Helicobacter ISSN 1523-5378 doi: 10.1111/hel.12211
Autophagy in Helicobacter pylori Infection and Related Gastric Cancer ~ o-Rodrıguez,* Nadeem O. Kaakoush,* Khean-Lee Goh,† Kwong Ming Fock‡ and Hazel M. Natalia Castan Mitchell* *School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia, †Department of Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia, ‡Division of Gastroenterology, Department of Medicine, Changi General Hospital, Singapore City, Singapore
Keywords stomach neoplasm, Helicobacter pylori, autophagy, polymorphism, gene expression. Reprint requests to: Hazel M. Mitchell, School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Lab301A, Biological Sciences Building (D26), Sydney, NSW 2052, Australia. E-mail: [email protected]
Abstract Background: Autophagy, a degradation pathway in which cytoplasmic content is engulfed and degraded by lysosomal hydrolases, plays a pivotal role in infection and inflammation. Given that defects in autophagy lead to increased susceptibility to infection, we investigated the role of autophagy in Helicobacter pylori-related gastric cancer (GC). Materials and Methods: Gene expression of 84 molecules was examined through quantitative real-time PCR in gastric epithelial cells (AGS) and macrophages (THP-1) upon exposure to H. pylori GC026 (GC) and 26695 (gastritis). Further, ATG16L1 rs2241880, IRGM rs13361189, and IRGM rs4958847, polymorphisms that have been investigated in relation to H. pylori infection or GC in Caucasians, were detected by MALDI-TOF mass spectrometry in 304 ethnic Chinese (86 noncardia GC cases/218 functional dyspepsia controls). Results: Gene expression analyses showed twenty-eight molecules involved in vesicle nucleation, elongation, and maturation to be significantly downregulated in H. pylori GC026-challenged AGS cells. Further, core autophagy proteins and autophagy regulators were differentially expressed in H. pylorichallenged THP-1-derived macrophages. Analyses of the selected polymorphisms showed that ATG16L1 rs2241880 increased the risk of GC (OR: 2.38, 95% CI: 1.34–4.24) and H. pylori infection (OR: 1.49, 95% CI: 1.02–2.16) while IRGM rs4958847 decreased GC risk (OR: 0.26, 95% CI: 0.09–0.74) in ethnic Chinese, these effect sizes being especially strong in H. pylori-infected individuals (ATG16L1 rs2241880 and IRGM rs13361189). Conclusions: Our findings indicate that highly virulent H. pylori strains markedly modulate autophagy in the host cell. Further, for the first time, autophagy polymorphisms were associated with GC in Chinese, a high GCrisk population.
Gastric cancer (GC) is one of the most commonly diagnosed cancers in the world, occupying the fifth position after lung, breast, colorectal, and prostate neoplasias [1]. Furthermore, it is the third leading cause of cancer-related deaths worldwide, with an estimated 723,000 GC-related deaths in 2012, accounting for 8.8% of total deaths from cancer [1]. Almost two-thirds of GC cases occur in East Asia, Eastern Europe, and Central and South America, these regions being classified as high GC-risk populations. The incidence in Chinese individuals not only represents 42% of the above worldwide estimation, but Chinese ethnicity has also
© 2015 John Wiley & Sons Ltd, Helicobacter
been identified as an independent risk factor for the development of GC in multiracial studies [2,3]. According to latest global estimates, the age-standardized incidence rate (ASR) of GC is 22.7 per 100,000 habitants in China. Further, in multiracial countries such as Singapore and Malaysia, Chinese have the highest ASR (25.7 per 100,000 men) as compared with the Malays and Indians who have ASRs of 6.6 per 100,000 and 8.4 per 100,000 men, respectively [1]. Given the significant morbidity and mortality associated with GC and the short survival time following diagnosis, what is clearly required is the identification
1
~o-Rodrıguez et al. Castan
Autophagy in Helicobacter pylori-Related GC
of factors that can predict those at high risk of GC development. While H. pylori infection has been established as the most important risk factor for GC, the etiology of GC also involves host and environmental factors [4]. This is evidenced by the fact that only 1–3% of H. pylori-infected patients develop GC and that progression to GC in some subjects occurs even after eradication of the bacterium [5]. Macroautophagy (autophagy) is defined as a degradation pathway in which cytoplasmic content is engulfed and degraded by the lysosome. Thus, autophagy acts as an intracellular mechanism to eliminate protein aggregates, damaged organelles, and intracellular microbes and, subsequently, maintain cellular homeostasis [6]. This highly conserved process that initiates with the formation of a double membrane compartment called the phagophore and culminates with the generation of a structure called the autolysosome
has been well characterized (Fig. 1A). Autophagy involves the assembly of proteins that are essential for autophagic vacuole formation including autophagyrelated gene (Atg) 9 (possible “membrane shuttle”), the UNC-51-like kinase (ULK) complex (comprises Atg13, FIP200, Atg101, and ULK1 or ULK2 and is involved in the initiation of autophagy), the Vps34 complex (comprises Vps34, Vps15, Beclin-1, UVRAG, and Atg14L and generates phosphatidylinositol 3-phosphate (PI3P), an essential lipid component of autophagosomes), PI3Pbinding proteins (WIPI1, WIPI2, DFCP1, and Alfy), the Atg12 ubiquitin-like conjugation system (comprises Atg12, Atg5, and Atg16L1 and mediates formation and elongation of the autophagosome), and the Atg8 ubiquitin-like conjugation system (comprises LC3 A/B/C and c-aminobutyric acid receptor-associated proteins (GABARAPs), and mediates autophagosome closure and determines autophagosome size) [7,8].
A
B
D
C
E
Figure 1 The autophagy pathway and its modulation in Helicobacter pylori-infected gastric epithelial cells. (A) Schematic diagram of the various stages of autophagy. First, the phagophore, which is an isolation membrane, encloses cytoplasmic material. Subsequently, the phagophore elongates to form a double membrane vacuole known as the autophagosome. The autophagosome then fuses with the endosome, which delivers cargo, components of the membrane fusion machinery, and lowers the pH of the autophagic vesicle before delivery of lysosomal acid proteases. Finally, the autophagosome fuses with the lysosome to form an autolysosome and degrade the enclosed cytoplasmic material [6]. (B) Differential expression of genes involved in autophagy induction, (C) vesicle nucleation, (D) vesicle elongation, and (E) vesicle maturation, in H. pylori GC026challenged AGS cells compared to the noninfected control group. Fold change (2 DDCt ) is the normalized gene expression (2 DCt ) in AGS cells challenged with H. pylori GC026 divided the normalized gene expression (2 DCt ) in the control group. Fold regulation represents fold-change results in a biologically meaningful way. Fold-change values greater than one indicate up-regulation, and the fold regulation is equal to the fold change. Fold-change values less than one indicate down-regulation, and the fold regulation is the negative inverse of the fold change. Fold difference compared to the control group showing a *p-value < .05 and a **p-value < .01.
2
© 2015 John Wiley & Sons Ltd, Helicobacter
~o-Rodrıguez et al. Castan
In the context of H. pylori infection, a recent paper by Raju et al. [9] showed that autophagy is induced in response to the virulence factor vacuolating cytotoxin A (VacA) and thus serves a cytoprotective role. However, prolonged exposure to VacA causes disruptive autophagy with autophagosomes that lack cathepsin D and thus have reduced catalytic activity [9]. In addition, although H. pylori has been considered to be an extracellular pathogen, emerging evidence demonstrates that it may be internalized and can subsequently replicate within the autophagosomes of macrophages, and dendritic and gastric epithelial cells [10–13]. From a host perspective, Raju et al. [9] have shown that the presence of a known genetic risk factor for Crohn’s disease (CD), ATG16L1 rs2241880, increases the risk of H. pylori infection in Caucasian individuals from Scotland and Germany [9]. ATG16L1, located in chromosome 2q37.1, encodes a protein that consists of 580– 630 amino acids, including an extreme N-terminal region that mediates interaction with the Atg12-Atg5 conjugate and a coiled-coil domain that mediates Atg16L1 dimerization. Autophagy plays a dual role in carcinogenesis. Evidence supporting the protective role against cancer is based on studies that have identified molecules involved in this process, including Atg4c, Bif-1, and UVRAG, as tumor suppressors [14]; the identification of anti-oncogenic signaling pathways, including PTEN, TSC1/2, LKB1, and p53, that stimulate autophagy [15]; and evidence that autophagy de-regulation mediates mitochondrial dysfunction, increased oxidative stress, and susceptibility to pro-inflammatory stimuli, conditions that all lead to DNA damage and thus genetic instability [14]. However, in established tumors, autophagy may confer a survival advantage to tumor cells under metabolic stress as a result of a high proliferation rate and exposure to hypoxia from insufficient vascularisation [14]. In addition, it has been reported that tumor cells under selective pressure from therapeutic interventions might be protected by autophagy, which acts as an adaptive cellular response that leads to chemoresistance and cancer cell survival [14]. In the context of gastric carcinogenesis, recent studies have indicated that increased expression of beclin-1 is associated with a favorable prognosis of GC [16]. Autophagy has also been linked to the protective effect of matrine, a natural opioid found in Sophora flavensces, against GC [17]. In addition, IFN-c has been shown to inhibit gastric carcinogenesis by inducing epithelial cell autophagy and T-cell apoptosis in a murine model [18]. Interestingly, investigation of the association between IRGM polymorphisms (rs4958847 and rs13361189) and GC in a Caucasian population by Burada et al. [19] has
© 2015 John Wiley & Sons Ltd, Helicobacter
Autophagy in Helicobacter pylori-Related GC
shown IRGM rs4958847 to decrease the risk of GC in these individuals. IRGM is located at chromosome 5q33.1 and encodes the immunity-related GTPase family M protein (IRGM), an IFN-inducible GTPase that is involved in autophagic vacuole formation. Given the recent reported interaction between H. pylori and autophagy, a comprehensive investigation of H. pylori recognition by this host intracellular mechanism and its role in GC is clearly warranted. Thus, we investigated the role of autophagy in H. pylori-related GC, by analyzing the gene expression of 84 molecules involved in autophagy in nonimmune (gastric epithelial) and immune (monocytic) cell lines upon exposure to H. pylori and by examining, for the first time, the association between polymorphisms in ATG16L1 (rs2241880) and IRGM (rs13361189 and rs4958847) and risk of GC in ethnic Chinese individuals, a high GC-risk population.
Materials and Methods Gene Expression of Molecules Involved in Autophagy in Mammalian Cells Mammalian cell culture. The human GC cell line AGS (American Type Culture Collection (ATCC); Manassas, VA, USA; code: ATCC CRL-1739) was cultured in F12K medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen, Mulgrave, Vic., Australia) and 100 lg/mL penicillin–streptomycin solution (Invitrogen). The human monocytic leukemia cell line THP-1 (code: ATCC TIB-202) was cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS (Invitrogen), 1 mmol/L sodium pyruvate (Invitrogen), 2500 mg/L sodium bicarbonate (Invitrogen), and 100 lg/mL penicillin–streptomycin solution (Invitrogen). Cells were maintained in 25-cm2 tissue culture flasks (Greiner-Bio-On, Frickenhausen, Germany) at 37 °C with 5% CO2.
Bacterial culture. The H. pylori strain 26695 (cagA+ and vacA s1m1+) was isolated from a patient with gastritis (code: ATCC 700392) [20]. The H. pylori strain GC026 (cagA+, cagE+, cagL+, cagT+, vacA s1m1+, babA+, oipA+, dupA+, and sabA+) was isolated from a GC patient undergoing endoscopy at the University Hospital of Malaysia, Kuala Lumpur, in a previous study conducted by our group [21,22]. Both H. pylori strains were grown for 48 hours on horse blood agar plates (Blood Agar Base No. 2 supplemented with 10% sterile defibrinated horse blood) (Oxoid, Heidelberg West, Vic.,
3
Autophagy in Helicobacter pylori-Related GC
Australia) at 37 °C in an anaerobic jar containing a gas generating kit (Oxoid) to provide a microaerobic atmosphere of 6% O2, 10% CO2, and 84% N2.
Infection assay. AGS and THP-1 cells were seeded into 6-well culture plates at a concentration of 5 9 105 cells/mL. THP-1 cells were subsequently differentiated into macrophages with phorbol myristate acetate (Sigma-Aldrich, Castle Hill, Australia) for 72 hours [23]. Prior to bacterial infection, mammalian cells were incubated in their respective antibiotic-free medium. The H. pylori strains 26695 and GC026 were then added to the medium at a multiplicity of infection (MOI) of 1 and incubated for 6 hours at 37 °C and 10% CO2. The bacterial concentration added was established based on a standard curve and optical density (OD) readings at 595 nm using a Bio-Rad microplate reader model 550 (Bio-Rad, Hercules, CA, USA). To determine the actual bacterial concentration added, a drop-plate method was employed.
RNA extraction, cDNA synthesis, and PCR arrays. After infection, nonchallenged and H. pylori-challenged mammalian cells were used for the isolation of mRNA using the Qiagen RNeasy Mini Kit as described by the manufacturer (Qiagen, Chadstone, Australia). For analysis of the gene expression of 84 molecules involved in autophagy, cDNA was synthesized from 0.8 lg total RNA using the RT2 First Strand cDNA Kit (Qiagen) and further analyzed using the Human Autophagy RT2 Profiler PCR array (PAHS-084Z) (Qiagen) as recommended by the manufacturer. Gene expression profiles were obtained from three independent experiments of H. pylori 26695-infected, H. pylori GC026-infected, and corresponding noninfected (control) samples. The experiments were performed employing the Rotor-Gene Q cycler (Corbett Life Sciences, Doncaster, Vic., Australia). Data analysis by means of the DDCt-based method of relative quantification was implemented using the Web-based RT2 Profiler PCR Array Data Analysis version 3.5 (http://pcrdataanalysis.sabiosciences.com/pcr/ arrayanalysis.php). At least twofold changes (≥2, ≤0.5) and p-values of
Autophagy in Helicobacter pylori Infection and Related Gastric Cancer ~ o-Rodrıguez,* Nadeem O. Kaakoush,* Khean-Lee Goh,† Kwong Ming Fock‡ and Hazel M. Natalia Castan Mitchell* *School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia, †Department of Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia, ‡Division of Gastroenterology, Department of Medicine, Changi General Hospital, Singapore City, Singapore
Keywords stomach neoplasm, Helicobacter pylori, autophagy, polymorphism, gene expression. Reprint requests to: Hazel M. Mitchell, School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Lab301A, Biological Sciences Building (D26), Sydney, NSW 2052, Australia. E-mail: [email protected]
Abstract Background: Autophagy, a degradation pathway in which cytoplasmic content is engulfed and degraded by lysosomal hydrolases, plays a pivotal role in infection and inflammation. Given that defects in autophagy lead to increased susceptibility to infection, we investigated the role of autophagy in Helicobacter pylori-related gastric cancer (GC). Materials and Methods: Gene expression of 84 molecules was examined through quantitative real-time PCR in gastric epithelial cells (AGS) and macrophages (THP-1) upon exposure to H. pylori GC026 (GC) and 26695 (gastritis). Further, ATG16L1 rs2241880, IRGM rs13361189, and IRGM rs4958847, polymorphisms that have been investigated in relation to H. pylori infection or GC in Caucasians, were detected by MALDI-TOF mass spectrometry in 304 ethnic Chinese (86 noncardia GC cases/218 functional dyspepsia controls). Results: Gene expression analyses showed twenty-eight molecules involved in vesicle nucleation, elongation, and maturation to be significantly downregulated in H. pylori GC026-challenged AGS cells. Further, core autophagy proteins and autophagy regulators were differentially expressed in H. pylorichallenged THP-1-derived macrophages. Analyses of the selected polymorphisms showed that ATG16L1 rs2241880 increased the risk of GC (OR: 2.38, 95% CI: 1.34–4.24) and H. pylori infection (OR: 1.49, 95% CI: 1.02–2.16) while IRGM rs4958847 decreased GC risk (OR: 0.26, 95% CI: 0.09–0.74) in ethnic Chinese, these effect sizes being especially strong in H. pylori-infected individuals (ATG16L1 rs2241880 and IRGM rs13361189). Conclusions: Our findings indicate that highly virulent H. pylori strains markedly modulate autophagy in the host cell. Further, for the first time, autophagy polymorphisms were associated with GC in Chinese, a high GCrisk population.
Gastric cancer (GC) is one of the most commonly diagnosed cancers in the world, occupying the fifth position after lung, breast, colorectal, and prostate neoplasias [1]. Furthermore, it is the third leading cause of cancer-related deaths worldwide, with an estimated 723,000 GC-related deaths in 2012, accounting for 8.8% of total deaths from cancer [1]. Almost two-thirds of GC cases occur in East Asia, Eastern Europe, and Central and South America, these regions being classified as high GC-risk populations. The incidence in Chinese individuals not only represents 42% of the above worldwide estimation, but Chinese ethnicity has also
© 2015 John Wiley & Sons Ltd, Helicobacter
been identified as an independent risk factor for the development of GC in multiracial studies [2,3]. According to latest global estimates, the age-standardized incidence rate (ASR) of GC is 22.7 per 100,000 habitants in China. Further, in multiracial countries such as Singapore and Malaysia, Chinese have the highest ASR (25.7 per 100,000 men) as compared with the Malays and Indians who have ASRs of 6.6 per 100,000 and 8.4 per 100,000 men, respectively [1]. Given the significant morbidity and mortality associated with GC and the short survival time following diagnosis, what is clearly required is the identification
1
~o-Rodrıguez et al. Castan
Autophagy in Helicobacter pylori-Related GC
of factors that can predict those at high risk of GC development. While H. pylori infection has been established as the most important risk factor for GC, the etiology of GC also involves host and environmental factors [4]. This is evidenced by the fact that only 1–3% of H. pylori-infected patients develop GC and that progression to GC in some subjects occurs even after eradication of the bacterium [5]. Macroautophagy (autophagy) is defined as a degradation pathway in which cytoplasmic content is engulfed and degraded by the lysosome. Thus, autophagy acts as an intracellular mechanism to eliminate protein aggregates, damaged organelles, and intracellular microbes and, subsequently, maintain cellular homeostasis [6]. This highly conserved process that initiates with the formation of a double membrane compartment called the phagophore and culminates with the generation of a structure called the autolysosome
has been well characterized (Fig. 1A). Autophagy involves the assembly of proteins that are essential for autophagic vacuole formation including autophagyrelated gene (Atg) 9 (possible “membrane shuttle”), the UNC-51-like kinase (ULK) complex (comprises Atg13, FIP200, Atg101, and ULK1 or ULK2 and is involved in the initiation of autophagy), the Vps34 complex (comprises Vps34, Vps15, Beclin-1, UVRAG, and Atg14L and generates phosphatidylinositol 3-phosphate (PI3P), an essential lipid component of autophagosomes), PI3Pbinding proteins (WIPI1, WIPI2, DFCP1, and Alfy), the Atg12 ubiquitin-like conjugation system (comprises Atg12, Atg5, and Atg16L1 and mediates formation and elongation of the autophagosome), and the Atg8 ubiquitin-like conjugation system (comprises LC3 A/B/C and c-aminobutyric acid receptor-associated proteins (GABARAPs), and mediates autophagosome closure and determines autophagosome size) [7,8].
A
B
D
C
E
Figure 1 The autophagy pathway and its modulation in Helicobacter pylori-infected gastric epithelial cells. (A) Schematic diagram of the various stages of autophagy. First, the phagophore, which is an isolation membrane, encloses cytoplasmic material. Subsequently, the phagophore elongates to form a double membrane vacuole known as the autophagosome. The autophagosome then fuses with the endosome, which delivers cargo, components of the membrane fusion machinery, and lowers the pH of the autophagic vesicle before delivery of lysosomal acid proteases. Finally, the autophagosome fuses with the lysosome to form an autolysosome and degrade the enclosed cytoplasmic material [6]. (B) Differential expression of genes involved in autophagy induction, (C) vesicle nucleation, (D) vesicle elongation, and (E) vesicle maturation, in H. pylori GC026challenged AGS cells compared to the noninfected control group. Fold change (2 DDCt ) is the normalized gene expression (2 DCt ) in AGS cells challenged with H. pylori GC026 divided the normalized gene expression (2 DCt ) in the control group. Fold regulation represents fold-change results in a biologically meaningful way. Fold-change values greater than one indicate up-regulation, and the fold regulation is equal to the fold change. Fold-change values less than one indicate down-regulation, and the fold regulation is the negative inverse of the fold change. Fold difference compared to the control group showing a *p-value < .05 and a **p-value < .01.
2
© 2015 John Wiley & Sons Ltd, Helicobacter
~o-Rodrıguez et al. Castan
In the context of H. pylori infection, a recent paper by Raju et al. [9] showed that autophagy is induced in response to the virulence factor vacuolating cytotoxin A (VacA) and thus serves a cytoprotective role. However, prolonged exposure to VacA causes disruptive autophagy with autophagosomes that lack cathepsin D and thus have reduced catalytic activity [9]. In addition, although H. pylori has been considered to be an extracellular pathogen, emerging evidence demonstrates that it may be internalized and can subsequently replicate within the autophagosomes of macrophages, and dendritic and gastric epithelial cells [10–13]. From a host perspective, Raju et al. [9] have shown that the presence of a known genetic risk factor for Crohn’s disease (CD), ATG16L1 rs2241880, increases the risk of H. pylori infection in Caucasian individuals from Scotland and Germany [9]. ATG16L1, located in chromosome 2q37.1, encodes a protein that consists of 580– 630 amino acids, including an extreme N-terminal region that mediates interaction with the Atg12-Atg5 conjugate and a coiled-coil domain that mediates Atg16L1 dimerization. Autophagy plays a dual role in carcinogenesis. Evidence supporting the protective role against cancer is based on studies that have identified molecules involved in this process, including Atg4c, Bif-1, and UVRAG, as tumor suppressors [14]; the identification of anti-oncogenic signaling pathways, including PTEN, TSC1/2, LKB1, and p53, that stimulate autophagy [15]; and evidence that autophagy de-regulation mediates mitochondrial dysfunction, increased oxidative stress, and susceptibility to pro-inflammatory stimuli, conditions that all lead to DNA damage and thus genetic instability [14]. However, in established tumors, autophagy may confer a survival advantage to tumor cells under metabolic stress as a result of a high proliferation rate and exposure to hypoxia from insufficient vascularisation [14]. In addition, it has been reported that tumor cells under selective pressure from therapeutic interventions might be protected by autophagy, which acts as an adaptive cellular response that leads to chemoresistance and cancer cell survival [14]. In the context of gastric carcinogenesis, recent studies have indicated that increased expression of beclin-1 is associated with a favorable prognosis of GC [16]. Autophagy has also been linked to the protective effect of matrine, a natural opioid found in Sophora flavensces, against GC [17]. In addition, IFN-c has been shown to inhibit gastric carcinogenesis by inducing epithelial cell autophagy and T-cell apoptosis in a murine model [18]. Interestingly, investigation of the association between IRGM polymorphisms (rs4958847 and rs13361189) and GC in a Caucasian population by Burada et al. [19] has
© 2015 John Wiley & Sons Ltd, Helicobacter
Autophagy in Helicobacter pylori-Related GC
shown IRGM rs4958847 to decrease the risk of GC in these individuals. IRGM is located at chromosome 5q33.1 and encodes the immunity-related GTPase family M protein (IRGM), an IFN-inducible GTPase that is involved in autophagic vacuole formation. Given the recent reported interaction between H. pylori and autophagy, a comprehensive investigation of H. pylori recognition by this host intracellular mechanism and its role in GC is clearly warranted. Thus, we investigated the role of autophagy in H. pylori-related GC, by analyzing the gene expression of 84 molecules involved in autophagy in nonimmune (gastric epithelial) and immune (monocytic) cell lines upon exposure to H. pylori and by examining, for the first time, the association between polymorphisms in ATG16L1 (rs2241880) and IRGM (rs13361189 and rs4958847) and risk of GC in ethnic Chinese individuals, a high GC-risk population.
Materials and Methods Gene Expression of Molecules Involved in Autophagy in Mammalian Cells Mammalian cell culture. The human GC cell line AGS (American Type Culture Collection (ATCC); Manassas, VA, USA; code: ATCC CRL-1739) was cultured in F12K medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen, Mulgrave, Vic., Australia) and 100 lg/mL penicillin–streptomycin solution (Invitrogen). The human monocytic leukemia cell line THP-1 (code: ATCC TIB-202) was cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS (Invitrogen), 1 mmol/L sodium pyruvate (Invitrogen), 2500 mg/L sodium bicarbonate (Invitrogen), and 100 lg/mL penicillin–streptomycin solution (Invitrogen). Cells were maintained in 25-cm2 tissue culture flasks (Greiner-Bio-On, Frickenhausen, Germany) at 37 °C with 5% CO2.
Bacterial culture. The H. pylori strain 26695 (cagA+ and vacA s1m1+) was isolated from a patient with gastritis (code: ATCC 700392) [20]. The H. pylori strain GC026 (cagA+, cagE+, cagL+, cagT+, vacA s1m1+, babA+, oipA+, dupA+, and sabA+) was isolated from a GC patient undergoing endoscopy at the University Hospital of Malaysia, Kuala Lumpur, in a previous study conducted by our group [21,22]. Both H. pylori strains were grown for 48 hours on horse blood agar plates (Blood Agar Base No. 2 supplemented with 10% sterile defibrinated horse blood) (Oxoid, Heidelberg West, Vic.,
3
Autophagy in Helicobacter pylori-Related GC
Australia) at 37 °C in an anaerobic jar containing a gas generating kit (Oxoid) to provide a microaerobic atmosphere of 6% O2, 10% CO2, and 84% N2.
Infection assay. AGS and THP-1 cells were seeded into 6-well culture plates at a concentration of 5 9 105 cells/mL. THP-1 cells were subsequently differentiated into macrophages with phorbol myristate acetate (Sigma-Aldrich, Castle Hill, Australia) for 72 hours [23]. Prior to bacterial infection, mammalian cells were incubated in their respective antibiotic-free medium. The H. pylori strains 26695 and GC026 were then added to the medium at a multiplicity of infection (MOI) of 1 and incubated for 6 hours at 37 °C and 10% CO2. The bacterial concentration added was established based on a standard curve and optical density (OD) readings at 595 nm using a Bio-Rad microplate reader model 550 (Bio-Rad, Hercules, CA, USA). To determine the actual bacterial concentration added, a drop-plate method was employed.
RNA extraction, cDNA synthesis, and PCR arrays. After infection, nonchallenged and H. pylori-challenged mammalian cells were used for the isolation of mRNA using the Qiagen RNeasy Mini Kit as described by the manufacturer (Qiagen, Chadstone, Australia). For analysis of the gene expression of 84 molecules involved in autophagy, cDNA was synthesized from 0.8 lg total RNA using the RT2 First Strand cDNA Kit (Qiagen) and further analyzed using the Human Autophagy RT2 Profiler PCR array (PAHS-084Z) (Qiagen) as recommended by the manufacturer. Gene expression profiles were obtained from three independent experiments of H. pylori 26695-infected, H. pylori GC026-infected, and corresponding noninfected (control) samples. The experiments were performed employing the Rotor-Gene Q cycler (Corbett Life Sciences, Doncaster, Vic., Australia). Data analysis by means of the DDCt-based method of relative quantification was implemented using the Web-based RT2 Profiler PCR Array Data Analysis version 3.5 (http://pcrdataanalysis.sabiosciences.com/pcr/ arrayanalysis.php). At least twofold changes (≥2, ≤0.5) and p-values of
