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

Respiratory Research volume 25, Article number: 396 (2024) Cite this article

Highlights

FOXO3 was decreased, while LA and FBXO22 were increased in NSCLC patients.

LA promoted cisplatin resistance in NSCLC in vitro and in vivo.

SQFZ inhibited LA-induced cisplatin resistance in NSCLC by regulating FOXO3.

FBXO22 affected p53 ubiquitination to reverse the inhibitory effect of SQFZ.

SQFZ inhibited cisplatin resistance in NSCLC by FOXO3/FBXO22/p53 axis.

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.

Introduction

Non-small cell lung cancer (NSCLC) accounts for about 80% of the total number of lung cancers [1]. Most patients with NSCLC are in the late stage of clinical diagnosis and lack effective therapeutic drugs [2]. Additionally, many NSCLC patients are resistant to commonly used chemotherapy or radiotherapy, resulting in a low 5-year survival rate [3,4,5]. Cisplatin (DDP) is a commonly used anti-tumor drug of NSCLC in clinical practice. It has a wide anti-cancer spectrum and synergistic effect with a variety of anti-tumor drugs [6]. However, the long-term use of DDP will cause drug resistance in patients [7]. Therefore, a better understanding of the mechanism and drug resistance of DDP will improve clinical outcomes of NSCLC patients. Tumors oppose chemotherapeutic agents through a variety of mechanisms, and research indicates that the tumor microenvironment plays a pivotal role in facilitating this resistance [8].

There is evidence that lactic acid (LA) is a product derived from the tumor microenvironment, and its content in tumor tissues is higher than that in normal tissues, which is also positively correlated with tumor metastasis [9]. Accumulation of LA can lead to acidosis, angiogenesis, immunosuppression, tumor cell proliferation and survival, which is harmful to human health [10]. LA acts as a critical immunoregulatory molecule involved in suppressing immune effector cell proliferation and inducing immune cell de-differentiation [11]. The secreted LA orchestrates the activation of MRP1/ABCC1 protein expression in NSCLC by coordinating the TGF-β1/Snail and TAZ/AP-1 pathways, thereby promoting chemoresistance [12].

In recent years, Chinese medicine treatment of cancer has become a major clinical feature. Shenqifuzheng injection (SQFZ) is prepared from Codonopsis pilosula and Astragalus membranaceus as the main raw materials, which can play a role in promoting blood circulation and resolving blood stasis [13]. A multi-center randomized trial indicated that the combination of SQFZ and chemotherapy demonstrates both safety and efficacy in the treatment of NSCLC [14]. SQFZ mainly exerts anti-tumor and enhances immune function, thereby increasing the quality of life of tumor patients [15]. SQFZ can reprogramme the immunosuppressive melanoma microenvironment in vivo to enhance the cytotoxicity of tumour-infiltrating immune cells [16]. Notably, SQFZ plays a pivotal role in treating NSCLC [17]. For example, SQFZ had definite toxicity relieving effects on treating elder patients with advanced NSCLC [18]. Moreover, SQFZ could improve the efficacy of chemotherpay and function of cellular immunity in NSCLC patients [19]. However, it is unclear whether SQFZ is a reversal agent for chemotherapy resistance and the potential mechanism by which SQFZ increases NSCLC chemotherapy sensitivity.

Forkhead box O3, also known as FOXO3 or FOXO3a, is the most active member of the FOXO family and is crucial in inhibiting tumor formation, cell proliferation and metabolism [20]. As a transcription factor, the abnormal expression of FOXO3 is associated with many cancers, including NSCLC. For instance, FOXO3 accelerated CASC11 transcription to regulate NSCLC progression [21]. Besides, FOXO3 modulated nm23-H1 during the metastasis process of NSCLC [22]. It was also reported that lactic acid regulated the m6A level of FOXO3 and affected the expression of FOXO3 [23]. Our previous results showed that FOXO3 had a binding site with FBXO22 promoter region. F-box only protein 22 (FBXO22), as one of the F-box proteins, its deletion will lead to the instability of the ubiquitination complex, thereby regulating progression and metastasis of tumors [24]. Interestingly, FBXO22 was reported to sensitize NSCLC cells to DDP [25]. However, the regulation of FBXO22 on NSCLC and its related molecular mechanisms are still unclear. P53 is an important tumor suppressor gene, and its wild type leads to cancer cell apoptosis, thereby preventing carcinogenesis [26]. Notably, FBXO22 was first identified as a p53-targeting gene [27]. Therefore, we speculate that FOXO3 may participate in regulating NSCLC through FBXO22/p53 axis.

The effect of SQFZ on DDP resistance in NSCLC was explored in this study. We propose a novel mechanism in which SQFZ upregulates FOXO3, thus making FOXO3 transcriptionally inhibit FBXO22 expression, while blocks the degradation of p53 due to FBXO22 ubiquitination, thereby hindering cancer cell proliferation and migration and inhibiting DDP resistance in lactate-induced NSCLC. Therefore, SQFZ may represent a novel therapeutic strategy for NSCLC patients administered with DDP-based therapies.

Materials and methods

Clinic samples

Paired NSCLC and adjacent normal tissues (located at a distance of more than 5 cm from the lung tumor) were taken from 30 patients who underwent primary surgical resection of NSCLC between January 2020 and December 2022. All patients did not receive adjuvant treatment before surgery. Tissue samples were snap frozen in liquid nitrogen for subsequent analysis. 30 healthy volunteers were also enrolled. We collected the serum samples of NSCLC patients and healthy control. Written informed consent was obtained from all subjects.

Cell culture and treatment

Human NSCLC cell lines (A549, H1975) were acquired from Procell (Wuhan, China) and propagated in RPMI 1640 medium enriched with 10% FBS (Invitrogen, Carlsbad, CA, USA) and 100 µg/mL of penicillin-streptomycin (Sigma, St. Louis, MO, USA) at 37 °C with CO2. DDP-resistant cells (A549/DDP, H1975/DDP) were established by continuous cultivation for 8 months in the selection of DDP (Sigma) from 0.3 to 20 µg/mL. To preserve the drug-resistant phenotype, 2 𝜇g/mL of DDP was added to the culture medium. Lactic acidosis conditions were generated by adding pure l-lactic acid (Solarbio, Shanghai, China) in normal culture medium until the final lactic acid level of 0–30 mM. SQFZ [National drug standard No.: WS3-387(Z-50)-2003(Z)] was obtained from Livzon Pharmaceutical Corporation (Guangdong, China). A concentrated solution of 10 mL was prepared, which corresponded to 5 g of Codonopsis pilosula and 5 g of crude drugs from Astragalus membranaceus. Cells were treated with SQFZ with different concentrations of 0, 50, 100, 200 mg/mL, respectively. For MG132 treatment, cells were treated with MG132 (30 µM, Sigma) for 6 h.

Cell transfection

The empty vector and plasmids containing overexpressed FBXO22 (oe-FBXO22) and p53 (oe-p53) were synthesized by Sangon Biotech (Shanghai, China). Specific shRNAs against FOXO3-1 (sh-FOXO3-1), FOXO3-2 (sh-FOXO3-2) , FOXO3-3 (sh-FOXO3-3) and FBXO22 (sh-FBXO22) and controlled shRNA (sh-NC) were purchased from Ribobio (Guangzhou, China). Transfection was performed utilizing Lipofectamine ™2000 (Invitrogen) following the manufacturer’s protocol for 48 h.

Quantitative real‑time PCR

TRIZOL reagent (Invitrogen) was adopted to extract total RNA from tissues or cells. Reverse transcription was performed utlizing a cDNA synthesis kit (Toyobo, Osaka, Japan). DNA was quantified by Fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) under LC480 Real-Time PCR Detection System (Roche, Basel, Switzerland). Primer sequences are as follows: FOXO3 F: 5′-CGGACAAACGGCTCACTC-3′, R: 5′-GGACCCGCATGAATCGACTAT-3′; FBXO22 F: 5′-ATGGGATCAGGTAGCAATCGAC-3′, R: 5′-CCACACGAAGTTCAGGGTTATC-3′; p53 F: 5′-GCAACTATGGCTTCCACCTG-3′, R: 5′-CAGAGAGCACCGCGACCACG-3′. Data analysis was conducted by 2−∆∆Ct method. GAPDH was utilized as internal reference.

Western blot

Total protein was extracted using RIPA lysis buffer (Beyotime, Shanghai, China). Protein concentrations were tested utilizing BCA protein assay kit (Beyotime). Equal amount of protein was loaded in gels for SDS-PAGE and moved on PVDF membranes (Roche). We then blocked membranes utilizing 5% non-fat dry and incubated membranes with primary antibodies at 4 °C overnight: FOXO3 (ab109629, 1:1000, Abcam, Cambridge, MA, USA), FBXO22 (ab230395, 1:1000, Abcam), p53 (ab227655, 1:1000, Abcam), followed by with horseradish peroxidase (HRP)-conjugated secondary antibodies (#7074, 1:1,000, Cell Signaling Technology, Danvers, MA, USA) for 1 h. Proteins were viewed with enhanced chemiluminescence (ECL) reagents (Thermo Fisher Scientific, MA, USA). Protein bands were quantified by ImageJ software (NIH, Bethesda, MD, USA). β-actin served as an endogenous reference.

Lactate measurement

NSCLC cells were plated (1 × 105 cells/well) in six well plates and incubated at 37 °C. After 36 h incubation, cells were isolated by centrifugation. Lactate levels in the culture medium were determined utilizing a Lactate Assay Kit (BioVision, Inc, Milpitas, CA, USA) according to instruction provided by manufacturer.

Cell counting Kit-8 (CCK-8) assay

A CCK-8 kit (Dojindo, Japan) was utilized to assess cell chemoresistance capacity and viability. A549/DDP cells or A549 cells (5 × 103 /well) were seeded into 96 well plates and incubated for 24 h, and then treated with DDP and different concentrations of LA. After incubation for 24 h, cell viability was tested by adding CCK-8 (10 µL/well) to cells for 2 h under 37 °C. Optical density was measured in 450 nm on a microplate reader (BioTek, Winooski, VT, USA). The half‑maximal inhibitory concentration (IC50) value was calculated to evaluate cell chemoresistance capacity.

Colony formation assay

Cells were seeded in 6 well plates in triplicate (500–1000 cells/well) and cultivated in 5% CO2 at 37 °C for 2 weeks. Afterwards, cells were fixed with methanol and stained with 0.1% crystal violet (Beyotime) for 20 min. The colonies were photographed and counted using a microscope (Olympus, Tokyo, Japan).

Flow cytometric detection of apoptosis

Cells from each group were collected by trypsinization, and resuspended at a density of 1 × 106 cells/mL within 1× binding buffer. After double staining with annexin V - FITC and propidium iodide (PI), utilizing Annexin V-FITC/PI apoptosis detection kit (KeyGEN BioTECH, Nanjing, China), cells were analyzed using FACS flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA).

Dual luciferase reporter assay

FBXO22 promoter was amplified by PCR (Promega, Madison, WI, USA). The FBXO22 reporter constructs containing the wild-type (WT-FBXO22) or mutated binding sites (MUT-FBXO22) of FOXO3 were synthesized and cloned into pGL3 vector (Promega). A549 cells were plated in a 24-well plate (2 × 104/well) for 24 h. Next, cells were co-transfected with FOXO3 or NC plasmids and FBXO22 promoter plasmids utilizing LipofectamineTM2000 (Invitrogen). After 48 h, luciferase activity was detected using a Dual‑Luciferase Reporter assay system (Promega).

Chromatin immunoprecipitation (ChIP) assay

ChIP assay was performed with the EZ-ChIP kit (Millipore, Bedford, MA, USA). A549 cells were cross-linked with 1% formaldehyde. A cell lysis buffer was used to lyses cells. After sonication, lysates were adopted to immunoprecipitation with anti-FOXO3 (ab109629, Abcam) or IgG (#2729, Cell Signaling Technology) antibodies. Afterwards, immune complexes were incubated with protein A/G-Sepharose beads (Roche). Next, PCR was conducted with FBXO22 primers.

Immunoprecipitation (IP) detection of p53 ubiquitination levels

Transfected cells pretreated with 30 µM MG132 (Sigma) for 6 h were lysed and incubated with anti-p53 antibody (ab227655, Abcam) conjugated with agarose beads (Roche) overnight at 4 °C. Beads were then washed and boiled with SDS loading buffer. The immunoprecipitated protein complexes were analyzed by western blot analysis utilizing an anti-ubiquitin antibody (ab134953, Abcam).

Animal experiments

BALB/c nude mice (6 weeks old) were obtained from Hunan SJA Laboratory Animal Co., Ltd (Changsha, China). Mice were randomly assigned into LA group, LA + SQFZ (10, 20, 30, 40 mL/kg ) groups (n = 5 per group). A549/DDP cells were subcutaneously injected (1 × 106 cells per injection) into the right back of nude mice. From the next day, intraperitoneal injection of DDP was performed (3.0 mg/kg, 3 times/week). After four weeks, nude mice were sacrificed and tumor weight was recorded. Tumor tissues were fixed with 4% paraformaldehyde for 24 h and subjected to TUNEL and IHC staining. All animal experiment protocols were approved by the Institutional Ethics Committee for the Administration of Laboratory Animals of Liaoning University of Traditional Chinese Medicine.

Immunohistochemistry (IHC)

Paraffin tissue sections were 4% paraformaldehyde fixed. Tissues were cut to 4-µm sections, dewaxed and rehydrated. Endogenous peroxidase was blocked with 3% hydrogen peroxide for 30 min. Primary antibodies were added and incubated at 4 °C overnight: Ki67 (ab15580, Abcam), FOXO3 (ab23683, Abcam), FBXO22 (ab230395, Abcam) and p53 (ab227655, Abcam). After washing with PBS, sections subjected to corresponding secondary antibodies. Finally, sections were incubated with diaminobenzidine (DAB) staining for 1 min and visualized utilizing microscope (Olympus). At least five fields were randomly selected for each section and integrated optical density was calculated by ImageJ (NIH).

TUNEL staining

Tissue Sect. (4 μm thick) were dewaxed with xylene and dehydrated with graded ethanol series. According to supplier’s protocol, TUNEL staining was used to detect cell apoptosis using an apoptosis detection kit (cat. no. ZK-8005; ZSJQB Co. Ltd., Beijing, China). Images were captured under a microscope (Olympus). Five fields were randomly selected from each section. Brown or brownish yellow cells with apoptotic cell morphology were identified as apoptotic cells.

Statistical analysis

Statistical testing was performed with SPSS version 16.0 (SPSS Inc, Chicago, IL, USA). Values were summarized as means ± standard deviation (SD). Differences were tested by t-test between two sets of data. One-way analysis of variance (ANOVA) was adopted to monitor differences for multiple comparisons. Every experiment was repeated 3 times. A P < 0.05 was considered statistically significant.

Results

The differential expression of LA, FOXO3, FBXO22 and p53 in NSCLC patients

We first compared the expression of LA, FOXO3, FBXO22 and p53 in 30 NSCLC patients. ELISA analysis showed high lactate levels in NSCLC patients compared to healthy control (Fig. 1A). RT-qPCR analysis revealed decreased expression of FOXO3 together with p53 and increased expression of FBXO22 in 30 cases of NSCLC cancer tissues compared to adjacent normal tissues (Fig. 1B). Additionally, Pearson analysis showed that in 30 NSCLC samples, there was a negative correlation between FOXO3 and FBXO22, a negative correlation between FBXO22 and p53, and a positive correlation between FOXO3 and p53 (Fig. 1C). The above data suggested that LA, FOXO3, FBXO22 and p53 were aberrantly expressed in NSCLC patients.

Fig. 1
figure 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

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LA promoted DDP resistance in NSCLC in vitro

Next, IC50 of DDP treatment in A549 and A549/DDP cells was measured utilizing CCK-8 assay. Compared to A549 cells exposed to DDP, IC50 values of A549/DDP cells after DDP treatment were greatly higher, suggesting DDP resistant (Fig. 2A). In addition, high lactate levels were discovered in A549/DDP cells compared to A549 cells (Fig. 2B). Next, cancer cell proliferation was remarkably decreased after DDP treatment compared to control cells, while LA treatment reversed the effects of DDP in a concentration dependent manner (Fig. 2C and D). Meanwhile, Flow cytometry and Western blot further indicated that cell apoptosis and FOXO3 protein level in DDP-treated cells were much higher than that in control cells, while LA treatment overturned the effects of DDP in a concentration dependent manner (Fig. 2E and F). Thus, DDP resistance in NSCLC was accelerated by LA treatment.

Fig. 2
figure 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

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SQFZ inhibited LA-induced DDP resistance by regulating FOXO3

Subsequently, function of SQFZ in DDP resistance of NSCLC cells was explored. A549/DDP cells were exposed to different combinations of DDP, LA and SQFZ as follows: DDP group, DDP + LA group, DDP + LA + SQFZ (50, 100 or 200 mg/ml) groups. Western blot results showed that in DDP-exposed A549/DDP cells, FOXO3 expression was decreased with LA administration, and FOXO3 level was increased after SQFZ addition in a concentration dependent manner, with the highest expression at 100 mg/ml and 200 mg/ml (Fig. 3A). Moreover, proliferation ability of A549/DDP cells was enhanced with LA treatment, which decreased with the addition of SQFZ in a concentration dependent manner (Fig. 3B and C). Additionally, A549/DDP cell apoptosis was decreased in response to LA treatment, which was further increased after treating with SQFZ in a concentration dependent manner (Fig. 3D). Taken together, these findings revealed that SQFZ increased the cytotoxicity of DDP in LA-induced DDP resistant NSCLC cells through upregulating FOXO3.

Fig. 3
figure 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

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SQFZ regulated LA-triggered DDP resistance in NSCLC via regulating FOXO3 transcription and down-regulating FBXO22 expression

To further investigate the potential mechanism of SQFZ on DDP resistance in NSCLC, JASPAR predicted that transcription factor FOXO3 and FBXO22 promoter had binding sites (Fig. 4A). Next, Dual luciferase assay found that FOXO3 decreased enzyme activity of WT-FBXO22, while MUT-FBXO22 had no effect (Fig. 4B). Furthermore, ChIP assay verified the targeted regulation relationship between FOXO3 and FBXO22 promoter (Fig. 4C). Subsequently, A549/DDP cells were exposed to different combinations of DDP, LA and SQFZ as follows: DDP + LA, DDP + LA + SQFZ group, DDP + LA + SQFZ + sh-NC group, DDP + LA + SQFZ + sh-FOXO3 group. Transfection efficiency of FOXO3 silencing was detected using RT-qPCR, and sh-FOXO3-2 was selected for the subsequent experiments (Fig. 4D). Western blot results showed that after LA treatment, FOXO3 was down-regulated and FBXO22 was up-regulated. SQFZ increased FOXO3 protein level and reduced FBXO22 protein expression; while sh-FOXO3 reversed the effect of SQFZ on FOXO2 and FBXO22 (Fig. 4E). LA treatment promoted cell proliferation, whereas SQFZ inhibited cell proliferation. However, sh-FOXO3 overturned the effect of SQFZ on cell proliferation (Fig. 4F and G). Additionally, LA treatment suppressed apoptosis, but SQFZ promoted apoptosis. FOXO3 knockdown reversed the effect of SQFZ on apoptosis (Fig. 4H). To conclude, these results implied that SQFZ enhanced FOXO3 transcription to inhibit FBXO22 expression in LA-induced DDP resistance in NSCLC.

Fig. 4
figure 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

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Overexpression of FBXO22 affected p53 ubiquitination to reverse the inhibitory effect of SQFZ on LA-induced DDP resistance in NSCLC

We next investigated whether FBXO22 could regulate p53 ubiquitination during LA-induced DDP resistance in NSCLC treated by SQFZ. When cells were exposed to proteasome inhibitor MG132, the ability of FBXO22 to reduce p53 level was reversed (Fig. 5A). In addition, Immunoprecipitation assay showed that FBXO22-driven p53 downregulation required 26 S proteasome activity. No increase in p53 level was observed in the control group after MG132 treatment (Fig. 5B). Next, transfection efficiency of FBXO22 was detected by RT-qPCR, and oe-FBXO22-2 was selected for follow-up experimentals (Fig. 5C). Afterwards, A549/DDP cells were treated with different combinations of DDP, LA and SQFZ as follows: DDP + LA group, DDP + LA + SQFZ group, DDP + LA + SQFZ + oe-NC group, DDP + LA + SQFZ + oe-FBXO22 group. FBXO22 protein was increased and p53 was decreased after LA treatment. SQFZ decreased FBXO22 protein and increased p53 protein expression, while oe-FBXO22 reversed the effect of SQFZ on FBXO22 and p53 expression (Fig. 5D). Furthermore, after SQFZ treatment, cell proliferation was significantly decreased in LA-treated cells, which could be rescued by FBXO22 overexpressing (Fig. 5E and F). In addition, in LA-treated cells, apoptosis was enhanced in response to SQFZ treatment; oe-FBXO22 ameliorated the effect of SQFZ (Fig. 5G). Taken together, these findings revealed that FBXO22 overturned the inhibitory effect of SQFZ on LA-induced DDP resistance in NSCLC through regulating p53 ubiquitination.

Fig. 5
figure 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

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SQFZ inhibited LA-induced DDP resistance in NSCLC through FOXO3/FBXO22/p53 axis

We next explored whether SQFZ could regulate DDP resistance in NSCLC via FOXO3/FBXO22/p53 axis. A549/DDP cells were exposed to different combinations of DDP, LA and SQFZ as follows: 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. As shown in Fig. 6A, Western blot demonstrated that after SQFZ treatment in DDP-treated A549/DDP cells, FOXO3 and p53 protein levels were increased, and FBXO22 expression was decreased. Sh-FOXO3 reversed the effect of SQFZ on FOXO3, FBXO22 and p53 expression. Sh-FBXO22 reduced the high expression of FBXO22 and increased the low expression of p53 induced by sh-FOXO3. Additionally, oe-P53 increased the expression of p53. Further, after SQFZ treatment in DDP-treated A549/DDP cells, cell proliferation ability decreased. Sh-FOXO3 reversed the inhibitory effect of SQFZ on cell proliferation, while sh-FBXO22 and oe-P53 inhibited cell proliferation (Fig. 6B and C). In addition, after SQFZ treatment in DDP-treated A549/DDP cells, apoptosis was increased. Sh-FOXO3 overturned the promoting effect of SQFZ on apoptosis, whereas sh-FBXO22 and oe-P53 promoted apoptosis (Fig. 6D). Altogether, LA-induced DDP resistance in NSCLC was suppressed by SQFZ via FOXO3/FBXO22/p53 axis.

Fig. 6
figure 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

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SQFZ regulated LA-mediated DDP resistance in NSCLC nude mice

To investigate the role of SQFZ in tumorigenesis of DDP-resistant NSCLC in vivo, 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. As shown in Fig. 7A and B, LA promoted tumor growth. With the increase of SQFZ concentration, tumor growth was inhibited, and the inhibitory effect was enhanced with the increase of concentration. TUNEL staining showed that with the increase of SQFZ concentration, the tissue apoptosis rate was increased (Fig. 7C). In addition, as the SQFZ concentration increases, Ki67 and FBXO22 levels in tissues were decreased, but FOXO3 and p53 expressions were increased (Fig. 7D). To sum up, SQFZ suppressed LA-mediated DDP resistance in vivo.

Fig. 7
figure 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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Discussion

SQFZ is a pure Chinese medicine made mainly from Codonopsis pilosula and Astragalus membranaceus [13]. It has a good effect in the adjuvant treatment of various malignant tumors, mainly playing the role of anti-cancer and enhancing immune function [15]. NSCLC is the most common malignant tumor in lung cancer. Its incidence is increasing year by year, which seriously affects the quality of life of patients [28]. Formononetin is one of the main SQFZ components. Recently, it has been found that Formononetin effectively counteracted osimertinib resistance in NSCLC cells [29]. A recent study has shown that combination of SQFZ and DDP enhanced NSCLC cell cycle arrest in G2/M phase, accompanied by upregulation of p53 expression and induced mitochondrial apoptosis [30]. This study provided the first demonstration that SQFZ inhibited LA-triggered DDP resistance in NSCLC via affecting FBXO22/p53 axis through FOXO3, suggegsting a potential role of SQFZ in chemoresistance of NSCLC. Thus, this study provided some new insights into the function and mechanism of SQFZ with regard to regulating the NSCLC response to chemotherapy.

At present, NSCLC is mainly treated by chemotherapy to kill cancer cells and prolong the survival time of patients [31]. Chemotherapy with DDP is a classic regimen to treat patients with NSCLC, but overall efficacy of advanced NSCLC is not ideal due to factors such as drug resistance and toxic side effects [32]. The effective rate of DDP chemotherapy is less than 40%, and the median survival time of patients is only 7–9 months [33]. Therefore, how to enhance the efficacy of chemotherapy, reduce chemotherapy resistance and side effects is of great importance. This study aimed to explore the effect of SQFZ combined with DDP on NSCLC treatment. We discovered that SQFZ increased the cytotoxicity of DDP in LA-induced DDP resistant A549/DDP cells. SQFZ inhibited LA-triggered DDP resistance in vitro and in vivo both in a concentration dependent manner.

Transcription factor FOXO3 acts as a tumor suppressor in various cancers [34]. Many anticancer drugs reduce drug resistance and enhance the efficacy of drugs by enhancing the expression of FOXO3. For example, by down-regulating Akt phosphorylation, FOXO3 is increased in nucleus, thereby promoting tumor cell apoptosis [35]. In addition, activated FOXO3 can also elevate levels of pro-apoptotic genes Bim and PUMA, thereby accelerating the apoptosis of drug-resistant cells [36]. Recently studies have shown that FOXO3 is a master regulator in NSCLC. For instance, lactate promoted DDP resistance in NSCLC through downregulating FOXO3 [23]. Moreover, FOXO3 sensitized NSCLC cells to DDP-induced apoptosis [37]. In current study, FOXO3 was found to decrease in NSCLC patients. We also showed that knockdown of FOXO3 significantly abrogated effect of SQFZ on LA-triggered DDP resistance in NSCLC, suggesting a crucial role of FOXO3 in mediating cytotoxic effects of SQFZ in NSCLC.

It is worth mentioning that, through Pearson analysis in NSCLC clinic samples, we revealed there was a negative correlation between FOXO3 and FBXO22, a negative correlation between FBXO22 and p53, and a positive correlation between FOXO3 and p53. However, the mechanism of how they functionally linked with each other in NSCLC remains unclear. FBXO22 is a newly discovered F-box E3 ubiquitin ligase and a target gene for p53 [27]. It has been shown that FBXO22 facilitated the polyubiquitination and subsequent inactivation of LKB1 to promote the growth of NSCLC cells [38]. In addition, FBXO22 degraded PD-L1 and sensitized cancer cells to DNA damage [39]. Of note, overexpressed FBXO22 promoted NSCLC growth by suppressing LKB1/AMPK/mTOR axis [38]. Our results identified FBXO22 acted as a direct target of FOXO3 and FOXO3 downregulated FBXO22 expression. Previous studies have shown that p53 induction increased sensitivity to immunotherapy in an autochthonous mouse lung cancer model [40]. LncRNA ITGB2-AS1 promoted DDP resistance in NSCLC by inhibiting p53-mediated ferroptosis [41]. We further showed that FBXO22 decreased p53 level by ubiquitin-dependent degradation. Moreover, reinforced FBXO22 reversed the inhibitory effect of SQFZ on LA-induced DDP resistance in NSCLC, indicating that targeting the FOXO3/FBXO22/p53 pathway may be an attractive approach to enhance SQFZ therapy.

In summary, SQFZ increased the cytotoxicity of DDP in LA-induced DDP resistant A549/DDP cells by upregulating FOXO3. Mechanically, FOXO3 interacted with FBXO22 to inhibit FBXO22-mediated p53 ubiquitination, then in turn enhanced p53 activity and thereby suppressed LA-induced DDP resistance in NSCLC. SQFZ may serve as a potential therapeutic reagent for reversal of DDP chemoresistance in NSCLC.

Data availability

All data generated or analyzed during this study are included in this article. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

CCK-8:

Cell Counting Kit-8

ChIP:

Chromatin Immunoprecipitation

DAB:

Diaminobenzidine

ECL:

Enhanced chemiluminescence

FBXO22:

F-box only protein 22

FOXO3:

Forkhead box O3

FOXO:

Forkhead box subfamily O

IHC:

Immunohistochemistry

IP:

Immunoprecipitation

LA:

Lactic acid

NSCLC:

Non-small cell lung cancer

PI:

Propidium iodide

SQFZ:

Shenqifuzheng injection

SD:

Standard deviation

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Acknowledgements

We thank everyone, who supports us to finish this study.

Funding

National Natural Science Foundation of China (Grant No. 81973735, No. 82174254), Liaoning Province Science and Technology Plan Project (No. 2022-NLTS-14-04) and NATCM’s Project of High-level Construction of Key TCM Disciplines (No. zyyzdxk-2023034).

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Authors and Affiliations

  1. College of Integrated Chinese and Western Medical, Liaoning University of Traditional Chinese Medicine, No. 79, Chongshan East Road, Huanggu District, Shenyang City, Liaoning Province, 110847, China

    Wei Bo, Ning Yu, Chun Wang & Chunying Liu

  2. Pathology Department, Shenyang Key Laboratory for Screening Biomarkers of Tumor Progression and Targeted Therapy of Tumors, Shenyang Medical College, Shenyang City, Liaoning Province, China

    Wei Bo & Xiaokai Wang

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Contributions

Guarantor of integrity of the entire study: Wei Bo. Study concepts: Chunying Liu. Study design: Chun Wan. gDefinition of intellectual content: Wei Bo. Literature research: Wei Bo. Clinical studies: Wei Bo. Experimental studies: Ning Yu. Data acquisition: Xiaokai Wang. Data analysis: Ning Yu. Statistical analysis: Wei Bo. Manuscript preparation: Wei Bo. Manuscript editing: Wei Bo. Manuscript review: Chunying Liu. All authors have read and approved the final version of this manuscript to be published.

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Correspondence to Chun Wang or Chunying Liu.

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Ethical approval

This study was approved by the Ethics Committee of Liaoning University of Traditional Chinese Medicine (No. 202131007).

The Animal Experiments was approved by the Animal Experimental Ethical Inspection of Shenyang Medical University of TCM (No. 2021.9.1).

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Bo, W., Wang, X., Yu, N. et al. Shenqifuzheng injection inhibits lactic acid-induced cisplatin resistance in NSCLC by affecting FBXO22/p53 axis through FOXO3. Respir Res 25, 396 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12931-024-03013-8

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Keywords

Respiratory Research

ISSN: 1465-993X

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