Enhancing the radiosensitivity of colorectal cancer cells by reducing spermine synthase through promoting autophagy and DNA damage

Figure 1 Up-regulation of spermine synthase in colorectal cancer cells HCT116 promotes cell proliferation and inhibits apoptosis. A and B: Western blot analysis for spermine synthase (SMS) protein expression levels in FHC, HCT116, SW62, SW480, HT-29 and LoVo cells groups (A), as well as in negative control shRNA (shNC), SMS shRNA (sh-SMS), negative control over-expression vector (OE-NC), and OE-SMS HCT116 cell groups (B); C: MTT assay to detect the viability of cells in shNC, sh-SMS, OE-NC, and OE-SMS HCT116 groups; D: Colony formation assay to assess the colony formation ability of cells in shNC, sh-SMS, OE-NC, and OE-SMS HCT116 groups; E: Flow cytometry analysis of cell apoptosis levels in shNC, sh-SMS, OE-NC, and OE-SMS HCT116 groups. ^aP< 0.05, ^bP < 0.01 vs FHC, shNC; ^dP < 0.01 vs OE-NC; SMS: Spermine synthase; sh-SMS: Spermine synthase shRNA; shNC: Negative control shRNA; OE-SMS: Spermine synthase over-expression vector; OE-NC: Negative control over-expression vector.

Figure 2 Down-regulation of spermine synthase enhances the radiosensitivity of HCT116 cells, inhibits cell proliferation, and promotes apoptosis. A: MTT assay to detect the viability of HCT116 cells in the negative control shRNA (shNC) group, shNC + IR group, spermine synthase shRNA (sh-SMS) group, and sh-SMS + IR group; B: Colony formation assay to assess the colony formation ability of HCT116 cells in the shNC group, shNC + IR group, sh-SMS group, and sh-SMS + IR group; C: Flow cytometry analysis to measure the apoptosis levels of HCT116 cells in the shNC group, shNC + IR group, sh-SMS group, and sh-SMS + IR group. ^bP < 0,01 vs shNC, ^dP < 0.01 vs shNC + IR, ^fP < 0.01 vs sh-SMS; SMS: Spermine synthase; sh-SMS: Spermine synthase shRNA; shNC: Negative control shRNA; OE-SMS: Spermine synthase over-expression vector; OE-NC: Negative control over-expression vector.

Figure 3 Knockdown of spermine synthase enhances the radiosensitivity of HCT116 cells by advancing DNA damage. A-C: Comet assay for assessing DNA damage levels in HCT116 cells of the negative control shRNA (shNC), shNC + IR, spermine synthase shRNA (sh-SMS), and sh-SMS + IR groups (A), and analysis of comet tail DNA levels (B) and comet tail moments (C); D and E: Western blot analysis of γH2AX, p-γH2AX, ATR, p-ATR, and P53 protein levels in HCT116 cells of the shNC group, shNC + IR group, sh-SMS group, and sh-SMS + IR group. ^bP < 0,01 vs shNC, ^dP < 0.01 vs shNC + IR, ^fP < 0.01 vs sh-SMS; SMS: Spermine synthase; sh-SMS: Spermine synthase shRNA; shNC: Negative control shRNA; OE-SMS: Spermine synthase over-expression vector; OE-NC: Negative control over-expression vector.

Figure 4 Knockdown of spermine synthase enhances the radiosensitivity of HCT116 cells by promoting autophagy. A and B: Immunofluorescence detection for LC3 puncta in HCT116 cells of the negative control shRNA (shNC) group, shNC + IR group, spermine synthase shRNA (sh-SMS) group, and sh-SMS + IR group; C and D: Western blot analysis for protein levels of LC3-I, LC3-II, p62, and Beclin 1 in HCT116 cells of the shNC group, shNC + IR group, sh-SMS group, and sh-SMS + IR group. ^bP < 0.01 vs shNC, ^dP < 0.01 vs shNC + IR, ^fP < 0.01 vs sh-SMS; SMS: Spermine synthase; sh-SMS: Spermine synthase shRNA; shNC: Negative control shRNA; OE-SMS: Spermine synthase over-expression vector; OE-NC: Negative control over-expression vector.

Figure 5 Knocking down spermine synthase enhances radiosensitivity of HCT116 cells by suppressing the mammalian target of rapamycin pathway. Western blot analysis for detecting the protein expression levels of mammalian target of rapamycin (mTOR), p-mTOR, S6K1, p-S6K1, S6, and p-S6 in HCT116 cells under the following conditions: Negative control shRNA (ShNC), shNC + IR, spermine synthase shRNA (sh-SMS), and sh-SMS + IR. ^bP < 0.01 vs shNC, ^dP < 0.01 vs shNC + IR, ^fP < 0.01 vs sh-SMS; SMS: Spermine synthase; sh-SMS: Spermine synthase shRNA; shNC: Negative control shRNA; OE-SMS: Spermine synthase over-expression vector; OE-NC: Negative control over-expression vector; mTOR: Mammalian target of rapamycin.