Dual Roles of Nrf2 in Adaptive Survival Response to Oxidative and Inflammatory Stresses in Normal vs Cancer Cells

다국어 초록 (Multilingual Abstract)

Inflammation has been implicated in multi-stage carcinogenesis. In effect of helping chronic inflammation to develop an incipient neoplasia is contributed with largely of the innate immune system which is demonstrated functionally important in inflammation status. Prostaglandins play a key role in inflammation response in inflammation-associated carcinogenesis. COX-2-derived prostaglandins are highly elevated in inflamed tissue, and it provokes cardinal signs of acute inflammation, proliferation, apoptosis, immunity, angiogenesis, invasion, metastasis and surveillance. Intratumoral levels of PGE2 depend not only upon the rate of production but also on the rate of its degradation. The nicotinamide adenine dinucleotide positive-dependent catabolic enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH) metabolizes PGE2 to the biologically inactive 15-keto-prostaglandins. To take account of strong link between inflammation and its associated carcinogenesis, it is important to control the 15-PGDH expression in inflammation status. In present study, we show that the regulation of COX-2 on 15-PGDH expression both in colitis and in its-related carcinogenesis.
To consider the logic of 15-PGDH as a function of prostaglandins degradation, it should be happened more severe inflammation in 15-PGDH knockout conditions. Contrary to our expectation, there were less inflammatory protein expression and similar pathological appearances in 15-PGDH knockout inflamed tissues compared with wild type one on mouse colitis and gastritis model. From these data, we hypothesized that accumulated prostaglandins in 15-PGDH knockout conditions may conduct an adaptive response and be ready for protect the tissues or cells from exogenous stimulus.
As a cellular deference mechanism against various exogenous stresses, certain molecules are involved in its adaptive response. Among them, nuclear factor-erythroid 2p45 (NF-E2)-related factor (Nrf2), a master regulator of the basal and inducible expression of diverse cytoprotective proteins is mainly important on cellular adaptive response. Nrf2 and its target protein HO-1 were highly elevated in chemical- or bacterial-induced inflammation tissues on 15-PGDH knockout mouse than wild type mouse. These findings suggest that Nrf2/HO-1 upregulation was resulted from accumulated prostaglandins in blocking of degrading pathway and this signal transduction was prepared to respond to the oxidative stress and capable of coping with it by inducing an adaptive response to maintain homeostasis or even to stimulate ourselves to promote a healthy state.
Multiple lines of evidence support those Helicobacter pylori infection is one of the primary causes of gastritis and peptic or duodenal ulcer, which are provoked by oxidative stress(Hahm, Kim et al. 2003). In spite of the intimate relationship between Helicobacter pylori infection and gastritis (or its related gastric carcinogenesis), not all infected individuals developed clinical diseases(Uemura, Okamoto et al. 2001). This is a report about 10 year’s follow-up of Helicobacter pylori-infected individuals (Uemura, Okamoto et al. 2001), and according to the results of this seminal paper, only 2.9% of Helicobacter pylori positive patients developed the gastric cancer, while the remaining subjects were asymptomatic. The other 97% of individuals did not developed gastric cancer. It reflects some of defense mechanism under Helicobacter pylori infected condition, and several host and environmental determinants are important in Helicobacter pylori-related disease. Helicobacter pylori infection induced Nrf2 and concomitant HO-1 expression in both of cells and animals. And Helicobacter pylori also stimulated nuclear accumulation of Nrf2, as well as its interaction with antioxidant response elements located in the PIAS2 promoter and regulating degradation of STAT3 signaling. PIAS2 is one of STAT3 negative regulator, E3 SUMO-protein ligase, binding to STAT3 dimer and leading its degradation. PIAS2 expression was also lowered in Nrf2 knockout MEF cells, lending further support to Nrf2-dependent PIAS2 expression. To evaluate the protective role of Nrf2 in Helicobacter pylori induced gastritis in mice, nrf2 knockout mice were utilized. Helicobacter pylori induced gastritis was much severe in nrf2 knockout mice compared with its wild type littermate. The Nrf2 and HO-1 expression levels were coordinately increased in Helicobacter pylori infected human stomach. These observations suggest that Helicobacter pylori infection can trigger the cellular defense mechanism through Nrf2-mediated upregulation of PIAS2, and consecutively related with STAT3 degradation.
Although a wide body of evidence indicates that activation of Nrf2 protects against a variety of toxicants and diseases, the prolonged activation of Nrf2 has been shown to favor the progression of several types of cancers. Nrf2 has been shown to be constitutively elevated in lung, breast, head and neck, ovarian, and endometrial carcinomas. Abnormally enhanced Nrf2 ability resulted cancer cell proliferation and promote chemoresistance and radioresistance. Survival of nearly all types of tumor cells depend on glycolysis, which proceeds at a much higher rate in tumor cells than that in the noncancerous tissues. Thus, tumor cells are exposed frequently and maybe constantly to glucose deprived conditions. In particular, the microenvironments of solid tumor tissues are often confronted with unfavorable conditions such as hypoxia and hypoglycemia(Warburg 1956, Weber 1977, Weber 1977). Thus, cancer cells tend to develop self-defense mechanisms to survive such adverse conditions. Although prolonged glucose deprivation induced apoptotic cell death in HepG2 cells, there was upregulated HO-1 expression before apoptosis manifested. We found that the induction of Nrf2-mediated HO-1 upregulation in glucose deprived HepG2 cells confers an adaptive survival response before the manifestation of cell death.
Taken together, these observations suggest that the Nrf2 might protect the cells and tissues from Helicobacter pylori-induced gastritis or DSS-treated colitis, particularly regulating inflammatory environment through inhibiting the IL6/STAT3 activated signaling. While activated Nrf2 in cancer cells obtained the survival response under hypoglycemia. Thus, better understanding of the mechanisms by which inflammation mediators cause Nrf2 activation and induce HO-1 expression will be useful in the development of novel therapeutic strategies for effective treatment or prevention of cancer.

목차 (Table of Contents)

• Table of Contents
• Abstract -------------------------------------------------------------------------------------------- i
• Table of Contents -------------------------------------------------------------------------------- v
• List of Figures ----------------------------------------------------------------------------------- ix
• List of Tables ----------------------------------------------------------------------------------- xii
• List of Abbreviations ------------------------------------------------------------------------- xiii
• Chapter I
• General Overview ------------------------------------------------------------------------------- 1
• Part 1. Enzymes involved in synthesis and catabolism of prostaglandin: A link of
• inflammation and cancer------------------------------------------------------------------------- 2
• 1.1. Introduction -------------------------------------------------------------------------------------------- 2
• 1.2. Inflammation and cancer ----------------------------------------------------------------------------- 3
• 1.3. Metabolism of prostaglandins ----------------------------------------------------------------------- 5
• 1.4. Involvement of key enzymes regulating prostaglandin metabolism in inflammation -------- 9
• 1.5. Involvement of key enzymes regulating prostaglandin metabolism in cancer --------------- 12
• 1.6. Conclusion -------------------------------------------------------------------------------------------- 16
• Part 2. Janus-faced functions of Nrf2 in Tumorigenesis------------------------------------------- 17
• 2.1. Introduction ------------------------------------------------------------------------------------- 17
• 2.2. The Nrf2 signaling pathway ------------------------------------------------------------------------ 18
• 2.3. Dual roles of Nrf2 in cancer------------------------------------------------------------------------- 19
• 2.4. Conclusion -------------------------------------------------------------------------------------------- 22
• Statement of Purpose -------------------------------------------------------------------------- 24
• Chapter II
• Part 1
• Down-regulation of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) expression by COX-2: Implications for inflammation-associated colon carcinogenesis----------25
• 1. Abstract -------------------------------------------------------------------------------------------- 26
• 2. Introduction --------------------------------------------------------------------------------------- 28
• 3. Materials and Methods -------------------------------------------------------------------------- 31
• 4. Results --------------------------------------------------------------------------------------------- 36
• DSS treatment reduces 15-PGDH expression and its activity------------------------------ 36
• Celecoxib restores 15-PGDH expression in DSS-induced colitis-------------------------- 36
• 15-PGDH expression is negatively regulated by COX-2 in inflammation conditions-- 36
• 15-PGDH is downregulated in colitis-induced colon carcinogenesis in mice------------ 37
• 15-PGDH may play a role of protection on inflammation-associated carcinogenesis
• model in COX-2 knockout mice---------------------------------------------------------------- 38
• 15-PGDH downregulation in human colitis and colon cancer specimens----------------- 38
• 5. Discussion ----------------------------------------------------------------------------------------- 40
• Part 2
• Nrf2 as a cellular adaptive signaling in 15-hydroxyprostaglandin dehydrogenase (15-PGDH) knockout system----------------------------------------------------------------- 52
• 1. Abstract -------------------------------------------------------------------------------------------- 53
• 2. Introduction --------------------------------------------------------------------------------------- 55
• 3. Materials and Methods -------------------------------------------------------------------------- 57
• 4. Results --------------------------------------------------------------------------------------------- 62
• Knockdown of 15-PGDH gene has merely intensified the inflammation----------------- 62
• Adaptive response in 15-PGDH knockout mouse under the inflammatory stresses----- 63
• 5. Discussion --------------------------------------------------------------------------------------- 68
• Chapter III
• Nrf2-mediated adaptive response to Helicobacter pylori-induced inflammation -71
• 1. Abstract ------------------------------------------------------------------------------------------- 72
• 2. Introduction -------------------------------------------------------------------------------------- 74
• 3. Materials and Methods ------------------------------------------------------------------------- 77
• 4. Results -------------------------------------------------------------------------------------------- 82
• Helicobacter pylori infection induces Nrf2 and concomitant HO-1 expression---------- 82
• Nrf2 regulates STAT3 signaling under Helicobacter pylori infection -------------------- 82
• Nrf2 regulates STAT3 via PIAS ---------------------------------------------------------------- 83
• Downstream of STAT3 signal is also related with Nrf2 expression------------------------ 84
• Helicobacter pylori induced gastritis is much severe in nrf2 knockout mice----------- 84
• Nrf2 and HO-1 are coordinately overexpressed in Helicobacter pylori infected human
• stomach--------------------------------------------------------------------------------------------- 85
• 5. Discussion ---------------------------------------------------------------------------------------- 100
• Chapter IV
• Nrf2-mediated Hemeoxygenase-1 upregulation as adaptive survival response to glucose deprivation-induced apoptosis in HepG2 Cells------------------------------- 103
• 1. Abstract ------------------------------------------------------------------------------------------- 104
• 2. Introduction -------------------------------------------------------------------------------------- 105
• 3. Materials and Methods ------------------------------------------------------------------------- 107
• 4. Results -------------------------------------------------------------------------------------------- 113
• Glucose deprivation causes apoptosis in HepG2 cells-------------------------------------- 113
• Glucose deprivation induces HO-1 expression as an adaptive response----------------- 113
• HO-1 upregulation induced by glucose deprivation is mediated by transient activation of
• Nrf2 ----------------------------------------------------------------------------------------------- 114
• Glucose deprivation enhances ROS production responsible for Nrf2 activation and HO-1
• induction ----------------------------------------------------------------------------------------- 115
• 5. Discussion --------------------------------------------------------------------------------------- 126
• Conclusion ------------------------------------------------------------------------------------ 130 References--------------------------------------------------------------------------------------- 132
• Abstract in Korean --------------------------------------------------------------------------- 155
• Biographical Data ---------------------------------------------------------------------------- 159
• List of Figures
• Chapter I (Part 1)
• Fig. 1-1. NSAIDs involvement in prostaglandin metabolism pathway----------------------------- 8
• Chapter I (Part 2)
• Fig. 1-2. Dual role of Nrf2 ------------------------------------------------------------------------------ 23
• Chapter II (Part1)
• Fig. 2-1. Expression of COX-2 and 15-PGDH protein level in DSS-treated mouse colon ----- 40
• Fig. 2-2. Effect of celecoxib on colon cancer cells and DSS-treated mouse colon --------------- 41
• Fig. 2-3. Negative regulation of 15-PGDH expression by COX-2 in colon cancer cells and COX2 knockout mice ------------------------------------------------------------------------------------- 42
• Fig. 2-4. Expression of COX-2 and 15-PGDH in two-stage mouse colon carcinogenesis model------------------------------------------------------------------------------------------------------------------ 44
• Fig. 2-5. Histological analysis of colonic mucosa on COX-2 genomic mutant mice ------------ 47
• Fig. 2-6. Expression of COX-2 and 15-PGDH in human colitis and colon tumor tissues ------- 48
• Chapter II (Part2)
• Fig. 2-7. DSS-induced colitis in 15-PGDH knockout mouse --------------------------------------- 64
• Fig. 2-8. Helicobacter pylori inoculation and high salt diet consumption induced gastritis in 15-PGDH knockout mouse----------------------------------------------------------------------------------- 66
• Fig. 2-9. Adaptive response signals in 15-PGDH knockout mouse ---------------------------------67
• Chapter III
• Fig. 3-1. Helicobacter pylori induced Nrf2 and HO-1 expression -------------------------------- 87
• Fig. 3-2. Helicobacter pylori induced HO-1 expression is regulated by Nrf2 ------------------- 89
• Fig. 3-3. Transfection of cagA induced Nrf2 signaling as Helicobacter pylori does ----------- 90
• Fig. 3-4. Helicobacter pylori activated stat3 signaling is negatively regulated by Nrf2 ------- 91
• Fig. 3-5. Nrf2 regulated STAT3 activation via PIAS ------------------------------------------------ 93
• Fig. 3-6. Downstream of STAT3 signal was also related with Nrf2 gene expression ---------- 94
• Fig. 3-7. Absence of Nrf2 resulted more severe inflammation by Helicobacter pylori infection----------------------------------------------------------------------------------------------------------------- 95
• Fig. 3-8. Expression levels of Nrf2 and HO-1 in Helicobacter pylori infected human patients ------------------------------------------------------------------------------------------------------------------98
• Fig. 3-9. A proposed pathway for Helicobacter pylori-induced Nrf2 activation and PIAS2 expression, which confers an anti-inflammatory response as suppressing STAT3 activity ------99
• Chapter IV
• Fig. 4-1. Glucose deprivation-induced apoptotic cell death in HepG2 cells --------------------- 117
• Fig. 4-2. Effect of glucose deprivation on HO-1 expression --------------------------------------- 118
• Fig. 4-3. Effect of HO-1 induction or inhibition of HO-1 activity on survival against glucose deprivation-induced cytotoxicity ---------------------------------------------------------------------- 119
• Fig. 4-4. Glucose deprivation-induced transient activation of Nrf2 in HepG2 cells ------------ 120
• Fig. 4-5. Effect of glucose deprivation on HO-1 expression in Nrf2-/- MEFs------------------- 122
• Fig. 4-6. Involvement of ROS accumulation in glucose deprivation-induced HO-1 expression through Nrf2 activation --------------------------------------------------------------------------------- 123
• Fig. 4-7. Effect of Nrf2 on glucose deprivation-induced cytotoxicity as an adaptive response ------------------------------------------------------------------------------------------------------------------ 124
• Fig. 4-8. A proposed pathway for glucose deprivation-induced Nrf2 activation and HO-1 expression, which confers an adaptive survival response in HepG2 cells ----------------------- 125
• List of Tables
• Chapter II (Part 1)
• Table 2-1. Incidence and multiplicity of colonic adenocarcinoma on ICR mice ----------------- 43
• Table 2-2. Incidence and multiplicity of colonic adenocarcinoma on COX2 wild type and knockout mice --------------------------------------------------------------------------------------------- 46