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A substantial body of evidence supports the conclusion that inflammation is a crucial component of tumour progression. Many malignancies arise from sites of chronic irritation and inflammation. It is now becoming clear that the tumour microenvironment, which is largely organizes by inflammatory cells, is an essential participant in the turmogenesis process like increase proliferation, migration and survival (Mantovani et al, 2008). For example, many studies have proof the strong association between chronic inflammatory bowel diseases and the risk of developing colon cancer (Jain SK et al, 1997). In addition, both sporadic and hereditary forms of chronic pancreatitis are linked with an increased risk of developing pancreatic cancer (Lowenfels AB et al, 1993; Whitcomb DC, 1999). The combined increase in cellular proliferation and genomic damage, both of which are seen with inflammation, are strongly stimulating the malignant transformation of pancreatic cells. The mediators of the inflammatory pathway like (COX-2, NF-kB), cytokines and reactive oxygen species have been observed to increase cell proliferation, stimulate oncogene expression and cause loss of tumor suppressor function; all of which may lead finally to pancreatic malignancy (McDade TP et al, 999). Diabetes has been associated with an increased risk of pancreatic cancer and is also characterised by a smouldering chronic inflammatory process (Huxley R et al, 2005). Along this line, anti diabetic drugs such as rosiglitazone and metfomin are best characterized by their insulin-sensitizing action and have been used in the treatment of diabetes where they also have anti-inflammatory activity (Currie et al., 2009; Evans et al, 2005). This may play a role in prevention and treatment of pancreatic cancer. This study will assess the impact of the two drugs on pancreatic cancer cells line to address the changes in inflammatory and metabolic processes on these cells.
1.1. Pancreatitis and the risk of cancer:
Pancreatic cancer is one of the most causes of death in all kinds of the solid tumor. The five-year survival of the disease is only 5 %. The symptoms of pancreatic cancer are generally nonspecific and may occur late in the course of the disease (Gullo L et al, 2001). As a result, pancreatic cancer is normally diagnosed at an advanced stage, usually after the tumor has already metastasized and spread to other parts of the body (Lillemoe KD et al, 2000). Pancreatic cancer is consider as insensitive disease to pharmacological and radiological intervention and often comes back after curative surgery (Warshaw AL et al, 1990). All these factors result to the bad prognosis of the cancer. Chronic pancreatic inflammation may represent at an early step in the development of malignancy. Current epidemiological data suggests an increased risk of pancreatic cancer in patients with hereditary and sporadic chronic pancreatitis. Persistent inflammation in pancreatic cells could lead to malignant transformation of ductal cells causing dysplasia and ultimate leads to cancer formation (Fig.1.). Similar to other cancer, in pancreatic cancer the mutation of proto-oncogenes and the loss of tumor suppressor genes have been implicated in pancreatic cancer development (Rozenblum El et al, 1997). K-ras mutations are found in most of cases of pancreatic adenocarcinomas as 90% of cases have this kind of mutation with the additional loss of the tumor suppressor protein p53 (Hruban RH et al, 1993). In addition, P16 and DPC4/SMAD mutation are consider to be an important etiologic factors involved with pancreatic cancer formation (Hahn SA et al, 1996).
Fig. 1. Represents the mechanism of inflammation formation and the development of
pancreatic cancer. The image is adopted from www.elsevier.com/locate/suronc
Inflammatory mechanisms of pancreatic cancer development:
A number of transcription factors and cytokines are associated with pancreatic cancer formation. These proteins play a momentous role in the inflammatory response that may end in cancer formation. Cytokines and peroxisome proliferator-activated receptor-γ are one of these factors.
Pancreatic cancers frequently exhibit an intense desmoplastic reaction around the primary tumor (Ryu B et al, 2001). New studies have exposed that these stromal cells release growth factors, cytokines, and angiogenic factors, which can influence tumor growth and migration (McCawley LJ et al, 2001). The mechanisms that contribute to this desmoplastic reaction are not entirely identified. In chronic pancreatitis cytokines are released, together with reactive oxygen species result in inflammation and also cellular damage (Norman J et al, 1998). Some of these cytokines are (e.g., IL-6, IL-8, TNF-α, and interferon γ) are increase in pancreatic cancer microenvironment (Friess H et al, 1999; Le X, Shi Q et al, 2000). Growth factors, like transforming growth factor beta (TGF-β) and platelet-derived growth factor (PDGF) are also release during the early stages of inflammation. Transforming growth factor-alpha (TGF-α) is upregulated in certain kinds of pancreatic cancer while PDGF is known to strongly stimulate fibrogenesis (Friess H et al, 1999). In the tumor infiltrate tumor associated macrophages are a common component which initially noted by Virchow (Balkwill F et al, 2001). These inflammatory cells are able to produce angiogenic and growth factors as well as proteolytic enzymes which can lead to degrade the extracellular matrix (Leibovich SJ et al, 1975). Macrophages may be then lead to tumor proliferation, angiogenesis, and metastasis. Macrophage proinflammatory human chemokine-3alpha (Mip-3α) is upregulated in human pancreatic cancer cells, which act to promote pancreatic cancer growth and enhances tumor migration (Kleeff J, et al, 1999).
1.2.2. Peroxisome proliferator-activated receptor-γ (PPARγ):
PPARγ is a nuclear receptor that mediating cellular differentiation, glucose homeostasis, and apoptotic pathways (Kliewer SA et al, 1999). Ligand for this receptor is anti-diabetic thiazolidinediones which sensitize tissues to insulin and making the cellular body more responsive to insulin action. PPARγ ligand is also exerting anti-inflammatory action by antagonizing the activity of NF-kB and inhibiting cytokine production leading to inhibition of iNOS (Escher p et al, 2000). In recent studies, the scientists have found that activation of the PPARγ receptor induces anti-tumor effects in lung and colon cancer (Chang TH et al, 2000; Wachtershauser A et al, 2000). In addition, PPARγ ligand activity decreases the severity of pancreatitis by inhibiting COX-2 and NF-kB activity (K. Hashimoto, unpublished data). In the pancreatic cell, this transcription factor is related to these inflammatory pathways which related pancreatic cancer development. In resent experimental studies the researchers have found that induction of apoptosis and reduced MMP-2 and MMP-9 activity suggesting a role for PPARγ in metastasis and invasion (K. Hashimoto, unpublished data). The fact that TZDs may have both antidiabetic and an anti-tumor action is important, particularly in light of the hypothesis that pancreatic cancer could be arising from islet cells. As result of its relationship to inflammatory and cell cycle pathways, PPARγ can be involved in the development of malignancy in the pancreas. Moreover, thiazolidinediones that activate PPARγ are safe for use in humans as antidiabetic therapies and may also represent novel antineoplastic agents against pancreatic cancer development.
1.3. Role of antidiabetic drug rosiglitazone in cancer:
Rosiglitazone are a novel class of oral antidiabetic drug now used to treat patients with type 2 diabetes mellitus as it works as an insulin sensitizer, by binding to the PPAR receptors in fat cells and other cells making the them more responsive to insulin action. In addition to that rosiglitazone has important anti- inflammation and anti-atherosclerotic properties (Jiang et al, 1998; Ricote et al, 1998; Kersten et al, 2000). Moreover, the peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-dependent transcription factor that is an important regulator of cellular proliferation and differentiation. PPARγ has been implicated in tumorigenesis as PPAR γ ligands, such as thiazolinediones (ie, rosiglitazone, troglitazone, and pioglitazone), can prevent tumorigenesis in animal models by inhibiting growth and induce redifferentiation in malignant human cells, and also cause cell cycle arrest and apoptosis (Schmidt S et al, 2004; Hirase N et al, 1999). In addition, PPARγ expression is dysregulated in several human malignancies including thyroid and lung cancer (Martelli ML et al, 2002; Theocharis S et al,2002).
1.4. Role of antidiabetic drug Metformin in cancer:
Metformin is an oral antidiabetic agent from biguanide class. It is the first drug of choice for treatment type 2-diabetes. It works by increasing liver and tissue sensitivity to insulin action as well as reduces hepatic glucose production. Many studies have suggested that metformin can decrease the inflammatory reaction in some cases of polycystic ovary syndrome by reducing the inflammatory markers in the plasma, including soluble intercellular adhesion molecules, vascular cell adhesion molecules-1, macrophages migration inhibitory factor, and C-reactive protein (CRP), which indicate modulation of inflammation of this drug (Morin-Papunen L et al, 2003; Diamanti-Kandarakis E et al, 2001). Metformin activates AMP-activated protein kinase, which act to induce glucose uptake in muscles. Targeting AMP-activated protein kinase requires LKB1, a well known tumor suppressor. The relationship between metformin and LKB1 might be then an explanation for the possible beneficial effects of metformin on cancer development (Ben Sahra I et al, 2008). Previous clinical studies showed that cancer risk was lower in patients exposed to metformin compare with unexposed patients (Evans JM et al, 2005; Libby G et al, 2009). Metformin has also been found to be beneficial in patients with specific kinds of cancer. For instance, type 2 diabetic patients who receiving neoadjuvant chemotherapy for breast cancer as well as metformin were more likely to have a complete remission compare with patients not receiving metformin (Jiralerspong S et al, 2009). In addition, patients getting metformin seem to have a lower incidence of pancreatic and prostate cancer (Wright JL et al, 2009; Li D et al, 2010).
3. Material and Methods
3.1. Cell culture
We provided pancreatic cell lines (PanIN and PDAC) which have a characteristic K-Ras and p53 mutation. These cells are taken from embryonic pancreas progenitor of Pdx1Cre LSL-KrasG12D mice model (The cell lines are obtained from Dr David Tuveson, Cambridge University).
The growth medium was prepared by adding 50ml heat inactivated (56oc for 1 hour) fetal calf serum (FCS) and 5 ml of 100x penicillin/streptomycin mixed solution to Dulbecco's modified Eagle's medium (DMEM) media (500ml) supplemented with 4.5 g/liter glucose and 4 mM L-glutamine purchased form ( PAA company). The final concentration of ingredients are 10% FCS, 2.0Mm L-glutamine, 1% penicillin/ streptomycin. Cell cultures were grown in 5% CO2 atmosphere at 37°C. The cell lines were sub-cultured in tissue culture flask (T175 flask) and were grown under standard conditions 370c, 5% co2. The cells were passaged every 3-4 days with the prepared cell culture medium. Cells number was counted by using a Vi-cell XR automated cell viability analyzer (Beckman Coulter).
3.1.4 Cell proliferation assay
PanIN and PDAC cells were plated at a density of 2x105 per well in 6-well plates and the cells were treated with various concentration of rosiglitazone and metformin (5,10,15,20μM) the treated cells then incubated in 5% CO2 atmosphere at 37C0 for 24hr and 48hr. In the next day, the old media was removed and replaced with about 500μl of trypsin, after 5min of incubation, the action of trypsin was stopped by adding 500 μl of fresh media containing 10% Fcs and 1% of penicillin/streptomycin. The number of cells were counted by using Vi-cell XR automated cell viability analyzer (Beckman Coulter).