Tumor Biol. (2013) 34:1523–1530 DOI 10.1007/s13277-013-0679-1
RESEARCH ARTICLE
Lactate dehydrogenase A is overexpressed in pancreatic cancer and promotes the growth of pancreatic cancer cells Yefei Rong & Wenchuan Wu & Xiaoling Ni & Tiantao Kuang & Dayong Jin & Dansong Wang & Wenhui Lou
Received: 26 November 2012 / Accepted: 28 January 2013 / Published online: 13 February 2013 # International Society of Oncology and BioMarkers (ISOBM) 2013
Abstract The prognosis for pancreatic cancer is very poor, and developing new therapeutic strategies for this cancer is needed. Recently, the Warburg effect (aerobic glycolysis) has attracted much attention for its function in the tumorigenesis. Lactate dehydrogenase A (LDHA) executes the final step of aerobic glycolysis and has been reported to be involved in the tumor progression. However, the function of LDHA in pancreatic cancer has not been studied. Here, we found that the expression of LDHA was elevated in the clinical pancreatic cancer samples. Forced expression of LDHA promoted the growth of pancreatic cancer cells, while knocking down the expression of LDHA inhibited cell growth dramatically. Moreover, silencing the expression of LDHA inhibited the tumorigenicity of pancreatic cancer cells in vivo. Mechanistically, knocking down the expression of LDHA activated apoptosis pathway. Taken together, our study revealed the oncogenic role of LDHA in pancreatic cancer and suggested that LDHA might be a potential therapeutic target. Keywords Pancreatic cancer . LDHA . Cell apoptosis . Proliferation
Yefei Rong and Wenchuan Wu contributed equally to this work. Y. Rong : W. Wu : X. Ni : T. Kuang : D. Jin : D. Wang (*) : W. Lou (*) Pancreatic Cancer Group, General Surgery Department, Zhongshan Hospital, Fudan University, 180th Feng Lin Road, Shanghai 200031, China e-mail:
[email protected] e-mail:
[email protected]
Introduction Pancreatic ductal adenocarcinoma (PDAC) ranks fourth among cancer-related death and the prognosis is very poor [1]. Although the efforts in the diagnosis and therapy of pancreatic cancer are made, the survival has not been improved over the past 25 years [2]. Therefore, it is very urgent to understand the underlying mechanisms and design novel therapeutic strategies for pancreatic cancer. The Warburg effect, also known as aerobic glycolysis, is one of the characteristics of tumor cells in which high rate of glycolysis is executed even in the presence of adequate oxygen [3]. In the final step of the Warburg effect, lactate dehydrogenase A (LDHA) converts the pyruvate to lactate. In various types of human cancers, the expression of LDHA was dysregulated [4, 5]. In esophageal squamous cell carcinoma, LDHA was upregulated in cancer tissues and promoted the survival of tumor cells [6]. Additionally, LDHA was reported to enhance the growth and migration of gastric cancer cells [7]. However, the expression status and the function of LDHA in pancreatic cancer still remain unknown. Here, the expression status and the function of LDHA in the pancreatic cancer were studied. We found that the expression of LDHA was upregulated in the clinical pancreatic cancer samples. Forced expression of LDHA promoted the growth of pancreatic cancer cells, while knocking down the expression of LDHA inhibited cell growth dramatically. Moreover, downregulation of LDHA decreased the tumorigenicity of pancreatic cancer cells in vivo. Mechanistically, knocking down the expression of LDHA linked the pancreatic cancer cells to apoptosis. Taken together, our study revealed the oncogenic function of LDHA in pancreatic cancer and suggested LDHA as a potential therapeutic target.
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Materials and methods Primary pancreatic cancer samples Primary tissues were collected from patients who received surgery for pancreatic cancer at Zhongshan Hospital of Fudan University. All patients had given informed consent. The fresh pancreatic cancer tissues and paired normal tissues were frozen immediately after surgery and stored at −80 °C until needed.
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3 min; 40 cycles of 95 °C for 30 s, 58 °C for 20 s, and 72 °C for 30 s; and 72 °C for 10 min. To confirm specificity of amplification, the PCR products from each primer pair were subjected to a melting curve analysis and electrophoresis in 2 % agarose gel. Immunohistochemistry
Pancreatic cancer cell lines PANC-1, ASPC-1, BXPC-3, Suit, and MIAPaCa-2 were obtained from ATCC and cultured in DMEM (Invitrogen) with 10 % fetal bovine serum, 10 units/ml penicillin G, and 10 mg/ml streptomycin. Cells were incubated at 37 °C in 5 % CO2-humidified air. To generate the LDHA expression vector, the open reading frame of human LDHA cDNA was cloned into the eukaryotic expression vector pcDNA3.1-myc. Cells were transfected with the LDHA expression vector and empty pcDNA3.1-myc vector using Lipofectamine 2000 reagent (Invitrogen). The transfected cells were selected in the presence of 600 μg/mlG418. The resistant clones were further confirmed by Western blot analysis.
Consecutive sections of 10-μm-thick pancreatic tumor tissues and paired normal tissues were cut and mounted on glass slides. After deparaffinizing, rehydrating, antigen retrieval, and blocking endogenous peroxidases, the sections were washed thrice with 0.01 mol/l phosphatebuffered saline (PBS) (8 mmol/l Na2HPO4, 2 mmol/l NaH2PO4, and 150 mmol/l NaCl) for 5 min each and blocked for 1 h in 0.01 mol/l PBS supplemented with 0.3 % Triton X-100 and 5 % normal goat serum, followed by the addition of anti-LDHA (1:200) antibody at 4 °C overnight. After brief washes in 0.01 mol/l PBS, sections were exposed for 2 h to 0.01 mol/l PBS containing horseradish peroxidase-conjugated rabbit anti-goat IgG (1:500), followed by development with 0.3 % H2O2 and 0.03 % 3,30-diaminobenzidine in 0.05 mol/l Tris– HCl (pH7.6). Immunohistochemistry for each sample was performed at least three separate times, and all sections were counterstained with hematoxylin.
RNA extraction and real-time PCR analysis
Western blot analysis
Total RNA was isolated from pancreatic cancer tissues and paired normal tissues using TRIzol reagent (Invitrogen). The RNA samples were separated in 2 % agarose gels containing ethidium bromide, and their quality was then determined by the visibility of 18S and 28S RNA bands under UV light. Using the reverse transcription kit, 2 μg of total RNA with high quality was processed directly to cDNA in a total volume of 25 μl (Promega, Madison, WI) following the manufacturer’s instructions. The primer used for the amplification of the human LDHA gene was as follows: forward primer 5′-CTCCTGTGCAAAATGGCAAC-3′ and reverse primer 5′-CCTAGAGCTCACTAGTCACAG-3′. As an internal control, a fragment of human beta-actin was amplified by PCR using the following primers: forward primer 5′-GATCATTGCTCCTCCTGAGC3′ and reverse primer 5′-ACTCCTGCTTGCTGATCCAC3′. The amplification reactions were performed in a 20-μl volume of the LightCycler-DNA Master SYBR Green I mixture from Roche Applied Science with the following: 10 pmol of primer, 2 mM MgCl2, 200 μM dNTP mixture, 0.5 units of Taq DNA polymerase, and universal buffer. All of the reactions were performed in triplicate in an iCycler iQ System (Bio-Rad). The thermal cycling conditions were as follows: 95 °C for
Western blot analysis was performed as previously described [7]. Primary antibodies to LDHA, poly(ADP-ribose) polymerase (PARP), X-linked inhibitor of apoptosis protein (XIAP), B cell lymphoma 2 (Bcl-2), and B cell lymphomaextra large (Bcl-XL) were purchased from Santa Cruz Biotechnology. Antibody to beta-actin was purchased from Sigma. Secondary antibodies, rabbit anti-mouse IgG (Sigma) and goat anti-rabbit IgG (Cell Signaling Technology), were used at a dilution of 1:2,000. Primary antibodies were diluted in TBST containing 1 % BSA and NaN3. The immunoreactive protein bands were visualized by an ECL kit (Pierce).
Cell culture and transfection
RNAi-mediated knockdown of LDHA Two target sequences for LDHA small interfering RNA were listed as follows: 5′-TTGTTGATGTCATCGAAG-3′ and 5′-GGGTCCTTGGGGAACATG-3′. The control nucleotide sequence of small interfering RNA was 5′-GTACATAGGGACGTAACG-3′, which was the random sequence that was not related to LDHA mRNA. FG12 RNAi vector was used to produce small double-stranded RNA (small interfering RNA) to inhibit target gene expression in pancreatic cancer cells.
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Crystal violet assay
Results
Equal number of control cells and cells knocking down the expression of LDHA (or overexpressing LDHA) were seeded in six-well plates and cultured in medium supplemented with 10 % fetal bovine serum (FBS) for 7 days. The medium was changed every other day. Cell growth was stopped after 7 days in culture by removing the medium and adding 0.5 % crystal violet solution in 20 % methanol. After staining for 5 min, the fixed cells were washed with PBS, photographed, and dissolved with 1 % SDS. The absorbance at 600 nm was evaluated using a microplate reader.
The expression of LDHA was elevated in pancreatic cancer
Soft agar assay The soft agar assay was performed to evaluate the effect of LDHA on the tumorigenicity of pancreatic cancer cells in vitro. Briefly, cells (1×104) were resuspended in a medium containing 10 % FBS with 0.3 % agarose and laid on the top of 0.6 % agarose in a medium supplemented with 20 % FBS in 60-mm plates. After 14 days of culture at 37 °C, colonies were photographed. Cell cycle analysis Cell cycle measurement was performed to evaluate the effect of LDHA on the cell cycle progression of pancreatic cancer cells. Briefly, cells (5×105) were seeded in six-well plates and allowed to adhere. The cells were harvested 24 h later by centrifugation at 1,000 rpm for 5 min. The cell pellets were washed twice with PBS followed by fixation with ice-cold 70 % ethanol and stored at −20 °C overnight. Then, the pellets were washed with cold PBS; suspended in 500 ml PBS containing 50 mg/ml propidium iodide, 0.1 mg/ml RNase A, and 0.05 % Triton X-100; and incubated at 37 °C for 40 min in the dark. The cell cycle distribution was determined on the Becton Dickinson FACSCalibur. The experiment was repeated thrice under the same conditions.
Firstly, we used real-time PCR analysis to examine the mRNA level of LDHA in the randomly selected pancreatic cancer tissues and the paired normal tissues. It was found that LDHA mRNA level was elevated in pancreatic cancer samples compared with the paired normal tissues (Fig. 1a, b). In addition, Western blot and immunohistochemistry analyses showed the increased protein level of LDHA in human pancreatic cancer tissues (Fig. 1c, d). Moreover, strong expression of LDHA was observed in the pancreatic cancer cell lines (Fig. 1e). These results indicated the oncogenic role of LDHA in the progression of pancreatic cancer. Forced expression of LDHA promoted the growth of pancreatic cancer cells To examine the effects of LDHA on the growth of pancreatic cancer cells, BXPC-3 and MIAPaCa-2 cells were stably transfected with either a pcDNA3.1/LDHA vector containing full-length LDHA or an empty vector pcDNA3.1. The G418-resistant clones were screened for LDHA expression by Western blot analysis. Two clones with a high expression of LDHA (LDHA #1 and #2) were shown in Fig. 2a, which were used for further study. The effect of LDHA on cell growth was evaluated by crystal violet assay. It was found that the growth rate of BXPC-3/LDHA cells and MIAPaCa2/LDHA cells was greater than that of the control cells (Fig. 2b). Next, we tested whether LDHA induced cell growth by changing the cell cycle. Indeed, flow cytometry analysis showed that overexpression of LDHA significantly induced S phase transition (Fig. 2c). Moreover, clonogenic assay showed that BXPC-3/LDHA cells formed more colonies than the control cells (Fig. 2d). These results indicated that forced expression of LDHA promoted the growth of pancreatic cancer cells. Knockdown of LDHA inhibited the growth of pancreatic cancer cells
Tumorigenicity assay Four 4-week-old nude mice were used in this study, and 1× 106 LDHA knockdown cells and the control cells were subcutaneously injected into the opposite flanks of the same mouse. The resulting tumors were measured every 5 days, and tumor volumes were calculated using the standard formula: length× width× height × 0.5326. Tumors were harvested 25 days after injection and individually weighted. Data were presented as tumor volume (mean ± SD) and tumor weight (mean ± SD). Statistical analysis was performed using the Student’s t test.
In the next study, we performed loss-of-function assay to determine whether endogenous LDHA played an important role in cell growth. We first knocked down the expression of LDHA in BXPC-3 cells and MIAPaCa-2 cells. The target sequence decreased the expression of LDHA effectively (Fig. 3a). In the crystal violet assay, silencing the expression of LDHA inhibited the growth of BXPC-3 cells and MIAPaCa-2 cells (Fig. 3b, c). Moreover, silencing the expression of LDHA dramatically attenuated the anchorage-independent growth of BXPC-3 cells and MIAPaCa-2 cells (Fig. 3d, e). These
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Fig. 1 The expression of LDHA was elevated in pancreatic cancer. a Relative expression of LDHA mRNA in human pancreatic cancer samples and paired normal tissues. Semiquantitative real-time PCR was performed on 40 pancreatic cancer samples and paired normal tissues. The LDHA expression was normalized to that of beta-actin. Data were calculated from triplicates. Each bar was the log2 value of the ratio of LDHA expression levels between pancreatic cancer tissues (C) and matched normal tissues (N) from the same patient. Because log22=1, bar value >1 represents a greater than twofold increase (C>N), whereas bar value
