Xian-Zhong Ding

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.

The mechanisms of lipoxygenase inhibitor-induced apoptosis in human breast cancer cells

Previous experimental studies have shown that high dietary fat intake is associated with mammary carcinogenesis. In the current study, the effect of 5-LOX or 12-LOX inhibitors on human breast cancer cell proliferation and apoptosis, as well as the possible mechanisms were investigated. The LOX inhibitors, NDGA, Rev-5901, and baicalein all inhibited proliferation and induced apoptosis in MCF-7 (ER+) and MDA-MB-231 (ER-) breast cancer cell in vitro. In contrast, the LOX products, 5-HETE and 12-HETE had mitogenic effects, stimulating the proliferation of both cell lines. These inhibitors also induced cytochrome c release, caspase-9 activation, as well as downstream caspase-3, caspase-7 activation, and PARP cleavage. LOX inhibitor treatment also reduced the levels of anti-apoptotic proteins Bcl-2 and Mcl-1 and increased the levels of the pro-apoptotic protein bax. In conclusion, blockade of both 5-LOX and 12-LOX pathways induces apoptosis in breast cancer cells through the cytochrome c release and caspase-9 activation, with changes in the levels of Bcl-2 family proteins.

Lipoxygenase and cyclooxygenase metabolism: new insights in treatment and chemoprevention of pancreatic cancer

The essential fatty acids, linoleic acid and arachidonic acid play an important role in pancreatic cancer development and progression. These fatty acids are metabolized to eicosanoids by cyclooxygenases and lipoxygenases. Abnormal expression and activities of both cyclooxygenases and lipoxygenases have been reported in pancreatic cancer. In this article, we aim to provide a brief summary of (1) our understanding of the roles of these enzymes in pancreatic cancer tumorigenesis and progression; and (2) the potential of using cyclooxygenase and lipoxygenase inhibitors for pancreatic cancer treatment and prevention.

Resveratrol inhibits proliferation and induces apoptosis in human pancreatic cancer cells.

Introduction Because of lack of early diagnosis and poor therapeutic responsiveness, median survival in patients with pancreatic cancer is <6 months, and survival beyond 5 years is rare. Thus, another dimension in chemotherapeutic agents for pancreatic cancer would be beneficial to control metastatic and unresectable disease. Resveratrol, a natural product from grapes, has been shown to be chemopreventive for carcinogen-induced skin cancer and also to inhibit proliferation of oral squamous, breast, colonic, and prostate cancer cells. Aim To investigate the effect of resveratrol in pancreatic cancer. Methodology To evaluate the potential role of resveratrol on pancreatic cancer cell proliferation, two human pancreatic cancer cell lines, PANC-1 and AsPC-1, were used. Results Resveratrol inhibited proliferation of both PANC-1 and AsPC-1 in a concentration- and time-dependent manner as measured by [3H]thymidine incorporation. Cell number of both PANC-1 and AsPC-1 was also significantly decreased following 48 and 72 hours of treatment with 100 &mgr;mol/L resveratrol. The growth inhibition induced by resveratrol was accompanied by apoptotic morphologic changes, characterized by cell rounding and cell membrane blebbing suggesting apoptosis. Propidium iodide staining of DNA, measured by flow cytometry, showed a dramatic increase in the fraction of sub-G0/G1 cells following resveratrol treatment in both PANC-1 and AsPC-1. The substantial apoptosis inducted by resveratrol on these two cell lines was confirmed by the terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling assay. Conclusion These findings suggest that the natural product resveratrol may have a potent antiproliferative effect on human pancreatic cancer with induction of apoptosis. Resveratrol is likely to be valuable for the management and prevention of human pancreatic cancer.

Physiological concentrations of insulin augment pancreatic cancer cell proliferation and glucose utilization by activating MAP kinase, PI3 kinase and enhancing GLUT-1 expression.

Pancreatic carcinoma is characterized by poor prognosis and lack of response to conventional therapy for reasons that are not clear. Because of the structural relationship between the exocrine and endocrine pancreas and high concentrations of islet hormones bathing pancreatic tissue, we hypothesized that pancreatic cancer cell proliferation and glucose utilization are regulated by pancreatic islet hormones, particularly insulin. Based on this, the effect of islet hormones on pancreatic cancer cells in vitro was investigated. Five pancreatic cancer cell lines, CD11, CD18, HPAF, PANC-1, and MiaPaCa2 were used to investigate the effect of islet hormones on cell proliferation, glucose utilization, and GLUT-1 expression. Insulin, but not somatostatin and glucagon, induced pancreatic cancer cell growth in a concentration-and time-dependent manner. At concentrations within the range of those in the intrapancreatic vasculature, insulin (10−10–10−8 mol/L) markedly increased [3H]-thymidine incorporation. Insulin significantly enhanced glucose utilization of pancreatic cancer cells before it enhanced cell proliferation. The MAPK kinase inhibitor PD 098059 abolished insulin-stimulated DNA synthesis and partially reduced insulin-stimulated glucose uptake. In contrast, the PI3 kinase inhibitor wortmannin substantially inhibited insulin-induced glucose uptake and partially blocked thymidine incorporation. Furthermore, after 24-hour treatment with insulin, GLUT-1 expression in pancreatic cancer cells was markedly increased, indicating that insulin enhances glucose utilization partly through increasing glucose transport. These findings suggest that insulin stimulates proliferation and glucose utilization in pancreatic cancer cells by two distinct pathways. Insulin augments DNA synthesis mainly by MAP kinase activation and glucose uptake mainly by PI3 kinase activation and enhancement of GLUT-1 expression. High intrapancreatic concentrations of insulin are likely to play an important role in stimulating pancreatic cancer growth indirectly by increasing substrate availability as well as by direct action as a trophic factor.

Cyclooxygenases and Lipoxygenases as Potential Targets for Treatment of Pancreatic Cancer

Pancreatic adenocarcinoma is characterized by poor prognosis, late diagnosis and lack of response to conventional therapies. The incidence of this disease shows no sign of declining in the Western world. Thus, new targets need to be identified for pancreatic cancer treatment. In particular, new chemotherapeutic agents would be extremely beneficial for control of unresectable cancer and metastatic lesions as well as for prevention of this deadly disease. Mounting evidence suggests that both lipoxygenases (LOXs) and cyclooxygenases (COXs), the key enzymes for arachidonic acid metabolism, have a profound influence on the development and progression of several human cancers. Recent evidence suggests that both COX and LOX pathways are important in pancreatic cancer. Results from immunocytochemical, RT-PCR, and Western blotting studies have shown that COX, specifically COX-2, is upregulated in human pancreatic cancer cell lines as well as human pancreatic cancer tissues compared with normal ductal cells and normal pancreas specimens. Agents that block COX enzymes significantly inhibit pancreatic cancer growth both in vitro and in vivo, in parallel with induction of apoptosis. Expression of both 5-LOX and 12-LOX is also seen in pancreatic cancer, although compared to the expression of COX this has not been extensively investigated. Chemical inhibitors or antisense oligonucleotides that block either 5-LOX or 12-LOX cause marked inhibition of pancreatic cancer cell proliferation. On the other hand, LOX metabolites stimulate growth of the tumor cells and reverse LOX-inhibitor-induced growth inhibition, suggesting the specific role of LOX in regulating pancreatic cancer cell proliferation. Although questions still need to be answered, such as the underlying mechanisms for COX and LOX-induced growth inhibition, both COX and LOX pathways are potential targets for pancreatic cancer treatment and chemoprevention. COX and LOX enzyme inhibitors are available and have been shown to be relatively safe in the treatment of other diseases.

12‐lipoxygenase metabolite 12(S)‐HETE stimulates human pancreatic cancer cell proliferation via protein tyrosine phosphorylation and ERK activation

We previously reported that inhibition of the 12‐lipoxygenase pathway abolished proliferation and induced apoptosis in several pancreatic cancer cell lines. Furthermore, the 12‐lipoxygenase product 12(S)‐HETE stimulated pancreatic cancer cell proliferation and reversed 12‐lipoxygenase inhibitor–induced growth inhibition. We investigated the underlying mechanism for 12(S)‐HETE–induced pancreatic cancer cell proliferation, using 2 human pancreatic cancer cell lines, PANC‐1 and HPAF. Cell proliferation was monitored by both thymidine incorporation and cell number. Western blotting was used to investigate the effect of 12(S)‐HETE on cellular protein tyrosine phosphorylation as well as ERK, P38 MAPK and JNK/SAPK phosphorylation. 12(S)‐HETE markedly stimulated proliferation of pancreatic cancer cells in a time‐ and concentration‐dependent manner. In parallel, 12(S)‐HETE induced tyrosine phosphorylation of multiple cellular proteins, while inhibition of tyrosine kinase by genestein abolished 12(S)‐HETE–induced proliferation, indicating that intracellular protein tyrosine kinase activation is involved in the mitogenic effects of 12(S)‐HETE. Following treatment with 12(S)‐HETE, both ERK and P38 MAPK, but not JNK/SAPK, were phosphorylated. The specific MEK inhibitors PD098059 and U0126, which in turn suppress ERK, abolished 12(S)‐HETE–stimulated proliferation. In contrast, inhibition of P38 MAPK with SB203580 did not affect 12(S)‐HETE–stimulated pancreatic cancer cell proliferation. Furthermore, 12(S)‐HETE–stimulated ERK phosphorylation was inhibited by genestein, indicating that tyrosine phosphorylation is essential for ERK activation. These findings suggest that both ERK and cellular protein tyrosine kinase activation are involved in 12(S)‐HETE–induced pancreatic cancer cell proliferation but P38 and JNK/SAPK are not involved in this mitogenic effect.

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.

BLT2 is expressed in PanINs, IPMNs, pancreatic cancer and stimulates tumour cell proliferation.

Pancreatic cancer has an abysmal prognosis. Targets for early detection, prevention and therapy are desperately needed. Inflammatory pathways have an important impact on tumour growth and progression. Expression of BLT2 (the second leukotriene B4 receptor) was investigated by real-time RT–PCR and immunohistochemistry. Cell proliferation was studied after stable transfection with BLT2, after treatment with siRNA and Compound A as an agonist. BLT2 is expressed in all pancreatic cancer cell lines. Results from real-time RT–PCR revealed significant overexpression of BLT2 in malignant intraductal papillary mucinous neoplasias (IPMNs) and pancreatic adenocarcinoma. Intense staining was evident in IPMNs, infiltrating tumour cells and advanced pancreatic intraepithelial neoplasias (PanINs) but not in normal ductal cells. Overexpression of BLT2 as well as stimulation of Colo357, Panc-1 and AsPC1 cells with Compound A caused a significant increase in tumour cell proliferation, an effect reversed after siRNA treatment. This study demonstrates for the first time the expression of BLT2 in the pancreas and overexpression in pancreatic cancers and malignant IPMNs in particular. Upregulation of BLT2 is already evident in precursor lesions (PanINs, IPMNs). Overexpression of this receptor leads to significant growth stimulation. Therefore, we suggest BLT2 as a new target for chemoprevention and therapy for pancreatic cancer.

Review of the Apoptosis Pathways in Pancreatic Cancer and the Anti-apoptotic Effects of the Novel Sea Cucumber Compound, Frondoside A

Pancreatic cancer cells are resistant to the growth‐inhibitory and apoptosis‐inducing effects of conventional chemotherapeutic agents. There are multiple genetic and epigenetic events during the process of carcinogenesis that enable the cancer cells to avoid normal growth constraints and apoptosis. Investigation of the mechanisms involved has led to multiple strategies that encourage cell death and apoptosis to occur. The pathways involved are summarized in this review, together with some recently developed strategies to promote cell death in this cancer and with a particular focus on the frondoside A, a novel triterpenoid glycoside isolated from the Atlantic sea cucumber, Cucumaria frondosa. Frondoside A inhibited proliferation of AsPC‐1 human pancreatic cancer cells in a concentration‐ and time‐dependent manner, as measured by 3H‐thymidine incorporation and cell counting. In concert with inhibition of cell growth, frondoside A induced significant morphological changes consistent with apoptosis. Propidium iodide DNA staining showed an increase of sub‐G0/G1 cell population of apoptotic cells induced by frondoside A. Frondoside A–induced apoptosis was confirmed by annexin V binding and TUNEL assay. Furthermore, western blotting showed a decrease in expression of Bcl‐2 and Mcl‐1, an increase in Bax expression, activation of caspases 3, 7, and 9, and an increase in the expression of the cyclin‐dependent kinase inhibitor, p21. These findings show that frondoside A induced apoptosis in human pancreatic cancer cells through the mitochondrial pathway and activation of the caspase cascade. Finally, a very low concentration of frondoside A (10 μg/kg/day) inhibited growth of AsPC‐1 xenografts in athymic mice. In conclusion, new chemotherapeutic agents are desperately needed for pancreatic cancer because of the poor responsiveness to currently available treatment options. Frondoside A has potent growth inhibitory effects on human pancreatic cancer cells, and the inhibition of proliferation is accompanied by marked apoptosis. Frondoside A may be valuable for the treatment or chemoprevention of this devastating disease.