| With the wide application of agrochemicals and industrial preservatives, ammonia nitrogen,nitrate nitrogen and nitrite nitrogen in natural water and soil increased year by year. In recent years, theload of nitrite, the commonly seen nitrite nitrogen, and ammonium sulfate, the commonly used fertilizer,in environment increased dramatically. At the same time, epidemiological studies have shown that themorbidity and mortality of cancer especially lung cancer rise gradually in areas with serious ammoniacontamination. Deep study of the relationship between ammonia nitrogen and the occurrence anddevelopment of lung cancer is significantly important.Objective To explore the biological effects of ammonia nitrogen and nitrite nitrogen in theevolution of lung cancer cells through taking lung cancer as the object of study. To research the effects ofammonia nitrogen and nitrite nitrogen in the invasion of lung cancer cells in simulated tumormicroenvironment and supply basis to further understand the relationship between nitrite and lungcarcinogenesis.Methods Mouse fibroblasts L929and lung cancer cells LLC were co-cultured to stimulatethe tumor microenvironment in vitro and set up the cell models. Using sodium nitrite on behalf of nitritenitrogen and ammonium sulfate on behalf of ammonia nitrogen, the expression of α-smooth muscle actin(α-SMA) was detected by immunocytochemistry. Cell proliferation index in vitro were determined inmethyl thiazolyl tetrazolium (MTT) assay; PI staining was used to detect the cell cycling wasinvestigated by flow cytometry; then the mitotic index was counted by Hoechst33258staining. Cellmigration and invasion were determined by scratch test and Transwell invasion assay, combined with cellmorphological changes in the cell climbing piece the occurrence of epithelial-to-mesenchymal transition (EMT) was determined. The content of reactive oxygen species (ROS) was detected withimmunofluorescence.Results MTT result showed that sodium nitrite, ammonium sulfate, sodium nitrite andammonium sulfate mixture had a concentration-dependent inhibition of proliferation effect on LLC(P<0.05). Cell proliferation increased slightly with the concentration of sodium nitrite at0.8mg L-1-1.0mg L-1or the concentration of ammonium sulfate at1.0mg L-1-1.8mg L-1. The cell proliferationinhibition effected by mixture of sodium nitrite and ammonium sulfate is significantly lower than puresodium nitrite or ammonium sulfate group (P<0.05). For L929cells, sodium nitrite in0.8mg L-1-1.0mg L-1and ammonium sulfate in the1.0mg L-1-1.8mg L-1range can promote cell proliferation. Forthe proliferation of LLC or L929, in the same dose and the same time condition, the effect of ammoniumsulfate is higher than sodium nitrite (P<0.05). Ammonium sulfate or sodium nitrite had a stronger effecton L929than that on LLC (P<0.05). The L929and LLC cells were co-cultured (L929-LLC) to stimulatethe tumor microenvironment, and the expression of α-SMA in some cells in co-culture system is positive,which demonstrated that these cells have been transformed into tumor associated fibroblasts(carcinoma-associated fibroblasts, CAFs). The MTT results showed that sodium nitrite in theconcentration of0.8mg L-1-1.0mg L-1and ammonium sulfate in the concentration of1.0mg L-1-1.8mg L-1could promote the proliferation of L929-LLC cells in48hours and sodium nitrite or ammoniumsulfate had a stronger effect on L929-LLC cell growth than that on single cultured L929or LLC cells(P<0.05). Sodium nitrite and ammonium sulfate mixed solution had a stimulating effect on cellproliferation in the concentration of0.1mg L-1-1.8mg L-1, especially the effect was most significantwith the concentration of1.8mg L-1(P<0.05). The sodium nitrite and ammonium sulfate mixed solution(1:1) in the concentration of1.8mg L-1had a stronger effect than single ammonium sulfate, meanwhile the ammonium sulfate group had a higher performance than the sodium nitrite group (P<0.05). Thesodium nitrite, ammonium sulfate, sodium nitrite and ammonium sulfate mixture had no inhibited effecton L929-LLC cell proliferation.0.8mg L-1sodium nitrite,1.0mg L-1ammonium sulfate and1.8mg L-1sodium nitrite and ammonium sulfate mixture were added into L929-LLC cells for culture for48hours.After PI staining, then flow cytometry analysis showed that the proportion of cells in G2and S phasewere (9.13±1.54)%,(10.17±2.10)%and (16.32±1.13)%respectively, and were all significantly higherthan control group (P<0.05). The proportion of L929-LLC cells with sodium nitrite and ammoniumsulfate mixture in G2and S phase was higher than the ammonium sulfate group, and that withammonium sulfate is higher than sodium nitrite group (P<0.05). After Hoechst33258staining, andcounted under microscopy, the index of cell division were (13.41±4.17)%,(15.2±3.75)%and (18.78±4.91)%, which were all significantly higher than control group (P<0.05). The cell division index ofL929-LLC cells with sodium nitrite and ammonium sulfate mixture was higher than ammonium sulfategroup, and that with ammonium sulfate was higher than sodium nitrite group (P<0.05). Under theinverted microscope, we have found that a large number of L929-LLC cells with sodium nitrite andammonium sulfate mixture for48hours were fusiform, and the spindle cell number was obviously largerthan that in pure ammonium sulfate group, while the spindle cell was less in sodium nitrite group. Thescratch test showed that the scratch healing relative distance of L929-LLC cells in sodium nitrite andammonium sulfate mixture group was longer than that of the ammonium sulfate group, and it was longerin ammonium sulfate group than the sodium nitrite group (P<0.05). Transwell cell invasion assay showedthat the number of cells thread basement membrane were90±2.94,114±3.43and132±3.96respectively,and were all significantly higher than control group (P<0.05). The transmembrane cells in sodium nitriteand ammonium sulfate mixture group were more than that in pure ammonium sulfate group, while it was more in ammonium sulfate group than that in sodium nitrite group (P<0.05). The immunofluorescentdetection showed that the content of ROS in sodium nitrite group, ammonium sulfate group and theammonium nitrite and ammonium sulfate mixture were significantly higher than control group, and thefluorescence intensity was higher in the mixture than the ammonium sulfate group, while it was higher inammonium sulfate group than sodium nitrite group (P<0.05).Conclusions1. Both single and mixed applications of ammonium sulfate and sodium nitrite could promote theproliferation of L929-LLC co-cultured cells, and the effect of the mixture is stronger thanammonium sulfate, while the effect of sodium nitrite is relatively weak.2. Both single and mixed applications of ammonium sulfate and sodium nitrite could induce L929-LLCco-cultured cells fusiform transformation and enhance their migration and invasion, while the effectin migration and invasion enhancement of the sodium nitrite and ammonium sulfate mixture isstronger than ammonium sulfate, and the effect of sodium nitrite is relatively weak.3. The ability to promote cells proliferation and enhance cells invasion of both single and mixedapplications of ammonium sulfate and sodium nitrite is positively related with increased ROS inL929-LLC co-cultured cells induced by them. |
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