PADI4 expression was detected by immunohistochemistry in all malignant tumor types examined including breast carcinomas, lung adenocarcinomas, hepatocellular carcinomas, esophageal squamous cancer cells, colorectal adenocarcinomas, renal cancer cells, ovarian adenocarcinomas, endometrial carcinomas, uterine adenocarcinomas, bladder carcinomas, chondromas, and other metastatic carcinomas (IRS ≥ 9). Furthermore, PADI4 was also expressed in over 40% of cells in malignant lymphomas (IRS ≥ 8). In contrast, no significant levels of PADI4 were detected in various benign tumors including leiomyomas of the stomach, uterine myomas, endometrial hyperplasias, cervical polyps, teratomas, hydatidiform moles, trophoblastic cell hyperplasias, hyroid adenomas, hemangiomas, lymph hyperplasias, schwannomas, neurofibromas, lipomas, and cavernous hemangiomas of the liver (IRS: 0–1). In breast fibroadenoma, thyroid adenoma, and pleomorphic adenoma samples, PADI4 was observed in the endothelial cells of capillaries and gland structures (IRS: 1–6), both of which mainly consist of benign tissues. PADI4 expression was not detected in most non-tumor inflamed tissues including cholecystitis, cervicitis and synovitis of osteoarthritis (IRS ≤ 1), although half of gastric ulcer and acute appendicitis samples displayed smeared PADI4 immunosignals (IRS: 3–6). In addition, more than 60% of the liver cirrhosis samples showed PADI4 expression (IRS: 3–8). PADI4 was also expressed in several mesenchymal cells in both tumor and non-tumor inflamed tissues. Immunohistochemical results are shown and listed in Figure and Table , respectively.
Figure 1 Immunohistochemistry showing PADI4 expression in various malignant tumors, benign tumors and non-tumor inflamed tissues. 1. breast cancer, 2. breast fibroadenoma. 3. esophageal squamous cell cancer, 4. gastric adenocarcinoma, 5. leiomyosarcoma of stomach, (more ...)
By western blot analysis, a 67 kDa band was detected in IP extracts of gastric adenocarcinomas, lung adenocarcinomas, hepatocellular carcinomas, esophageal squamous cell cancers and breast cancers as well as in their corresponding healthy tissues (see Figure ). Immunosignal densities were significantly higher in malignant tissues than in corresponding healthy tissues or in breast fibroadenomas. Since IP extracts were purified from equivalent tissue weights for each sample, the density of the immunosignals directly corresponded to PADI4 expression levels. In addition, PADI4 was detected in total protein extracts from A549, SKOV3 and U937 tumor cell lines.
Figure 2 Western blot analysis of PADI4 expression in tumor cell lines, tumors and corresponding healthy tissues. Extracts were fractioned by electrophoresis and probed with anti-PADI4 antibody. PADI4 immunosignals (67 kDa) were detected in malignant tissues, (more ...)
TaqMan quantitative PCR was used to measure PADI4 transcription in tumor samples. Levels of PADI4 mRNA were determined from standard curves and amplification plots (based on the Ct and copy number). As cDNA templates were synthesized from an equivalent amount of total RNA for each sample, the copy number/ug total RNA directly corresponds to PADI4 expression levels in the tissue samples. PADI4 transcripts were detected in gastric adenocarcinomas, lung adenocarcinomas, hepatocellular carcinomas, esophageal squamous cell cancers, breast cancers and breast fibroadenomas as well as their corresponding healthy tissues. PADI4 mRNA levels were significantly elevated in malignant tissues compared to those in corresponding healthy tissues. The PADI4 mRNA level in breast fibroadenomas was lower than that in breast cancers, but higher than that in corresponding healthy tissues. TaqMan quantitative PCR results are shown in Figure .
Figure 3 PADI4 transcription levels in tumors and corresponding healthy tissues by TaqMan quantitative PCR. cDNA templates were synthesized using an equivalent amount of total RNA for each sample. Lanes 1–2, PADI4 mRNA levels in gastric adenocarcinomas (more ...)
Plasma PADI4 levels of patients with malignant tumors were measured by ELISA and compared with those with benign and healthy controls. PADI4 levels were significantly increased in the blood of patients with breast carcinomas, hepatocellular carcinomas, lung cancer, esophageal carcinomas, gastric cancer, colon cancer, rectal cancer, pancreatic cancer, ovarian carcinomas, renal cell carcinomas, cervical cancer, prostate carcinomas and bladder carcinomas (p < 0.01), but not in those with endometrial carcinomas, uterine myomas and thyroid carcinomas. After tumor excision surgery, PADI4 levels in the blood decreased considerably in breast carcinomas, hepatocellular carcinomas, lung cancer, gastric cancer, colon cancer, rectal cancer and pancreatic cancer samples (p < 0.05). Meanwhile, plasma PADI4 levels in patients with chronic gastritis, chronic nephritis and cervicitis did not considerably change, or even declined, compared to samples from healthy control patients. However, patients with breast fibroadenomas, thyroid adenomas, hepatitis A and B, liver cirrhosis, pulmonitis, acute pancreatitis and acute appendicitis had higher plasma PADI4 levels than those of healthy controls, with some samples showing even higher levels than that observed for patients with malignancies. ELISA results of PADI4 are shown in Figure .
Figure 4 Levels of PADI4 and cAT in plasma of patients with various tumors. Plasma samples are listed as the followings: lane 1–4 breast carcinoma (n = 112), after receiving surgery (n = 86), breast fibroadenomas controls (n = 30), and health controls (more ...)
Plasma cAT levels of patients with malignant tumors were measured by sandwich ELISA and compared to those with benign tumors and healthy controls. Plasma cAT levels were significantly elevated in most patients with malignant tumors, including those with breast carcinomas, hepatocellular carcinomas, lung carcinomas, esophageal carcinomas, gastric cancer, colon cancer, rectal cancer, pancreatic cancer, ovarian carcinomas, bladder carcinomas, uterine myomas, thyroid carcinomas and prostate carcinomas (p < 0.01), but not in those with cervical cancer or endometrial carcinomas. After tumor excision surgery, cAT levels in the blood declined for most malignancies. Plasma cAT expression was not considerably altered in patients with breast fibroadenomas, acute pancreatitis, chronic nephritis and cervicitis, and displayed slightly diminished levels in patients with chronic gastritis and acute appendicitis when compared to those of healthy control subjects. The cAT content was higher in samples from patients with thyroid adenomas, hepatitis A and B, liver cirrhosis and pulmonitis compared to healthy controls (see Figure ). cAT levels associated significantly with PADI4 levels in patients with hepatocellular carcinomas, lung cancer, ovarian cancer, endometrial carcinomas and thyroid adenomas (p < 0.01). In addition, cAT levels clearly associated with CEA levels in the blood of patients with breast carcinomas (p = 0.032), CA199 levels in those with gastric cancer (p = 0.025), CEA levels in those with pancreatic cancer (p = 0.05), CA125 levels in those with ovarian cancer (p = 0.03), CEA levels in those with bladder cancer (p = 0.016), CEA in those with renal cell carcinomas (p = 0.046), and PSA in those with prostate cancer (p = 0.028). PADI4 levels also significantly associated with CEA levels in patients with gastric cancer (p = 0.05) and prostate cancer (p = 0.028).