRNA were cultured in medium containing 5 µg/ml Sunitinib for 24 hours, then subjected to assess cell apoptosis by flow cytometry. Suntinib treatment caused more apoptotic cells(Annexin V-PE+/7-AAD+/−)in 786-0 cells stably expressing EpoR siRNA than in 786-0 cells stably expressing negative siRNA (★★★:
786-0 cells stably expressing EpoR siRNA were inoculated in nude mice, using 786-0 cell stably expressing negative siRNA and the parental 786-0 cells as controls. Tumor xenograft of 786-0 cells with knockdown EpoR grew significantly slower than the xenografts of the two control groups (
Nude mice were subcutaneously inoculated with 2×106 parental 786-0 cells (Control, n = 6), 786-0 cells stably expressing negative siRNA (Neg SiRNA, n = 7), or 786-0 cells stably expressing EpoR siRNA (EpoR siRNA, n = 10). (A) (B) Tumor sizes after the inoculation for 10 weeks. (C) Changes of tumor size within 10 weeks (★★:
Epo and EpoR expression has been reported in many tumors including RCC, but the function of Epo/EpoR pathway is largely unknown in cancer cells. Subsequently, it has been a controversial issue whether rhEpo and its analog erythropoietin stimulating agents are detrimental for the treatment of anemia in cancer patients by promoting cancer cell survival and angiogenesis
Using quantitative real-time reverse-transcription-PCR (qRT-PCR) and western blot, we found that primary renal cancer cells and the 6 RCC cell lines including 786-0, 769P, A498, OS-RC-2, Caki-1 and Caki-2 cells expressed EpoR and Epo, consistent with the findings from human RCC surgical samples
To evaluate the function of EpoR in RCC cells, we used RNA interference method to down-regulate EpoR in 786-0 cells, and examined the changes of signaling molecules, proliferation, invasion, apoptosis, xenograft growth
Sunitinib is a multi-targeted receptor tryrosine kinases inhibitor effective for the treatment of RCC. However, this drug only extends patients’ life, drug resistance usually occurs after a median of 6–15 months of treatment
In conclusion, co-expression of Epo and EpoR was found in RCC cells. EpoR and its signaling molecules STAT5, Akt, and Erk1/2 were constitutively activated in primary renal cancer cells, 786-0 cells and Caki-1 cells. Exogenous Epo had no additional effects on proliferation and invasion ability of the cells. Down-regulation of EpoR expression in 786-0 cells attenuated the proliferation and invasion
The cell lines used in this study included six human RCC cell lines (786-0, 769P, A498, OS-RC-2, Caki-1 and Caki-2). HK-2 (a normal human proximal tubular cell line), HepG-2 (a human hepatoma cell line expressing Epo), and UT-7 cells (an Epo-dependent human leukemia cell line) were used as the control cell lines. 786-0 cells, a human renal cell line carrying a mutation in
Samples of RCC were obtained from a patient who underwent nephrectomy in Peking University First Hospital. The renal tumor block was cut into small pieces, digested with collagenase for 4 hours, and then with trypsin for 1 hour. The cells were washed twice in RPMI 1640, and then cultured at 37°C in an atmosphere of 5% CO2. Cells were retrieved by trypsinization and passage when they reached a confluence of 90–95%. Cells were collected at 2nd passage. The protocol for the primary renal tumor cell culture was approved by the Medical Ethics Committee of Peking University First Hospital.
We designed oligonucleotides targeting human EpoR mRNA of
Total RNA was extracted from cells using Trizol reagent (Invitrogen, Carlsbad, USA). Reverse-transcription (RT) was performed using 2 µg total RNA, oligo (dT) primer and AMV reverse transcriptase in a volume of 20 µl. Quantitative RT-PCR was carried out in a 7300 real-time PCR system (ABI, Foster City, USA) using TaqMan probes as the indicator and the default condition set in the instrument. For measurement of Epo cDNA, we used primer Epo-F:
AGCCGAGCCACA-TAMRA.
When RCC cells grew to confluence, the medium was used for Epo determination. We used the Human Epo Elisa Assay kit (RB, CA, USA) following the manufacturer’s recommendation. Each sample was performed in triplicate.
Cells were washed twice with ice cold phosphate-buffered saline (PBS) and lysed in RIPA buffer (50 mmol/l Tris-HCl pH 7.4, 150 mmol/l NaCl, 1 mmol/l EDTA, 0.25% sodium deoxycholate, 1% NP-40, 0.1 mg/ml PMSF, 10 µg/ml aprotinin, and 1 mmol/l sodium orthovanadate). Lysate containing 40 µg protein quantified by BCA Protein Assay Kit (keyGEN, Nanjing, China) for each sample was subjected to SDS-PAGE, transferred to nitrocellulose membrane, and blotted by the primary antibodies against EpoR (M-20, sc-697, Santa Cruz, CA, USA), phosphorylated-EpoR (p-EpoR) (rabbit polyclonal antibodies, Santa Cruz, Santa Cruz, CA; 1∶500), STAT5, p-STAT5, Akt (PKB), p-Akt, Erk1/2 or p-Erk1/2 (rabbit polyclonal antibodies, Cell Signaling, Danvers, MA; 1∶1000), or GAPDH (mouse monoclonal antibody, Santa Cruz, CA, USA) as a loading reference. Blotted primary antibodies were detected by 1∶5000 HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibody and enhanced chemiluminescence (ECL), and visualized by Imaging Station 4000 mm Pro (Kodak).
Viable cells were assessed using a modified MTT assay kit (keyGEN, Nanjing, China) following the manufacturer’s protocol. To determine the stimulating effect of rhEpo on cell proliferation, RCC cells were plated in 96-well plates at a density of 1,000 cells per well, incubated to adhesion, cultured in serum-free medium for 24 hours, and then cultured in medium containing 10% FBS and 0 to 100 U/ml rhEpo. The amount of viable cells was determined every 24 hours using six wells per time point. For color development, 10 µl dye solution was added to each well and the plate was incubated at 37°C for 3 hours. Absorbance at 450 nm was determined using a 96-well plate reader. Each sample was performed in triplicate.
To assay the apoptosis effect of down-regulated EpoR, 786-0 cells were transiently transfected with the recombinant lentivirus vector for 3 days for down-regulation of EpoR mRNA, then gently trypsinized and washed with PBS, re-suspended in 50 µl 1× binding buffer (10 mmol/l HEPES buffer pH 7.4, 150 mmol/l NaCl, 1 mmol/l CaCl2), and stained with PE-conjugated annexin V and 7-Aminoactinomycin D (7-AAD). To determine the anti-apoptosis effect of rhEpo, 786-0 cells were plated in six-well plates at a density of 5×105/well, cultured to sub-confluence, treated with Sunitinib with or without rhEpo in the medium for 24 hours, trypsinized and washed twice with PBS, re-suspended in 50 µl 1 × binding buffer, and stained with FITC-conjugated annexin V and propidium iodide (PI). Cells were then incubated at room temperature for 10 minutes in dark, analyzed in a flow cytometer (FACSAria, BD) within one hour. Each sample was done at least in triplicate.
The 24-well Biocoat Matrigel Invasion Chamber with an 8 µm pore polycarbonate filter (Becton Dickinson, Bedford, MA, USA) was used for the assay. Cells in a growing phase were trypsinized, re-suspended in serum-free medium with or without Epo, and plated in upper chamber at a density of 2×104 cells. The lower compartment was filled with medium containing 10% FBS. After incubation for 36 hours, cells on filter were stained and counted under a microscope.
Matrix metallopeptidase (MMP) activities in 786-0 cells were determined by gelatin zymography. The sample extracted from cell supernatant was diluted in a buffer (0.12 M Tris–HCl, 20% glycerol, 0.1% bromophenol blue, 10% SDS), and 10 mg total protein was separated in gelatin-impregnated zymogram gel (10% zymogram gelatin gel) at 120 V for 90 min. The gel was incubated at room temperature in a zymogram renaturing buffer (Invitrogen, CA) for 30 min, washed in zymogram developing buffer for 30 min, incubated in fresh zymogram developing buffer overnight at 37°C, developed by staining with 0.5% Coomassie blue for 90 min, and washed in destaining solution until clear bands of MMPs appeared against a blue background.
Thirty BALB/C nude mice were randomly divided into three groups and then subcutaneously injected into left groin area with 2×106 786-0 cells stably expressing EpoR siRNA, 786-0 cell expressing scrambled negative siRNA and parental 786-0 cells. Tumor size was recorded every week for 10 weeks. At the end point, 6, 7 and 10 nude mice remained in parental cell group, negative siRNA group, and EpoR siRNA group, respectively. The animal experiment protocol was approved by Peking University Institutional Animal Care and Use Committee.
Data are expressed as mean ± SD. Comparisons among groups were conducted by one-way ANOVA followed by the least square difference test. Paired
We thank Prof. Ding-fang Bu, Center for Medical Experiments, Peking University First Hospital, for his technical support and editorial assistance.