Surufatinib achieves radiosensitivity in cholangiocarcinoma by suppressing GINS4 expression to induce Ca2+/cAMP signaling activation

Figure 1 Surufatinib is associated with enhanced radiosensitivity of NOZ and TFK-1 and increased DNA damage. A: Cell viability assays were performed by Cell Counting Kit-8. Concentrations are shown in μg/mL. The anti-proliferative effect of surufatinib on NOZ and TFK-1 cells; B: Representative colony formation staining images and survival curves of NOZ and TFK-1 with different treatments. The survival curves were plotted using the multi-target single-hit model; C: Surufatinib increased the fluorescence foci of γ-H2AX in tumor cells after radiotherapy. Representative fluorescence foci images and quantitative detection of γ-H2AX foci in NOZ and TFK-1 cells following different treatments; scoring was performed blindly by two investigators; D and E: Apoptosis assessment by flow cytometry in NOZ and TFK-1 cells following different treatments. Apoptotic rates were calculated as the percentages of annexin-V FITC-positive cells. All columns indicate mean and error bars indicate SEM from three independent experiments. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. SF: Surufatinib; RT: Radiotherapy.

Figure 2 Surufatinib is associated with radiation sensitization in a nude mouse tumor transplantation model by inhibiting tumor proliferation and angiogenesis. A: Tumor growth curves of different treatment groups. Data are mean values from 4 mice in control, surufatinib, and radiotherapy groups, and 3 mice in the combination group (one mouse died from anesthesia); B: Representative tumor images of different treatment groups (control, surufatinib, radiation, and combination); C: Histogram of tumor weight of each mouse in the 4 groups; D: Representative immunohistochemistry staining image for Ki-67 in the 4 groups and the histogram for Ki-67 positive proportions. All immunohistochemical scoring was performed blindly; E: Representative immunohistochemistry staining image for CD31 in the 4 groups and the histogram for number of tumor vessels. ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. Ctrl: Control; SF: Surufatinib. RT: Radiotherapy alone.

Figure 3 GINS4 is involved in the radiosensitizing effect of surufatinib on cholangiocarcinoma. A: A Venn diagram from RNA sequencing (RNA-seq) data identifies 23 genes associated with surufatinib (SF) and 6 Gy irradiation; B: Another Venn diagram highlights five genes differentially expressed in SF-induced radiosensitization; C: Heatmap analysis shows expression patterns of these genes across various treatment groups; D: RNA-seq analysis reveals significant downregulation of GINS4 following SF + 6 Gy treatment compared to radiotherapy or SF alone; E: Western blot confirms the changes in GINS4 expression across different groups; F and G: Analysis of GSE10072 and The Cancer Genome Atlas RNA-seq data indicates elevated GINS4 expression in tumors; H: High GINS4 expression correlates with poor prognosis; I: Western blot assesses GINS4 protein levels in NOZ and TFK-1 cells across four groups: Si-NC, si-NC + SF + 6 Gy, si-GINS4, si-GINS4 + SF + 6 Gy. Control (Ctrl) in panels J-L represents untreated control cells; si-NC served as the transfection control (data not shown separately due to similarity to Ctrl); J: Cell proliferation of NOZ and TFK-1 cells measured using the Cell Counting Kit-8 assay; K and L: Apoptosis rates determined by flow cytometry; M: Clonogenic survival rates evaluated using the clonogenic assay. All data are presented as mean ± SEM from three independent experiments. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. NS: Not significant; Ctrl: Control; SF: Surufatinib; RT: Radiotherapy; TCGA: The Cancer Genome Atlas.

Figure 4 Overexpression of GINS4 promotes tumor proliferation and inhibits apoptosis, while surufatinib suppresses GINS4 expression and is associated with radiosensitization. A: The pLenti-GINS4 (OE-GINS4) construct was generated using standard molecular biology techniques. Western blot analysis was performed to detect GINS4 expression levels among different treatment groups in NOZ and TFK-1 cells; control = empty vector control; B: Under conditions of GINS4 overexpression, cell viability was assessed across various treatment groups; C and D: Flow cytometry was employed to evaluate apoptosis in NOZ and TFK-1 cells in OE-GINS4 compared to control cells, with or without SF + 6 Gy treatment. Quantitative analysis of apoptosis rates among different treatment groups is presented in D; E: Clonogenic assays were conducted to assess the proliferative capacity of OE-GINS4 and control cells, with or without SF + 6 Gy treatment. Representative images of colony formation and quantitative analysis of colony counts for different treatment groups in NOZ and TFK-1 cells are shown in E, respectively. All data are presented as mean ± SEM from three independent experiments. ^bP < 0.01, ^cP < 0.001. SF: Surufatinib.

Figure 5 GINS4 expression is correlated with Ca²⁺/cAMP signaling in surufatinib-treated cells. A: Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of GINS4 reveals significant pathways; B and C: Gene Set Enrichment Analysis highlights the impact of GINS4 on calcium signaling pathways; D and E: The Fluo-4 calcium ion detection kit was employed to measure changes in intracellular calcium ion concentration across different intervention groups, and quantitative analysis was presented in a histogram; F: Enzyme-linked immunosorbent assay was utilized to quantitatively analyze cAMP levels in different treatment groups for both OE-GINS4 and control (Ctrl) samples; G: Western blot analysis was performed to assess the expression of cAMP downstream-related proteins in different treatment groups for both OE-GINS4 and Ctrl samples. All data are presented as mean ± SEM from three independent experiments. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. Ctrl: Control; GINS4 overexpression: OE-GINS4; SF: Surufatinib.