| 【Background and Objective】Epidemiological evidence suggests that diabetes increases the risk of multiple cancers. The consensus report of the American Diabetes Association(ADA) and the American Cancer Society(ACS) in 2010 has reported that type 2 diabetes mellitus(T2DM) could increase the risk of colorectal carcinoma, breast cancer, endometrial carcinoma, liver cancer, pancreatic cancer and bladder caner, et al. And the relation between T2 DM and cancer has been proved in a Meta analysis published on the British Medical Journal. With the increased precalence of diabetes, the populations of diabetes with cancer will also rising. This population also needs anti-diabetic therapy, but the mechanism of anti-diabetic agents affect the tumor behavior is not clear.Dipeptidyl peptidase IV inhibitors(DPP-4i) are the commonly used anti-diabetic agents in clinic, and have been recommended as the first line anti-diabetic agents in addition to metformin by American Association of Clinical Endocrinologists(AACE)-clinical practice guidelines. As mentioned in our Meta analysis by my colleague, DPP-4i could not increase the risk of tumorgenesis, so that DPP-4i can be used in diabetes. But there need to be further study the safety and mechanism of DPP-4i used in diabetic patients with cancer.Alpha lipoic acid(ALA) can protect the cells from oxidative stress with that can increase levels of endogenous antioxidants and Phase II detoxification enzyme. So ALA always used in prevention of oxidative stress caused by diabetes, hypertension and other diseases. ALA also been recommended as the anti-tumor agents in clinic. The anti-tumor effect of ALA is mainly to inhibit tumor cells proliferation and accelerate their apoptosis, but few studies have investigated the effects that ALA have on tumors.The aim of the study was to explore the effects of the anti-diabetic DPP-4i, especially saxagliptin(Sax) and sitagliptin(Sit), and the antioxidant ALA on the biological behavior of tumors. We explored the effects and mechanisms of DPP-4i on tumor cells migration and invasion in vitro. In vivo experiments were performed to investigate the effects of Sax and Sit on tumors in spontaneous and experimental metastasis mouse models. Additionally, in vivo and in vitro experminents were performed to explore the mechanmism of DPP-4i and ALA promoting tumor metastasis with the silenced or ovexpressed Nrf2. With the research, we will reveal the biological behaviour links between DPP-4i and tumors, analysis the molecular cascade mechanism, and explore the rational medication for diabetic patients with cancer.【Methods】1. In vitro experiments1.1 Effects of DPP-4i on cancer cells:0.1 μM Sax,0.6 μM Sit treated cancer cells:(1) Transwell/Transwell+Matrigel characterized the effects of DPP-4i on cells migration and invasion.(2) Cell Time-Lapse Microscopy and Track-Plot Analysis used to evaluate the persistence and forward migration index(FMI).1.2 Effects of DP-4i on DPP-4 activity, oxidative stress and Nrf2 pathway:(1) DPP-4 activity Fluorometric Assay Kit used to detect DPP-4 activity.(2) DCFH-DA used for reactive oxygen species(ROS) measurement. Intracellular GSH/GSSG was measured with GSH/GSSG assay kit. Immunofluorescent staining of 8-oxo-d G was performed to assess cancer cell DNA injury.(3) Immunohistochemical staining and Western Blot analyzed the protein expression of Nrf2 and Phase II detoxification enzyme.(4) Ubiquitination assay was performed to examine the effect of cysteine 151 in Keap1(Keap1-C151S) in Nrf2 induction by Sax.1.3 Western Blot analyzed metastasis-associated protein expression:(1) Common metastasis-associated protein, including HIF-1, VEGF, COX-2.(2) Lymphatic metastasis(colon caner)- associated protein APRIL and BAMBI.(3) Hematogenous metastasis(liver caner)- associated protein Cortactin.1.4 Effects of silenced, ovexpressed or pharmacologic(sulforaphane SF, ALA) activated Nrf2 on cancer cells:(1) Transwell/Transwell+Matrigel characterized the effects of DPP-4i on cells migration and invasion.(2) Cell time-lapse microscopy and Image J used to evaluate the persistence and FMI.1.5 Immunohistochemical staining and Western Blot analyzed Nrf2, Phase II detoxification enzyme and metastasis-associated protein expression(HIF-1, VEGF, COX-2, APRIL, BAMBI and CORTACTIN).2. In vivo experiments2.1 Effects of DPP-4i on tumor metastasis:(1) 8-week old Pathogen-free athymic female BALB/c nude mice were footpad(FP)-inoculated with HCT116-Luc+ cells or tail vein(TV)-injected with HuH-7-Luc+ cells to generate a spontaneous or experimental metastasis model, respectively. Mice were randomly allocated to the indicated treatment groups and received normal saline(control), 15 mg/kg saxagliptin, or 120 mg/kg sitagliptin(via oral gavage) for 6 weeks.(2) Bioluminescence imaging used to measure tumor burden in vivo.(3) H&E staining used to observe the morphological changes in liver and lung.(4) Immunohistochemical staining and Western Blot analyzed Nrf2, Phase II detoxification enzyme and metastasis-associated protein expression in liver and lung.2.2 Effects of silenced Nrf2 on tumor metastasis:(1) 8-week old Pathogen-free athymic female BALB/c nude mice were TV-injected with HuH-7-Luc+ cells to generate an experimental metastasis model. Mice were randomly allocated to the indicated Control(Ctl) group and Nrf2 shRNA group, and fed by normal diet for 3 weeks. Bioluminescence imaging used to measure tumor burden in vivo.(2) The former two groups were randomly allocated to the Sax groups and Sit groups, received 15 mg/kg Sax, or 120 mg/kg Sit(via oral gavage) daily for 3 weeks. Test methods and indexes were same with 2.1.2.3 Effects of ALA and SF on tumor metastasis:(1) 8-week old Pathogen-free athymic female BALB/c nude mice were TV-injected with HuH-7-Luc+ cells to generate an experimental metastasis model. Mice were randomly allocated to the indicated treatment groups and received normal saline(control), 80 mg/kg ALA(i.p) daily, or 10 mg/kg SF(i.p) three times per week for 3 weeks. Test methods and indexes were same with 2.1.3. Human tissue microarray(TMA) chips for scanned images of H&E staining. A total of 195 samples, including 105 non-metastatic, 69 with lymph node metastasis, 2 with distant metastasis, and 19 metastatic lesions were analyzed. The stained TMA slides were scored to analyze the Nrf2 and GCLM expression.【Results】1.1 Effects of DPP-4i on cancer cells:(1) Transwell/Transwell+Matrigel characterized DPP-4i promoting cancer cells migration and invasion, and increased the cells persistence and FMI.(2) Western Blot indicated that levels of tumor metastasis-associated protein increased with DPP-4i treatment.1.2 Bioluminescence imaging indicated Sax and Sit treatment accelerated metastatic capacity in metastasis models. By Immunohistochemical staining and Western Blot, we observed higher expression of metastasis-associated proteins in livers and lungs with DPP-4i treatments.1.3 DPP-4i greatly reduced ROS production, increased GSH/GSSG ratios, and decreased 8-oxo-dG in cancer cells, suggesting that DPP-4i accelerated tumor metastasis maybe through reducing oxidative stress in cancer cells.1.4 Levels of Nrf2 and downstream genes expression increased with DPP-4i treatments, suggesting that DPP-4i accelerated tumor metastasis maybe related with Nrf2 activation. And Sax induced Nrf2 activation was inhibited in Keap1-C151 S mutant cells, indicating that Sax induced Nrf2 activation may depend on Keap1-C151.1.5 Silenced Nrf2 could significantly ameliorate DPP-4i induced tumor metastasis in vitro and in vivo.1.6 Overexpressed or pharmacologic activated Nrf2 promoted tumor metastasis in metastasis models.1.7 Analyzing 195 human liver cancer tissue microarrays, we observed that Nrf2 and GCLM expression were associated with tumor metastasis and stage.【Conclusion】Our study shows that the anti-diabetic DPP-4i and ALA accelerate tumor metastasis via Nrf2 activation by inhibition of Nrf2 ubiquitination in the Keap1-C151 dependent manner. In human liver cancer tissues microays, Nrf2 expression positively associated with tumor metastasis, suggesting that Nrf2 may be a potential biomarker and therapeutic target for tumors. Our findings also indicate that antioxidants that activate Nrf2 signaling should be administered with caution in cancer patients. |
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