| BackgroundArsenic and benzo[a]pyrene are the most common environmental contaminants to which humans are exposed.Arsenic is a natural and widely distributed chemical substance.Longterm low-dose arsenic exposure can lead to serious cardiovascular diseases,neurobehavioral disorders,skin lesions,liver and kidney dysfunction,reproductive system diseases.Benzo[a]pyrene is a kind of high molecular weight polycyclic aromatic hydrocarbon,which is a persistent organic pollutant.Benzo[a]pyrene is lipophilic,and excessive accumulation of benzo[a]pyrene in the body can have serious toxic effects on the liver,heart,kidneys,neurodevelopment,reproductive system,immune system,placenta,etc.In addition to their general toxic effects,arsenic and benzo[a]pyrene are classified as Group 1 carcinogens(known human carcinogen)by the International Agency for Research on Cancer and are responsible for lung cancer,skin cancer,liver cancer,bladder cancer and other types of cancer.Because millions of people are exposed to arsenic by drinking arsenic-contaminated water,and there are a lot of benzo[a]pyrene in cigarette smoke and barbecue,thus,human exposure to both arsenic and benzo[a]pyrene at the same time is very common.However,the effects and potential toxic mechanisms of arsenic and benzo[a]pyrene co-exposure are currently unknown.Cancer stem cells are a small group of cancer cells,which have the ability to self-renewal and differentiate into a variety of cancer cell "progeny" that can form the bulk of the tumors.Cancer stem cells are thought to be a major cause of tumor growth,heterogeneity,recurrence,metastasis and drug resistance.Recent studies have shown that chronic low-dose exposure to arsenic or other metal carcinogens induces normal cell transformation to produce cancer stem cells or cancer stem cell-like cells,which is considered a novel mechanism of metal carcinogenesis.However,whether arsenic and benzo[a]pyrene co-exposure can produce cancer stem cells or cancer stem cell-like cells(more or less)remains to be determined.Integrin α4(ITGA4)is a heterodimer transmembrane protein,which is a member of Integrin family.Studies have shown that ITGA4 plays an important role in cell survival,proliferation,migration,invasion,tumor growth and metastasis.The up-regulation of ITGA4 has been found in many types of cancer.However,few studies focused on the role of ITGA4 in tumorigenesis and development.Our gene microarray results revealed that was highly expressed in arsenic and benzo[a]pyrene co-exposure transformed normal human lung epithelial cells(BEAS-2B).This is the start of the thesis,which try to explore the mechanism of ITGA4 in lung cancer induced by co-exposure to arsenic and benzo[a]pyrene.Objectives1.To study the characteristics of the arsenic and benzo[a]pyrene co-exposure.2.To explore the effect of ITGA4 on lung cancer induced by co-exposure to arsenic and benzo[a]pyrene.3.To explore the molecular mechanism of lung cancer caused by co-exposure to arsenic and benzo[a]pyrene.Methods1.Establishing an in vivo mouse model of co-exposure to arsenic and benzo[a]pyrene,and to detect the occurrence of lung cancer after co-exposure to arsenic and benzo[a]pyrene by pathological analysis of the lungs.2.Establishing an in vitro BEAS-2B cell model was exposed to arsenic and benzo[a]pyrene.To detect the effects of co-exposure to arsenic and benzo[a]pyrene on cell malignant transformation and cancer stem cell-like properties by using serum-free suspension culture sphere formation assay,soft agar colony formation assay,nude mouse xenograft tumorigenesis assay and ALDEFLUOR assay.3.Stable knockdown of ITGA4 on arsenic and benzo[a]pyrene co-exposure transformed BEAS-2B cells.The effects of knockdown of ITGA4 on cell malignant transformation and cancer stem cell-like properties were detected by serum-free suspension culture sphere formation assay,soft agar colony formation assay,nude mouse xenograft tumorigenesis assay,molecular biology experiment and survival analysis of lung cancer patients.4.Using q-PCR experiments and gene set enrichment analysis to screen the signaling pathway(Hedgehog pathway)associated with both ITGA4 expression and tumor stem cell-like properties.To identify the changes in cancer stem cell-like properties on arsenic and benzo[a]pyrene co-exposure BEAS-2B cells by using si RNA interference techniques,inhibitors,and agonists to inhibit or activate Hedgehog pathway.5.The role of SUFU protein in the Hedgehog pathway regulated by ITGA4 was detected by si RNA interference,gene overexpression and small molecule drugs.6.To explore the regulatory relationship among ITGA4,Hedgehog pathway and PI3K/Akt pathway in arsenic and benzo[a]pyrene co-exposure transformed BEAS-2B cells using molecular biology experiments.Then the changes of proteins expression were observed by treating corresponding protein inhibitors.Results1.In vivo arsenic and benzo[a]pyrene co-exposure to A/J mouse model showed that arsenic(20 ppm)and benzo[a]pyrene(24 μmol)used in this study did not result in significant changes in mice body weight(P>0.05).Tumors developed in the lungs of all mice exposed to benzo[a]pyrene alone and the mice exposed to arsenic and benzo[a]pyrene.Mice lung tissues were used to performed HE staining and pathological analysis.It was found that the the lung tumor multiplicity and tumor burden in mice exposed to arsenic and benzo[a]pyrene were significantly higher than mice exposed to benzo[a]pyrene alone(P<0.05).2.In vitro arsenic and benzo[a]pyrene co-exposure to BEAS-2B cell model showed that the number of cell clones formed in soft agar and the formation of spheres in arsenic and benzo[a]pyrene co-exposure transformed cells(BEAS-2B-As+Ba P)were significantly higher than that in BEAS-2B cells exposed to arsenic or benzo[a]pyrene alone(P<0.05).The results of ALDEFLUO assay showed that the positive rate of ALDH in BEAS-2B-As+Ba P cells was significantly higher than that in BEAS-2B cells exposed to arsenic or benzo[a]pyrene alone(P<0.05).Nude mouse xenograft tumorigenesis assay also showed that significantly more tumors grew in nude mice injected with BEAS-2B-As+Ba P cells than in nude mice injected with arsenic or benzo[a]pyrene alone transformed BEAS-2B cells(P<0.05).3.Effects of knockdown of ITGA4 on the cancer stem cell-like properties and tumorigenic capacity of BEAS-2B-As+Ba P cells.The expression levels of ITGA4 m RNA and protein in BEAS-2B-As+Ba P cells were significantly higher than that in BEAS-2B cells exposed to arsenic or benzo[a]pyrene alone(P<0.01).The cells formed less clones in soft agar and spheres after stable knockdown ITGA4 in BEAS-2B-As+Ba P cells(P<0.01).The tumor incidence rate in mice injected with ITGA4 knockdown cells was significantly lower than mice injected with control sh RNA cells(P<0.01).Further tumor histology analysis showed that the Ki-67 positive staining in tumors from ITGA4 knockdown cells is significantly less than the tumor from control sh RNA cells(P<0.01).4.Effects of Hedgehog signaling pathway on cancer stem cell-like properties of BEAS-2B-As+Ba P cells.The results of q-PCR and gene set enrichment analysis showed that Hedgehog pathway was highly activated in BEAS-2B-As+Ba P cells,and was positively correlated with the expression of ITGA4.Interference of GLI-1 by using si RNA and Hedgehog pathway inhibitors significantly reduced the number of spheres formed by BEAS-2B-As+Ba P cells(P<0.01).In contrast,up-regulation of GLI-1 by using Hedgehog pathway agonists increased the number of spheres formed by ITGA4 knockdown cells(P<0.01).5.The role of SUFU protein in the Hedgehog pathway regulated by ITGA4 in BEAS-2B-As+Ba P cells.Interference of SUFU by using si RNA significantly increased the number of spheres formed by ITGA4 knockdown cells(P<0.01).However,overexpression of SUFU significantly reduced the number of clones in soft agar and the number of spheres formed by BEAS-2B-As+Ba P cells(P<0.01).The results of q-PCR and Western blot showed that knockdown of ITGA4 did not alter the level of SUFU m RNA(P>0.05),but up-regulated SUFU protein expression(P<0.01).MG132 and cycloheximide treatment experiment showed that knockdown ITGA4 could inhibit its degradation by proteasome and increase the stability of SUFU protein,thereby up regulating the expression of SUFU protein.6.The role of PI3K/Akt pathway in the Hedgehog pathway regulated by ITGA4 in BEAS-2B-As+Ba P cells.Western blot showed that PI3K/Akt pathway was highly activated in BEAS-2B-As+Ba P cells and was positively correlated with the expression of ITGA4.It was found that the Hedgehog pathway was inhibited while the PI3K/Akt pathway was inhibited by using PI3K/Akt pathway inhibitor.PI3K/Akt pathway inhibitors,cycloheximide,okadaic acid were used to treat cells and found that knockdown ITGA4 increased the stability of SUFU protein by inactivate PI3K/Akt pathway,and ultimately inhibit the Hedgehog pathway.Conclusions:In summary,our study is the first to establish a mouse model of arsenic and Ba P exposure via drinking water and oral ingestion of Ba P,as well as a BEAS-2B cell model,and confirmed the synergistic lung carcinogenic effects of As and Ba P by using both models.In addition,we identified for the first time that ITGA4 is plays an important role in lung cancer caused by As and Ba P co-exposure: Up-regulation of ITGA4 could down regulate SUFU protein and up regulate GLI-1 protein expression by activating PI3 K / Akt signaling pathway,then activates Hedgehog signaling pathway to enhance the cancer stem cell-like properties and tumorigenesis. |
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