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Reactive Oxygen Species Are Involved in the Development of Gastric Cancer and Gastric Cancer-Related Depression
- Thread starter ginfreely
- Start date
Published15 Jul 2019
Abstract
Background. The mechanisms of crosstalk between depression and gastric cancer (GC) remain ill defined. Given that reactive oxygen species (ROS) is involved in the pathophysiology of both GC and depression, we try to explore the activities of ROS in the development of GC and GC-related depression. Methods. 110 patients with newly diagnosed GC were recruited in our study. The clinical characteristics of these patients were recorded. Inflammation and oxidative stress markers were detected by ELISA. The depression status of patients with GC was assessed during follow-up. The association between ROS, ABL1, and inflammation factors was evaluated in H2O2-treated GC cell lines and The Cancer Genome Atlas (TCGA) database. The effect of ABL1 on inflammation was detected with Imatinib/Nilotinib-treated GC cell lines. A chronic mild stress- (CMS-) induced patient-derived xenograft (PDX) mice model was established to assess the crosstalk between depression and GC. Results. Depression was correlated with poor prognosis of patients with GC. GC patients with depression were under a high level of oxidative status as well as dysregulated inflammation. In the CMS-induced GC PDX mice model, CMS could facilitate the development of GC. Additionally, tumor bearing could induce depressive-like behaviors of mice. With the treatment of ROS, the activities of ABL1 and inflammatory signaling were enhanced both in vitro and in vivo, and blocking the activities of ABL1 inhibited inflammatory signaling. Conclusions. ROS-activated ABL1 mediates inflammation through regulating NF-κB1 and STAT3, which subsequently leads to the development of GC and GC-related depression.
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1. Background
Patients with gastric cancer (GC) are susceptible to mood disorder, especially to depression. We demonstrated that about 50% of investigated patients with GC suffered from depression and experienced poor quality of life [1]. Unfortunately, the unequivocal mechanism leading to the occurrence of depression for patients with GC remains poorly understood. We found that depression was associated with an impairment of immunologic functions and oxidative imbalance [2]. Oxidative stress (OS), characterized as excessive production of reactive oxygen species (ROS) [3], is involved in the initiation and development of cancer and depression. ROS lead to damage to tissues by attacking the DNA or disrupting cellular signaling [4, 5]. As a marker of OS, 8-hydroxy-2-deoxyguanosine (8-OHdG) is dramatically elevated in serum samples of patients with GC. Furthermore, we have shown that levels of ROS are upregulated in serum and tissue samples of GC patients [2]. As a recognized risk factor of GC, infection of Helicobacter pylori (H. pylori) contributes to the generation of ROS by cytotoxin-associated gene A (CagA) [6], subsequently activating Wnt, Ras, and mechanistic targets of rapamycin (mTOR) to drive the initiation of gastric carcinogenesis [7–10].Increasing evidences indicate that oxidative stress is implicated in the pathological process of major depressive disorder (MDD). Similar with patients with GC, 8-OHdG increases in serum samples of patients with depression [11]. Low levels of antioxidant uric acid are associated with the occurrence of depression because of decreased levels of antioxidant properties [12]. The elevated levels of ROS are capable of decreasing the expression of brain-derived neurotrophic factor (BDNF), partially contributing to the progress of depression [13, 14]. With sparse whole-genome sequencing, Cai et al. identified sirtuin 1 (SIRT1) as a contributor to major depressive disorder [15]. SIRT1 functions by means of its antioxidant effects through enhancing antioxidant enzyme activity and inhibiting free radical-mediated oxidative injuries via decreasing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation [16, 17]. All these suggest that oxidative stress plays crucial role in the process of depression.
With a high level of ROS, numerous oxidative stress-related proteins are dysregulated. SIRT1 and phosphoinositide 3-kinase (PI3K), for instance, are activated in both GC and depression [16, 18]. In our previous work, we found that ABL1 was upregulated in patients with both GC/colorectal carcinoma and depression [19, 20], and some kinds of cytokines like Leptin-LepRb and COX2 are abnormally expressed [21, 22]. Given the role of ROS in depression, GC, and crosstalk with inflammation, we speculated that ROS, ABL1, and inflammation are involved in the development of GC as well as GC-related depression. In this study, we designed series experiments to validate our hypothesis.
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2. Patients and Methods
2.1. Patients
110 patients newly diagnosed with gastric adenocarcinomas were recruited in our study. They were diagnosed and received treatment from January 1, 2008 to December 31, 2016 in the Department of Oncology, the First Affiliated Hospital of Xi’an Jiaotong University. All the GC patients received examination and treatment following the National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines in Oncology. Follow-up was performed 3 times per year for the first 5 years, and annually thereafter. The project was terminated on December 31, 2016.2.2. Instruments
During treatment and follow-up, assessment of the mood status among these patients was performed by physicians in the Department of Psychology, the First Affiliated Hospital of Xi’an Jiaotong University. The psychology assessment was performed every 6 months for 5 years and annually thereafter, and terminated when the patient died or when the study was terminated, which ever came earlier. We measured psychiatric symptoms with the method described in our previous work [23].2.3. Ethical Standards
This study was approved by the First Affiliated Hospital of Xi’an Jiaotong University Ethics and Scientific Committee and met international standards for informed consent. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1964 and later versions. Informed consent to be included in the study, or the equivalent, was obtained from all patients. All animal experiments were approved by the review board of Zhongnan Hospital of Wuhan University, and all institutional and national guidelines for the care and use of laboratory animals were followed.2.4. Cell Culture
GC cell lines SGC-7901 and AGS were cultured in RPMI-1640 (HyClone) medium containing 10% fetal bovine serum (HyClone) and 1% penicillin/streptomycin (HyClone) in a 37°C incubator with a constant temperature and high humidity under 5% CO2 atmosphere.2.5. MTT Assay
The MTT assay was used for evaluating cell proliferation. Briefly, cells in log phase were trypsinized into a single cell. Then, the GC cell lines were seeded into a 96-well plate ( cells/well) and cultured in 200 μl of culture medium. 72 h later, the MTT solution (Sigma-Aldrich) (1 : 10 to complete culture medium) was added into each well. After incubation for 4 h, the supernatant of each well was aspirated carefully, and the crystals were dissolved with 100 μl of DMSO (Sigma-Aldrich) by shaking for 5 min. The absorbance of each well was read by a microplate reader. The study was performed in triplicate.2.6. Migration Assay
A wound-healing assay was used to detect cell migration. Briefly, the proper density of cells was passaged into a 6-well plate to ensure the 90%–100% monolayer cell confluence on the next day. Then, a 200 μl pipette tip was used to form a wound. 48 hours later, the number of cells that migrated into the wound were recorded.2.7. Drug Treatment
GC cell lines SGC-7901 and AGS were seeded into 96-well plates and treated with a serial concentration of Imatinib and Nilotinib (a gift from Novartis) to determine the half-maximal inhibitory concentration (IC50). Then, GC cells were treated with the two drugs at a dose of IC50 to explore the effect of ABL1 on inflammation signaling.2.8. Flow Cytometric Analysis
Flow cytometry was accomplished with a FACS flow cytometer (BD Biosciences). SGC-7901 cells were stained with anti-E-cadherin (rabbit, Cell Signaling Technology) and anti-N-cadherin (mouse, Abcam) for 45 min, then stained with Alexa Fluor 488- and Alexa Fluor 594-labeled secondary antibody for 30 min after washing with PBS. All procedures were performed on ice. The analysis for the data was done with the FlowJo software (TreeStar Inc., Ashland, OR, USA).2.9. PDX Model
The generation of the PDX mice model was established as described before [24]. Briefly, fresh surgical PDX tissue fragments (from GC patients of the Zhongnan Hospital of Wuhan University) (approximately 15 mm3) were implanted subcutaneously directly into male nude (8-week-old) mice (Hubei Provincial Laboratory Animal Public Service Center, Wuhan, China) and serial transplantation was conducted. The animal experiments in this study were performed according to the policies approved by IACUC. Tumor size was monitored twice per week, and tumor volumes () were calculated with the following formula: .2.10. CMS
Mice were randomly exposed to several stressors in the morning and in the afternoon. The stressors included dampened sawdust, removal of sawdust, replacement of sawdust with water at 21°C, repeated alteration of sawdust, inclining the cage with 45°C, restraint stress, change of the dark and light cycle, and predator voice [25].2.11. Behavioral Tests
2.11.1. Open-Field Test (OFT)
OFT was adopted for measuring the spontaneous locomotor activities of mice. Briefly, mice were carefully placed in an open-field apparatus ()(Chengdu Techman Software Co. Ltd., Chengdu, China). The movement traces (5 min) of the mice were monitored by infrared cameras and analyzed with corresponding software [26].2.11.2. Sucrose Preference Test (SPT)
As described before, both water and food were denuded 24 h before SPT. During the test, two bottles with the same size were supplied to each animal: one with clean water and the other one with 1% sucrose solution. After 1 h, fluid consumptions were checked. Sucrose preference proportion was calculated with the following formula: [27].2.11.3. Tail Suspension Test (TST)
TST was performed for 6 minutes by suspending a mouse 30 cm from the floor. The immobile duration was monitored for a 6 min period. Mice were defined immobile only when they hung passively and were totally motionless [28].2.12. Enzyme-Linked Immunosorbent Assay (ELISA)
The levels of 8-OHdG in patients’ or mice’ blood samples were quantitatively assayed with a DNA-damage ELISA kit (Ann Arbor, MI, USA). A hydrogen peroxide assay kit and a catalase assay kit were used to detect the levels of hydrogen peroxide and catalase in patients’ blood samples (Nanjing Jiancheng Biotechnology Co., Nanjing, China). The concentrations of IL-6, IL-1β, COX2, and 5-HT of mice’ blood samples were determined with a commercial ELISA kit for mouse IL-6, IL-1β, COX2, and 5-HT (Elabscience Biotechnology Co. Ltd., China). Procedures were performed based on the manufacturers’ protocols, and the absorbance values were read at the recommended wave lengths.2.13. Western Blot Analysis
Proteins from both the hippocampus and tumor tissue samples and cell lines were extracted. The concentration of proteins was detected with the BCA protein assay reagent (Shenzhen Wolsen Technology Co. Ltd., China). A total 15 μg of proteins from each sample was loaded on 12% gel, then the proteins were transferred from the gel to the polyvinylidene difluoride membranes (EMD Millipore Corporation, Billerica, MA, USA), followed by blocking the membrane with 5% nonfat milk at room temperature for 2 h. The membrane was then incubated with a primary antibody specific for ABL1/p-ABL1(y412), IL-6, IL-1β, and COX2 (Abcam, Shanghai, China); NF-κB1/p-NF-κB1 (ser933) (Cell Signaling Technology, Danvers, MA, USA); STAT3/p-STAT3 (Tyr 705) (Santa Cruz Biotechnology (Shanghai) Co. Ltd., Shanghai, China); and β-actin antibody (Proteintech Group, Wuhan, China) at 4°C for 12 h. The membranes were washed 5 times with TBST (6 min each time), followed by 1 h incubation with the secondary antibody at room temperature. Image Lab software was used for the quantification of protein levels. Western blots were repeated three times for each sample.2.14. Measurement of Serum Levels of ROS
The sera of mice were harvested, and serum levels of ROS were determined by the OxiSelect™ In Vitro ROS/RNS Assay Kit (Cell Biolabs Inc., California, USA) in accordance with the protocol provided. The fluorescence was read with a fluorescence plate reader at excitation 480 nm and emission 530 nm.2.15. Statistical Analyses
Statistical analysis was performed by GraphPad Prism. Data are presented as . Significant difference at each comparison point is shown as and .
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This is exactly what Ccb Malaysian Cantonese dogs and bitches in Nusa Duta and elsewhere did to me using firecrackers, knocks, whistle isn’t it? Knnbccb may all Malaysian criminals and their descendants who used and using the firecrackers whistles knocks etc be gangraped. Again unlike Cantonese dogs sons of dirty prostitutes @musclepower @gsbslut etc fabricated stories to harm me dare not swear, if I have fabricated any stories to harm them I die a violent death. That’s the difference between good human and dogs Pui!
Up up up Cantonese dogs @musclepower @gsbslut @Cottonmouth etc proving themselves dogs and proud murderers scammers bullies with not a shred of human kindness. May they be reincarnated as women and be gangraped. Pui!!
Tsk tsk tsk everything you accused me is what your cheap Cantonese mother wife daughters are. I am a virgin with annulled marriage of course means I am a virgin prior to marriage. You Cantonese dog son of prostitute hide in rat hole do defamation crimes to me that’s why you dare not swear scared to die a violent death. Hey proud murderer you think you don’t swear only heaven got no eyes to see you are doing daily stress to cause me cancer and will serve you karma? I will see how you die. Pui!
Tsk tsk tsk you Cantonese dog son of Malaysian pahang prostitute proud murderer criminal bully gangster hiding in rat hole doing defamation crimes to Gansiokbin and extorting evidence from your victom of crimes i see how you die. All evidence of science pointing to the FACT you Cantonese dogs are murderer. Pui!
WHOEVER DARE NOT SWEAR DARE NOT TAKE UP VIRGINITY TEST CHALLENGE IS THE ONE CAUGHT LYING AND AS USUAL WHATEVER YOU MURDERER CANTONESE DOG SON OF PROSTITUTE ACCUSE ME IS WHAT YOU AND YOUR SELF ADMITTED CHEAP CANTONESE DIRTY SLUT PROSTITUTE MOTHER ARE. PUI!
You shameless murderer criminal pretend no see my threads with scientific evidence that you Malaysian Cantonese dog son of prostitute are proven murderer using evil dirty tricks of daily multiple chronic stress to cause me cancer and accuse your victim of crimes as playing victim. Full of lies narcissist is you Cantonese dog that think you are emperor I am slave for you to trample. Pui!
Tsk tsk tsk you cancer Cantonese dog son of prostitute keep spewing lies dare not swear. 贱货是你。
Tsk tsk tsk you MURDERER cancer Cantonese dog son of prostitute keep spewing lies dare not swear. 小人贱货骗子都是你
Tsk tsk tsk you EVIL MURDERER Cantonese and son of Cantonese prostitute say until like that then why dare not swear? So evil coward LIAR CRIMINAL BULLY DOGS WITH ZERO HUMAN CONSCIENCE PUI!
Tell that to yourself change topic is you Cantonese dog son of prostitute pretend don’t know you are the MURDERER KILLING ME. YOU HAVE NO EVIDENCE OF ME AS CHICKEN AND YOU DONT EVEN DARE TO SWEAR. 假装不知道是你贱货广东狗没证据又不敢发誓。呸！
Look at how shameless you are Malaysian Cantonese dog son of prostitute hide in rat hole do crimes to murder me is called winning. You will get your retribution because not every women is like yours so cheap open leg big big sell nude buy Porsche or fabricate stories of kena thrown eggs in Taiwan to harm people. As cheap and evil as you LOSER SON OF CANTONESE CHICKEN. Pui!
@musclepower why you dare not swear you are not Malaysian? Btw the way you write in Chinese already gave away you are Malaysian. You write in low class Chinese that you think very good but actually so evil dirty shit that smear and accuse people I see a lot of Malaysians in my neighborhood wrote like that. China Chinese in my neighborhood wrote in much more high class way. One look I can tell who is Malaysian who is China Chinese in my neighborhood just by the Chinese written.
Up up up Cantonese dogs @musclepower@gsbslut @Cottonmouth etc proving themselves dogs and proud murderers scammers bullies with not a shred of human kindness. May they be reincarnated as women and be gangraped. Pui!!
You Malaysian @nightsafari clone way of ceating issues out of no issues again. Say I scale down when I did nothing different say nothing different for years that you Cantonese dogs sons of prostitutes smear me hokkien virgin as chicken. 无中生有 fabricate stories tell lies put words into my mouth is your proud Cantonese cancer way of attack. 真是贱！
See everyone? Cantonese dog son of prostitute @musclepower proved me right again that he is sneaky always appear soon after I logoff to smear hokkien virgin as chicken. And see how sneaky and despicable he is to appear in this thread instead of the proud thread that he always posted to defame Gansiokbin?
As usual Cantonese dog self admitted son of DIRTY SLUT @gsbslut KENA SLAPPED BY ME dare not answer and just cancer repeat lies that have been answered. Why so shameless double standard you @gsbslut and your fellow Cantonese got Cantonese cancer is not called do evil tio cancer but I got cancer is called evil? Yikes! As usual whatever Cantonese sons of prostitute accused me is what they are themselves double standard. Pui!
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