Parviz M. Pour

Islet Amyloid Polypeptide in Patients with Pancreatic Cancer and Diabetes

BACKGROUND The diabetes mellitus that occurs in patients with pancreatic cancer is characterized by marked insulin resistance that declines after tumor resection. Islet amyloid polypeptide (IAPP), a hormonal factor secreted from the pancreatic beta cells, reduces insulin sensitivity in vivo and glycogen synthesis in vitro. In this study, we examined the relation between IAPP and diabetes in patients with pancreatic cancer. METHODS We measured IAPP in plasma from 30 patients with pancreatic cancer, 46 patients with other cancers, 23 patients with diabetes, and 25 normal subjects. IAPP immunoreactivity and IAPP messenger RNA were studied in pancreatic cancers, pancreatic tissue adjacent to cancers, and normal pancreatic tissue. RESULTS Plasma IAPP concentrations were elevated in the patients with pancreatic cancer as compared with the normal subjects (mean [+/- SD], 22.3 +/- 13.6 vs. 8.0 +/- 5.0 pmol per liter; P < 0.001), normal in the patients with other cancers, and normal or low in the patients with diabetes. Among the patients with pancreatic cancer, the concentrations were 25.0 +/- 8.7 pmol per liter in the 7 patients with diabetes who required insulin, 31.4 +/- 12.6 pmol per liter in the 11 patients with diabetes who did not require insulin, and 12.2 +/- 2.4 pmol per liter in the 9 patients with normal glucose tolerance (3 patients had impaired glucose tolerance; their mean plasma IAPP concentration was 11.7 +/- 5.5 pmol per liter). Plasma IAPP concentrations decreased after surgery in the seven patients with pancreatic cancer who were studied before and after subtotal pancreatectomy (28.9 +/- 16.4 vs. 5.6 +/- 3.4 pmol per liter, P = 0.01). Pancreatic cancers contained IAPP, but the concentrations were lower than in normal pancreatic tissue (17 +/- 16 vs. 183 +/- 129 pmol per gram, P < 0.001). In samples from the patients with both pancreatic cancer and diabetes, immunostaining for IAPP was reduced in islets of pancreatic tissue surrounding the tumor; in situ hybridization studies suggested that transcription occurred normally in these islets. CONCLUSIONS Plasma IAPP concentrations are elevated in patients with pancreatic cancer who have diabetes. Since IAPP may cause insulin resistance, its overproduction may contribute to the diabetes that occurs in these patients.

5-Lipoxygenase and Leukotriene B4 Receptor Are Expressed in Human Pancreatic Cancers But Not in Pancreatic Ducts in Normal Tissue

The 5-lipoxygenase (5-LOX) pathway is critical for pancreatic cancer cell growth and escape from apoptosis. Inhibition of 5-LOX blocks proliferation and induces apoptosis in human pancreatic cancer cells. However, the expression of 5-LOX and its downstream signaling pathway have not been investigated in human pancreatic adenocarcinoma. Reverse transcriptase-polymerase chain reaction revealed expression of 5-LOX mRNA in all pancreatic cancer cell lines tested including, PANC-1, AsPC-1, and MiaPaCa2 cells, but not in normal pancreatic ductal cells. The expression of 5-LOX protein in pancreatic cancer cell lines was demonstrated by Western blotting. Finally, 5-LOX up-regulation in human pancreatic cancer tissues was verified by intense positive staining in cancer cells by immunohistochemistry. Staining for the 5-LOX protein was particularly evident in the ductal components of the more differentiated tumors but not in ductal cells in normal pancreatic tissues from cadaver donors. Immunohistochemistry also revealed strong staining of cancer tissues with an antibody to the receptor of the downstream 5-LOX metabolite, leukotriene B(4). The current study demonstrated marked expression of 5-LOX and the leukotriene B(4) receptor in human pancreatic cancer tissues. These findings provide further evidence of up-regulation of this pathway in pancreatic cancer and that LOX inhibitors are likely to be valuable in the treatment of this dreadful disease.

Current knowledge of pancreatic carcinogenesis in the hamster and its relevance to the human disease

Syrian hamsters present a unique species for induction of pancreatic tumors that in many aspects resemble human pancreatic cancer. The specific response of Syrian hamsters, in contrast to many other rodents, for development of pancreatic ductal (ductular) tumors is not yet known. All pancreatic carcinogens thus far tested show certain common features. They are all nitrosamines that possess or can be metabolized to compounds with 2‐oxopropyl‐ or 2‐hydroxypropyl substituents. All but one, N‐nitrosomethyl(2‐oxopropyl)amine, occur or metabolize to nitrosamines with the ability to cyclize and form structures resembling glucose. Hence it is suggested that this cyclic structure may be responsible for the pancreatic carcinogenicity of these nitrosamines, as has been proposed for the pancreatotropic effect of streptozotocin. It is also of further interest that one pancreatic ductal (ductular) carcinogen, N‐nitroso‐2‐methoxy‐2,6‐dimethylmorpholine, which possesses a totally cyclic structure, acts, like streptozotocin, as β‐cell cytotoxic and diabetogenic when given in a high single dose. Modification of pancreatic tumor induction has been demonstrated by specific procedures. A high fat diet significantly increases both the incidence and number of induced cancers. Methods for early diagnosis and therapy are being developed and their significance and applicabilities for clinical use will be of major importance. Compared with the other most common types of human cancer, pancreatic cancer has extraordinary characteristics, which make the disease one of the most mysterious of maladies. Consequently, pancreatic cancer represents a serious international problem and requires urgent resolution, especially with regard to its etiology, early diagnosis, prevention, and therapy.

Early diagnosis of pancreatic cancer: neutrophil gelatinase-associated lipocalin as a marker of pancreatic intraepithelial neoplasia.

Pancreatic cancer is a highly lethal malignancy with a dismal 5-year survival of less than 5%. The scarcity of early biomarkers has considerably hindered our ability to launch preventive measures for this malignancy in a timely manner. Neutrophil gelatinase-associated lipocalin (NGAL), a 24-kDa glycoprotein, was reported to be upregulated nearly 27-fold in pancreatic cancer cells compared to normal ductal cells in a microarray analysis. Given the need for biomarkers in the early diagnosis of pancreatic cancer, we investigated the expression of NGAL in tissues with the objective of examining if NGAL immunostaining could be used to identify foci of pancreatic intraepithelial neoplasia, premalignant lesions preceding invasive cancer. To examine a possible correlation between NGAL expression and the degree of differentiation, we also analysed NGAL levels in pancreatic cancer cell lines with varying grades of differentiation. Although NGAL expression was strongly upregulated in pancreatic cancer, and moderately in pancreatitis, only a weak expression could be detected in the healthy pancreas. The average composite score for adenocarcinoma (4.26±2.44) was significantly higher than that for the normal pancreas (1.0) or pancreatitis (1.0) (P<0.0001). Further, although both well- and moderately differentiated pancreatic cancer were positive for NGAL, poorly differentiated adenocarcinoma was uniformly negative. Importantly, NGAL expression was detected as early as the PanIN-1 stage, suggesting that it could be a marker of the earliest premalignant changes in the pancreas. Further, we examined NGAL levels in serum samples. Serum NGAL levels were above the cutoff for healthy individuals in 94% of pancreatic cancer and 62.5% each of acute and chronic pancreatitis samples. However, the difference between NGAL levels in pancreatitis and pancreatic cancer was not significant. A ROC curve analysis revealed that ELISA for NGAL is fairly accurate in distinguishing pancreatic cancer from non-cancer cases (area under curve=0.75). In conclusion, NGAL is highly expressed in early dysplastic lesions in the pancreas, suggesting a possible role as an early diagnostic marker for pancreatic cancer. Further, serum NGAL measurement could be investigated as a possible biomarker in pancreatitis and pancreatic adenocarcinoma.

Immortalization with telomerase of the Nestin-positive cells of the human pancreas

Cells expressing the neuronal stem cell marker Nestin are present in the human pancreas but the biological role of these cells has yet to be resolved. We report here the establishment with the catalytic subunit of human telomerase (hTERT) of a line of normal human cells representing this cell type. Primary human cells derived from the ducts of the pancreas were transduced with an hTERT cDNA. The infected cells became positive for telomerase, failed to senesce, and were still proliferating after more than 150 doublings. The immortalized cells were positive for the expression of Nestin (at both the mRNA and protein levels) and were found to be free of cancer-associated changes: diploid and expressing wild type p16(INK4a), p53, and K-Ras. An established line of normal human cells representing this cell type should be of great value to help define the biological properties of this novel cell type.

Patterns of growth and metastases of induced pancreatic cancer in relation to the prognosis and its clinical implications

To understand high malignancy of pancreatic cancer, the growth and metastatic patterns of pancreatic cancer induced in Syrian hamsters were examined. In this model, induced tumors resemble the human disease morphologically, clinically, biologically, and immunologically. In the current study, primary-induced cancer and transplants of pancreatic cancer cell line (PC-1) into the SC tissue or pancreas of homologous hosts were used. In the primary-induced pancreatic cancer, perineural invasion was the most common path (88%), followed by lymphogenic (31%) or vascular (2%) metastases. Inoculation of PC-1 cells into the pancreas resulted in 100% tumor take within 3 weeks. Of 19 intrapancreatic allografts, all showed peritoneal invasion, 5 (26%) liver metastases, 3 (16%) lymph node metastases, 17 (89%) perineural invasion, and none vascular invasion. Even microscopic tumors were found to metastasize primarily via perineural spaces. It was also demonstrated, for the first time, that cancer cells take this route to reach distant tissues, including the lymph nodes. Intraductal spreading occurred in both primary cancers and intrapancreatic allografts either continuously or discontinuously. The patterns of discontinuous intraductal tumor expansion imitated tumor multicentricity. Although perineural invasion was the most common feature of primary cancer and intrapancreatic allografts, lymphatic, hepatic, and vascular invasion and metastases usually occurred in advanced cases. Environmental factors seem to influence expansion and metastases, as evidenced by differences in growth and in metastatic patterns between SC and intrapancreatic allografts.

Pancreatic neoplasms in an animal model: Morphological, biological, and comparative studies

After a short latency (15 weeks), a 100% incidence of pancreatic neoplasms was induced in Syrian golden hamsters, following the administration of 2,2′‐dihydroxy‐di‐n‐propylnitrosamine (DHPN). Data extracted from reports of human pancreatic neoplasms were compared with findings relative to the induced neoplasms. The latter resembled human pancreatic tumors, in both biological and morphological aspects.

Neural invasion in the staging of pancreatic cancer.

The natural history of pancreatic ductal adenocarcinoma makes it one of the most malignant human diseases. Unknown etiology, lack of early symptoms, explosive outcome, short survival, and resistance to therapy are hallmarks of this cancer. Although surgery has been shown to be an effective therapeutic approach, the inevitable tendency for recurrence, even after apparently curative operation, has remained a mystery. Ironically, the reasons for this recurrence, which usually leads to the death of the patient within 1 or 2 years after surgery, have not been a focus of research. Several studies highlighting this problem and the possible explanations have been performed by Japanese investigators.

Selective induction of pancreatic ductular tumors by single doses of N-nitrosobis(2-oxopropyl)amine in syrian golden hamsters*

The selectivity of response by the Syrian hamster pancreas to the neoplastic effect of N-nitrosobis(2-oxopropyl)amine (BOP) was demonstrated by single subcutaneous injections of the compound. Only occasional tumors were induced simultaneously with those of the pancreas in the biliary ducts, kidneys and lungs of a few hamsters. In the pancreas, the ductules, especially those of an intrainsular location, were the cells primarily affected. The responded to doses as low as 1/40 of the LD50, whereas ductal lesions were found only in some hamsters treated with doses above 1/5 of the LD50. Hence it is concluded that hamster ductular cells are the most responsive to the carcinogenic action of BOP.

Transdifferentiation of human islet cells in a long-term culture

It has been established that ductal cells or precursor cells within the ductal tree of the pancreas can differentiate into islet cells. Although islet cells can also form exocrine cells, it is unclear whether they arise from precursor (stem) cells or from mature endocrine cells by transdifferentiation. Using a defined culture medium and technique for islet purification, for the first time we were able to maintain human islets in culture for more than a year. Multilabeling immunohistochemical and immunoelectron microscopic examination of the islets at different days of culture using islet cell markers (antibodies to hormones, neuron-specific enolase, chromogranin A) and ductal cell markers (cytokeratins 7 and 19, carbonic anhydrase II, DU-PAN2, CA 19-9, and MUC1) revealed that endocrine cells gradually transdifferentiate to ductal, acinar, and intermediary cells. Although islet hormone secretion ceased after day 28 in culture, endocrine cells were still detectable at day 60. However, later, all endocrine and exocrine cells were replaced by undifferentiated cells that expressed neuron-specific enolase, chromogranin A, laminin, vimentin, cytokeratin 7 and 19, &agr;-1-antitrypsin, transforming growth factor-&agr;, and epidermal growth factor receptor. Our data thus show that, under proper conditions, human islets can be maintained in vitro over a long period and that, in the culture condition, islet cells seem to transdifferentiate to exocrine cells and undifferentiated cells, which may be considered pancreatic precursor (stem) cells.