Procell Life Science & Technology Co., Ltd. (Wuhan, China) supplied the lung cancer cell lines A549, H1299, and H838. All cell lines were validated by STR profiling and tested negative for mycoplasma. These cells were cultured at 37 °C with 5% CO₂ in RPMI 1640 (lot: WH0021A021, Procell, Wuhan, China) supplemented with 10% fetal bovine serum (FBS; Lot. No. 2364724, Gibco, Carlsbad, CA, USA) and 1% penicillin-streptomycin (Lot. No. 080421211115, Beyotime, Shanghai, China). Cell viability was assessed using a Cell Counting Kit-8 (CCK-8; Lot. No. 23002K1018, APExBIO, China). Rosuvastatin calcium was obtained from Shanghai Yuanye Technology Co., Ltd. (Shanghai, China) with a purity of $>$98%.

Each well of a 96-well plate received 100 µL of complete RPMI 1640 medium with 10% FBS, and each well was seeded with 3 $\times$ 10³ lung adenocarcinoma cells (A549, H1299, and H838). The plate was incubated at 37 °C in a cell culture incubator for 24 h to allow the cells to adhere and stabilize. Following stabilization, the cells were treated and incubated for an additional 72 h. Subsequently, 10 µL of CCK-8 reagent was added to each well, and the plate was incubated for 2 h. Absorbance was measured at 450 or 630 nm using a microplate reader (SpectraMax M5, Thermo Fisher Scientific, Waltham, MA, USA).

An EdU kit was acquired from RiboBio (C10310-1, Lot. No. V1104, Guangzhou, China). After 24 h of treatment with varying concentrations of rosuvastatin, A549, H1299, and H838 cells were harvested. To ensure a density of 3 $\times$ 10³ cells per well, a cell suspension (100 µL) was added to each well of a 96-well plate. After EdU was diluted to 20 µM, 100 µL of this working solution was added to each well to reach a final concentration of 10 µM. For 2 h, the cells were incubated at 37 °C. Following incubation, the culture medium was discarded, and fixation of the cells was carried out with 100 µL of 4% paraformaldehyde for 15 minutes. Following fixation, the cells were rinsed three times with a wash solution and then permeabilized with 100 µL of permeabilization solution for 15 minutes at room temperature. The cells underwent three rounds of washing after the permeabilization solution was removed. Each well was then filled with 50 µL of the reaction solution, followed by incubation for 30 minutes at room temperature in the dark. After discarding the reaction fluid, the cells underwent three rounds of washing. Nuclear staining was performed with 100 µL of 1$\times$ Hoechst 33342 solution, and the cells were incubated for 10 minutes in the dark at room temperature. The cells were photographed and examined using a Leica SP8 confocal fluorescence microscope (Leica Microsystems, Wetzlar, Germany) after removal of the staining solution and three additional washes.

A 24-well Transwell plate was used for the migration assay. The lower chamber received 800 µL of complete RPMI 1640 supplemented with 10% FBS, while the upper chamber received serum-free RPMI 1640 medium (200 µL). After treating with rosuvastatin after 48 h, lung adenocarcinoma cells (A549, H1299, or H838) were seeded in the upper chamber at 4 $\times$ 10⁴ cells per well, followed by incubation at 37 °C for 24 h. An inverted microscope (CKX41, Olympus Corporation, Tokyo, Japan) was used to photograph the fixed and stained migrated cells on the lower surface after the non-migrated cells in the upper chamber were removed.

For the invasion assay, 50 µL of Matrigel diluted 1:4 with serum-free RPMI 1640 medium was added to the upper chamber, and allowed to solidify for 2 h at 37 °C. After that, 50 µL of serum-free RPMI 1640 was used to hydrate the Matrigel-coated chamber for 30 min. The subsequent procedures were identical to those described for the migration assay.

Using a sterile 10 µL pipette tip, a straight-line scratch was made in a six-well plate once the cells attained approximately 80% confluence. After aspirating the medium, unattached cells were removed from the wells by washing them with phosphate-buffered saline (PBS). After adding 2 mL of fresh serum-free medium, the plate was incubated for 15 min at 37 °C with 5% CO₂. Images were acquired at 200$\times$ magnification using an inverted microscope (CKX41, Olympus Corporation, Tokyo, Japan), which was designated the 0-hour time point. The plate was returned to the incubator and observed at subsequent time points (e.g., 24 h and 48 h), with images captured for analysis.

Cell apoptosis was assessed using a Cell Apoptosis Detection Kit (C1065L, Lot. No. 090921211105, Beyotime, China) according to the instructions outlined by the manufacturer. Briefly, cells (4 $\times$ 10⁵) were seeded in six-well plates and cultured overnight. Cells were then treated with rosuvastatin or dimethyl sulfoxide (DMSO) for 48 h. Following treatment, the cells were trypsinized, washed with PBS, and then resuspended in annexin V binding buffer. After the addition of propidium iodide (PI) and fluorescein isothiocyanate (FITC)-annexin V, the samples were left in the dark for 20 min at room temperature. Flow cytometric analysis was conducted using a FACSCalibur flow cytometer (Beckman Coulter, USA).

A Reactive Oxygen Species Assay Kit (ID3130, Lot. No. 320A011, Solarbio, Beijing, China) was used to measure intracellular reactive oxygen species (ROS) levels. Cell seeding and drug treatment were performed using the same procedures as described for the apoptosis assay. After treatment, the culture medium were removed, and cells were incubated with 1 mL of serum-free medium containing DCFH-DA (diluted 1:1000, final concentration 10 µM) at 37 °C for 20 min in a CO₂ incubator. Before flow cytometric analysis, the cells were washed three times with serum-free medium.

For 48 h, H838 cells were exposed to DMSO or 30 µM rosuvastatin. Following treatment, 1 mL of TRIzol reagent was used to lyse the samples. RNA sequencing was performed by Metware Biotechnology Co., Ltd. (Wuhan, China).

SiRNA sequences that target PLK1 were synthesized by GenePharma Co., Ltd. (Suzhou, China). Lipofectamine 2000 reagent (Invitrogen, MA, USA) was used for transfection according to the manufacturer’s instructions. Before transfection, A549, H1299, and H838 cells were seeded to achieve 50–60% confluence on the day of transfection. For transfection, two 1.5 mL Eppendorf tubes were prepared: 250 µL of Opti-MEMI and 5 µL of Lipofectamine 2000 were in Tube A, and 250 µL of Opti-MEMI and 5 µL of siRNA were in Tube B. After 5 min of room temperature incubation, both tubes were mixed and allowed to stand for a further 20 min. Next, 500 µL of the transfection mixture was added to 1.5 mL of medium (serum-free) and carefully mixed. After removing the culture medium from the six-well plate and performing two PBS washes, 2 mL of the prepared transfection mixture was added. Before the subsequent studies, the cells were cultured for an additional 48 h after the medium were changed to a complete serum-containing medium at 6 h. The specific interfering sequences employed for PLK1 knockdown are listed in Table 1.

Following a 48 h treatment with different doses of rosuvastatin in A549, H1299, and H838 cells, total protein was extracted, and Western blotting was performed. The following antibodies were employed in this investigation: anti-CDK1 (abs159273, rabbit, 1:1000, Absin); anti-CDK2 (10122-1-AP, rabbit, 1:1000, Proteintech); anti-cyclin B1 (55004-1-AP, rabbit, 1:5000, Proteintech); anti-cyclin A2 (18202-1-AP, rabbit, 1:5000, Proteintech); anti-GAPDH (60004-1-Ig, mouse, 1:5000, Proteintech); anti-cyclin D1 (AF0931, rabbit, 1:1000, Affinity); anti-P21 (S0B2351, rabbit, 1:1000, Starter); anti-PLK1 (S0B0035, rabbit, 1:10,000, Starter); anti-Bcl-2 (S0B2181, rabbit, 1:1000, Starter); anti-survivin (S0B6183, rabbit, 1:1000, Starter); anti-rabbit secondary antibody (98164S, goat, 1:5000, Cell Signaling Technology); anti-mouse secondary antibody (91186S, goat, 1:5000, Cell Signaling Technology).

Male BALB/c nude mice (3–5 weeks old, weighing 18–20 g) were purchased and kept in a specified pathogen-free (SPF) environment. A549 cells (2.5 $\times$ 10⁶ cells/mouse) were subcutaneously injected into the left axillary region, slightly dorsal, to establish a lung cancer xenograft model. Once tumors reached approximately 200 mm³, the mice were randomly assigned to three groups: control, low-dose treatment, and high-dose treatment, each consisting of five mice. Intraperitoneal injections of 10 mg/kg or 20 mg/kg of rosuvastatin were administered to treatment groups. On the other hand, an equivalent volume of vehicle solution (10% DMSO, 40% PEG300, 5% Tween 80, and 45% normal saline, ~0.1 mL/mouse) was given to the control group. Using the formula V = (L $\times$ W²) $\times$ 0.5, where L stands for tumor length and W for width, the tumor volume was measured every other day. Throughout the experiment, body weight was also tracked. The mice were put to death after the tumor volume reached about 2000 mm³. In this trial, no anesthetics were employed. The mice were put to death by cervical dislocation, which involved fastening the head and neck with one hand while holding the base of the tail with the other. The cervical vertebrae were then quickly and forcefully separated by backward traction. To verify death, the absence of spontaneous breathing and heartbeat was then observed for a minimum of five minutes. Tumors were removed and weighed once the mice’s deaths were confirmed. Half of the tissue was kept in formalin for immunohistochemistry and hematoxylin and eosin (H&E) staining. On the other hand, Western blot analysis and protein extraction were performed on the other half. The Animal Ethics Committee of Soochow University (202312A016) approved and oversaw all animal procedures.

The Human Protein Atlas, GEPIA, and TIMER databases were used to validate differential PLK1 expression between tumor and adjacent normal tissues in patients with human lung adenocarcinoma. Additionally, survival analysis was performed using these platforms.

Every experiment was conducted three times. The statistical significance between the two groups was evaluated using an unpaired Student’s t-test. The mean $\pm$ SD was used to express the results. GraphPad Prism 8.0 (GraphPad Software, Inc., San Diego, CA, USA) was employed for the statistical analyses, and p 0.05 was deemed statistically significant.

The chemical structure of rosuvastatin is shown in Fig. 1A. A549, H1299, and H838 lung adenocarcinoma cells were treated with rosuvastatin at various doses, and cell viability, cytotoxicity, and growth inhibition were assessed using the CCK-8 assay. The results demonstrated that rosuvastatin reduced cell viability in a dose-dependent way. Following treatment for 72 h, the IC50 values for A549, H1299, and H838 cells were 159.1 µM, 150.4 µM, and 47.5 µM, respectively (Fig. 1B). To further evaluate its effect on proliferation, an EdU assay was performed. The results showed that rosuvastatin treatment significantly decreased the number of EdU-positive A549, H1299, and H838 cells (Fig. 1C–E). Collectively, these findings indicate that rosuvastatin effectively suppresses the proliferation of lung adenocarcinoma cells.

Fig. 1.

Rosuvastatin inhibits the proliferation of lung adenocarcinoma cells. (A) Chemical structure of rosuvastatin. (B) The effect of rosuvastatin on the proliferation of the A549, H1299, and H838 human lung adenocarcinoma cell lines was assessed using the CCK-8 assay. (C–E) EdU uptake was used to assess the frequency of EdU-positive cells with or without rosuvastatin treatment. The drug concentrations for each cell line were as follows: A549 (0, 50, and 100 µM); H1299 (0, 40, and 80 µM); and H838 (0, 15, and 30 µM). A 48-hour treatment time point was used for all cell lines. Scale bar = 200 µm. *p 0.05, **p 0.01, ***p 0.001.

Transwell migration and invasion assays revealed that rosuvastatin significantly suppressed the migratory and invasive abilities of A549, H1299, and H838 cells in a dose-dependent manner (Fig. 2A–C). The wound-healing assay demonstrated that as the drug concentration increased, wound closure was significantly delayed in A549, H1299, and H838 cells, further confirming the anti-migratory effects of rosuvastatin (Fig. 2D–F). These results show that rosuvastatin suppressed lung adenocarcinoma cell invasion and migration.

Fig. 2.

Rosuvastatin inhibits the migration and invasion of lung adenocarcinoma cells. (A–C) Transwell migration and invasion assays assessing the changes in the migration and invasion abilities of A549, H1299, and H838 cells after treatment with different concentrations of rosuvastatin. The drug concentrations for each cell line were as follows: A549 (0, 50, and 100 µM); H1299 (0, 40, and 80 µM); and H838 (0, 15, and 30 µM). A 48-hour treatment time point was used for all cell lines. Scale bar = 200 µm. (D–F) Wound-healing assay-based evaluation of the effects of different concentrations of rosuvastatin on the migration and invasion of A549, H1299, and H838 cells. The drug concentrations and treatment timing were the same as in (A–C). Scratch wounds were imaged and measured at defined time points for each cell line: A549 (0, 48, and 72 h); H1299 (0, 24, and 48 h); and H838 (0, 24, and 48 h). Scale bar = 200 µm. ***p 0.001.

The pro-apoptotic effects of rosuvastatin were evaluated by flow cytometry (Fig. 3A–C), and Western blotting was used to assess apoptosis-related protein expression. The findings demonstrated that in A549, H1299, and H838 cells, rosuvastatin administration resulted in the dose-dependent downregulation of Bcl-2 and survivin (Fig. 3D–F). Moreover, rosuvastatin treatment significantly increased ROS levels in these cells (Fig. 3G–I). This suggests that rosuvastatin may induce apoptosis in lung adenocarcinoma cells, at least in part, by promoting oxidative stress. Overall, our data show that rosuvastatin can trigger apoptosis and inhibit the progression of lung adenocarcinoma.

Fig. 3.

Rosuvastatin promotes apoptosis in lung adenocarcinoma cells. (A–C) Flow cytometry-based quantification of the frequencies of apoptotic A549, H1299, and H838 cells following treatment with various rosuvastatin concentrations for 48 h. (D–F) Changes in apoptosis-related Bcl-2 and survivin protein expression in lung adenocarcinoma cells following treatment with various concentrations of rosuvastatin for 48 h, as detected by Western blotting. (G–I) Effects of treatment with different concentrations of rosuvastatin for 48 h on reactive oxygen species (ROS) levels in A549, H1299, and H838 cells. ns, no significance; *p 0.05, **p 0.01, ***p 0.001.

RNA sequencing (RNA-seq) analysis of H838 cells treated with 30 µM rosuvastatin for 48 h was conducted further to explore the molecular mechanisms underlying rosuvastatin’s effects. A total of 1700 differentially expressed genes (DEGs) were discovered using a criterion of FDR 0.05 and $|$log2FC$|$ $\ge$1, with 761 upregulated and 939 downregulated (Fig. 4A). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were used to evaluate the functions of these DEGs. The results revealed that cell cycle-related pathways were the most significantly enriched (Fig. 4B), indicating that rosuvastatin may primarily modulate the cell cycle to exert its anti-tumor effects. After 48 h of rosuvastatin treatment, cell cycle-related marker proteins were measured by Western blotting in A549, H1299, and H838 cells. The cell cycle-related CDK1, CDK2, cyclin A2, cyclin B1, and cyclin D1 proteins were all downregulated in response to treatment in H1299 and H838 cells, while P21 was increased, as seen in Fig. 4C,D. These findings imply that rosuvastatin may primarily affect lung cancer cell growth by preventing progression through the cell cycle.

Fig. 4.

Rosuvastatin modulates the cell cycle in lung adenocarcinoma cells to exert anti-tumor effects. (A) A volcano plot highlighting the differentially expressed genes (DEGs) identified by RNA sequencing (RNA-seq) in the transcriptional profiling of rosuvastatin-treated H838 cells. (B) Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses of DEGs identified by RNA-seq. (C,D) CDK1, CDK2, cyclin A2, cyclin B1, cyclin D1, and P21 protein levels were detected in H1299 and H838 cells by Western blotting before and after rosuvastatin treatment. **p 0.01, ***p 0.001.

We then examined the transcriptional patterns of rosuvastatin-treated H838 cells using these RNA-seq results to find DEGs associated with the cell cycle. Fig. 5A displays the heatmap that was produced. This conclusion was further supported by immunohistochemical data from the Human Protein Atlas, which showed elevated PLK1 expression levels in lung cancer tissues (Fig. 5B). Furthermore, analysis using the GEPIA and TIMER databases revealed that PLK1 was significantly overexpressed in lung adenocarcinoma tissues and negatively correlated with patient survival (Fig. 5C–E). We treated A549, H1299, and H838 cells with varying concentrations of rosuvastatin for 48 h to further explore its impact on PLK1. Next, we collected protein and total RNA for Western blot and qRT-PCR analysis, respectively. The findings demonstrated that rosuvastatin significantly reduced PLK1 expression at the mRNA and protein levels (Fig. 5F–I).

Fig. 5.

PLK1 may be a key target for rosuvastatin’s anti-cancer effects. (A) Heatmap showing DEGs related to the cell cycle identified by RNA-seq analysis of H838 cells treated with rosuvastatin. (B) Immunohistochemical staining from the Human Protein Atlas showing PLK1 expression in lung adenocarcinoma and adjacent normal tissues. (C,D) GEPIA database analysis results show that PLK1 is significantly overexpressed in lung adenocarcinoma patients (C) and is negatively correlated with survival (D). (E) TIMER database analysis results show differential PLK1 expression between lung adenocarcinoma tissues and adjacent normal tissues. (F) qRT-PCR analysis of PLK1 mRNA levels in A549, H1299, and H838 cells treated with increasing concentrations of rosuvastatin for 48 h. The concentrations used were 0, 50, and 100 µM for A549; 0, 40, and 80 µM for H1299; and 0, 15, and 30 µM for H838. (G–I) Western blot analysis of PLK1 protein expression in A549 (G), H1299 (H), and H838 (I) cells treated with the indicated concentrations of rosuvastatin for 48 h. *p 0.05, **p 0.01, ***p 0.001.

To investigate whether PLK1 is a key mediator of rosuvastatin’s anti-cancer actions, we used RNA interference to inhibit PLK1 expression in A549 and H838 cells. Western blotting and qRT-PCR were used to confirm the knockdown’s effectiveness (Fig. 6A–D). Two highly effective siRNA sequences were selected for PLK1 knockdown in A549 and H838 cells. PLK1 knockdown significantly reduced the growth of these lung adenocarcinoma cells, as shown by EdU staining. Furthermore, rosuvastatin and PLK1 knockdown showed synergistic effects, resulting in an even greater reduction in proliferation (Fig. 6E, F). Compared with the control group, the PLK1-silenced lung adenocarcinoma cells showed a significant decrease in their ability to migrate and invade in Transwell assays. Although rosuvastatin treatment further inhibited migration and invasion, its effects were partially attenuated in PLK1-knockdown cells, suggesting a potential additive interaction (Fig. 6G,H). Taken together, these findings demonstrate rosuvastatin inhibits lung adenocarcinoma cell proliferation, migration, and invasion by downregulating PLK1. This demonstrates that PLK1 is an essential mediator of rosuvastatin’s anti-cancer effects.

Fig. 6.

PLK1 knockdown reduces the proliferation, migration, and invasion of lung adenocarcinoma cells. (A–D) qRT-PCR and Western blot validation of PLK1 knockdown efficiency in A549 and H838 cells transfected with PLK1-targeting siRNA. (E,F) An EdU assay-based analysis of the effect of PLK1 knockdown and rosuvastatin treatment on the proliferation of lung adenocarcinoma cells. Scale bar = 200 µm. (G,H) Transwell migration and invasion assays of A549 (G) and H838 (H) cells after PLK1 knockdown, rosuvastatin treatment, or their combination. Scale bar = 200 µm. ns, no significance; *p 0.05, **p 0.01, ***p 0.001.

To evaluate rosuvastatin’s in vivo anti-tumor activities, a BALB/c nude mouse xenograft model was established (Fig. 7A). After the tumors grew to a volume of around 200 mm³, the mice were randomly divided into three groups (n = 5 per group) and given daily intraperitoneal injections of vehicle control, 10 mg/kg rosuvastatin, or 20 mg/kg rosuvastatin. The xenograft tumors were removed, and the mice were euthanized after 12 days of treatment. The results demonstrated that rosuvastatin significantly inhibited lung adenocarcinoma cell growth in vivo (Fig. 7B–D), with no significant changes in body weight observed among the treated mice (Fig. 7E). Western blotting and immunohistochemical staining analyses of the excised tumor tissues were conducted to further investigate the effect of rosuvastatin on PLK1 expression in xenograft tumors. Both studies confirmed the significant downregulation of PLK1 at the protein level following rosuvastatin treatment (Fig. 7F,G).

Fig. 7.

Rosuvastatin inhibits lung adenocarcinoma tumor growth in vivo. (A) Schematic overview of the establishment of a xenograft tumor model in nude mice. Created using Adobe Illustrator 2024. (B) Images of excised tumors after treatment (n = 5). (C) Endpoint tumor weights. (D) Tumor volume was monitored every 2 days during the treatment period. (E) Changes in murine body weight were measured every 2 days during the treatment period. (F) Western blotting analysis of PLK1 protein expression levels in tumors from the control (DMSO), low-dose treatment (rosuvastatin, 10 mg/kg), and high-dose treatment (rosuvastatin, 20 mg/kg) groups. (G) Immunohistochemical staining of PLK1 protein expression in tumors from different treatment groups and hematoxylin and eosin (H&E) staining of lung adenocarcinoma tissue. Scale bar = 100 µm. ns, no significance. **p 0.01, ***p 0.001.