Synergistic Anti‐Tumor Effect of Toosendanin and Paclitaxel on Triple‐Negative Breast Cancer via Regulating ADORA2A‐EMT Related Signaling

Triple negative breast cancer (TNBC) is an aggressive cancer with very poor prognosis. Combination therapy has proven to be a promising strategy for enhancing TNBC treatment efficacy. Toosendanin (TSN), a plant‐derived triterpenoid, has shown pleiotropic effects against a variety of tumors. Herein, it is evaluated whether TSN can enhance the efficacy of paclitaxel (PTX), a common chemotherapeutic agent, against TNBC. It is found that TSN and PTX synergistically suppress the proliferation of TNBC cell lines such as MDA‐MB‐231 and BT‐549, and the combined treatment also inhibits the colony formation and induces cell apoptosis. Furthermore, this combination shows more marked migratory inhibition when compared to PTX alone. Mechanistic study shows that the ADORA2A pathway in TNBC is down‐regulated by the combination treatment via mediating epithelial‐to‐mesenchymal transition (EMT) process. In addition, the combined treatment of TSN and PTX significantly attenuates the tumor growth when compared to PTX monotherapy in a mouse model bearing 4T1 tumor. The results suggest that combination of TSN and PTX is superior to PTX alone, suggesting that it may be a promising alternative adjuvant chemotherapy strategy for patients with TNBC, especially those with metastatic TNBC.


Introduction
Triple-negative breast cancer (TNBC) is pathologically defined as estrogen receptor (ER)-, progesterone receptor (PR)-, and HER2-negative malignancy. It accounts for 12-17% of all breast cancers. [1] Although general improvements have been made in DOI: 10.1002/adbi.202300062 the past several decades in the management of breast cancer, TNBC still represents a daunting challenge. TNBC is known to have increased aggressiveness, and higher risk of local invasion and distant metastasis, therefore higher frequency of recurrence, shorter disease-free survival, and poorer overall survival. [2] Chemotherapy is the most common therapeutic option for treating TNBC following surgery, with paclitaxel (PTX) being the most frequently used chemotherapeutic agent. However, the associated adverse effects and inevitable drug resistance are frequently observed with the PTX-based treatment. [3,4] Hence, treatment for TNBC still remains an unmet medical need, and alternative therapeutic strategies are urgently needed. One such strategy to circumvent this challenge is to combine adjuvant therapy with cytotoxic chemotherapeutic drugs such as PTX to enhance the treatment efficacy while reducing the associated side effects. [5] Agents derived from natural sources have been demonstrated to play a promising role in cancer treatment, owing to their better safety and efficacy profile, and they are believed to be a good source for novel drug discovery. [6][7][8] The use of herbal medicines in cancer patients purportedly aim to alleviate the side effects and complications during chemotherapy or radiotherapy, to enhance Combination index (CI) of TSN and PTX co-treatment at a ratio of 2:1 on the cell lines. CI < 1, = 1, and>1 indicates synergism, additive effect and antagonism, respectively. Data presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001. the body's general immunity, thereby improving the quality of life of patients and prolonging survival. [9,10] Accumulating evidence has shown that combination therapy with natural compounds has the potential to maximize the efficacy, while minimizing drug resistance and reducing side effects. [11] These findings implicate that novel natural compounds with good therapeutic potential could be used to develop novel remedies against TNBC. [4] Toosendanin (TSN), a tetracyclic triterpenoid isolated from the dried mature fruit of Melia toosendan Sieb et Zucc, exhibits analgesic, insecticidal and anti-inflammatory activities. [12] It is commonly used for the treatment of stomachache, hepatodynia, gastritis and mastitis. [13] Preclinical studies indicate that TSN is also effective against a variety of human cancers such as gastric cancer [14] and hepatocellular carcinoma. [15] In addition, it is well tolerated and has the ability to reverse the chemo-resistance on anticancer drug Adriamycin. [16] Thus, TSN has good potential as a novel adjuvant drug for cancer chemotherapy and chemoprevention. However, the potential synergistic effects of TSN with PTX on TNBC has not yet been reported till now. Therefore, it is worthwhile to characterize whether TSN could strengthen the anti-cancer activity of PTX and to elucidate its action mechanism.
In this study, we found that TSN and PTX could act synergistically to suppress TNBC cell proliferation, migration and induce apoptosis. These findings suggest that the combination of TSN and PTX may be an attractive strategy for TNBC treatment.

TSN Effectively Inhibits Cell Proliferation and Acts Synergistically with PTX on TNBC Cell Inhibition
TSN has previously been reported to significantly inhibit the growth of different cancer cells. [14,[17][18][19][20][21][22][23] We then assessed the effect of TSN on the viability of human MDA-MB-231 and BT-549 TNBC cells, and also investigated whether a combination of TSN and PTX elicits a synergistic effect on cell proliferation in TNBC cells. We treated MDA-MB-231 and BT-549 cells with indicated concentrations of TSN, PTX or their combination, and then cell viabilities were assessed by CCK8 assay. Our results showed that TSN effectively inhibited the proliferation of TNBC cells, and the combination treatment more markedly reduced the cell viability when compared to treatment with either TSN or PTX alone in MDA-MB-231 cells (Figure 1c-f), suggesting that TSN combined with PTX enhanced the anti-proliferative effects of PTX against MDA-MB-231 cells. The drug combination indices (CI) for the 50-80% growth inhibition were then calculated using the Loewe Additivity model. Isobologram analysis showed that the CI for every combination treatment was < 1 (Figure 1g,h), indicating significant synergistic effects of these combination treatments. These results clearly demonstrated that combination of TSN with the lower doses of PTX elicited greater inhibition in the cancer cell growth than either TSN or PTX alone.

TSN Enhances the Effects of PTX-Induced Apoptosis in TNBC Cells
We then performed the colony formation assay that allowed us to repeat the treatments for a relatively long period of time. The combination of TSN and PTX effectively inhibited the formation and growth of colonies of TNBC cells (Figure 2a-c). Furthermore, we examined if the combination of TSN and PTX could function in a similar way in enhancing apoptosis in MDA-MB-231 and BT-549 cells. We first conducted Hoechst 33342 nuclear staining and Annexin V-FITC/PI flow cytometry assays to determine whether TSN could have synergistic cytotoxic effect with PTX on TNBC cells and whether the effects were associated with the induction of apoptosis. As shown in Figure 2d, cells treated with the combination of TSN and PTX exhibited typical apoptotic features such as cellular shrinkage and cell wall blebs. Apoptotic induction was further confirmed by Annexin V-FITC/PI staining, and the results showed that the combination treatment enhanced the Annexin V-positive cell populations in MDA-MB-231 and BT-549 cells (Figure 2e,f). In addition, we analyzed the apoptotic pathway by western blot. We found that the combination treatment decreased the protein expression levels of pro-caspase-3 and PARP, we also observed coordinated appearance of cleaved forms of caspase-3 (Figure 2g,h), indicating an augmented effect on apoptotic induction. These results amply indicate that the enhanced apoptosis as a result of the combined TSN and PTX treatment was closely associated with the caspase-dependent pathway.

TSN Sensitizes the Anti-Metastatic Effects of PTX in TNBC Cells
Transwell migration and scratch assays were performed to examine the effect of TSN, PTX and their combination on the migratory ability of MDA-MB-231 and BT-549 cells. As shown in Figure  3a-c, the cells migrated from the upper to the lower surface of the chamber was significantly inhibited in the combination treatment group when compared with either TSN or PTX alone. In addition, the results of the scratch assay indicated that the combination treatment of TSN and PTX markedly decelerated the healing of TNBC cell wound gaps at 24 h (Figure 3d,e). Taken together, the above results verified that TSN significantly enhanced the migration inhibitory effect of PTX in MDA-MB-231 and BT-549 cells, and the combination of TSN and PTX more markedly suppressed the cell migration than either TSN or PTX alone.

TSN Decreases the PTX-Induced Upregulation of ADORA2A
In view of the above finding that TSN has multiple effects on the TNBC, we then explored the molecular mechanism underlying the action of TSN. Based on our previous RNA-seq analysis, we identified several differentially expressed genes (DEGs) induced by TSN, and found that ADORA2A was significantly altered by TSN treatment (data not shown). To determine whether the ADORA2A was involved in the TSN-mediated synergistic antitumor effect of PTX in TNBC cells, we first treated TNBC cells with PTX, and found that the level of ADORA2A was elevated following treatment with PTX (Figure 4a,b). Then we treated the cells with PTX in the presence of TSN, and found that TSN inhibited the PTX-mediated upregulation of ADORA2A in the two cell lines (Figure 4c,d). Concordantly, the mRNA level of ADORA2A was also significantly decreased as a result of the combined TSN and PTX treatment ( Figure 4e). In addition, the endogenous ADORA2A immunostaining further confirmed that combination treatment reduced the upregulation of ADORA2A protein level induced by PTX in MDA-MB-231 and BT-549 cells (Figure 4f  downregulate the PTX-mediated accentuation in ADORA2A expression in TNBC cells.

TSN Alters the Expression of EMT Process Related Protein
EMT is known to be associated with cancer metastasis and antitumor drug resistance in breast cancer. [24] TWIST1 is a basic helix-loop-helix transcription factor that induces EMT, and is thought to be involved in EMT initiation in various pathological environments. [25] To examine whether the TSN-mediated reduction of ADORA2A in TNBC cells was a result of EMT process inactivation, we treated these cells with TSN and PTX. The result showed that combined treatment of TSN and PTX markedly suppressed the expression of the PTX-induced EMT marker proteins, such as N-cadherin, -catenin, SNAI1 and TWIST1 with slightly increased E-cadherin expression in MDA-MB-231 (Figure 5a,b) and BT-549 cells (Figure 5c,d), suggesting that TSN was able to downregulate the ADORA2A expression caused by PTX, which is believed to contribute, at least partially, to EMT process.

TSN Treatment Synergizes with PTX Therapy on TNBC Cells In Vivo
Following the in vitro findings, we subsequently examined the possible synergy between these two drugs in vivo. We used  4T1-fluc-red cells to construct orthotopic TNBC mouse model for real-time monitoring of tumor growth. We first evaluated the in vivo anti-TNBC activity of the combination treatment using fluorescent imaging in this orthotopic TNBC tumor model ( Figure  6a). We performed in vivo imaging system (IVIS; MS FX PRO, USO) 10 min after injection of D-luciferin ( Figure 6b). The 4T1luc tumor-bearing mice were randomly divided into four groups and respectively received treatment of 0.05% DMSO (sham), TSN (3 mg kg −1 ), PTX (5 mg kg −1 ) or their combination to determine the therapeutic efficacy of these treatments in inhibiting orthotopic tumor growth ( Figure 6d). As shown in Figure 6e,f, after a 25-day treatment regimen, the combination groups exhibited the highest inhibitory effect on TNBC tumor growth among all treatment groups. The quantitative measurement of the tumor mass revealed that TSN, PTX and their combinations all significantly reduced the tumor weight, as compared with the vehicle control ( Figure 6g). Besides, as shown in Figure 6h, the H & E staining depicted a dramatic reduction in cell density in combination treatment group, as compared with all other treatment groups.
To further elucidate the molecular mechanisms of TSN and PTX co-treatment in vivo, we used immunohistochemistry (IHC) to measure the levels of ADORA2A and EMT related markers, such as vimentin and TWIST1, we found that the expression of ADORA2A was greatly decreased (Figure 6i). For EMT marker proteins, we also observed the declined levels of vimentin and TWIST1 in the tumor tissues after TSN-PTX co-administration ( Figure 6i). Overall, the combination treatment group exhibited significantly lower fluorescent area and lower tumor growth when compared to the treatment by either agent alone.

The Effect of TSN on the Toxicity of the Conjugated PTX
We further investigated the toxicity of TSN and PTX alone or their combination in mice. As shown in Figure 6j, the body weight of the mice did not change significantly during the experimental period. Furthermore, TSN induced no treatment-related abnormality concerning the histological morphology ( Figure  S1a, Supporting Information). In addition, we evaluated the liver and renal toxicity of TSN treatment via blood chemistry analysis. At the end of the treatment (day 25), we collected the serum from each dosage group and measured AST and ALT levels to test liver toxicity. As shown in Figure 6k,l, among all dosage, none of them caused any elevated level in either AST or ALT, as compared with the vehicle group. Similarly, we evaluated the renal toxicity of TSN by measuring the levels of UA, Cre and BUN, and we observed no renal toxicity among all treatment groups (Figure 6m-o). These in vivo data demonstrated that the www.advancedsciencenews.com www.advanced-bio.com Taken together, the results from the in vivo TNBC orthotopic xenograft experiment confirmed our in vitro studies and demonstrated that combination treatment more effectively suppressed tumor growth than either monotherapy alone.

Discussion
TNBC represents a refractory subtype of breast cancer with the poorest prognosis due to its inherent resistance to chemotherapy. The standard treatment strategy for TNBC includes a combination of chemotherapy, surgery, and radiation therapy based on the clinicopathological features of the disease. [26] Although the outcome of the treatment has improved steadily over the past decades, the curative rate for TNBC is unlikely to improve further with current therapies, especially for patients with metastatic TNBC. PTX is the most commonly used chemotherapeutic drug for the late stages of TNBC, but is cytotoxic and poses serious adverse effects to the patients. Besides, PTX is also prone to development of drug-resistance, thus limits its therapeutic application among TNBC patients. Therefore, in an effort to find effective alternative strategies that can enhance the therapeutic efficacy while minimize the systemic toxicity of chemotherapeutic agents, we explored the possible synergistic effects of phytochemical-based anti-cancer agents when combined with anticancer drugs on combating TNBC. [5] The past decades have witnessed significant progress in the use of herbal medicines or natural products for the treatment of various human malignancies. Approximately 50% of the new drugs introduced since 1994 were either natural products or derivatives thereof. [6][7][8]27,28] Nature still is the most attractive source for new anticancer drug discovery and development. [6,8] TSN is a naturally occurring triterpenoid compound that possesses anti-tumor effects in several cancers. [14,21,23,29,30] However, it remains unclear up to now whether TSN could exert synergistic effects with PTX on TNBC. Our results for the first time showed that TSN could significantly inhibit the proliferation, migration, and induce apoptosis in TNBC cells. Moreover, it could markedly potentiate the anti-cancer effect to PTX. Furthermore, the in vivo study showed that the combination of TSN and PTX significantly attenuated the tumor growth when compared to either agent alone in an orthotopic model of mice bearing 4T1-flucred TNBC cells. It should be stressed that combination of TSN and PTX carried with low toxicity, suggesting a promising safety profile.
Metastatic progression and tumor recurrence pertaining to TNBC are undoubtedly the leading cause of breast cancerrelated mortality. [26] EMT is a well-documented mechanism that describes the alteration of cancer cell adhesion to promote metastasis, which is more active in TNBC compared to other breast cancer subtypes. Therefore, TNBC is more prone to metastasis. [26,31,32] Several studies have reported that the mesenchymal marker protein vimentin is required for TNBC invasion and metastasis. [33] Additionally, a set of pleiotropically acting transcriptional factors, such as SNAI1, Slug and TWIST1 have been reported to orchestrate the EMT and related migratory processes during embryogenesis. Our studies uncovered that TSN could inhibit the migration of TNBC cells and then enhanced the PTX-mediated migration inhibition.
Mechanically, based on our previously RNA-seq analysis of TSN-treated MDA-MB-231 cells, 1860 DEGs was identified and induced by TSN (unpublished data). By further analysis of these DEGs, we found that Adenosine A2a receptor (ADORA2A) was significantly affected by TSN (data not shown).
As a novel immunotherapeutic target gene, immunecheckpoint ADORA2A was reported to be highly expressed in recurrent head and neck squamous cell carcinoma (HNSCC), and HNSCC tissues collected after chemotherapy. [34][35][36] In addition, research has shown that ADORA2A could play a crucial role in metastasis and EMT process. Activation of ADORA2A can promote EMT in gastric cancer cells, leading to increased cell migration and invasion. [37] Inhibition of ADORA2A signaling could increase immune effector functions to retard metastatic progression. [38,39] But little is known about its association with TNBC. Hence, we elucidated that whether metastasis inhibition by the combination of TSN and PTX was mediated by the regulation of ADORA2A signaling in TNBC. We found that ADORA2A expression was increased in a dose-dependent manner following PTX treatment, whereas it decreased with cotreatment of TSN, at both protein and mRNA levels. We further tested the expression of EMT related marker protein after combination treatment of TSN and PTX, and the result showed that Vimentin, SNAI1, TWIST1 and -catenin were also markedly downregulated, as compared with the PTX alone treatment.
Collectively, this study for the first time unraveled that TSN plus PTX markedly exerted synergistic anti-cancer effects on TNBC cells. The in vivo study further confirmed that TSN potentiated the anti-tumor effect of PTX. The underlying mechanisms of TSN with PTX were through activation of the intrinsic apoptotic pathway to induce apoptosis and further to inhibit metastatic TNBC through downregulating ADORA2A-EMT associated signaling pathway. The results obtained from this study would provide valuable experimental evidence for further developing TSN into a new adjuvant therapy in conjunction with PTX for TNBC treatment.
Cell Culture: Human breast cancer cells MDA-MB-231, BT-549, and murine breast cancer cells 4T1 cells were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). All of the cell lines were cultured in Dulbecco's Modified Eagle medium (DMEM) supplied with 10% fetal bovine serum (FBS) and 10 U mL −1 penicillin-streptomycin in an incubator at 37°C, 5% CO 2 .
Cell Survival and Colony Formation Assays: Cells were seeded in 96 well plates overnight and then exposed to drugs for 24 or 48 h. Cell viabilities were determined using CCK8 assay (Cell Counting Kit-8; Beyotime, China) according to the manufacturer's instruction. The absorbance was measured at 490 nm using a FLUOstar OPTIMA microplate reader (BMG Labtech, Offenbury, Germany).
The interaction effect between TSN and PTX was analyzed using the combination index (CI). The CI value calculated by Loewe Additivity For colony assay, cells were plated into 6-well plates at 500 cells per well, and the plates were incubated overnight. Subsequently, cells were treated with drugs for 24 h and then changed to drug-free growth medium and further incubated for 14 days before being fixed with 4% formaldehyde and stained with 0.5% crystal violet.
FACS Apoptosis Assay: For the cell apoptosis assay, Annexin V/PI staining kit (BD Pharmingen, USA) was used to detect the cell apoptosis as per the manufacturer's instruction. Apoptotic cells were counted by a Cytomics FC500 flow cytometer (Beckman Coulter, Fullerton, CA, USA). All the data generated by flow cytometer were analyzed using the Kaluza software (Beckman Coulter). Apoptosis was also evaluated by using Hoechst 33342 (Invitrogen, Carlsbad, CA, United States) staining according to the manufacturer's protocol.
Transwell Migration and Scratch Assay: Cell migrations were estimated using transwell chambers (Corning, NY, USA) with a pore size of 8 μm. A total of 3 × 10 4 cells resuspended in 100 μL serum-free medium with TSN, PTX alone and their combination would be seeded in the upper chamber, and 600 μL of complete medium was added to the lower chamber. The chamber was incubated in 5% CO 2 at 37°C for 24 h. Then, the cells on the upper surface of the chamber were removed using cotton swabs, and the migrated cells on the bottom surface fixed in 4% paraformaldehyde, stained with 0.1% crystal violet and scored under a light microscope in five random fields.
In the Scratch assay, TNBC cells were seeded in 24-well plates and cultured until they reached 90% confluence. A scrape was made through the confluent monolayer with a sterile plastic pipette tip. The plates were then washed twice with PBS and incubated in fresh complete medium at 37°C in the presence or absence of the indicated concentrations of TSN, PTX or TSN+PTX. Cells were monitored at 0 and 24 h after scratching and images were captured using an inverted phase contrast light microscope at 50× magnification.
Western Blot Analysis: Proteins were extracted from cell lysates or tumor tissues with lysis buffer, supplemented with a complete protease inhibitor cocktail (Thermo Scientific, USA). The concentrations of the protein extracts were determined by bicinchoninic acid (BCA) test. Protein samples were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. After blocking nonspecific binding with TBS/T (0.1%) containing 5% non-fat milk for 1 h at room temperature (RT), the membranes were then incubated overnight at 4°C with primary antibody diluted in 3% BSA in TBS/T (0.1%). The membrane was washed with TBS/T 4 times to remove the unbound antibody and then incubated with the secondary antibody (HRP-conjugated goat anti-mouse IgG or goat anti-rabbit IgG, 1: 2500; Cell Signaling Technology) for 1 h at room temperature. Protein bands were visualized with an ECL kit (Invitrogen, Carlsbad, CA, USA). All antibodies and their dilutions used in this experiment were listed in the Additional file 1, Table S1 (Supporting Information).
Immunofluorescence Staining: MDA-MB-231 and BT-549 cells were plated on coverslips and allowed to adhere overnight and exposed to TSN (1.5 μm), PTX (0.5 μm), or their combination for 24 h. Then the cells were washed twice with PBS and subsequently fixed with 4% paraformalde-hyde/PBS for 20 min. The cells were permeabilized with 0.5% TritonX-100 for 15 min, followed by blocking with 5% BSA (in PBS) for 30 min. The cells were incubated with ADORA2A primary antibody (1:100; Invitrogen, # PA1-042) in PBS containing 3% BSA overnight, then the cells were incubated with Cy3-labeled secondary antibody (1:1000 dilution, Abcam, United Kingdom) at room temperature for 1.5 h in dark. Nuclei were stained with DAPI (Santa Cruz, Texas, USA). Images were visualized using an inverted fluorescent microscope (Carl Zeiss, Germany).
Animal Xenograft and Treatments: Six-week-old female BALB/c nude mice were obtained from the Laboratory Animal Services Centre, The Chinese University of Hong Kong (CUHK). All animals were kept in a pathogen-free environment with free access to food and water. All animal experiments were conducted according to the ethical policies and procedures approved by the Animal Experimentation Ethics Committee of CUHK (Ref. No.: 21-224-HMF).
For the orthotopic implantation, 2×10 5 4T1-fluc-red cells were inoculated in a mammary fat pat of each mouse. One week after tumor implantation (day 1), 24 mice were divided into 4 groups as follows: Group 1, vehicle control (0.05% DMSO, intraperitoneally (i.p.)); Group 2, TSN (3 mg kg −1 , daily, i.p.); Group 3, PTX (5 mg kg −1 twice a week, i.p.), Group 4, the combination TSN (3 mg kg −1 , daily, i.p.) and PTX (5 mg kg −1 twice a week, i.p.). TSN or PTX was given to the mice for 25 consecutive days. At the end of the drug treatment, the mice were sacrificed by cervical dislocation. The tumor tissues were removed and subjected to immunohistochemical analysis and biochemical analysis. Tumor volume was calculated using the following formula: (length × width 2 ) / 2.
Prior to taking the in vivo bioluminescence imaging, the mice were anesthetized with Ketamine-Xylazine (100 -10 mg kg −1 ) and injected i.p. with D-Luciferin (150 mg kg −1 ) weekly for 4 weeks, and bioluminescence imaging (BLI) was captured by an In-Vivo MS FX Pro Imaging System (Bruker BioSpin, Woodbridge, CT, USA) to confirm that all tumors were of similar size.
Immunohistochemistry (IHC): Tumor tissues were fixed in 4% paraformaldehyde and embedded in paraffin. The paraffin blocks were then dissected at 5 μm. For the IHC, the tissues were incubated with ADORA2A (1:50, Invitrogen), Vimentin (1:50, Abclonol) and TWIST1 (1:50, Proteintech) overnight at 4°C. Secondary antibody was then applied for 30 min after washing the slides in three 5-min rounds of 1× PBS. Slides were then subjected to DAB Advanced Chromogenic Kit (Ivitrogen; 8801-4965-72) for 1-3 min to measuring the brownish-red precipitate to visualize the localization of a HRP-conjugated antibody. Counterstaining was performed using hematoxylin. Dehydration was performed via immersing the slides in ethanol and xylene. Slides were mounted using 2 drops of mounting medium.
Statistical Analysis: All data were expressed as the mean ± SEM, and analyzed using one-way ANOVA, followed by post-hoc Bonferroni test to detect intergroup differences. Statistically significance was defined as p < 0.05 or p < 0.01. Statistical analyses were carried out using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA).

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.