Mouse B16 melanoma cells purchased from EK Biosciences GmbH were cultured in Gibco Dulbecco's modified Eagle's medium supplemented (cat. no. C1995500BT) with 1% penicillin-streptomycin and 10% gibco fetal bovine serum (cat. no. 10099-141) (Thermo Fisher Scientific, Inc.) in an incubator at 37°C and 5% CO₂. The medium was changed every 1–2 days, and when the cells covered 90% of the culture flasks, they were trypsinized and passaged. All cell lines tested negative for Mycoplasma contamination.

The Annexin V-FITC/PI double staining cell apoptosis detection kit (cat. no. BD556547) was purchased from BD Biosciences. Mouse GAPDH monoclonal antibody (cat. no. 60004-1-Ig), mouse Beclin-1 monoclonal antibody (cat. no. 66665-1-Ig), mouse caspase 3/p17/p19 monoclonal antibody (cat. no. 66470-2-Ig), mouse Bax monoclonal antibody (cat. no. 60267-1-Ig), mouse Bcl2 monoclonal antibody (cat. no. 68103-1-Ig), mouse cyclin D1 monoclonal antibody (cat. no. 60186-1-Ig), mouse CDK4 monoclonal antibody (cat. no. 66950-1-Ig), mouse CDK6 monoclonal antibody (cat. no. 66278-1-Ig), mouse CDK2 monoclonal antibody (cat. no. 60312-1-Ig), rabbit cyclin E1 polyclonal antibody (cat. no. 11554-1-AP), rabbit microtubule-associated protein light chain 3 (LC3) polyclonal antibody (cat. no. 14600-1-AP), goat anti-rabbit IgG-HRP (cat. no. SA00001-2) and goat anti-mouse IgG-HRP (cat. no. SA00001-1) antibodies were purchased from ProteinTech Group, Inc. Rabbit sequestosome 1/p62 (cat. no. AF5384), mTOR (cat. no. AF6308), p70 ribosomal S6 kinase (p70-S6k; cat. no. AF6226), eukaryotic initiation factor 4E-binding protein (4E-BP1; cat. no. AF6432), phosphorylated (p)-mTOR (cat. no. AF3308), p-p70-S6k (cat. no. AF3228) and p-4E-BP1 (cat. no. AF3830) polyclonal antibodies were purchased from Affinity Biosciences. Thiazolyl Blue (MTT; cat. no. HY-15924), rapamycin (cat. no. HY-10219) and chloroquine (CQ; cat. no. HY-17589A), an autophagy inhibitor that can block the degradation of LC3 and p62 by inhibiting the fusion of autophagosomes and lysosomes, were purchased from MedChemExpress. Rapamycin was configured with DMSO into 10°, 10¹, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷ and 10⁸ nM concentrated reservoirs (later diluted), and stored frozen at −20 or −80°C for in vitro cell experiments. For in vivo animal studies, the drug solution was prepared in anhydrous ethanol and phosphate-buffered saline (PBS) at a concentration of 1 mg/ml and stored at −20°C, ready to use.

Cell suspensions made from logarithmic cells were inoculated into 96-well culture plates (20,000 cells/well). Solubilized rapamycin solution (final drug concentrations, 10⁻³, 10⁻², 10⁻¹, 10°, 10¹, 10², 10³, 10⁴ and 10⁵ nM) was added. After 48 h at 37°C, 20 µl MTT working solution (5 g/l) was added to each well and the cells were incubated at 37°C for 4 h. After discarding the supernatant, the purple formazan crystals were solubilized with 150 µl DMSO. Finally, the optical density was measured at 492 nm using a plate reader (Multiskan FC; Thermo Fisher Scientific, Inc.).

B16 cells pretreated with different concentrations (0, 0.1, 1, 10 and 100 nM) of rapamycin for 48 h at 37°C were collected. The supernatant was collected from the 6-well plate, and ~1×10⁵ cells/well were digested with 0.25% EDTA-free trypsin after washing with PBS. After termination of digestion, the cells were centrifuged at 500 × g for 5 min at 4°C. The cells were then washed twice with PBS, 500 µl binding buffer was added, and the cells were incubated with Annexin V-FITC (5 µl) and PI (5 µl) at room temperature for 15 min in the dark. Finally, the stained cells were detected by flow cytometry (NovoCyte 2040R with FlowJo v10.9; ACEA Bioscience, Inc.) within 2 h (25).

The culture fluid was collected from the cells into a centrifuge tube and set aside for termination of digestion, and 1×10⁵ cells/well were digested with 0.25% trypsin after PBS rinsing. After the cells were collected into a centrifuge tube, they were centrifuged at 4°C for 3 min at 1,000 × g. The cells were washed twice with pre-cooled PBS and added to 1 ml pre-cooled 70% ethanol. The cells were gently aspirated and blown with a spiking gun to prevent clumps, then mixed and fixed at 4°C for 12 h. The cells were centrifuged for a further 3 min at 1,000 × g and 4°C. The supernatant was then aspirated and washed once with PBS. Subsequently, 500 µl staining buffer (Cell Cycle Kit; cat. no. C1052; Beyotime Institute of Biotechnology), 25 µl PI staining solution (20 X) and 10 µl RNase A (50X) was added to each sample. After incubation at room temperature for 30 min, PI staining was examined by flow cytometry (NovoCyte 2040R with FlowJo v10.9; ACEA Bioscience, Inc.).

Total RNA was extracted from the B16 cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and the A260/A280 of total RNA in each group was determined. A PrimeScript^® RT Reagent Kit (cat. no. RR047A; Takara Bio, Inc.) was used to reverse transcribe RNA into cDNA. qPCR was performed on a real-time PCR detection instrument (Light Cycler 480II; Roche Diagnostics) with a TB Green^® Premix Ex Taq™ II (Tli RNaseH Plus) cat. no. RR820A; Takara Bio, Inc.) following standard procedures. The thermal cycling conditions were as follows: 95°C for 30 sec of pre-denaturation; followed by 40 cycles at 95°C for 5 sec and 60°C for 30 sec; and a final incubation at 95°C for 5 sec and 60°C for 1 min. Finally, the reaction was cooled to 50°C in 30 sec. The relative expression levels of autophagy-related genes were quantified using the 2^−ΔΔCq (26) method using GAPDH as a control. The mRNA expression levels were normalized to those of GAPDH. The primer sequences are shown in Table I.

A total of 200,000 B16 cells per well were inoculated in 6-well culture plates and allowed to grow for 48 h. TEM was used to examine the morphology of B16 cells under 0, 1 and 10 nM rapamycin treatment for 48 h at 37°C. Briefly, cells were immediately fixed in electron microscopy fixative (3% osmium tetroxide) for 2 h at 4°C. After low-speed centrifugation for 3 min at 1,000 × g and 4°C, they were rinsed three times with PBS (pH 7.4). Subsequently, the cells were dehydrated in a graded ethanol series, then permeabilized and embedded in acetone, and cut into 60–80 nm ultrathin sections. Sections were double-stained with uranium-lead (2% uranyl acetate saturated alcohol solution and 3% lead citrate, each for 15 min at room temperature 25°C), and sections were dried at room temperature overnight. The sections were finally observed under TEM (HT7700; Hitachi, Ltd.) and images were collected for analysis.

Lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) containing 1 mM phenylmethanesulfonyl fluoride was used to extract total cellular or mouse tumor tissue proteins after treatment, and the bicinchoninic acid protein assay kit (cat. no. P0010; Beyotime Institute of Biotechnology) was used to measure protein concentration. SDS-PAGE at a 12% concentration was used to separate equal amounts of 0.8 µg/µl of protein samples, which were then transferred to PVDF membranes. The membranes were blocked for 15 min at room temperature in Rapid Blocking Solution (cat. no. P0252; Beyotime Institute of Biotechnology) and incubated for 2 h at room temperature with different primary antibodies against Beclin-1, LC3, p62, CDK1, caspase 3, Bax, Bcl2, cyclin D1, CDK4, CDK6, CDK2, cyclin E1, mTOR, p-mTOR, p70-S6k, p-p70S6k, 4E-BP1, p-4E-BP1 (dilution for all, 1:1,500) and GAPDH (dilution, 1:10,000). Membranes were then washed three times with PBS-0.05% Tween 20 and then incubated with HRP-coupled anti-rabbit or anti-mouse secondary antibodies (dilution of both, 1:10,000) for 2 h at room temperature. After PBS washing three times, protein bands were observed using a protein blotting assay kit (Applygen Technologies Inc.) with ultrasensitive enhanced chemiluminescence, and ImageJ software (National Institutes of Health) was used for semi-quantitative analysis of protein bands (27).

A total of 32 male C57BL/6 mice (age, 8 weeks; weight, 18 g) were purchased from the Guangdong Medical Laboratory Animal Centre. The mice were housed in an environment of 50–60% relative humidity and 21–25°C, with an alternating light and dark cycle of 10/14 h. The mice had free access to clean water and food. The present study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, Eighth Edition, 2010, and also complied with the ARRIVE guidelines and the AVMA euthanasia guidelines 2020 (28,29). The animal experiments were approved by the Ethics Committee of Hainan Medical University (Haikou, China; approval no. HYLL: 2022-227). All protocols were in accordance with the approved guidelines and regulations.

The abdominal hair of the mice was first shaved and sterilized, and then B16 cells (1×10⁶ in 100 µl) were injected subcutaneously into the right abdomen of 8-week-old male C57BL/6 mice to establish a subcutaneous tumor transplantation model. These mice were randomly and evenly grouped into four groups (n=8 mice/group). To ensure successful inoculation, tumor length was regularly observed and recorded daily. To assess the inhibitory effect of rapamycin on B16 melanoma cells in vivo, the mice were treated with different doses of rapamycin (1, 1.5 and 2 mg/kg/day) for 12 days, and the control group was injected intraperitoneally with an equal amount of PBS. When the tumor diameter of control mice reached the execution criteria (15 mm), all mice were euthanized by intravenous injection with an overdose of anesthetic (sodium pentobarbital, 150 mg/kg). Humane endpoints included malignancy, ulceration and necrosis. Tumors were removed and weighed. Tumor volume was measured as follows: Tumor volume (mm³)=length × width × width/2.

The tumor tissues were fixed with 4% paraformaldehyde at room temperature for 24 h, paraffin embedded and cut into 4-µm sections. The sections were deparaffinized with xylene for 15 min twice and subsequently placed in 100, 95 and 80% ethanol, and sequentially rehydrated for 10 min. PBS-rinsed sections were immersed in 0.1 mol/l citrate (pH 6.0) and incubated for 15 min in a microwave oven for antigen repair. After the sections were cooled at room temperature for 10 min, incubation with 3% H₂O₂-PBS for 10 min at room temperature was used to eliminate endogenous peroxidase activity. Next, 5% BSA (cat. no. ST023; Beyotime Institute of Biotechnology) was applied at 37°C to close the sections for 1 h. Subsequently, the sections were incubated with primary antibodies against the autophagy marker proteins LC3 (dilution, 1:200) and p62 (dilution, 1:200) at 37°C for 1 h. PBS was used to replace the first antibodies in the blank control. After incubating with HRP-conjugated goat anti-rabbit (dilution, 1:300) at 37°C for 20 min, tissue sections were stained with 3,3-diaminobenzidine and hematoxylin at room temperature for 5 min in turn. After being dehydrated and mounted, the tissues were observed under a fluorescence inverted microscope. Relative expression was assessed by measuring the average optical density of positive responses in each group using ImageJ software.

The paraffin-embedded sections were deparaffinized and washed twice with PBS (5 min/wash). After shaking the sections dry, proteinase K working solution (cat. no. 11684795910; MilliporeSigma) was applied dropwise and the sections were incubated for 25 min at 37°C before being washed twice with PBS. The sections underwent two PBS washes after being dried once more and treated with a membrane-breaking working solution for 10 min at room temperature. The sections were treated with a 50-µl 1:9 mixture of reagents TdT and Cy3-dUTP from the TUNEL kit (cat. no. 11684817910; Roche Diagnostics) at 37°C for 1 h. After adding the DAPI staining solution and washing the sections three times in PBS (5 min/wash), they were maintained for 10 min at room temperature in the dark. After submerging the slides in PBS and shaking them three times (5 min each), the sections were blocked with an anti-fluorescence quenching sealing agent at 37°C for 2 min once the sections had somewhat dried. Using a fluorescence microscope (DS-Fi3; Nikon Corporation), sections were examined and images were captured.

All experiments were conducted at least three times and data are presented as the mean ± standard deviation. GraphPad 9.5 software (Dotmatics) was used for statistical analysis. Multiple groups were compared using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effects of rapamycin on B16 melanoma cell viability, the MTT assay was performed. B16 melanoma cells were treated with a range of rapamycin concentrations (0, 10⁻³, 10⁻², 10⁻¹, 10°, 10¹, 10², 10³, 10⁴ and 10⁵ nM) for 48 h. As shown in Fig. 1A, rapamycin at a concentration of 10⁻¹ nM significantly reduced the viability of B16 melanoma cells compared with that in the control group. In addition, the half-maximal inhibitory concentration of rapamycin in B16 cells was 84.14 nM (Fig. 1B).

To investigate the possible mechanism by which rapamycin inhibits the viability of B16 cells, the present study examined the apoptosis of B16 cells following rapamycin treatment using flow cytometry. As shown in Fig. 2A and B, cell apoptosis was increased by rapamycin in the concentration range of 0.1–100 nM (10⁻⁷−10⁻⁴ mM). In addition, rapamycin increased the protein expression levels of cleaved caspase 3 and Bax, and decreased the protein expression levels of Bcl2 compared with those in the control group (Fig. 2C-E). These results indicated that rapamycin can induce the apoptosis of melanoma cells in vitro.

After treating B16 cells with different concentrations of rapamycin, the cell cycle distribution of B16 cells was assessed using flow cytometry. As shown in Fig. 3A and B, rapamycin induced cell cycle arrest in B16 cells, with an increased proportion of cells in G₁ phase and a decreased proportion of cells in G₂/M phase compared with that in the control group. Furthermore, rapamycin reduced the protein expression levels of CDK1, cyclin D1 and CDK4, whereas it did not affect CDK6, cyclin E1 and CDK2 expression, compared with in the control group (Fig. 3C-I). These results suggested that rapamycin may induce B16 cell cycle arrest at G₀/G₁ phase.

Autophagic vesicles observed by TEM are the gold standard for autophagy detection. In order to understand the effect of rapamycin on cellular autophagy, TEM was performed to observe the structure of the cells. The results revealed that the control cells had intact cell membranes, intact nuclear membranes, and a small number of autophagic vesicles and autophagic lysosomal structures (Fig. 4A). By contrast, B16 cells in the prominent 1 and 10 nM rapamycin groups had notably more autophagic vesicles and autophagic lysosomes than the cells in the control group. Moreover, cells in the 1 and 10 nM rapamycin groups exhibited marked cell membrane breaks and mitochondrial ridge breaks. Furthermore, at the gene and protein levels, rapamycin increased LC3 and Beclin-1 expression, and decreased p62 expression compared with that in the control group (Fig. 4B-H). To determine whether rapamycin enhanced autophagy, western blotting was used to detect the changes in LC3 and p62 expression following treatment with CQ (an autophagy inhibitor that blocks the degradation of LC3 and p62 by inhibiting the fusion of autophagosomes and lysosomes). Rapamycin in combination with CQ further increased LC3 and p62 expression compared with that in the CQ group, suggesting that rapamycin indeed induced an increase in autophagy in B16 cells (Fig. 4I-K).

The mTOR/p70-S6k signaling pathway serves an important role in the regulation of autophagy, cell proliferation and cell survival in eukaryotic cells (30). To investigate the molecular mechanism underlying rapamycin-induced cellular autophagy in B16 cells, the expression and activation of important components of the mTOR signaling pathway, including p-mTOR, p-p70-S6k and p-4EBP1, were assessed after 48 h of rapamycin treatment. As shown in Fig. 5, the phosphorylation of mTOR and p70-S6k was downregulated by rapamycin compared with that in the control group, whereas there was no change in 4EBP1 phosphorylation. This result suggested that the mTOR/p70-S6k signaling pathway may be inhibited by rapamycin.

To determine whether rapamycin inhibits melanoma growth in vivo, B16 cells were subcutaneously injected into the abdomen of 8-week-old male mice, and rapamycin was administered at 1, 1.5 or 2 mg/kg/day. As shown in Fig. 6A-C, rapamycin at 1, 1.5 and 2 mg/kg/day effectively inhibited B16 melanoma growth compared with that in the control group. Western blotting results showed that the protein expression levels of LC3 II were increased, whereas the protein expression levels of p62 were decreased in rapamycin-treated tumors compared with those in the control group (Fig. 6D-F). Immunohistochemistry results showed an increase in the area of LC3 II-positive regions in rapamycin-treated tumors, along with a decrease in the area of p62-positive regions compared with that in the control group (Fig. 6G-I), which was consistent with the western blotting results. These findings suggested that rapamycin may induce B16 cell autophagy and suppress B16 melanoma growth in vivo.

TUNEL fluorescent labeling and western blotting were performed to detect apoptosis in tumor tissues to examine whether rapamycin may trigger apoptosis in mice with melanoma in vivo. TUNEL immunofluorescence results showed that rapamycin promoted apoptosis of tumor cells in tumor tissues compared with that in the control group (Fig. 7A). The results of western blotting demonstrated that rapamycin induced a reduction in Bcl2 expression, and an increase in the expression levels of cleaved caspase 3 and Bax compared with those in the control group (Fig. 7B-D). These findings indicated that rapamycin may cause mouse melanoma tumor cells to undergo apoptosis.

To determine if proteins associated with the mTOR/p70-S6k signaling pathway were altered in tumors, western blotting was performed. The administration of 1, 1.5 and 2 mg/kg/day rapamycin resulted in a decrease in the protein expression levels of p-mTOR, p-P70 S6k and p-4E-BP1 in comparison to those in the control group (Fig. 8A-D). These findings suggested that rapamycin can inhibit the mTOR/p70-S6k/4E-BP1 signaling pathway in tumor tissues.