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. 2024 Nov 8;14:27169. doi: 10.1038/s41598-024-73736-7

Enhancing DOX efficacy against NSCLC through UDCA-mediated modulation of the TGF-β/MAPK autophagy pathways

Ying Li 1,2,#, Helian Zhao 3,#, Zhoumin Shen 1, Yao Zheng 2, Yuanyuan Jiang 3, Ying Song 2,✉, Yimin Cai 1,✉
PMCID: PMC11544154  PMID: 39511265

Abstract

Lung carcinoma, predominantly manifested as non-small cell lung cancer (NSCLC), significantly contributes to oncological mortality, underscoring an imperative for novel therapeutic paradigms. Amidst this context, the present investigation delineates the synergistic potentiation of doxorubicin (DOX)—a canonical chemotherapeutic—by Ursodeoxycholic acid (UDCA), a compound with a historical pedigree in hepatobiliary medicine, now repositioned within oncological pharmacotherapy due to its dichotomous cellular modulation—affording cytoprotection to non-malignant epithelia whilst eliciting apoptotic cascades in neoplastic counterparts. This study, through a rigorous methodological framework, elucidates UDCA’s capacity to inhibit NSCLC cellular proliferation and induce apoptosis, thereby significantly amplifying DOX’s chemotherapeutic efficacy. Notably, the co-administration of UDCA and DOX was observed to attenuate DOX-induced autophagy via the modulation of the TGF-β/MAPK signaling axis, a pathway pivotal in mediating cellular survival and autophagic mechanisms. Such findings not only underscore the therapeutic potential of UDCA as a chemosensitizer but also illuminate the molecular underpinnings of its modulatory effects, thereby contributing to the corpus of knowledge necessary to surmount chemoresistance in NSCLC. The implications of this research are twofold: firstly, it offers a compelling evidence base for the clinical reevaluation of UDCA in combinatory chemotherapeutic regimens; secondly, it posits a novel mechanistic insight into the modulation of chemotherapeutic efficacy and resistance. Collectively, these insights advocate for the expedited clinical translation of UDCA-DOX synergy, potentially heralding a paradigm shift in the management of NSCLC, thereby addressing a critical lacuna in contemporary oncological therapy.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-73736-7.

Keywords: Autophagy, Chemotherapy, Non-small cell lung cancer, Ursodeoxycholic acid, Doxorubicin, TGF-β/MAPK pathway

Subject terms: Cancer, Oncology

Introduction

Lung cancer constitutes a major component of cancer-related mortality, claiming the lives of 350 individuals daily, making it one of the deadliest malignancies1. The disease classification encompasses the less prevalent small cell lung cancer (SCLC), which makes up 14% of cases, to the more ubiquitous non-small cell lung cancer (NSCLC), representing a vast 82% of diagnose2. In clinical practice, radiotherapy and chemotherapy remain indispensable as first-line treatment modalities. Doxorubicin (DOX), a premier chemotherapeutic agent, has been employed in the treatment of an array of malignancies, including lung cancer3. Its primary antineoplastic mechanism revolves around the inhibition of both type I and type II topoisomerases and its intercalation into DNA, leading to DNA fragmentation4. Regrettably, tumor cells can enhance their resistance to DOX through mechanisms such as increased autophagy and modulation of the Fas/FasL signaling pathway, which allow them to survive under chemotherapeutic pressure5–7. Therefore, breaking the resistance of tumors to improve the efficacy of chemotherapy is of great importance to clinical treatment.

Autophagy is a crucial mechanism for maintaining cellular homeostasis. It ensures the stability of the intracellular environment by degrading and recycling damaged organelles and proteins, thereby promoting cell survival. When tumor cells face survival pressures, such as chemotherapy drugs, the level of autophagy significantly increases, enhancing the tumor cells’ resistance to DOX.

Ursodeoxycholic acid (UDCA), synthesized through the microbial biotransformation of chenodeoxycholic acid in the intestinal tract, boasts a venerable history of therapeutic application, predominantly for the dissolution of cholesterol gallstones, treatment of primary biliary cholangitis, and various hepato-biliary disorders8,9. Over the past two decades, the oncological potential of UDCA and its synthesized anti-neoplastic derivatives have garnered escalating attention within the scientific community10. Intriguingly, UDCA manifests a dichotomous impact on cellular phenotypes: it furnishes protection to epithelial cells against damage and apoptosis, whilst concurrently promoting apoptosis in neoplastic cells10–13. Preliminary investigations hint at the efficacy of UDCA in ameliorating conditions associated with colorectal14–16, gastric13,17,18, and hepatocellular carcinoma19,20. Yet, empirical studies elucidating its applicability and modulatory effects in non-small cell lung carcinoma (NSCLC) remain conspicuously absent. Therefore, this article aims to investigate the effects of UDCA on non-small cell lung cancer and its related regulatory mechanisms, as well as to study its antitumor efficacy in combination with doxorubicin (DOX), in hopes of providing a new avenue for combating chemotherapy resistance.

Materials and methods

Cell culture and animal care

All the cells used in this study were purchased from the ATCC cell bank. The cell lines A549 and H1299 were cultivated under rigorous conditions using distinct media. Specifically, A549 was cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM), while H1299 was cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium. This culture medium was supplemented with a 1% solution of penicillin-streptomycin to mitigate bacterial contamination, and additionally enriched with a 10% fetal bovine serum (FBS) to support cellular growth and viability. To optimize cell growth kinetics and maintain phenotypic stability, these cell cultures were incubated under a physiologically relevant atmosphere containing 5% CO2 at a consistent temperature of 37 °C.

All animal experiments in this study were conducted in accordance with the ARRIVE guidelines and were approved by the local Institutional Ethics Review Committee, ensuring strict adherence to the established ethical guidelines for animal use and care at Hunan Normal University. Male BALB/c athymic mice, aged 7–8 weeks, were obtained from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China). In order to establish tumor models, 2 × 106 A549 cells were orthotopically inoculated subcutaneously into the right axillary region of the mice, providing the basis for further investigation. Anesthesia for all experiments was administered using inhaled isoflurane, and euthanasia was performed by cervical dislocation following anesthesia.

Cell viability assay

Cell viability was examined in A549, H1299, and BEAS-2B cells using the CCK-8 assay after a 24-hour exposure to various treatment. All cells were cultured in appropriate growth media and plated in 96-well plates at a density of 5,000 cells per well. After overnight attachment, cells were treated with a range of DOX, UDCA, or combined drug concentrations for 24 h. Subsequently, CCK-8 solution was added to fresh medium, and the plates were incubated for an additional 2 h. Absorbance was measured at 450 nm using a microplate reader, and cell viability percentages were calculated relative to the untreated control group for each cell line. IC50 values were determined from the generated dose-response curves.

Drug synergy analysis

A549 and H1299 were treated with varying concentrations of UDCA (0, 0.01, 0.02, 0.05, 0.1, 0.5, 1 mmol/L) and DOX (0, 0.5, 1, 2, 5, 10 µmol/L), both individually and in combination. After 48 h of treatment, cell viability was measured using the CCK-8 assay. The viability data were analyzed using the SynergyFinder tool, applying the Highest Single Agent (HSA) model. Synergy scores were calculated, with values greater than 10 indicating a synergistic effect. The tool generated 2D and 3D synergy maps to visualize the interactions between UDCA and DOX across different dose combinations.

Cell apoptosis detection

In essence, A549 or H1299 cells were seeded onto 6-well plates at a density of 1 × 105 cells per well and allowed to adhere for 6 h. Subsequently, the cells were subjected to distinct experimental conditions in order to evaluate their apoptotic responses. After being treated, the cells were thoroughly washed with PBS, detached, and collected in centrifuge tubes. Apoptosis was subsequently determined using the Annexin V-FITC/PI apoptosis detection kit (Yeasen, Shanghai) in strict accordance with the manufacturer’s guidelines.

Cell cloning assay

Cells were meticulously seeded onto 6-well culture plates, maintaining a density gradient of 1500 cells per well, delineated by specific experimental conditions. Subsequent to the requisite treatments, an extended culturing phase of approximately 14 days ensued or persisted until the preponderance of the individual clones surpassed a cellular count of 50. Interim assessments, encapsulating cellular morphology and vitality, were diligently carried out every third day, synchronized with the periodic replenishment of the culture medium. Thereafter, a judicious rinse with Phosphate Buffered Saline (PBS) prefaced the fixation phase, wherein cells were treated with a 4% paraformaldehyde solution for an interval ranging between 30 and 60 min. A subsequent PBS rinse set the stage for a meticulous staining regimen, employing crystal violet for an optimal duration of 10 to 20 min. Ensuing the staining, cells were subjected to rigorous PBS washes to mitigate excess staining.

Real-time PCR

Total RNA was isolated using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and cDNA was synthesized employing TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA, USA). Quantitative real-time PCR (qRT-PCR) analysis was carried out using 50 ng of cDNA as a template in triplicate for each primer pair on an iCycler iQ™ Real-Time PCR Detection System. The primer sequences used in this study, synthesized by GenScript Biotech Corp (Nanjing, China), are detailed below.

mTgfb1: Forward: 5’- CTAATGGTGGAAACCCACAACG-3’.

mTgfb1: Reverse: 5’- TATCGCCAGGAATTGTTGCTG-3’.

Western blotting

A549 and H1299 Cells were subjected to diverse treatment modalities for 48 h, followed by sequential washing with ice-cold PBS twice on ice. Subsequently, cytoplasmic proteins were extracted using RIPA lysis buffer containing protease and phosphatase inhibitors. Post collection of cell lysates, cell debris was eliminated, and equal amounts of protein were mixed with sample loading buffer, denatured at 95 °C. Protein concentrations were determined utilizing the BCA protein assay kit. Thereafter, proteins were resolved via 12% SDS-PAGE and transferred to PVDF membranes. To prevent non-specific binding, membranes were blocked with a 5% skim milk solution at 4 °C. Consecutively, membranes were probed with primary antibodies specific to the target proteins, followed by incubation with AP-conjugated anti-rabbit secondary antibodies. Lastly, protein bands were visualized using the ECL method, and band intensities were quantitatively assessed employing Image J software. The antibodies used were as follows: Rabbit anti-baclin1(cat. 11306-1-AP; proteintech; USA), Rabbit anti-LC3B (cat. 14600-1-AP; proteintech; USA), Rabbit anti-P62 (cat. ab109012; abcam; UK), Mouse anti-JNK (cat. 66210-1-lg; proteintech; USA), Rabbit anti-P-JNK (cat. ab124956; abcam; UK), Rabbit anti-ERK (cat. 11257-1-AP; proteintech; USA), Rabbit anti-P-ERK (cat.28733-1-AP; proteintech; USA), Rabbit anti-P38 (cat.14064-1-AP; proteintech; USA), Rabbit anti-P-P38 (cat. 28796-1-AP; proteintech; USA) and Rabbit anti-β actin (cat. 66009-1-lg; proteintech; USA).

ELISA assay

To detect TGFβ1, TGFβ2, and TGFβ3 in tumor cell supernatants using ELISA, start by coating 96-well ELISA plates overnight at 4 °C with specific capture antibodies for each TGFβ subtype, diluted in carbonate-bicarbonate buffer. After three washes with PBS, block non-specific binding sites by adding a 2.5% BSA blocking buffer for 0.5 h at room temperature. Post-washing, add the appropriately diluted tumor cell supernatants and TGFβ standards to the wells, incubating for 2 h. Subsequently, introduce the horseradish peroxidase-conjugated secondary antibody and incubate for another hour. After another washing step, apply the 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate, allowing it to react in the dark for up to 30 min. Terminate the reaction using a stop solution. Absorbance is read at 450 nm with an ELISA reader, and concentrations are derived from a standard curve plotted using TGFβ standards.

Cyto-ID autophagy detection kit assay

A549 and H1299 cells underwent designated treatments before being subjected to a 30-minute incubation in phenol red-free DMEM (Enzo, ENZ-KIT175-0050). Subsequently, the cells were stained with Hoechst nuclear dye for 10 min and thoroughly rinsed with PBS. Capturing high-resolution images was facilitated by employing a confocal microscope21.

In vivo anti-tumor therapy

Balb/C nude mice laden with subcutaneous xenograft tumors were systematically partitioned into four experimental assemblies (n = 5), each earmarked for distinct therapeutic interventions: a control group receiving PBS; UDCA at a dosage of 30 mg/kg; DOX at 10 mg/kg; and a synergistic combination of UDCA and DOX. UDCA is administered via direct intratumoral injection, while DOX is administered through intravenous injection in the tail. Therapeutic agents were precisely delivered via intravenous injection at intervals on days 0, 2, 4, and 6, while a rigorous regimen of bi-daily examinations facilitated the assiduous cataloging of body mass and tumor dimensions V = (width^2 × length) / 2).

In vivo survival analysis

Tumor-afflicted mice were divided into four cohorts (n = 7), each subjected to one of the following treatments: PBS, UDCA at a dosage of 30 mg/kg, DOX at 10 mg/kg, or a combination of UDCA and DOX. Intravenous injections were performed on days 0, 2, 4, and 6. Subsequent monitoring of the mice over a 50-day period allowed for the observation of survival rates and any significant clinical symptoms or behavioral changes. This meticulous tracking was instrumental in the construction of a Kaplan-Meier survival curve, highlighting the potential effects of UDCA and DOX in the context of tumor management.

H&E and immunofluorescence staining of tumor tissue

Tumor tissues procured were immersed in 4% paraformaldehyde, followed by a dehydration process employing anhydrous ethanol at 56 °C. Subsequently, the samples were embedded in paraffin and sectioned meticulously. The 2.5 μm sections underwent both hematoxylin and eosin (H&E) staining and immunofluorescence staining with anti-TGF-β antibodies. Imaging was carried out utilizing a Nikon Ni-E microscope (Nikon, Minato, Japan). Moreover, TUNEL and caspase3 assays were conducted in adherence to established protocols. A series of high-resolution images were acquired employing a fluorescence microscope.

Statistical analysis

Quantitative data were presented as the average ± standard deviation (SD). Differences among various groups were evaluated using one-way ANOVA. Levels of significance were categorized as: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Results

UDCA suppresses lung cancer cell proliferation

Our initial analysis focused on the impact of UDCA on the proliferation of A549 and H1299 lung cancer cell lines, further examining its effect on normal HUEVC cells (Fig. 1A-C). Through CCK-8 assays, we observed a clear dose-response relationship, where a concentration of 1 mmol/L UDCA significantly reduced the viability of A549 and H1299 cells to 57.12% and 48.2%, respectively, while the viability of HUEVC cells was 64.88%. This finding elucidates the anti-proliferative properties of UDCA and its potential biosafety.

Fig. 1.

Fig. 1

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(A-C) The viability of Human NSCLC cells A549 H1299 and Human normal lung epithelial cells BEAS-2B was measured by CCK8 in cells treated with different concentration of UDCA. (D) Flow cytometry was employed to assess apoptosis in A549 and H1299 cells. (E, F) Quantitative assessment of the proportion of cells across distinct apoptotic stages. (G) Photos of the colony formation assays for both cell variants. (H, I) Semi-quantitative analysis of the area of colony-forming regions.

Cell status, including live, early apoptotic, late apoptotic, and necrotic cells, was assessed using Annexin V and PI staining. This delineation across different cell states revealed the effect of UDCA concentration on the apoptosis of A549 and H1299 lung cancer cells. With increasing UDCA concentrations, this effect was intensified, reducing the survival rate of A549 cells to 60.5% and H1299 cells to 62.27% (Fig. 1D-F), highlighting UDCA’s potential therapeutic promise in lung cancer intervention.

To further corroborate these results, we conducted clonogenic assays, which demonstrated a consistent dose-dependent inhibitory pattern (Fig. 1G-I). As the concentration of UDCA increased, a noticeable reduction in crystal violet-stained cell colonies was observed, further confirming UDCA’s potent anti-proliferative capability.

UDCA enhances chemotherapy efficacy and inhibits DOX-induced autophagy

We subsequently investigated the therapeutic efficacy of combining different concentrations of UDCA with varying concentrations of DOX on two human-derived non-small cell lung cancer (NSCLC) cell lines, A549 and H1299. The concentration gradients for UDCA were 0, 0.01, 0.02, 0.05, 0.1, 0.5, and 1 mmol/L, while those for DOX were 0, 0.5, 1, 2, 5, and 10 µmol/L. Using the CCK-8 assay to assess cell viability across different treatment conditions, we found that UDCA, at both low and high doses, consistently enhanced the cytotoxic effects of DOX on A549 and H1299 cells. Compared to the inhibition of tumor cell proliferation observed with either UDCA or DOX alone, their combination yielded significantly improved outcomes (Fig. 2A and B). SynergyFinder results show that the 2D (Fig. 2C) and 3D (Fig. 2D) synergy maps of UDCA and DOX highlight synergistic (red) and antagonistic (green) dose regions. The HSA synergy score of 21.011 indicates a strong synergistic effect, as scores above 10 suggest synergy. Further apoptosis assays also confirmed the cytotoxic effects of UDCA and DOX on lung cancer cells and their enhanced therapeutic efficacy when used in combination (Fig. 2E-G). The clonogenic assay, which assesses the clonogenic capability of the targeted cell lines, further supported the above findings (Fig. 2H-J).

Fig. 2.

Fig. 2

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(A, B) The viability of Human normal NSCLC cells A549 H1299 and was measured by CCK8 in cells with different treated. The 2D (C) and 3D (D) synergy maps of UDCA and DOX in SynergyFinder highlight the synergistic and antagonistic dose regions in red and green, respectively. (E). Flow cytometry was employed to assess apoptosis in A549 and H1299 cells with different treated. (F, G) Quantitative assessment of the proportion of cells across distinct apoptotic stages. (H) Photos of the colony formation assays. (I, J) Semi-quantitative analysis of the area of colony-forming regions. (K) The CYTO-ID Autophagy Detection Kit was used to examine autophagy in A549 cells under various treatments. (L) Quantitative assessment of the number of CYTO-ID puncta.

Numerous studies have confirmed the role of autophagy in chemotherapy resistance22,23, prompting us to further investigate the impact of UDCA and DOX on tumor autophagy to explore their potential mechanisms. The results showed a stark contrast between the weak fluorescence in the control group and the pronounced proliferation of green fluorescent spots in the DOX group, the latter indicating DOX-induced autophagy. However, in the combination group, this autophagy response was significantly inhibited (Fig. 2K and L). Therefore, we believe that UDCA may possess the potential to reduce DOX-triggered autophagy in non-small cell lung cancer, inhibiting this crucial resistance mechanism.

Autophagy manipulation with UDCA in cancer cells

As depicted in Fig. 3A-D, our data clearly delineate the relationship between UDCA concentration and Beclin1 expression, consistent with the trend of a decrease in the LC3B II/LC3B I ratio. The expression level of P62 increased with the rising concentration of UDCA.

Fig. 3.

Fig. 3

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(A) Western blotting was used to detect the expression levels of Beclin1, P62, and LC3B in response to various concentrations of UDCA treatment. (B-D) Quantitative assessment of the expression levels of Beclin1, P62, and LC3B. (E) Autophagy in A549 cells, subjected to varying concentrations of UDCA treatment, was evaluated using the CYTO-ID Autophagy Detection Kit. (F) Quantitative evaluation of the CYTO-ID puncta count.

To further validate these results, we performed immunofluorescence staining to observe the formation of autophagosomes. Consistent with the Western blot (WB) results, UDCA significantly reduced the formation of cellular autophagosomes (Fig. 3E and F). These findings help to elucidate the sensitizing effect of UDCA on DOX therapy and further suggest UDCA’s potential for clinical treatment.

UDCA suppresses autophagy via TGF-β inhibition

Our previous research indicated that autophagy might play a significant role in the effects of UDCA and DOX on non-small cell lung cancer. Transforming Growth Factor β (TGF-β) plays a major role in cell growth, differentiation, apoptosis, and immunity24,25. Numerous studies have confirmed that elevated levels of TGF-β are effective promoters of tumor cell autophagy26.

To explore the relationship between UDCA and TGF-β, we utilized ELISA to measure the expression of TGF-β1, TGF-β2, and TGF-β3 in the supernatant of A549 and H1299 cells (Fig. 4A, B), as well as the impact of UDCA or DOX on their expression. To further validate our research findings, we examined the changes in TGF-β1 mRNA and protein expression under different treatment regimens through PCR (Fig. 4C, D) and Western blotting (Fig. 4E-H). This analysis further confirmed UDCA’s ability to suppress the expression of TGF-β1.

Fig. 4.

Fig. 4

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(A, B) ELISA of TGF-β1, TGF-β2 and TGF-β3 proteins in supernatants of A549 and H1299 cells with different treatment. (C, D) real-time PCR analysis of TGF-β1 mRNA expression in A549 and H1299 cells with different treatment. (E, F) Western blot demonstrations of TGF-β1 protein alterations in treated A549 and H1299 cells. (G, H) Quantitative assessment of the expression levels of TGF-β1.

Our findings reveal that DOX induced the expression of related TGF-β, while UDCA significantly reduced the levels of TGF-β. This suggests a potential mechanism by which UDCA inhibits autophagy.

UDCA inhibits TGF-β-induced MAPK phosphorylation in lung cancer cells

Previous research has unequivocally illuminated the pivotal role of non-Smad signaling pathways, specifically the mitogen-activated protein kinase (MAPK) pathway—a sophisticated triptych comprising extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and the p38 MAPK cascade—in the orchestration of autophagy, often guided by transforming growth factor beta (TGF-β)27,28. With this foundation, we investigated UDCA’s potential regulatory influence in MAPK pathway, employing Western blotting to scrutinize UDCA’s effect on the phosphorylation of ERK and JNK29,30.

As shown in Fig. 5A-C, at a concentration of 0.5mmol/L, UDCA significantly reduced the phosphorylation of ERK and JNK. The addition of exogenous TGF-β was able to reverse the inhibitory effect of UDCA on the phosphorylation of ERK. Cellular fluorescence staining further demonstrated that the suppressive effect of UDCA on autophagy in A549 cells was restored by TGF-β (Fig. 5D, E).

Fig. 5.

Fig. 5

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(A) Western Blot analysis evaluated the influence of UDCA on the phosphorylation of ERK and JNK under conditions with or without the addition of TGF-β. (B, C) Quantitative assessment of the phosphorylation of ERK and JNK. (D) The CYTO-ID Autophagy Detection Kit was employed to assess autophagy levels in A549 cells. (E) Quantitative analysis of the count of CYTO-ID puncta. (F) Western Blot assesses the phosphorylation levels of ERK, JUK, and P38 under different treatment conditions. (G-I) Quantitative analysis of the phosphorylation levels of ERK, JUK, and P38.

We further investigated the impact of DOX and UDCA on the MAPK pathway, revealing that DOX alone enhances the phosphorylation of ERK and JNK, whereas UDCA inhibits this effect, thus suppressing the activation of the MAPK pathway (Fig. 5F-I). This further validates the sensitizing effect of UDCA on DOX and its potential mechanism.

Combined effects of UDCA and DOX in inhibiting tumor growth in vivo

To explore the effects of UDCA and DOX in vivo, we established a xenograft tumor model with A549 cells in BALB/c nude mice. When the tumor size reached an appropriate volume (V = 100 mm^3), we divided the experimental mice into four groups (Fig. 6A). Consistent with our in vitro experimental results, both UDCA and DOX were capable of inhibiting tumor growth independently. However, the combined treatment of UDCA and DOX more significantly suppressed tumor progression (Fig. 6B, C and F-I) and extended the survival time of the treated mice (Fig. 6D), but did not affect the body weight of the mice (Fig. 6E), indicating the safety of the combined treatment strategy. Fluorescence results from TUNEL and caspase 3 staining, along with H&E histological staining of the obtained tumor samples, further confirmed our research findings (Fig. 6O), showing that the combination treatment group more prominently induced apoptosis in the tumor region.

Fig. 6.

Fig. 6

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(A) Drug administration flowchart. (B) Photographs of tumors from different mouse groups. (C, F-I) Growth curves of mouse tumors. (D) Changes in mouse body weight over time. (E) Survival curve. (J) Immunofluorescence staining to detect TGF-β expression in tumor tissues. (K) Western Blot analysis of autophagy in tumor tissues. (L-N) Quantitative analysis of Beclin-1, LC3B, and P62. (O) H&E staining of mouse tumor tissues and fluorescence dual staining of caspase3/tunnel.

We also evaluated the impact of different treatment methods on the expression of TGF-β in tumor tissues (Fig. 6J). DOX significantly promoted the expression of TGF-β. However, following UDCA treatment, the expression of TGF-β was notably reduced. Analysis results from Western blot further validated the expression levels of autophagy-related proteins under different treatment methods (Fig. 6K-N). This also further validated in vivo that UDCA may inhibit the enhancement of autophagy induced by DOX via the TGF-β pathway, thereby amplifying the cytotoxicity of DOX against non-small cell lung cancer cells.

Discussion

In recent years, though both immunotherapy and targeted therapies have made significant strides in advancing the treatment landscape for non-small cell lung cancer (NSCLC)5,31–33, combination chemotherapy remains the frontline therapeutic approach for patients diagnosed with advanced NSCLC34. Enhancing the efficacy of chemotherapy and reducing tumor resistance are crucial for clinical treatment.

The intricate interplay of autophagy in the context of non-small cell lung cancer (NSCLC) garners significant attention, especially chemotherapeutic resistance mechanisms35,36. Autophagy is a sophisticated intracellular regulatory pathway, in response to a myriad of stressors, encompassing nutrient deprivation and organellar damage37–39. The precise role of autophagy in tumorigenesis and tumor progression remains a subject of contention40–42. While a plethora of studies posit autophagy at basal levels as a tumorigenic suppressor, functioning through the degradation of damaged cellular components and proteins and thereby maintaining cellular homeostasis, there exists a growing body of literature delineating its facilitative role in the sustenance and propagation of advanced-stage cancers22,43,44. Particularly under strenuous conditions marked by hypoxia and nutrient scarcity, autophagy emerges as a cellular salvage mechanism45. In the backdrop of chemotherapy with agents like DOX, a prevailing hypothesis underscores autophagy as a cytoprotective mechanism in a diverse array of tumors, potentially fostering resistance46,47. This mechanism likely involves modulation of autophagy-associated proteins, including ATGs, Beclin-1, mTOR, p53, KRAS, among others, and impinges on pathways like mTOR, PI3K, MAPK, EGFR, HIF, and NFκB48. Notably, TGFβ has the capability to activate the MAPK pathway, amplifying the phosphorylation of ERK, JNK, and P38, thereby potentiating autophagy and concomitant cancer progression49.

Ursodeoxycholic acid (UDCA) is traditionally known for its role in promoting bile flow and alleviating the formation of gallstones, while its potential for anticancer effects has gradually been unveiled. A plethora of studies have demonstrated its capability to inhibit cancer cell proliferation, induce apoptosis, and regulate various molecular pathways associated with tumorigenesis. For instance, UDCA has been shown to induce cell cycle arrest and apoptosis in melanoma cells through mitochondria-related pathways13. In prostate cancer, it has been demonstrated to effectively enhance apoptosis induction and sensitize cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)50. Furthermore, UDCA shows promise in disrupting the cancer stem cell phenotype in colorectal and pancreatic cancers, reducing self-renewal, and inhibiting tumor initiation51. Therefore, a comprehensive analysis of UDCA’s mechanism of action against tumors to explore its anticancer potential is crucial for its clinical translation.

Our research illuminates the capacity of UDCA to modulate the phosphorylation of the MAPK pathway instigated by DOX, thereby inhibiting autophagy in NSCLC cells and countervailing DOX resistance. Specifically, UDCA not only attenuates tumor cell proliferation but also modulates the phosphorylation of the MAPK pathway intricately associated with ERK, JNK, and P38. This modulation manifests in altered expression ratios of Beclin1, LC3B-II/LC3B-I, and levels of P62, consequently diminishing DOX-induced autophagy. The introduction of TGF-β appears to mitigate this effect. In this study we found that the synergistic interplay of UDCA and DOX in vivo significantly downregulates TGF-β expression, effectively curtailing tumor growth without eliciting additional toxicity. This unveils a possible mechanism wherein UDCA, by stifling TGF-β induced autophagy, can potentiate the sensitivity of NSCLC cells to DOX, thereby augmenting its therapeutic efficacy (Fig. 7).

Fig. 7.

Fig. 7

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This schematic depicts the mechanism by which UDCA suppresses autophagy through modulation of the TGFβ/MAPK signaling pathway, thereby sensitizing tumor cells to Doxorubicin (DOX) treatment.

In summary, our study provides reliable evidence for UDCA as a promising chemosensitizer, enhancing the therapeutic potential of DOX by inhibiting autophagy. Although our understanding of chemotherapy has rapidly evolved in recent years, studies on exogenously regulated drugs significantly enhancing the clinical efficacy of chemotherapy remain scarce52–54. To further elucidate the regulatory mechanisms related to autophagy, TGF-β, and the treatment response in NSCLC, it is necessary to conduct more in-depth research using more representative in vivo models, paving the way for potential clinical translation in the future.

Conclusion

In conclusion, our research demonstrates the role of UDCA in combination with DOX in the treatment of non-small cell lung cancer, with UDCA inhibiting DOX-induced autophagy enhancement through the regulation of the TGF-β-MAPK pathway as a potential mechanistic pathway for enhancing the efficacy of chemotherapy. This provides important preliminary evidence for the clinical therapeutic translation of UDCA.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (8MB, docx)

Acknowledgements

This research received no external funding.

Author contributions

Y.L. and H.Z. designed and conducted the experiments, analyzed the data, and wrote the main manuscript text. Z.S. and Y.Z. prepared Figs. 1, 2 and 3 and assisted in data interpretation. Y.J. conducted the literature review and assisted in the preparation of supplementary materials. Y.S. and Y.C. supervised the project, provided critical revisions, and corresponded with the journal. All authors reviewed and approved the final manuscript.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally to this work.

Contributor Information

Ying Song, Email: 2502870731@qq.com.

Yimin Cai, Email: 158460107@qq.com.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (8MB, docx)

Data Availability Statement

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

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