| Objective In order to reveal the influence of sodium nitrite(NaNO2) in environment on tumor progression and provide data support for institute the health standard of sodium nitrite in diet , the study is to investigate the effects of sodium nitrite on the proliferation,apoptosis and invasion in SMMC-7721 cells and evaluate the relationship between hepatoma and sodium nitrite in environment.Methods Human hepatocarcinoma SMMC-7721 cells were used as model. The cells were treated with a series of concentrations of NaNO2 ,the proliferation was measured by MTT assay and cell counting. The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression changes of c-fos mRNA. FITC-annexinⅴ/PI flow cytometry ,Hoechst 33258 staining and Hoechst 33258/ PI staining were used to detect the apoptotic cells. The cells'migration and invasion were measured by scratch wound assay and transwell migration assay.Colorimetric technique was performed to measure the activities of superoxide dismutase (SOD) and catalase (CAT) and the levels of malondialdehyde (MDA). Immunocytochemical method to observe the expression of the Metallothionein(MT). Western blotting analysis was used to assay the expression of apoptosis-related proteins.Results The results of MTT assay and cell counting indicated that NaNO2 pretreatment of SMMC-7721 cells at concentrations ranging from 3.13 mg·L-1 to 800 mg·L-1 could promote cell proliferation, the minimum dose of the effect is 17 mg·L-1.While NaNO2 and nitric oxide specific scavenger c-PTIO combined to treat the cells, the promotion effect was still exist in spite of the proliferation overall decline. NaNO2 inhibited the proliferation of SMMC-7721 cells within the range of 800 mg·L-13200 mg·L-1.The results of RT-PCR showed that NaNO2 could elevate the expression of c-fos mRNA within the range of 17 mg·L-1800 mg·L-1. The SMMC-7721 cells were pretreated with 17 mg·L-1 NaNO2 for 24 h or 4 W, followed with 200 mmol.L-1ethanol or 25μmol·L-1 cadmium chloride (CdCl2) for additional 12 h. Both temporal and chronic NaNO2 pretreat groups could prevent the cytotoxicity and inhibited the apoptosis which were induced by ethanol or CdCl2.Meanwhile cells pretreated with NaNO2 followed by ethanol or CdCl2 challenges showed significantly decreased the levels of malondialdehyde (MDA), elevated the levels of metallothionein(MT)as well as increased superoxide dismutase (SOD) and catalase (CAT) activities. Western blotting analysis revealed that ethanol treated cells pretreated with NaNO2, the HIF-1αand Bcl-2 were obviously increased, while the expression of pro-apoptotic proteins, including bax,caspase-9, caspase-3 were decreased.NaNO2 pretreatment of SMMC-7721 cells at concentrations ranging from 17 mg·L-1 to 800 mg·L-1 result in HIF-1αexpression increased. While NaNO2 and nitric oxide specific scavenger c-PTIO combined to treat the cells, the promotion was still exist in spite of the HIF-1αexpression overall decline.The results of wound healing assay indicated that the SMMC-7721 cells were pretreated with 17 mg·L-1 NaNO2 for 24h could increase the relative wound closure. Transwell migration assay showed after treated with the supernatant (come from 17 mg·L-1 NaNO2 that treat the cells for 48h) for 24h, the number of invasion cell through reconstituted basement membrane were increased.Conclusions1. NaNO2 pretreatment of SMMC-7721 cells at concentrations ranging from 3.13 mg·L-1 to 800 mg·L-1 could promote cell proliferation in a non-linear way, whereas proliferation inhibited within the range of 800 mg·L-13200 mg·L-1 in a dose-dependent fashion.2. The SMMC-7721 cells were pretreated with 17 mg·L-1 NaNO2 could increase anti-oxidant activities , expression of HIF-1αand resist apoptosis of the SMMC-7721 cells which induced by ethanol or CdCl2. 3. The migration of the SMMC-7721 cells were increased after treated with 17 mg·L-1 NaNO2 for 24 h.4. The supernatant was collected from the SMMC-7721 cells which were pretreated with 17 mg·L-1 NaNO2 for 48h and then treated with the supernatant for 24h, the invasion abilitity of the SMMC-7721 cells was increased. |
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