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. 2024 Nov 1;25(1):396.
doi: 10.1186/s12931-024-03013-8.

Shenqifuzheng injection inhibits lactic acid-induced cisplatin resistance in NSCLC by affecting FBXO22/p53 axis through FOXO3

Wei Bo  1   2 , Xiaokai Wang  2 , Ning Yu  1 , Chun Wang  3 , Chunying Liu  4
Affiliations

Shenqifuzheng injection inhibits lactic acid-induced cisplatin resistance in NSCLC by affecting FBXO22/p53 axis through FOXO3

Wei Bo et al. Respir Res. .

Abstract

Background: Non-small cell lung cancer (NSCLC) accounts for 80% of lung cancers. Cisplatin (DDP)-based combination chemotherapy is the main treatment of NSCLC. Due to resistance to DDP, 5-year overall survival rate of NSCLC patients is very low. Shenqifuzheng injection (SQFZ) is essential for lung cancer progression. However, whether SQFZ plays a role in DDP resistance in NSCLC and its molecular mechanism remains unclear.

Methods: Levels of FOXO3, FBXO22 and p53 in NSCLC tissues and cells were assessed by RT-qPCR and Western blot. Cell proliferation and apoptosis were analyzed utilizing CCK-8, Colony formation and Flow cytometry assays. Lactate (LA) levels were tested via ELISA. ChIP and Dual luciferase reporter assays validated regulatory relationship between FOXO3 and FBXO22. Immunoprecipitation assay evaluated p53 ubiquitination levels. The subcutaneous tumor model of nude mice was constructed. TUNEL staining detected apoptosis in tissues, and IHC assessed expression of Ki67, FOXO3, FBXO22 and p53.

Results: FOXO3 was decreased, whereas LA and FBXO22 were increased in NSCLC patients. LA led to a higher DDP resistance in A549/DDP cells, while SQFZ reversed this effect by upregulating FOXO3. Furthermore, FBXO22 was a downstream effecter of FOXO3 and FBXO22 affected p53 ubiquitination to reverse the inhibitory effect of SQFZ. We next found SQFZ inhibited LA-induced DDP resistance in NSCLC via FOXO3/FBXO22/p53 axis. Finally, SQFZ regulated LA-mediated DDP resistance in NSCLC nude mice.

Conclusion: SQFZ influences LA-induced DDP resistance in NSCLC via FOXO3/FBXO22/p53 pathway, providing a promising agent for NSCLC treatment.

Keywords: Cisplatin resistance; FBXO22; FOXO3; Non-small cell lung cancer; Shenqifuzheng injection; p53.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
The differential expression of LA, FOXO3, FBXO22 and p53 in NSCLC patients. (A) Serum lactate levels were detected by ELISA in 30 NSCLC patients and 30 healthy control. (B) RT-qPCR analysis of FOXO3, FBXO22 and p53 expression in cancer tissues and adjacent normal tissues of 30 NSCLC patients. (C) Pearson analysis detected correlation between FOXO3, FBXO22 and p53 expression in NSCLC patients. *P < 0.05,**P < 0.01,***P < 0.001
Fig. 2
Fig. 2
LA promoted DDP resistance in NSCLC in vitro. (A) The IC50 values were detected by CCK-8 after treatment with different concentrations of DDP in A549 and A549/DDP cells. (B) Lactate levels were detected by ELISA. (C) CCK-8 assay evaluated cell viability. (D) Colony formation experiments detected cell proliferation. (E) Flow cytometric detection of cell apoptosis. (F) FOXO3 expression was measured by Western blot. Results expressed as mean ± SD for three independent experiments. *P < 0.05,**P < 0.01,***P < 0.001
Fig. 3
Fig. 3
SQFZ inhibited LA-induced DDP resistance by regulating FOXO3. A549/DDP cells were treated with different drugs: DDP group, DDP + LA group, DDP + LA + SQFZ (50, 100 or 200 mg/ml) groups. (A) FOXO3 expression was measured by Western blot. (B) CCK-8 assay evaluated cell viability. (C) Colony formation experiments detected cell proliferation. (D) Flow cytometric detection of cell apoptosis. Results expressed as mean ± SD for three independent experiments. *P < 0.05,**P < 0.01,***P < 0.001
Fig. 4
Fig. 4
SQFZ regulated LA-induced DDP resistance in NSCLC by regulating FOXO3 transcription and down-regulating FBXO22 expression. (A) JASPAR predicted sites for the transcription factors FOXO3 and FBXO22 promoter. (B) Luciferase reporter assay examined luciferase activity of WT/MUT-FBXO22 after co-transfection with FOXO3 or vector. (C) ChIP assay validated the regulatory relationship between FOXO3 and FBXO22. Next, A549/DDP cells were treated with different drugs: DDP + LA, DDP + LA + SQFZ group, DDP + LA + SQFZ + sh-NC group, DDP + LA + SQFZ + sh-FOXO3 group. (D) FOXO3 was determined by RT-qPCR. (E) FOXO3 and FBXO22 levels were determined by Western blot. (F) CCK-8 assay evaluated cell viability. (G) Colony formation experiments detected cell proliferation. (H) Flow cytometric detection of cell apoptosis. Data are the means ± SD for three independent experiments. *P < 0.05,**P < 0.01,***P < 0.001
Fig. 5
Fig. 5
Overexpression of FBXO22 affected p53 ubiquitination to reverse the inhibitory effect of SQFZ on LA-induced DDP resistance in NSCLC. (A) Western blot detected effect of sh- FBXO22 on p53 protein expression in A549/DDP cells with MG132 treatment. (B) In vitro detection of p53 protein ubiquitination by IP. (C) FBXO22 was determined by RT-qPCR. Next, A549/DDP cells were treated with different drugs: DDP + LA group, DDP + LA + SQFZ group, DDP + LA + SQFZ + oe-NC group, DDP + LA + SQFZ + oe-FBXO22 group. (D) FBXO22 and p53 levels were determined by Western blot. (E) CCK-8 assay evaluated cell viability. (F) Colony formation experiments detected cell proliferation. (G) Flow cytometric detection of cell apoptosis. Results expressed as mean ± SD. *P < 0.05,**P < 0.01,***P < 0.001
Fig. 6
Fig. 6
SQFZ inhibited LA-induced DDP resistance in NSCLC through FOXO3/FBXO22/p53 axis. A549/DDP cells were treated with different drugs: DDP + LA group, DDP + LA + SQFZ group, DDP + LA + SQFZ + NC group, DDP + LA + SQFZ + sh-FOXO3 group, DDP + LA + SQFZ + sh-FOXO3 + sh-FBXO22 group, DDP + LA + SQFZ + sh-FOXO3 + oe-p53 group. (A) Expression of FOXO3, FBXO22 and p53 was detected by Western blot. (B) CCK-8 assay evaluated cell viability. (C) Colony formation experiments detected cell proliferation. (D) Flow cytometric detection of cell apoptosis. Data are the means ± SD for three independent experiments. *P < 0.05,**P < 0.01,***P < 0.001
Fig. 7
Fig. 7
SQFZ regulated LA-mediated DDP resistance in NSCLC nude mice. The subcutaneous tumor model of nude mice was constructed by subcutaneous injection of A549/DDP cells and treated with DDP. Mice (N = 5 per group) were then treated with different drugs: LA group, LA + SQFZ (10, 20, 30, 40 mL/kg) group. (A) Tumor size photographs of nude mice. (B) Changes in tumor weight. (C) TUNEL staining detected apoptosis. (D) Expression of Ki67, FOXO3, FBXO22 and p53 was detected by IHC. Values are mean ± SD. *P < 0.05,**P < 0.01,***P < 0.001
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