Corosolic acid reduces NSCLC cell proliferation, invasion, and chemoresistance via inducing mitochondrial and liposomal oxidative stress

Background: Corosolic acid is a pentacyclic triterpenoid isolated from Lagerstroemia speciosa, which is known to inhibit cancer cell proliferations. Whereas, it is unclear whether this compound has any effect on non-small cell lung cancer (NSCLC) cells. Methods: Here, we cultured A549 and PC9 cells in increasing corosolic acid concentrations, as well as treated mice with a physiologically relevant concentration of the compound, and used metabolomics analysis and high-throughput sequencing to examine its inuences on cell invasion and proliferation, chemoresistance, and metastasis. Results: We found that corosolic acid inhibited cell invasion and proliferation in vivo and in vitro, as well as increase the chemosensitivity of both cell types to cisplatin. Furthermore, we found that corosolic acid destabilized the glutathione peroxidase 2-mediated redox system, which increased mitochondrial and liposomal oxidative stress. Corosolic acid also decreased the targeting protein for Xklp2 level, which inhibited PI3K/AKT signaling and induced apoptosis. In addition, the accumulation of reactive oxygen species dissociated the CCNB1/CDK1 complex and induced G2/M cell cycle arrest. Conclusion: Taken collectively, the data indicate that corosolic acid reduces NSCLC cell invasion and proliferation, as well as chemoresistance, by inducing mitochondrial and liposomal oxidative stress. dose of corosolic acid for 24 h. The data are expressed as the mean ± SD. ***p < 0.001 vs control. (E and F) Flow cytometry detection show the apoptosis of A549-DPP cells after treatment with different dose of cis-platinum. Data are presented as the mean ± SD. ***p < 0.001 vs. control. (G and H) Flow cytometry detection show the apoptosis of A549-DPP cells after treatment with cis-platinum and corosolic acid single or combine. Data are presented as the mean ± SD. *p < 0.05, ***p < 0.001 vs. control. (I and Representative photographs of A549 tumor formation in the xenografts of nude mice. Tumor weight was measured at 30 days post-injection. Data are presented the mean


Background
NSCLC is a main cause of cancer-related mortality in US. Currently, overall ve-year survival rate of metastatic NSCLC patients is <5% [1]. Although signi cant advancements have been made in the diagnosis and treatment of NSCLC in the last two decades, more investigations are demanded to unravel the mechanisms underlying the development and progression of NSCLC. Application of small molecule tyrosine kinase inhibitors, combined with immunotherapy, is reported to be successful in patients with NSCLC, especially in those with metastatic disease. Therefore, there is an urgent need for new therapies [1,2].
Trees and plants are indispensable sources of bioactive compounds that possess anti-cancer properties and exhibit mild side effects [3][4][5]. Corosolic acid, also known as 2α-hydroxyursolic acid, is a pentacyclic triterpenoid that enriched in the Banaba tree (Lagerstroemia speciosa) leaves. Initially, this compound gained attention because of its anti-diabetic properties [6][7][8]. Subsequently, it was found to possess anticancer properties; for instance, corosolic acid can inhibit the development of colorectal cancer through inhibiting HER2/HER3 heterodimerization [8]. In addition, corosolic acid possesses anti-lymphangiogenic and anti-angiogenic properties, as shown in endothelial cells and a colon carcinoma mice model [9]. Corosolic acid is reported to block the transformation and to reactivate Nrf2 in epigenetically-altered TRAMP-C1 prostate cells [10]. However, it is unclear whether this compound has any effect on NSCLC cells. Here, we investigate the corosolic acid effects in lung cancer cells and identify it mechanism of action.

Metabolomics data analysis
We employed A549 cells treated with or not corosolic acid for metabolomics analysis. Our team seeded cells (1 × 10 6 cells/well) in 6-well plates and cultured for 2 days. Thereafter, we washed cells with phosphate buffered saline (PBS) three times, xed them with ice-cold methanol, collected them into microcentrifuge tubes, and homogenized them on ice. Our team centrifuged samples at 14,000 × g for 10 minutes and analyzed them by liquid chromatography-mass spectrometry. Our team processed data with Compound Discoverer Software (Thermo Fisher Scienti c), followed by orthogonal partial least squares discriminant analysis (OPLS-DA) and PCA with SIMCA-P Software (MKS Umetrics AB, Umeå, Sweden).
The metabolite characterization was based upon product ion spectra and accurate masses. We used MetaboAnalyst Software for pathway analysis.
Strand-speci c and high-throughput RNA-Seq library construction Our team extracted total RNA from A549 cells treated with and without corosolic acid as indicated above.
To remove ribosomal RNA, the VAHTS Total RNA-seq (H/M/R) Library Prep Kit (Vazyme Biotech Co., Ltd, Nanjing, China) following the instruction. Thereafter, our team treated RNA that puri ed with RNase R (Vazyme Biotech Co., Ltd, Nanjing, China), followed by puri cation with TRIzol Reagent. The KAPA Stranded RNA-Seq Library Prep Kit (Roche, Basel, Switzerland) was used to prepare libraries, which were subjected to deep sequencing using the Illumina HiSeq 4000 System (Aksomics, Inc., Shanghai, China).

Cell migration assay
Transwell cell culture inserts (Corning Inc., Corning, NY, USA) were employed for cell migration assay. One day after incubation, cells on upper surfaces of membranes were erased with cotton swabs, whereas our team xed those on lower surfaces with ice-cold methanol for 10 min and stained them with crystal violet solution. Our team photographed stained cells with an inverted light microscope (Olympus, Tokyo, Japan) and counted them in ve randomly selected elds.
Cell cycle analysis and apoptosis assay For the apoptosis assay, Annexin V/PI Staining Kit (Abcam, Cambridge, MA, USA) was used. We seeded cells (1 × 10 3 /well) in plates with 96 wells and incubated them with solasonine (15 μg/mL) for 1 day.
Thereafter, we trypsinized cells, washed them with PBS, resuspended them in binding buffer, and simultaneously stained them with annexin V-uorescein isothiocyanate and PI for 15 minutes at room temperature in dark conditions. The apoptotic rate was determined with the NovoCyte 2000 Flow Cytometer.
Wound healing assay PC9 and A549 cells (1 × 10 6 cells/well) transfected with corresponding vectors were seeded in 6-well plates and cultured for 1 days until con uence. We generated wounds by scratching cell layers with 100-mL pipet tips, and cells were cultured for an additional 1, 2, and 3 days, as indicated above. Cells were photographed with a light microscope, and images were compared to respective controls.

Ethynyldeoxyuridine analysis
Our team utilized 5-Ethynyl-2´-deoxyuridine (EdU) Detection Kit (RiboBio, Guangzhou, China) to assess cell proliferation. We seeded PC9 and A549 cells (5 × 10 3 cells/well) in 96-well plates and cultured them for 1 days until con uence. Thereafter, our team added EdU labeling reagent to each well, and incubated plates for another two hours. We xed cells with 4% [wt/vol] paraformaldehyde, permeabilized them with 0.5% [vol/vol] Triton X-100, stained them with Hoechst 33342, and viewed them under an inverted uorescence microscope (Olympus). We calculated EdU incorporation ratio as EdU-stained cell (red uorescence) numbers over the Hoechst 33342-stained cell (blue uorescence) numbers.

Cell proliferation assay
We utilized Cell Counting Kit-8 assay (Dojindo Laboratories, Kumamoto, Japan) to assess cell proliferation following the instruction. We seeded cells (1 × 10 3 /well) in 96-well plates containing 100 μL Complete Cell Culture media (Cell Biologics, Inc., Chicago, IL, USA). Absorbance readings were obtained at 570 nm, and the proliferation rate was determined at 1, 2, and 3 days after transfection.

Colony formation assay
We seeded PC9 and A549 cells (1 × 10 6 cells/well) in 6-well plates, cultured them for 10 days after treated them with corosolic acid for 24 hours. Ten days after treatment, we determined colony number by staining with crystal violet solution and counting stained colonies.
Establishment of tumor mouse model A549 cells (2 × 10 6 ) were resuspended in PBS and injected into the anks of 4-week-old male nude mice. Five days after injection, mice were randomly divided into 2 groups: (i) control (NC) group received PBS (100 mL) and (ii) corosolic acid treatment group received the compound at 5 mg/kg body weight in 100 mL. We determined tumor volume and body weight every 5 days.
For chemotherapy resistance experiment, A549-DDP cells (2 × 10 6 ) were resuspended in PBS and injected into the anks of 4-week-old male nude mice. Five days after injection, we randomly divided mice into 5 groups: (i) NC group received PBS (100 mL), (ii) the corosolic acid treatment group received the compound at 2.5 mg/kg body weight in 100 mL, (iii) the high DDP treatment group received the compound at 3 mg/kg body weight in 100 mL, (iv) the low DDP treatment group received the compound at 1.5 mg/kg body weight in 100 mL, and (v) the corosolic acid + DDP treatment group received 2.5 mg/kg body weight of the former compound and 1.5 mg/kg body weight of the latter compound in 100 mL. We determined tumor volume and body weight every 5 days.
For tumor metastasis experiment, luminescence-labeled A549 cells (1 × 10 5 ) were resuspended in PBS and injected into tail veins of 4-week-old male nude mice. Four weeks after injection, lung metastasis was assessed with a bioluminescence imaging system. The metastatic foci count was determined by hematoxylin and eosin staining of lung tissue cross-sections.
Fluorescence was observed with the ImageXpress HT.ai High-Content Imaging System (Molecular Devices, Sunnyvale, CA, USA).
Mitochondrial membrane potential measurement Our team measured mitochondrial membrane potential with the Mitochondrial Membrane Potential Assay (Beyotime Biotechnology, Shanghai, China). We analyzed cells using the NovoCyte 2000 Flow Cytometer.
Oxidative stress measurement The MitoSOX Red Mitochondrial Superoxide Indicator (Yeasen Biotech Co., Ltd., Shanghai, China) and the C11 BODIPY581/591 Lipid Peroxidation Sensor (MKBio, Shanghai, China) were used to measure oxidative stress in A549 and PC9 cells. Fluorescence was quanti ed with the ImageXpress HT.ai High-Content Imaging System.

Statistical analysis
Statistics analysis was conducted with GraphPad Prism Software (GraphPad, San Diego, CA, USA). Statistician determined statistical differences using Student's t-test for comparisons between two groups and by ANOVA for comparisons among three or more groups. Data are denoted by means ± standard deviation (SD). P-values ≤0.05 are regarded statistical signi cance.

Corosolic acid inhibits NSCLC proliferation
Corosolic acid is known as 2α-hydroxyursolic acid, which has a molecular formula C 30 H 48 O 4 and a molecular weight 472.70 g/mol (Fig. 1A). In in vitro experiments, we found that the proliferation of PC9 and A549 cells decremented dose-dependently after treatment with corosolic acid for 24 hours, with IC 50 values of 27.5 μg/mL and 12.5 μg/mL, respectively (Fig. 1B, C). Thereafter, we treated A549 cells with 25 and 27.5 μg/mL of corosolic acid and PC9 cells with 10 and 12.5 μg/mL of corosolic acid for 1, 2, and 3 days. Control cells were treated with the vehicle. Concordant with the results in Figure 1B and C, our team found that the A549 and PC9 cell proliferation decremented signi cantly after corosolic acid treatment at inhibitory concentrations (Fig. 1D, E). EdU analysis revealed that the IC 50 values for A549 and PC9 cells were 27.5 μg/mL and 12.5 μg/mL, respectively (Fig. 1F-H). Furthermore, we found that the colony number in both cell types after treatment with corosolic acid was lower than that in controls (Fig. 1I-K).
In in vivo experiments, we found that corosolic acid signi cantly reduced tumor growth, as evidenced by decreased tumor volumes and weights ( Fig. 2A-C). By immuno uorescence staining, Ki67 expression was decreased in corosolic acid-treated cells compared to controls (Fig. 2D, E), indicating that corosolic acid can suppress lung cancer cell proliferation.

Corosolic acid inhibits NSCLC cell migration and invasion
We found that the A549 and PC9 cell migration decremented dose-dependently after treatment with corosolic acid for 24 hours, with IC 50 values of 15 μg/mL and 7.5 μg/mL, respectively (Fig. 3A-C).
Furthermore, we found that the invasion of both cell types was decreased after treatment with corosolic acid at inhibitory concentrations ( Fig. 3D-G). Hematoxylin and eosin staining of lung tissue crosssections, coupled with imaging analysis, revealed that corosolic acid decreased the metastatic foci number compared with controls, indicative of decreased pulmonary metastasis (Fig. 4A-D). These results indicate that corosolic acid can inhibit lung cancer cell invasion and migration.

Corosolic acid suppresses NSCLC cell chemoresistance
Flow cytometric analysis revealed that treatment with corosolic acid at inhibitory concentrations for 24 hours induced apoptosis (Fig. 5A-D). The result also found that the apoptosis of A549-DDP cells were increased with the dose increased of cisplatin (Fig. 5E, F). No apoptosis was observed when we treated A549-DDP cells with corosolic acid (5 μg/mL) or cisplatin (6 μg/mL). However, apoptosis was observed when we simultaneously treated A549-DDP cells with both compounds at the indicated concentrations; the percentage of apoptotic cells treated with both compounds increased to 26% compared to controls (Fig. 5G, H). Furthermore, tumor growth was signi cantly decreased in mice treated with cisplatin (3 mg/kg), as well as in those treated with cisplatin (1.5 mg/kg) and corosolic acid (2.5 mg/kg), but not in mice treated with cisplatin (1.5 mg/kg) or corosolic acid (2.5 mg/kg) (Fig. 5I, J).

Corosolic acid induces mitochondrial and liposomal oxidative stress
We found that corosolic acid increased mitochondrial and liposomal oxidative stress in both A549 (27.5 μg/mL) and PC9 (12.5 μg/mL) cells (Fig. 6A, B), with liposomal oxidative stress triggering ferroptosis in lung cancer cells. By immuno uorecence staining, caspase-3 expression in corosolic acid-treated cells was increased compared to controls (Fig. 6C). mRNA high-throughput sequencing, metabolomics data collection and analysis Metabolite analysis revealed that corosolic acid increased the levels of many metabolites, including anatalline, L-carnitine, oxidized glutathione, suberic acid, lysylvaline, acetanilide, and L-arginine, in A549 cells (Fig. 7A, B). We used MetaboAnalyst Software (Fig. 7C) to pinpoint the pathways underlying these alterations in metabolites, and identi ed several pathways altered by corosolic acid such as glutathione (GSH) metabolism; asminoacyl-tRNA biosynthesis; tyrosine, phenylalanine and tryptophan biosynthesis; and D-glutamine and D-glutamate metabolism. Former investigations reported that disruption of glutathione peroxidase 2-mediated redox reactions can increase oxidized glutathione levels, thereby inducing ferroptosis. Flow cytometric analysis showed the mitochondrial membrane potential of A549 after treatment with 27.5 μg/ml corosolic acid with or without added 1 μM ferrostatin-1. The ferroptosis inhibitor, ferrostatin-1, reversed the corosolic acid-induced depolarization of the mitochondrial membrane potential (Fig. 8A). Furthermore, corosolic acid-induced apoptosis was partly reversed after treatment with ferrostatin-1 (Fig. 8B, C).
By high-throughput sequencing, we found that 1812 and 3441 genes in corosolic acid-treated A549 cells were up-and down-regulated, respectively, compared with controls (supplementary materials. 1). Furthermore, the levels of GPX2, AKT1, TPX2, CCNB1, and CDK1 were decreased after treatment with corosolic acid (Fig. 9A, B), and the data were con rmed via RT-qPCR (Fig. 9C). By RT-qPCR, we also found that corosolic acid increased the caspase-3 level.

Discussion
Despite signi cant advancements in the NSCLC diagnosis and treatment, the patient prognosis is often unsatisfactory due to metastasis [11], and there is an urgent need for new therapies. Corosolic acid is a pentacyclic triterpene that is enriched in medicinal plants such as Ugni molinae, Vaccinium macrocarpon, and Eriobotrya japonica [12]. Former investigations reported that corosolic acid can inhibit cancer cell proliferation and invasion, as well as chemoresistance [7,13,14]. While the corosolic acid effect on lung cancer cells is unknown. Current sutdy found that corosolic acid suppressed lung cancer cell proliferation and invasion dose-dependently, with IC 50 values of 27.5 μg/mL and 12.5 μg/mL for A549 and PC9 cell migration, and IC 50 values of 15 μg/ml and 7.5 μg/mL for A549 and PC9 cell invasion. Corosolic acid also halted the cell cycle at G2 phase. Taken collectively, these results indicate that corosolic acid affects cell migration.
Many patients with advanced NSCLC do not respond to treatment, and chemoresistance can be caused by gene alterations, epigenetic alterations, and tumor heterogeneity [15]. Here, the cisplatin-resistant strain, A549-DDP, was used. We found that corosolic acid induced A549 and PC9 cell apoptosis dosedependently. However, no apoptosis was detected in A549-DDP cells treated with corosolic acid (5 μg/mL) or cisplatin (3 μg/mL), although simultaneous treatment with both compounds promoted apoptosis. These results were con rmed in in vivo experiments, which showed that low concentrations of cisplatin and corosolic acid signi cantly inhibited tumor growth, indicating that corosolic acid can increase the sensitivity of lung cancer cells to drugs.
We performed metabolomics analysis to identify the mechanism of action of corosolic acid. We found that corosolic acid altered glutathione metabolism. Previous studies have reported that glutathione metabolism limits oxidative stress in cells , and ROS-induced oxidative damage induces ferroptosis [16,17]. Here, the ROS in A549 cell levels increased after treatment with corosolic acid, whereas ROS production was decreased after treatment with ferrostatin-1.
This study also revealed that corosolic acid can induce mitochondrial oxidative stress and disrupt the mitochondrial membrane potential. Mitochondrial dysfunction can cause apoptosis [18], and the release of su cient amounts of ROS into the mitochondria vicinity can activate local pools of redox-sensitive enzymes, such as the pro-apoptotic protein, caspase-3 [19,20]. The results of high-throughput sequencing and RT-qPCR revealed that treatment with corosolic acid decreased the levels of GPX2, AKT1, TPX2, and CCNB1. A previous study has reported that glutathione peroxidases (GPXs) 1-4 can protect against oxidative challenge, thereby inhibiting in ammation and apoptosis by maintaining the redox balance [21]. Corosolic acid decreased the GPX2 level and induced ferroptosis. Other research illustrated that the CCNB1/CDK1 complex dissociation can suppress cell invasion [22,23], and decreased levels of CCNB1 and CDK1 can induce G2/M cell cycle arrest and promote apoptosis [24]. Here, we found that corosolic acid down-regulated the TPX2 level, which inhibited PI3K/AKT signaling and promoted caspase-3-mediated apoptosis [25]. TPX2, microtubule-associated protein, regulates the dynamics of mitotic spindles, which is indicative of its important role in the cell cycle [26]. In other studies, TPX2 was demonstrated to function as an oncogene, and TPX2 knockdown promoted apoptosis and blocked tumor cell growth [27].

Conclusions
Taken collectively, our results show that corosolic acid destabilized the GPX2-mediated redox system, thereby increasing mitochondrial and liposomal oxidative stress. Corosolic acid also decreased the TPX2 level, inhibiting PI3K/AKT signaling and inducing apoptosis. The accumulation of ROS dissociated the CCNB1/CDK1 complex and induced cell cycle arrest at the G2/M phase, which further promoted apoptosis (Fig. 10). Further mechanistic studies are needed to expand these ndings.     Wound-healing assays showing that solasonine suppressed the invasive capacity of both A549 and PC9 cells after treatment with IC50 concentration of corosolic acid for 0, 24, 48 and 72 h. Data are presented as the mean ± SD. *p < 0.05, ***p < 0.001 vs. control.       after treatment with (27.5 μg/ml) corosolic acid with or without combine ferrostatin-1 (1 μM). Data are presented as the mean ± SD. ***p < 0.001 vs. control. ###p < 0.001 vs. corosolic acid. and 3441 were downregulated in A549 cells after treatment with Corosolic acid when comparted with control cells. (C) RT-qPCR detection show the differentially expressed genes. Data are presented as the mean ± SD. ***p < 0.001 vs. control.

Figure 10
A proposed mechanism of Corosolic acid-induced apoptosis in NSCLC cells.

Supplementary Files
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