Nobiletin enhances Doxorubicin sensitivity in osteosarcoma through ER stress-induced apoptosis mediated by the PI3K/AKT pathway

Abstract

Osteosarcoma (OS) is the most prevalent primary malignant bone tumor, and chemotherapy resistance represents one of the primary challenges in its treatment. Nobiletin (Nob), a natural compound with anti-tumor properties, has an unclear mechanism of action but may increase the sensitivity of tumor cells to chemotherapy agents. This study explores how Nob enhances the effects of Doxorubicin (Dox) on OS cells. Two OS cell lines (143B and U2OS) were used in vitro experiments, where different concentrations of Nob and Dox were combined to treat the cells. The CCK-8 was used to calculate the cell proliferation inhibition rate, and SynergyFinder 3.0 was employed to assess the synergistic concentration of the interventions. Colony formation and cell scratch assays were used to assess the proliferation and migration potential of the cells. Apoptosis levels were identified by flow cytometry, while the expression of proteins related to apoptosis, endoplasmic reticulum stress (ERS), and the PI3K-AKT pathway was detected by Western blotting and immunofluorescence. Moreover, qPCR was employed to measure the expression levels of associated mRNAs. GO and KEGG analyses were used to confirm the differential biological processes and signaling pathways in OS cells. A tumor xenograft model, complemented with H&E and immunohistochemistry staining, were employed to validate the anti-tumor effects and underlying biological mechanisms of the combined treatment with Nob and Dox. The combined treatment of Nob and Dox markedly inhibited both the proliferation and migration of OS cells, exhibiting a pronounced synergistic effect. The combination treatment led to an increased apoptosis rate, characterized by reduced expression of Bcl-2 and elevated levels of Bax and Caspase3. Additionally, the combination group significantly upregulated ERS-related proteins GRP78, CRT, CHOP and ATF6, while the activity of the PI3K-AKT pathway was notably diminished. In a tumor xenograft model, the combination of Nob and Dox significantly inhibited tumor growth, inducing ERS and apoptosis. This study reveals that Nob enhances Dox-induced ERS and apoptosis by inhibiting the PI3K-AKT pathway, thereby increasing the sensitivity of OS cells to Dox. This discovery offers a promising new strategy for the treatment of OS.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-44757-1.

Introduction

Osteosarcoma (OS) is the most prevalent primary malignant bone tumor, primarily affecting adolescents and children, with an incidence rate of approximately 8–11/million/year¹. It is characterized by high malignancy, early metastasis, and poor prognosis, frequently resulting in distant metastases, and the 5-year survival rate remains very low^2,3. Doxorubicin (Dox) is a broad-spectrum antitumor agent that has been used clinically for more than half a century⁴. While it has demonstrated effective results in some tumor types, the growing resistance of OS cells restricts its clinical efficacy. Additionally, the complexity of the resistance mechanisms poses significant challenges in the treatment of OS⁵. Research suggests that the resistance of OS cells may be associated with multiple factors, involving reduced cell membrane permeability that affects the transport of drugs from the extracellular environment into the cytoplasm⁶, as well as alterations in the tumor microenvironment⁷. Furthermore, the use of Dox is associated with considerable toxic adverse effects including cardiotoxicity and bone marrow suppression. These adverse effects not only impair patients’ quality of life but may also restrict the dosages and duration of treatment⁸. Consequently, there is an urgent need to develop novel therapeutic strategies to increase the sensitivity of OS cells to Dox and improve patient outcomes.

Nobiletin (Nob)is a polymethoxylated flavonoid extracted from citrus peels, with previous research showing its significant effects in various biological activities, including anti-tumor, anti-thrombotic, anti-inflammatory, and anti-atherosclerotic properties^9–12. Owing to its low toxicity and minimal adverse effects, Nob holds promise for overcoming resistance to conventional chemotherapeutics^9,13,14. Natural products may offer a potential therapeutic strategy for overcoming chemotherapy resistance in human OS cells¹⁵. While there are reports of Nob’s inhibitory effects and its ability to reverse chemotherapy resistance in breast and gastric cancer cells^16,17, its application in OS research has not been thoroughly explored. Given the chemotherapy resistance observed in OS cells, natural compounds such as Nob may represent a promising therapeutic strategy that deserves further exploration.

Endoplasmic reticulum stress (ERS) refers to a physiological condition that occurs when ER function is compromised due to environmental changes and metabolic stress. The endoplasmic reticulum is essential for maintaining cellular homeostasis and managing ERS through mechanisms such as endoplasmic reticulum-associated degradation and the unfolded protein response (UPR)¹⁸. However, prolonged ERS can result in cell apoptosis, especially within the tumor microenvironment, making the regulation of ERS and its associated pathways a new target for cancer therapy¹⁹. Accumulating evidence indicates that ERS is a key determinant of tumor-cell chemoresistance. On one hand, some chemotherapeutic agents can induce ERS, leading tumor cells to adapt to the stress of the drugs by activating the UPR and enhancing their viability through anti-apoptotic and autophagy pathways²⁰. Conversely, the ongoing presence of ERS may cause tumor cells to develop resistance to chemotherapy, resulting in a marked reduction in treatment effectiveness²¹. ERS can activate multiple signaling pathways, with the PI3K-AKT signaling pathway being particularly important for cell survival, proliferation, and metabolic regulation. Activation of the PI3K-AKT signaling pathway can improve cellular tolerance to ERS, facilitating cell repair and metabolic adaptation, thus mitigating the adverse effects of ERS²².

In this study, we investigated the potential of Nob to induce apoptosis in human OS cells and examined its mechanisms for increasing sensitivity to Dox. Additional studies revealed that the synergistic effects of Nob and Dox is intimately associated with ERS and is critically dependent on the PI3K–AKT pathway. These findings suggest that the combination of Nob and Dox could provide new therapeutic strategies for the clinical treatment of OS. Taken together, these results reveal a crucial role of Nob enhances the sensitivity of osteosarcoma cells to Doxorubicin by inducing endoplasmic reticulum stress and inhibiting the PI3K-AKT pathway.

Materials and methods

Materials

The following reagents were purchased from Shanghai Yishan Biotechnology Co: RPMI-1640 medium (ES-RG001), fetal bovine serum (ES-FBS001), penicillin-streptomycin (ES0015), 0.25% trypsin (ES0014), CCK-8 kit (ES-7011), and PBS buffer (ES-4011). Nobiletin (M4552), Doxorubicin (M1969), and DMSO (ST038-100 ml) were obtained from Aomei Biotechnology (Shanghai) Co., Ltd. The apoptosis detection kit (Goonie, 100-102-100) was sourced from Xin tian weng Biotechnology (Guangzhou) Co., Ltd.

Cells and culture

The human OS cell lines 143B and U2OS (obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. After thawing, the cells were incubated at 37 °C in a 5% CO₂ environment and passaged every 2–3 days, utilizing early passages (from the third to eighth generations) for all experiments.

Cell viability assay and screening for drug synergistic concentrations

143B and U2OS OS cells (0.8 × 10^4 cells/well) were plated in a 96-well plate and allowed to adhere overnight. Afterward, the cells were treated with various concentrations of Nob (0µΜ, 5µΜ, 10µΜ, 20µΜ, 30µΜ) in combination with different concentrations of Dox (0µΜ, 0.125µΜ, 0.25µΜ, 0.5µΜ, 1µΜ, 2µΜ) and incubated at 37 °C for 24 h. The absorbance of each well was measured at 450 nm using a microplate reader to calculate the cell proliferation inhibition rate. The SynergyFinder 3.0 software (https://synergyfinder.fimm.fi/) was utilized to construct dose-response tables and calculate zero interaction potency (ZIP) and highest single agent (HSA) synergy scores, which provids preliminary validation of the effectiveness of the drug combination and facilitated the screening of drug concentrations.

Colony Formation Assay

Logarithmically growing 143B and U2OS cells were seeded at a density of 700 cells/well in 6-well plates. After cell adhesion and growth, the drug-containing medium was replaced every 1–2 days according to the colony size observed under an inverted microscope, and the cells were cultured for 10 days. Cells were fixed with 4% paraformaldehyde for 30 min, followed by staining with 0.1% crystal violet in the dark. The wells were then gently washed with water, and scanned to record colony formation and calculate the colony formation rate.

Cell Scratch Assay

143B and U2OS cells were collected by centrifugation, and adjusted to a concentration of 1 × 10^6 cells/well, then seeded into 6-well cell culture plates. After routine culture until adhesion, a “cross” scratch was made at the bottom of the plates using a 200µL pipette tip. The cells were washed twice with PBS and then treated with the specified drug concentrations (added with 2% FBS) for 24 h. Images were captured using an inverted fluorescent microscope at 0 h, 12 h, and 24 h after drug treatment. The scratch areas were marked with ImageJ software to analyze the drug’s effect on the migration ability of OS cells.

Apoptosis detection

Logarithmically growing cells were treated with specific drugs for 24 h and then digested using trypsin without EDTA. The cells were centrifuged at 1000 r/min for 10 min, then stained with Annexin V-AF647 and PI according to the manufacturer’s instructions, and apoptosis levels were assessed using a flow cytometer.

Bioinformatics analysis

Gene expression data from the GSE12865 dataset in the GEO public database were obtained, which included 12 pediatric OS samples and 2 normal human osteoblasts (HOB). The ClusterProfiler package in R-limma (3.4.2) was used to analyze potential mRNA Gene Ontology (GO) functions and enrich Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, utilizing the KEGG database^23–25 for annotation. PCA plots were constructed using the ggord package in R, while expression bubble plot were illustrated using the ggplot2 package in R.

Western blotting

Proteins were extracted using RIPA (G2002; Servicebio) and quantified with a BCA assay kit (PC0020; Solarbio). The samples were separated by SDS-PAGE and transferred to a PVDF membrane. They were then incubated overnight at 4 °C with primary antibodies against Bcl-2 (BF9103, 1:800, Affinity), Bax (60267-1-1G, 1:800, Proteintech), caspase-3 (db12058, 1:800, Dakewe), GRP78 (AF5366, 1:800, Affinity), CRT (DF3139, 1:800, Affinity), CHOP (AF6277, 1:800, Affinity), ATF6 (DF6009, 1:800, Affinity), PI3K (ET1608-70, 1:800, Huabio), AKT (AF6261, 1:800, Affinity), and p-AKT (AFO016, 1:800, Affinity). After washing with TBST, the membrane was incubated with HRP-conjugated goat anti-mouse IgG secondary antibody (GB23301, 1:5000, Servicebio), and the target protein expression levels were normalized to β-actin (66009-1-lg, 1:20000, Proteintech).

Immunofluorescence (IF) Staining

The expression levels of ERS-related markers GRP78, CRT, ATF6, and CHOP were analyzed using IF. Briefly, osteosarcoma cells on the slides were fixed with 4% paraformaldehyde, and then permeabilized with 0.1% Triton X-100 in PBS. Subsequently, the cells were incubated overnight at 4 °C with primary antibodies against GRP78 (1:200), CRT (1:200), ATF6 (1:200), and CHOP (1:200). On the following day, an appropriate secondary antibody was added and incubated in the dark at room temperature for 60 min. After washing with PBS, the osteosarcoma cells were incubated with DAPI at room temperature. The slides were then photographed using a fluorescence microscope (Nikon, Tokyo, Japan).

Quantitative real-time polymerase chain reaction analysis (qPCR)

RNA was extracted from cells or tumor tissues using the Trizol reagent kit (R401-01, Vazyme) and reverse transcribed into cDNA following the manufacturer’s instructions with the HiScript II Q RT kit (R223-01, Vazyme). Quantitative PCR (qPCR) analysis was conducted using ChamQ SYBR qPCR Master Mix (R311-02; Vazyme) on a CFX 96 Touch (Bio-Rad, Hercules, CA, USA). The expression levels of the specified genes were quantified using the 2 − ΔΔCt method, with β-actin serving as the reference gene. The sequences of all primers are listed in Table 1.

Table 1.

Sequences of the primers used for qPCR.

Name	Primer	Sequence
BAX	Forward	5′-GTA CTC CAG CAA GCA GCA G-3′
	Reverse	5′-GGT CTC TGG CCT CTC TCA G-3′
Bcl-2	Forward	5′-GGC TGG GAG ATG TCT TCT G-3′
	Reverse	5′-TCC CTC TGC TGT TCC TCT C-3′
Caspase-3	Forward	5′-TGA GAC CTC GAA GAC CAA G-3′
	Reverse	5′-GGT TCT TGG GTC TTT GTC C-3′
GRP78	Forward	5′-GCC TGG CTT TCA GAA GAT G-3′
	Reverse	5′-GCT GGT TCT CTT GGT CTT G-3′
CRT	Forward	5′-GCC AAT GAC CCA CCT TGA T-3′
	Reverse	5′-GCA TCT GGT CCA GGT CTT C-3′
ATF6	Forward	5′-GAG CAG GAC CAA GGA GAA G-3′
	Reverse	5′-GCT GCT GTT GGA TTT GGT C-3′
CHOP	Forward	5′-GGA CAA CCA GGA GAC CAA G-3′
	Reverse	5′-GGA GAT GTT GGT GGT GGT G-3′
PI3K	Forward	5′-GGA GAA GGA ACG GAC TCA A-3′
	Reverse	5′-GCA GGA GCA ACC GTC GCA A-3′
AKT	Forward	5′-GCC TCA GAT GGA GAC GAA G-3′
	Reverse	5′-GGA GGT GCT GGT GAT GTT G-3′
β-actin	Forward	5′-CAT GTA CGT TGC TTA CCA TGA C-3′
	Reverse	5′-CTC CTT AAT GTC ACG CAA GTT G-3′

Tumor xenograft model in vivo

The BALB/c nude mice (5weeks, male, Vital River, Beijing, China) were maintained at 20–26 °C with 30–70% humidity under a 12-hour light/dark cycle. Following a minimum of one week’s adaptation to the environment, each mouse received a subcutaneous injection of 1 × 10^7 143B cells into the right axilla. When the tumor reached approximately 50 mm^3, the 20 mice were randomly assigned to four groups: the group NC: saline 3 mg/kg, the group Nob: 5 mg/kg Nob, the group Dox: 3 mg/kg Dox, and the group Nob + Dox: 5 mg/kg Nob alongside 3 mg/kg Dox, which were administered via intraperitoneal injection every two days. The health status of the mice was monitored every three days, and tumor volume was measured, and tumor growth curves were constructed. Fourteen days following drug administration, mice were weighed, euthanized by cervical dislocation, with tumors extracted and stored at -80 °C for subsequent qPCR, Western blotting, and IHC analyses. This study has been approved by the Animal Welfare and Ethics Committee of Affiliated Hospital of Shandong University of Traditional Chinese Medicine (2024-091-01). All the methods and treatments were in accordance with ARRIVE guidelines (https://arriveguidelines.org). All experiments were performed in accordance with relevant guidelines and regulations.

H&E staining

After paraffin sections were dewaxed and hydrated, then the nucleus was stained with hematoxylin for 3 min. Then the tissues were stained for 10 s, washed with distilled water, and immersed in eosin for 2 min. After sequential treatment with distilled water, alcohol, and xylene, images were captured using a microscope.

Immunohistochemical (IHC) Staining

IHC involved excising tumor samples, fixing them in 10% buffered formalin, and embedding the samples in paraffin, which included preparing 4 µM tissue sections were prepared for staining. The primary antibodies GRP78 and CRT were diluted to 1:500 and applied to the sections, which were incubated overnight at 4 °C in a humidified chamber. Secondary antibodies were then added, and nuclei were counterstained, and the sections were dehydrated and coverslipped for interpretation under a light microscope.

Statistical analysis

Statistical analysis was conducted using GraphPad Prism 9.3, with experimental data presented as mean ± standard deviation, and each experiment independently repeated a minimum of three times. One-way ANOVA was performed to assess the significance of differences between groups, utilizing Brown-Forsythe and Welch ANOVA when variances were unequal, with P < 0.05 regarded as statistically significant.

Results

Nob synergizes with dox to inhibit OS cell proliferation and migration

The chemical structure of Nob is shown in Fig. 1A. To investigate whether Nob can inhibit OS cell growth, we first applied different concentrations of Nob and Dox in the 143B and U2OS cell lines for combined intervention. The cell viability changes were assessed using the CCK-8 assay, which demonstrated a significant dose-dependent inhibition of OS cell growth in both cell lines (Fig. 1B).

Fig. 1.

Nob synergizes with dox to inhibit OS cell proliferation and migration. (A) The chemical structure of Nob. 143B and U2OS cell lines were treated with the indicated concentrations of Nob and Dox for 24 h, CCK-8 assessed cell viability (n = 5) (B). SynergyFinder 3.0 calculated ZIP, HSA synergy scores, and Bliss synergy scores (C, D), with ZIP > 10 representing significant synergistic effects (n = 5). (E) Untreated, Nob (20 µM), Dox (1 µM), and Nob (20 µM) + Dox (1 µM) treated for 10 days, with crystal violet staining used to detect colonies (n = 3). (F) Scratch wound assays were performed in 2% FBS medium to suppress proliferation and specifically evaluate migration of 143B and U2OS cells after 12- or 24-hour treatment with Nob and Dox (n = 3).

The data obtained were analyzed using SynergyFinder 3.0 software to calculate the ZIP, HSA synergy score, and Bliss synergy score (Fig. 1C and D). The combination showed strong synergy with a ZIP score of 10.173 and 12.513. Generally, a positive value (> 0) for ZIP, HSA, and Bliss indicates a synergistic effect of the drug combination. The combined application of Nob and Dox showed a significant synergistic effect, with concentrations of 20 µM for Nob and 1 µM for Dox chosen as the optimal synergy doses for subsequent experimental studies.

Additionally, colony formation and cell scratch assays validated that Nob, Dox, and their combination suppressed the progression of OS, with the combination group exhibiting more pronounced inhibitory effects (Fig. 1E, F). These results provide preliminary evidence for the synergistic interaction between Nob and Dox and their inhibitory effects on OS cells.

Nob and dox induce apoptosis in OS cells

Next, flow cytometry was used to evaluate the effects of the combination of Nob and Dox on OS cell apoptosis. The apoptosis levels were highest in the combination treatment and showed statistically significant differences compared to other groups (Fig. 2A, B). Western blotting analysis of apoptosis-related protein expression revealed an upregulation of the pro-apoptotic protein Bax, a downregulation of the anti-apoptotic protein Bcl-2, and a corresponding increase in the apoptosis marker Caspase-3 (Fig. 2C, D). Similarly, qPCR analysis, with β-actin as an internal control, indicated an increase in the relevant mRNA levels of apoptosis-related genes, which was statistically significant (Fig. 2E). These results suggest that the combination of Nob and Dox effectively promotes apoptosis in OS cells.

Fig. 2.

Nob and dox induce apoptosis in OS cells. (A, B) 143B and U2OS cells untreated, treated separately with Nob (20 µM), treated separately with Dox (1 µM), and treated with Nob (20 µM) + Dox (1 µM) for 24 h, followed by staining with Annexin V-AF647/PI and flow cytometry analysis. (C–E) Western blot analysis and qPCR measurements of Bax, Bcl-2, Caspase-3, and β-actin levels in each group post-treatment. Data are expressed as mean ± SD(n = 3). *p < 0.05, **p < 0.01, ***P < 0.001, ****P < 0.0001.

Nob and dox induce ERS

A bioinformatics analysis was first conducted to identify differentially expressed genes (DEGs) between osteosarcoma tissue and normal osteoblasts. GO enrichment analysis revealed that ERS is one of the main biological processes affected. The most significant differences were observed in cellular components such as nucleoplasm, cytosol, and nucleus, while protein binding was identified as the most prominent molecular function (Fig. 3A). KEGG pathway enrichment analysis indicated that the PI3K/AKT/mTOR signaling pathway is the primary pathway involved (Fig. 3B).

Fig. 3.

Nob and dox induce ERS. (A) GO analysis showing biological processes, cellular components, and molecular functions of DEGs between OS and osteoblasts. (B) KEGG enrichment analysis of signaling pathways associated with DEGs. 143B and U2OS cells were untreated or treated with Nob (20 µM), Dox (1 µM), and their combination for 24 h, (C) Western blotting and (D). qPCR measurements of ERS indicators, GRP78, CRT, ATF6, and CHOP. (E) 2 mM of 4-PBA was used with or without Nob (20 µM) + Dox (1 µM) to treat 143B and U2OS cells, with cell viability assessed by CCK-8. (F) Western blotting analysis of apoptotic markers Bax, Bcl-2, and Caspase-3. Data are presented as mean ± SD(n = 3). *p < 0.05, **p < 0.01, ***P < 0.001, ****P < 0.0001.

In this study, the changes in ERS levels following the aforementioned drug interventions were explored. Western blotting results demonstrated a significant accumulation of ERS-related proteins, including GRP78, CRT, ATF6, and CHOP, in the combination treatment group (Fig. 3C). Similarly, the corresponding mRNA levels showed consistent changes (Fig. 3D), indicating that Nob combined with Dox induces significant ERS. Furthermore, the addition of 4-PBA, an ERS inhibitor, reversed the reduction in cell viability caused by Nob and Dox treatment (Fig. 3E), and apoptosis-related protein expressions showed no significant difference compared to the untreated group (Fig. 3F). Collectively, these results suggest that Nob combined with Dox inhibits OS progression by inducing ERS.

Nob enhances dox-induced ERS via the PI3K-AKT pathway

To further elucidate the molecular mechanisms by which Nob and Dox induce apoptosis and ERS, the GO analysis results were considered. The PI3K/AKT pathway is a significant signaling pathway associated with tumor development and ERS. Previous studies have indicated that Nob can regulate tumor apoptosis and resistance by affecting AKT phosphorylation¹⁴. Our Western blotting results showed decreased levels of PI3K, AKT, and phosphorylated AKT proteins in the combination treatment group (Fig. 4A-B). qPCR results indicated a statistically significant reduction in mRNA expression levels of PI3K and AKT (Fig. 4C), suggesting that Nob enhances Dox’s ability to induce DNA damage by inhibiting AKT.

Fig. 4.

Nob enhances dox-induced ERS via the PI3K-AKT pathway. 143B and U2OS OS cell lines were subjected to treatment with Nob (20 µM), Dox (1 µM), or a combination of both for 24 h, with control groups receiving no treatment. Western blot analysis (A, B) and qPCR (C) were conducted to assess the expression levels of key molecules in the PI3K-AKT signaling pathway. The OS were treated with 5 µM IGF-1, both alone and in combination with Nob (20 µM) + Dox (1 µM). The viability of the cells after treatment was evaluated using the CCK-8 assay (D), while IF staining was used to determine the expression levels of ERS markers GRP78, CRT, ATF6, and CHOP (E). Data are expressed as mean ± SD(n = 3), with significance denoted as follows: *p < 0.05, **p < 0.01, ***P < 0.001, ****P < 0.0001.

To validate these findings, the pathway inhibitor IGF-1 was used in conjunction with the combination treatment on osteosarcoma cells. CCK-8 results confirmed that the inhibitor reversed the previous trend (Fig. 4D). To demonstrate that the drug effect mediates through the PI3K/AKT pathway regulating ERS, our IF experiments showed that the high expression state of ERS markers in the combination treatment group was reversed by IGF-1 (Fig. 4E). Thus, Nob combined with Dox induces ERS in OS cells via the PI3K-AKT pathway, thereby inhibiting tumor growth.

Nob combined with dox suppresses OS growth in an ectopic mouse model

For further validation in vivo, the subcutaneous xenograft mouse models were established. Once the tumor volume reached 50 mm³, mice were randomly assigned to four groups as previously described. The results demonstrated that either Nob or Dox alone inhibited OS growth to a certain extent, while the combination therapy exhibited superior efficacy (Fig. 5A-B). Notably, the mice in the Dox group experienced a significant decrease in body weight, which was not observed in the combination therapy group (Fig. 5C). The differences in tumor volume and weight among the four groups were statistically significant (Fig. 5D-E). IHC analysis of tumor tissue indicated elevated levels of ERS markers in the Nob and Dox combination group (Fig. 5F). Subsequent Western blotting and PCR analyses revealed increased levels of apoptosis markers, high expression of ERS indicators, and inhibition of the PI3K-AKT pathway following treatment (Fig. 5G-H).

Fig. 5.

Nob combined with dox suppresses OS growth in vivo. 143B cells were injected subcutaneously into the flank of nude mice. Once palpable tumors formed, the mice received specified drug treatments or combinations (normal saline 3 mg/kg, Nob 5 mg/kg, Dox 3 mg/kg, or Nob 5 mg/kg + Dox 3 mg/kg, administered intraperitoneally every other day for 14 days). Evaluation included mouse images (A), tumor images (B), body weight change curves (C), tumor growth volume curves (D), and tumor weights (E) (Group NC, Nob, Dox + Nob n = 5, Group Dox n = 4).H&E staining and IHC analysis for GRP78 and CRT were performed on tumor sections (F). Western blotting analysis (G) and qPCR analysis (H) provided insights into the expressions of proteins and mRNAs related to apoptosis, ERS, and the PI3K-AKT pathway in the xenograft tumor tissues (n = 3). Data are presented as mean ± SD, with significance denoted as follows: *p < 0.05, **p < 0.01, ***P < 0.001, ****P < 0.0001.

Discussion

With the increasing resistance to commonly used anticancer chemotherapy agents, numerous studies have investigated the potential of herbal medicines as adjuncts to chemotherapy^15,26 and explored their therapeutic advantages. Studies have shown that regular consumption of fruits and vegetables can lower the risk of various cancers²⁷, Nob, a flavonoid rich in citrus fruits, possesses antioxidant properties, inhibits cell proliferation, promotes apoptosis, and modulates the activation of carcinogenic pathways¹⁴. A prior study demonstrated that Nob inhibits the proliferation of gastric cancer cells, resulting in cell cycle arrest and apoptosis, while also inhibiting de novo fatty acid (FA) synthesis and activating ERS²⁸. Natural polyphenol and NOB can synergistically driven by a variety of interactions to achieve synergistic anti-tumor efficacy²⁹. Our initial findings also confirm that Nob exerts inhibitory effects on OS cells in both in vitro and in vivo settings. Utilizing single herbal medicines or combinations to enhance the sensitivity of cancer cells to conventional chemotherapy drugs represents an emerging strategy to address resistance³⁰. This study primarily investigates the potential of using Nob as a therapeutic agent for OS, aimed at enhancing sensitivity to Dox while minimizing the adverse effects of chemotherapy. While Nob alone exhibits inhibitory effects on OS cells, it does not present a significant dosage advantage relative to conventional chemotherapy agents. However, when Nob was combined with Dox, we observed a noteworthy synergistic effect.

Apoptosis is the most prevalent form of cell death, and the anticancer mechanisms of Dox are complex, primarily involving the induction of nucleosome dissociation that causes chromatin damage, the formation of DNA adducts, inhibition of DNA topoisomerase II activity, induction of lipid reactive oxygen species (ROS) accumulation, and mitochondrial dysfunction, all contributing to apoptosis³¹. Certain mutations in tumor cells may allow DNA damage to be repaired under specific conditions, promoting tumor progression and contributing to treatment resistance³². The BCL2 family is pivotal in endogenous apoptosis, essential for maintaining the balance between cell survival and death. When apoptotic signals occur within the cell, pro-apoptotic proteins (such as Bax) are activated, while anti-apoptotic proteins (like BCL2) are bound by sensitizer pro-apoptotic proteins, resulting in outer mitochondrial membrane permeabilization. This leads to the release of apoptotic factors such as cytochrome C and Smac/DIABLO, activating downstream caspase cascades, amplifying death signals, causing protein cleavage, and ultimately triggering apoptosis^33,34.Our results indicate that under the combined application of Nob and Dox, the pro-apoptotic protein Bax is upregulated while the level of the anti-apoptotic protein BCL2 significantly decreases. Correspondingly, the level of Caspase3, indicative of protein structural damage, is elevated. Flow cytometry further verifies that apoptosis can be effectively induced in osteosarcoma cells by the combination of Nob and Dox.

Conversely, ERS plays a crucial role in inducing apoptosis in cancer cells. In OS cells, ERS is maintained at a moderate level to support their survival and growth. However, severe ERS and overload can lead to programmed cell death, including apoptosis³⁵. Bioinformatics analyses reveal that endoplasmic reticulum stress (ERS) is a significant biological process in the progression of OS cells, with the PI3K-AKT pathway identified as one of the critical pathways. ERS levels are a significant factor contributing to chemotherapy resistance in OS cells, and disrupting ER functionality presents an effective strategy to overcome Dox resistance³⁶. GRP78 serves as a crucial regulatory factor in ERS signaling, with its accumulation in the unfolded protein response (UPR) correlated with cancer proliferation, chemotherapy resistance, and patient survival rates³⁷. Calreticulin (CRT), a chaperone molecule involved in ERS, generally maintains homeostasis by functioning as a Ca2 + buffer and aiding in protein folding³⁸. Previous studies have demonstrated that CRT levels are lower in tumor tissues compared to adjacent normal tissues³⁹. Activating transcription factor 6 (ATF6) is a classic downstream transcription factor of the unfolded protein response (UPR). Its upregulation in tumors induces the transcription of C/EBP homologous protein (CHOP)^40,41, downregulates members of the Bcl-2 family, and upregulates BIM, resulting in Bax and Bcl-2 antagonist 1-dependent apoptosis⁴². Experimental results show that the combination of Nob and Dox significantly upregulates ATF6 levels compared to Dox treatment alone, reflecting a heightened state of ERS that may initiate further apoptosis. In subsequent studies, we administered 4-PBA, an ERS inhibitor, and noted an increase in both the proliferative capacity and the levels of anti-apoptotic proteins in OS cells. Accordingly, the expression of Bax and Caspase3 decreased. The PI3K/Akt signaling pathway is implicated in the metastasis and invasion of OS. Inhibiting this pathway intensifies ERS in tumor cells via the downstream mediator mTOR, triggering a cascade of responses that facilitate apoptosis in OS cells. Our rescue experiments affirm that the PI3K-AKT pathway is a crucial avenue through which the combination of Nob and Dox exerts its therapeutic effects.

We further confirmed that the combination of Nob and Dox inhibits tumor growth while mitigating the weight loss induced by Dox alone in vivo experiments. In a study by Song et al., NOB was shown to mitigate cisplatin - induced ototoxicity by reducing apoptosis and oxidative stress⁴³. The attenuation of body-weight loss may be attributed to both diminished tumor burden and the plant-derived agent’s modulation of chemotherapy-induced gut dysbiosis⁴³. IHC results revealed a significant increase in the expression of endoplasmic reticulum stress markers surrounding the cell nucleus. Finally, Western blotting and PCR analyses were conducted to comprehensively compare the effects of the combined treatment on apoptosis, endoplasmic reticulum stress, and the PI3K-AKT pathway. Natural products and their derivatives are gaining attention for their anti-cancer effects mediated by endoplasmic reticulum stress. Combining them with traditional chemotherapy could significantly reduce dosages, enhance tumor suppression, and minimize adverse effects, thereby offering a highly promising cancer treatment strategy.

Our study has several limitations. Firstly, the mechanisms by which Nob inhibits tumors remain complex, with ERS-mediated apoptosis constituting only one aspect as we know. Additionally, we did not assess damage markers in critical organs, such as the heart and kidneys, due to logistical constraints. Although the bioinformatics findings are intended for hypothesis generation, we note that the GSE dataset contains only two control HOB samples, which limits the statistical power of the comparison.

Conclusion

In the present study, we revealed the combination of Nob and Dox for the treatment of OS works by downregulating the PI3K-AKT pathway, inducing ERS, and promoting apoptosis, demonstrating significant efficacy in both in vivo and in vitro models. This offers a novel strategy for combining natural products with classical chemotherapy agents in the treatment of OS, aimed at increasing chemotherapy sensitivity and reducing adverse effects.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

OS

Osteosarcoma

Nob

Nobiletin

Dox

Doxorubicin

ERS

endoplasmic reticulum stress

ER

endoplasmic reticulum

UPR

unfolded protein response

IF

immunofluorescence

IHC

immunohistochemistry

GO

Gene Ontology

KEGG

Kyoto Encyclopedia of Genes and Genomes

BCL-2

B-cell lymphoma-2

Bax

Bcl-2-associated X protein

GRP78

Glucose-regulated protein

CRT

Calreticulin

ATF6

Activating transcription factor 6

CHOP

C/EBP homologous protein

PI3K

Phosphoinositide 3-Kinase

Author contributions

Yongkui Zhang and Wenpeng Xie: Conceptualization, Funding acquisition, Supervision, Project administration, Writing–review & editing. Fei Liu, Daotong Yuan: Writing – original draft, Methodology, Data curation, Formal analysis. Zhimeng Zhang and Rui Gong: Resources, Software, Visualization. Ximin Jin and Chaolu Wang: Validation, Investigation.

Funding

This work was supported by the Natural Science Foundation of Shandong Province (NO. ZR2023MH236, ZR2019MH114), Medical and Health Science and Technology Development Project of Shandong Province (202404070421), Development Plan of Shandong Medical and Health Technology (2019WS580), and Cultivation Project of Qilu Health and Wellness Leading Talent.

Data availability

The raw data supporting the conclusions requests should be directed to the corresponding author at 71000356@sdutcm.edu.cn.

Declarations

Competing interests

The authors declare no competing interests.