Abstract:

Objective To investigate the effects of metformin on esophageal squamous cell carcinoma cell growth， migration and angiogenesis by regulating the expression of ALKBH3. Methods Human esophageal cancer TE-1 cells were treated with different concentrations （0， 0.5， 1.0， 2.0， 4.0， 8.0 mmol/L） of metformin， and they were divided into a blank control group， low- （0.5 mmol/L）， medium- （1.0 mmol/L）， and high- （2.0 mmol/L） concentration metformin groups， a metformin （2.0 mmol/L）+pcDNA-NC group， and a metformin （2.0 mmol/L）+pcDNA-ALKBH3 group. The cell viability was determined by the CCK-8 method. The cell proliferation ability was detected by the clone formation assay. The cell migration and invasion abilities were examined by the Transwell assay. The cell apoptosis was detected by flow cytometry. The tube formation ability of cells was detected by the angiogenesis assay. A xenograft tumor model was constructed using 4- to 6-week-old male BALB/c thymus-less nude mice， which were divided into a model control group， a metformin group， a metformin+pcDNA-NC group， and a metformin+pcDNA-ALKBH3 group using a random number table method， and with six in each group. And the volume and weight of the tumor were measured. The protein expression levels of apoptosis-related proteins Bcl-2， Bax， ALKBH3 and vascular endothelial growth factor A （VEGF-A） were detected by Western blotting. The expression of CD31 protein was detected by immunohistochemistry. Results After treating TE-1 cells with 0， 0.5， 1.0， 2.0， 4.0， and 8.0 mmol/L metformin for 48 hours， the cell viability was （100.00±0.00）%， （90.31±5.23）%， （81.25±8.65）%， （63.52±6.80）%， （54.64±5.35）%， and （31.48±4.21）%， respectively， with a statistically significant difference （F=98.11， P<0.001）. There were statistically significant differences in cell viability between 0.5， 1.0， 2.0， 4.0， 8.0 mmol/L and 0 mmol/L （all P<0.05）. The IC₅₀ of metformin for TE-1 cells was 4.46 mmol/L. The numbers of colony formations of TE-1 cells in the blank control group， low-， medium-， and high-concentration metformin groups， metformin+pcDNA-NC group， and metformin+pcDNA-ALKBH3 group were 153.15±13.55， 134.80±11.62， 116.24±10.43， 93.17±8.85， 89.39±8.46， 110.26±7.21， respectively， with a statistically significant difference （F=34.28， P<0.001）； the numbers of colony formations of TE-1 cells in the metformin groups at different concentrations decreased significantly with the increase in metformin concentration （both P<0.05）； compared with the metformin+pcDNA-NC group， the number of colony formations of cells in the metformin+pcDNA-ALKBH3 group increased （P<0.05）. The numbers of migration of TE-1 cells of 6 groups were 152.13±13.40， 133.85±10.72， 115.28±8.64， 91.16±7.89， 85.39±7.23， 116.85±8.36， the numbers of invasion were 135.22±10.77， 112.07±9.53， 86.30±7.45， 69.53±6.74， 65.81±5.65， 79.80±6.32， respectively， with statistically significant differences （F=41.35， P<0.001； F=69.06， P<0.001）； the numbers of migrated and invaded cells in the metformin groups at different concentrations decreased significantly with the increase in metformin concentration （all P<0.05）； compared with the metformin+pcDNA-NC group， the numbers of migrated and invaded cells in the metformin+pcDNA-ALKBH3 group increased significantly （both P<0.05）. The apoptosis rates of TE-1 cells in 6 groups were （3.22±1.13）%， （13.82±1.90）%， （22.67±2.53）%， （29.18±3.24）%， （26.84±2.75）%， and （16.36±1.63）%， respectively， with a statistically significant difference （F=103.66， P<0.001）； the apoptosis rates of cells in the metformin groups at different concentrations gradually increased with the increase in metformin concentration （both P<0.05）； compared with the metformin+pcDNA-NC group， the apoptosis rate of cells in the metformin+pcDNA-ALKBH3 group was relatively lower （P<0.05）. The tubular structure of cells in blank control group was intact， and there were different degrees of damage to the tubular structure of cells in the low-， medium-， high- concentration metformin groups， the degree of damage to the tubular structure of cells in the metformin+pcDNA-ALKBH3 group was reduced. The numbers of cellular tubular structures of TE-1 cells in the 6 groups were 38.35±3.20， 27.15±2.64， 15.92±3.14， 7.39±1.50， 8.61±1.37， and 29.33±4.20， respectively， with a statistically significant difference （F=113.92， P<0.001）； the number of cellular tubular structures in the low-， medium-， and high- concentration metformin groups gradually decreased （both P<0.05）； the number of cellular tubular structures in the metformin+pcDNA-ALKBH3 group was more than that in the metformin+pcDNA-NC group （P<0.05）. There were statistically significant differences in the protein expressions of Bcl-2， Bax， ALKBH3， and VEGF-A in TE-1 cells among 6 groups （F=56.36， P<0.001； F=57.26， P<0.001； F=159.30， P<0.001； F=132.89， P<0.001）； compared with the blank control group， the protein expressions of Bcl-2， ALKBH3， and VEGF-A in the metformin groups at different concentrations decreased， while the protein expression of Bax increased （all P<0.05）； compared with the metformin+pcDNA-NC group， the protein expressions of Bcl-2， ALKBH3， and VEGF-A in the metformin+pcDNA-ALKBH3 group increased， and the expression level of Bax decreased （all P<0.05）. The weights of tumors in the model control group， metformin group， metformin+pcDNA-NC group， and metformin+pcDNA-ALKBH3 group were （1.16±0.12）， （0.46±0.05）， （0.50±0.06）， （1.19±0.14） g， the volumes of tumors were （878.36±108.93）， （413.59±50.23）， （439.78±51.39）， （793.75±96.98） mm³， with statistically significant differences （F=96.61， P<0.001； F=51.90， P<0.001）； the weight of tumors were lower and the volume of tumors were smaller in the metformin group than those in the model control group （both P<0.05）， the weight of tumors were higher and the volume of tumors were bigger in the metformin+pcDNA-ALKBH3 group than those in the metformin group and the metformin+pcDNA-NC group （all P<0.05）. CD31 was mainly distributed in the cytoplasm and cell membrane of tumor cells. There were statistically significant differences in the positive rates of CD31 and the protein expression levels of VEGF-A in transplanted tumor tissues among 4 groups （F=7.12， P=0.002； F=48.81， P<0.001）； the positive rate of CD31 and the protein expression level of VEGF-A in the metformin group were lower than those in the model control group； the positive rate of CD31 and the protein expression level of VEGF-A in the metformin+pcDNA-ALKBH3 group were higher than those in the metformin group and the metformin+pcDNA-NC group （all P<0.05）. Conclusions Metformin may inhibit the proliferation， migration， and tumor angiogenesis of esophageal squamous cell carcinoma by reducing ALKBH3 expression.

Key words: Esophageal squamous cell carcinoma, Metformin, AlkB enzymes, Cell proliferation, Cell movement