The study group consisted of 65 patients with primary PDAC, 40 males and 25 females, ranged in age from 36 to 82 years, with a median age of 52 years. They underwent pancreaticoduodenectomy or distal pancreatectomy with lymph node dissection at Pancreatic Center of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, between January 2007 and December 2008. Sixty-five cases of pancreatic cancerous tissues and 38 cases of corresponding adjacent normal tissues were collected, the adjacent normal tissues were taken from the edge of the tumor and confirmed by pathology. All samples were divided into two parts immediately after removed from patients, one was put into liquid nitrogen for real-time PCR, the other was fixed in 4% buffered formaldehyde and embedded in paraffin wax for immunohistochemistry. None of the patients received preoperative chemotherapy or radiation therapy. Among 65 patients, 25 (38.5%) cases were moderately differentiated, 21 (32.3%) well differentiated, and 19 (29.2%) poorly differentiated. Forty cases (61.5%) had lymph node metastases. Nineteen cases (29.2%) had venous invasion. According to the sixth edition of the TNM classification of the International Union Against Cancer (UICC), 24 patients were at pathological stage I or II, and 41 patients were at stage III or IV. All patients received chemotherapy routinely after operation, and were followed up regularly for 5-35 mo (mean, 14 mo). The procedure had been approved by the Ethics Committee of Union Hospital, Tongji Medical College.

The total RNA of PDAC and adjacent tissues were extracted using Trizol (invitrogen, USA) according to the manufacturer’s protocol. The concentration was analyzed by ultraviolet spectrophotometer. Two micrograms of RNA was used for the reverse transcription. TLR4, HIF-1α and β-actin genes were amplified with SYBR Green in a fluorescence reader FTC-2000 (Feng Ling Bio-engineering Co., Ltd., Shanghai, China). The primer sequences for TLR4 gene were as follows: 5'-ACCTGTCCCTGAACCCTATGAA-3' (forward) and 5'-CTTCTAAACCAGCCAGACCTTG-3' (reverse); the primer sequences for HIF-1α gene were: 5'-TGAAGTGTACCCTAACTAGCCG-3' (forward) and 5'-AATCAGCACCAAGCAGGTCATAG-3' (reverse); and the primer sequences for β-actin gene were: 5'-GAAACTACCTTCAACTCCATC-3' (forward) and 5'-CGAGGCCAGGATGGAGCCGCC-3' (reverse). Cycling conditions were as follows: initial denaturation at 94°C for 5 min, followed by 30 cycles at 94°C for 30 s, at 58°C for 30 s and at 72°C for 30 s. The negative control was performed using normal pancreatic tissue taken far away from the cancerous tissues. The results were analyzed by the fluorescence reader program [calculation formula: folds = 2^-ΔΔCt, ΔΔCt = [(Ct target gene - Ct β-actin) experiment sample - (Ct target gene - Ct β-actin) control sample][14]. Each experiment was carried out in triplicate using β-actin as an internal standard; the results were expressed as mean ± SD of representative triplicates.

The tissue specimens were fixed in 10% formalin buffered at pH 7.0 for 24 h and paraffin-embedded. Histologic slides (5 μm) were deparaffined in xylene and rehydrated through a series of graded ethanol. Endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide in methanol for 10 min. Antigen retrieval was performed by microwaving the slides in citrate buffer (pH 6.0) for 20 min at 120°C. Then the sections were washed and blocked with normal goat serum (Streptavidin/Peroxidase immunohistochemical kit, Zhongshan Goldenbridge Biological Technology Co., LTD, Beijing, China) for 30 min at room temperature, subsequently exposed to the primary antibody (rabbit polyclonal anti-TLR4, 1:50, Santa Cruz, CA, USA; mouse monoclonal anti-HIF-1α, 1:50, Santa Cruz, CA, USA; NF-κB p65 rabbit monoclonal antibody, 1:50, Cell Signaling Technology Inc, MA, USA). overnight at 4°C. After washing in phosphate buffered saline (PBS), the slides were incubated with a biotinylated goat anti-mouse or anti-rabbit secondary antibody (1:200, Streptavidin/Peroxidase immunohistochemical kit) for 30 min at room temperature. After reactions with freshly prepared diaminobenzidine-tetrahydrochloride (DAB kit, Zhongshan Goldenbridge Biological Technology Co., LTD, Beijing, China), the slides were counterstained in haematoxylin and coverslipped in a systemic mounting medium. Negative controls were prepared without the primary antibodies.

The assessment of TLR4, HIF-1α and NF-κB p65 was based on previously described guidelines[15], which consist of the percentage of stained tumor cells and the staining intensity. At least 3 different high-power (× 400) fields of tumor infiltration were examined. The percentage of positive tumor cells was rated as follows: 1 point, ≤ 10% positive tumor cells; 2 points, 11%-50% positive tumor cells; 3 points, 51%-80% positive tumor cells; and 4 points, ≥ 81% positive tumor cells. The staining intensity was rated as follows: 1 point, weak intensity; 2 points, moderate intensity; and 3 points, strong intensity. Points for expression and percentage of positive cells were added, and specimens were attributed to four groups according to their overall score: negative, ≤ 10% of cells stained positive, regardless of intensity; weak expression, 3 points; moderate expression, 4-5 points; and strong expression, 6-7 points. Weak expression was rated as negative, and moderate and strong expressions were rated as positive for analysis. Two independent investigators (Zhang JJ and Tian Y) blinded to clinical data performed the analysis. Specimens scored differently by the two investigators were reinvestigated together using a multiheaded microscope.

For statistical analysis, SPSS 15.0 was used. The independent t test was used to compare the means of TLR4 or HIF-1α mRNA between PDAC and its adjacent tissues. The χ² test was used to analyze the difference in expression of TLR4, NF-κB p65 and HIF-1α protein between PDAC and its adjacent tissues, as well as correlation between TLR4, HIF-1α and clinicopathologic features. One-way analysis of variance was used for three groups. Survival curves were calculated using the Kaplan-Meier method and analyzed using the log-rank test. Differences at P < 0.05 were considered to be statistically significant.

To investigate whether TLR4 is expressed in PDAC, we first detected TLR4 mRNA by real-time PCR. The results showed that the mean level of TLR4 mRNA was 0.81 ± 0.10 in tumor tissues, and it was 0.70 ± 0.16 in adjacent normal tissues, with significant difference between them (P = 0.002, Figure 1A). We then detected TLR4 protein by immunohistochemistry, and found that TLR4 protein was localized in cytoplasm and membrane of tumor cells (Figure 2A and B), and the expression of TLR4 was 69.2% (45/65) in cancerous tissues, which was significantly higher than that in adjacent normal tissues (69.2% vs 39.5%, P = 0.003, Table 1). When analyzing the relationship between TLR4 and clinicopathological features, we found that the expression of TLR4 mRNA or protein was not correlated with age, gender, location and differentiation of the tumor (P > 0.05), but significantly correlated with tumor size, lymph node involvement, venous invasion and pathological stage (P < 0.05, Table 2).

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Figure 1 Toll-like receptor (TLR) 4 and hypoxia-inducible transcription factor-1α (HIF-1α) mRNA in pancreatic carcinoma and adjacent tissues. ^aP < 0.05 vs adjacent tissues. A: TLR4 mRNA in 30 cases of pancreatic carcinoma and adjacent tissues was determined by real-time polymerase chain reaction (PCR); B: HIF-1α mRNA in 30 cases of pancreatic carcinoma and adjacent tissues was determined by real-time PCR.

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Figure 2 Expression of TLR4 and HIF-1α protein in pancreatic carcinoma tissue. Brown color displays the positive expression. A: Expression of TLR4 protein in carcinoma tissue (SP, × 100); B: Expression of TLR4 protein in carcinoma tissue (SP, × 400); C: Expression of HIF-1α protein in carcinoma tissue (SP, × 100); D: Expression of HIF-1α protein in carcinoma tissue (SP, × 400); E: Expression of NF-κB p65 protein in carcinoma tissue (SP, × 100); F: Expression of NF-κB p65 protein in carcinoma tissue (SP, × 400).

We continued to detect HIF-1α in PDAC, and found that the mean level of HIF-1α mRNA in tumor tissues was 0.87 ± 0.11, being significantly higher than that in adjacent tissues, which was 0.68 ± 0.13 (P = 0.000, Figure 1B). Immunohistochemical results showed that HIF-1α was expressed in the nuclei and/or the cytoplasm of tumor cells (Figure 2C and D). The expression of HIF-1α was found in 46 of 65 (70.8%) cases of tumor tissues, and 14 of 38 (36.8%) cases of adjacent tissues, with significant difference between them (Table 1, P < 0.05). Furthermore, we also found that the expression of HIF-1α was correlated with tumor size, lymph node involvement, venous invasion and pathological stage (P < 0.05, Table 2).

In macrophages, LPS can raise the level of HIF-1α in a TLR4-dependent fashion[13], but it remains unknown in tumor cells. To study whether TLR4 is associated with HIF-1α in PDAC, we performed the correlative analysis. The results showed that there existed a significant correlation between TLR4 mRNA and HIF-1α mRNA (r = 0.423, P = 0.020, Figure 3), similar result was also found between TLR4 and HIF-1α protein (r_p = 0.451, P < 0.05, Table 3), suggesting that TLR4 has a positive correlation with HIF-1α in PDAC.

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Figure 3 Correlation between TLR4 mRNA and HIF-1α mRNA.

NF-κB is an essential downstream component of TLR signal pathway[16], and HIF-1α is a target gene of NF-κB[17]. To further explore the association between TLR4 and HIF-1α, we investigated NF-κB p65 expression in PDAC by immunochemistry. The results showed that NF-κB p65 was expressed in the nuclei and/or the cytoplasm of tumor cells (Figure 2E and F), and the positive expression of NF-κB p65 was found in 43 (66.15%) of 65 cases of tumor tissues, being significantly higher than in 12 (31.58%) of 38 cases of adjacent tissues (P = 0.001). Considering NF-κB is an essential component in TLR4 signal pathway and an important modulator of HIF-1α, we could speculate that NF-κB p65 may be a link in the association between TLR4 and HIF-1α.

In order to understand whether over-expression of TLR4 and HIF-1α in PDAC has an impact on patient survival, the relationship between the expression of TLR4 or HIF-1α and life span of patients was analyzed. The mean survival of patients without over-expression of TLR4 in tumor tissues was 19.7 mo, and it was 12.4 mo in those with over-expression of TLR4, log-rank analysis showed there was a significant difference (P = 0.011, Table 4 and Figure 4A). Similarly, the mean survival of patients without over-expression of HIF-1α in tumor tissues was 20.1 mo, being significantly longer than those with over-expression of HIF-1α, which was 12.8 mo (P = 0.005, Table 4 and Figure 4B). In addition, the survival time of group with neither TLR4 nor HIF-1α over-expression was 21.6 mo, significantly longer than those with both over-expressions, which was 11.8 mo (P = 0.014, Table 4 and Figure 4C).

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Figure 4 Kaplan-Meier analysis overall survival depending on TLR4 and HIF-1α expression in pancreatic ductal adenocarcinoma. A: The mean survival of patients with negative TLR4 expression in tumor tissues was significantly longer than those with TLR4 positive expression (P = 0.011); B: The mean survival of patients with negative HIF-1α expression in tumor tissues was significantly longer than those with HIF-1α positive expression (P = 0.005); C: Survival of patients with both negative TLR4 and HIF-1α expression was significantly longer than those with both positive TLR4 and HIF-1α expression (P = 0.014).

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant digestive tumor with a very poor prognosis. Hypoxia-inducible transcription factor-1α (HIF-1α) is involved in malignant progression in many solid tumors, including PDAC, up-regulation of HIF-1α accelerates PDAC progression, but the exact regulatory mechanisms of HIF-1α in PDAC has not been unequivocally addressed. Recently, an increasing number of studies reported that toll-like receptors (TLRs) were upregulated in epithelial malignancies and involved in tumor progression, but whether TLRs, such as TLR4, is expressed on PDAC cells remains unknown. In immune- related cells, TLR signal pathway may induce expression of HIF-1α, but it is also still unclear whether there exists some association between TLR4 and HIF-1α in tumor microenviroment, such as PDAC.

Activating TLRs pathway in tumor cells could promote the proliferation and inhibit the apoptosis, leading to migration, invasion and angiogenesis of tumor, but it remains unknown whether TLR4 is expressed and what role it plays in PDAC. In human cancers, HIF-1α is overexpressed as a result of intratumoral hypoxia. Recently, HIF-1α has also been shown to be up-regulated under normoxia in response to LPS, a potent agonist of TLR4, in immune-related cells, however, whether HIF-1α is regulated by TLR4 signal pathway in tumor is unknown. In this study, the authors demonstrate that TLR4 and HIF-1α are over-expressed in PDAC, and TLR4 expression is associated with HIF-1α. nuclear factor-κB (NF-κB) p65, an important component in both TLR pathway and HIF-1 pathway, is also over-expressed in PDAC. The expression of TLR4 and HIF-1α has a significant impact on survival of patients with PDAC. These results might indicate that TLR4 could be a potential factor for mediating HIF-1α in PDAC.

This is the first study to report that TLR4 is over-expressed in PDAC, and it is also the first report to show that TLR4 is associated with HIF-1α in PDAC.

The study demonstrated that TLR4, NF-κB p65 and HIF-1α were over-expressed in PDAC. The expression of TLR4 and HIF-1α was associated with tumor size, lymph node metastasis, venous invasion and pathological stage. Furthermore, the expression of TLR4 was positively correlated with expression of HIF-1α. Thus, TLR4 may be a novel marker for the progression and prognosis of PDAC, and may provide a new strategy for therapeutic intervention in the treatment of patients with PDAC in the future.

This is an interesting study with valuable information regarding the expression and clinical correlation of TLR4 and HIF-1α in pancreatic ductal adenocarcinoma.