A549/DDP cells obtained from JRDUN Biotech (Shanghai, China) were grown in RPMI 1640 medium (Hyclone, Logan, UT, USA) containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA) at 37°C under a humidified atmosphere containing 5% CO2.

To explore the effects of melittin on A549/DDP cell proliferation, A549/DDP cells were incubated with 0, 1, 2, 4, 8 or 16 μg/mL melittin (Sigma-Aldrich, St. Louis, MO, USA), and cell proliferation inhibition was assessed by Cell Counting Kit-8 (CCK-8) assay for 0, 24, 48, or 72 h. At the end of incubation, CCK-8 solution (Beyotime, Shanghai, China) was added to the palates. After 2 h of incubation, cell proliferation was determined by measuring optical density at 450 nm.

The study was approved by the Institute Research Ethics Committee of Shanghai Municipal Hospital of Traditional Chinese Medicine (Shanghai, China) and conducted in accordance with the Declaration of Helsinki. A total of 25 NSCLC patients were enrolled at Shanghai Municipal Hospital after all patients provided written informed consent. Paired NSCLC specimens and adjacent normal tissues were collected, snap-frozen, and stored at −80°C until use.

The relevance of TRIM8 mRNA expression to overall survival was determined by using www.kmplot.com (Győrffy et al., 2013). The tool is based on public NSCLC microarray data from Gene Expression Omnibus (GEO). The patients were divided into high and low expression groups, HR (95% CIs), log-rank P, and Kaplan–Meier survival plots were then displayed on the webpage.

Three short hairpin RNAs targeting TRIM8 were designed, synthesized, and cloned into pLKO.1 lentiviral vector (Addgene, Cambridge, MA, USA). The target sites were as follows: siTRIM8-1, 5’-CCAACATCGTGGAGAAGTT-3’; siTRIM8-2, 5’-CCAGCTGTACAAACTCGAG-3’.

Human TRIM8 full-length cDNA was amplified and cloned into the pLVX-puro vector (Clontech, Palo Alto, CA, USA). The primers were as follows: forward primer, 5’-CGGAATTCATGGCGGAGAATTGGAAGAAC-3’ and reverse primer, 5’-CGGGATCCTTAGCTCGTCACGTAGTGTTTGG-3’.

To package the lentivirus, the constructs were co-transfected with packaging plasmids into 293 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The supernatant was harvested 48–72 h later and used to infect A549/DDP cells.

Total RNA was isolated with TRIzol Reagent (Invitrogen) and reverse-transcribed with MMLV reverse transcriptase (Promega, Madison, WI, USA) in accordance with the manufacturers’ instruction. To determine the relative gene expression, real-time PCR was conducted with an ABI 7500 instrument (Applied Biosystems, Foster City, CA, USA). β-actin was applied as an internal control. All the primers are listed in Tab. S1.

The cells were lysed with ice-cold radioimmunoprecipitation assay (RIPA) buffer containing proteinase and phosphatase inhibitor cocktails (Sigma-Aldrich) at 4°C. Following centrifugation at 12,000 rpm for 15 min at 4°C, the supernatant was collected, and protein concentration was determined by BCA assay (Walker, 2009). Equal protein concentrations from the samples were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes (Millipore, Bredford, MA, USA). The membranes were blocked with 5% skim milk and then probed with primary antibodies against TRIM8 (Abcam, Cambridge, MA, USA), p-AKT (Cell Signaling Technology, Danvers, MA, USA), AKT (Cell Signaling Technology) or β-actin (Abcam). Following incubation with an HRP-conjugated secondary antibody (Beyotime), immunoreactive signals were detected using by Enhanced chemiluminescence (ECL) technique (Millipore). β-actin was detected for equal loading control.

Annexin V-FITC/propidium iodide (PI) staining kit (Beyotime) was used to stained cells undergoing apoptosis following the manufacturer’s protocol. In brief, A549/DDP cells in 6-well plates were harvest by trypsinization. Following washing, once with PBS, the cells were collected by centrifugation and re-suspended in 195 μL Annexin V-FITC binding buffer. Subsequently, the cells were mixed with 5 μL Annexin V-FITC and 5 μL PI, and then incubation at dark for 20 min at room temperature. The apoptotic rate (Annexin V positive and PI negative cells) was analyzed by a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

A549/DDP cells were cultured with glucose-free medium for 3 h and then incubated with 100 μM 2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose; Cayman, Ann Arbor, MI, USA) in glucose-free medium for 45 min. After washing with glucose-free Krebs-Ringer buffer (KRB), cells were collected by trypsinization and the fluorescent density was measured with a flow cytometer (BD Biosciences). The relative fluorescent density of treated samples was normalized to the control group, which was set as 100%.

Lactate production in the culture medium was measured with a lactic acid detection kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer’s protocol.

The experimental procedures were approved by the Committee on Animal Care and Use of Shanghai Municipal Hospital of Traditional Chinese Medicine (Shanghai, China) and performed in accordance with the Guidelines for Animal Experiments of Shanghai Municipal Hospital of Traditional Chinese Medicine. Subcutaneous xenografts were established in BALB/c athymic nude mice (4–5 weeks old) by subcutaneous injection of A549/DDP cells (5 × 10⁶ cells per mouse). After 7 days, the mice were randomly divided into 4 groups and there were 5 mice in each group: Control, DDP, melittin (MEL) and MEL + DDP. The mice in DDP and MEL group were intraperitoneally injected with cisplatin (5 mg/kg, Chinese medicine reagent, Beijing, China) and melittin (2 mg/kg) every 7 days for 3 weeks, respectively. The mice in the MEL + DDP group were administrated with cisplatin (5 mg/kg) and melittin (2 mg/kg) every 7 days for 3 weeks. The tumor volume was estimated every 3 days and calculated using the following formula: volume = (width² × length) / 2. After 3 weeks, the mice were euthanized, and the tumor xenografts were harvested for TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling) analyses (Roche, Indianapolis, IN, USA).

All data are presented as the mean ± SD. Statistical analysis was performed using Graphpad Prism Software (Graphpad Prism, San Diego, CA, USA). One-way analysis of variance (ANOVA) was performed for the in vitro experiments and xenograft experiments. A two-tailed Student’s t-test was conducted for comparing TIMIM8 expression in clinical samples. Differences were considered statistically significant at p < 0.05.

First, we confirmed that A549/DDP cells were resistant to cisplatin comparing to A549 cells (Fig. S1). Then, A549/DDP cells were incubated with 0–16 μg/mL melittin (MEL) and cell proliferation inhibition was assessed by CCK-8 assay for 24, 48, or 72 h. As shown in Fig. 1A, the inhibitory rate of proliferation was dose- and time-dependently increased by melittin exposure. The inhibitory rate of proliferation was not significant at a concentration of 1 μg/mL at 24 h post-treatment, while 16 μg/mL caused more than 50% inhibition at 72 h post-treatment. Therefore, 2, 4, and 8 μg/mL were chosen for the following experiments.

We used flow cytometry to determine the effect of melittin on A549/DDP cell apoptosis (Fig. 1B). Apoptosis rate of the A549/DDP cells was significantly increased at 48 h following melittin treatment (0 μg/mL, 1.40 ± 0.26%; 2 μg/mL, 7.07 ± 0.29%; 4 μg/mL, 8.47 ± 0.72%; 8 μg/mL, 11.40 ± 0.95%) in comparison with the control.

The Warburg effect, a key feature of tumors, has been implicated in chemoresistance (Bhattacharya et al., 2016); thus, we detected the effects of melittin on glucose uptake and lactate production. A549/DDP cells showed a dose-dependent decrease in 2-NBDG uptake (Fig. 1C) and lactate production (Fig. 1D) following melittin treatment.

AKT pathway plays an important role in regulating cell apoptosis and glycolysis (Cairns et al., 2011). As expected, the Western blotting analysis indicated that the level of phosphorylated AKT (p-AKT) was also reduced with melittin treatment (Fig. 1E). These results suggest that melittin induced cell apoptosis and inhibited AKT signaling and Warburg effect in A549/DDP cells.

To identify TRIM proteins possibly involving in the effects of melittin, mRNA levels of several TRIM proteins were assessed in A549 cells, and A549/DDP cells treated with melittin or not. As shown in Fig. 2, significant differences were observed in 9 TRIM proteins between A549 cells and A549/DDP cells, while only 3 TRIM proteins, TRIM8, TRIM31, and TRIM40, showed significant differences between melittin-treated and -untreated A549/DDP cells. The most significant change was observed in TRIM8, which may be a selective target of melittin, and further study was focused on TRIM8.

Western blotting results showed that TRIM8 protein was reduced with the treatment of melittin in A549/DDP cells in a dose-dependent manner (Fig. 3A). To determine whether TRIM8 is involved in the effect of melittin, A549/DDP cells were transduced with a virus overexpressing TRIM8 and then treated with 4 μg/mL melittin. As shown in Fig. 3B, TRIM8 expression was significantly increased in A549/DDP cells by lentivirus transduction. Consequently, TRIM8 overexpression reduced the induction of cell apoptosis (Fig. 3C) and the inhibition of 2-NBDG uptake (Fig. 3D), lactate production (Fig. 3E), and p-AKT level (Fig. 3F) caused by melittin exposure.

TRIM8 mRNA expression was found elevated in NSCLC specimens as compared to adjacent normal tissues (N = 25, Fig. 4A). The prognostic value of TRIM8 mRNA expression was then examined by using www.kmplot.com. Survival curves were plotted for all patients (N = 1,926) and adenocarcinoma (N = 720) with probe 22131_s_at. High expression of TRIM8 mRNA was correlated with poor overall survival for all patients and adenocarcinoma (Fig. 4B).

To explore the biological function of TRIM8, TRIM8 expression was suppressed in A549/DDP adenocarcinoma cells by RNAi. As shown in Fig. 4C, TRIM8 siRNAs obviously reduced TRIM8 expression, and siTRIM8-1 and siTRIM8-2 were then used in the following experiments. A549/DDP cells showed a notable increase in apoptosis (Fig. 4D) and a significant decrease in 2-NBDG uptake (Fig. 4E), lactate production (Fig. 4F), and p-AKT level (Fig. 4C) when TRIM8 expression was down-regulated. These data demonstrated the functions of TRIM8 in apoptosis and glycolysis of A549/DDP cells.

To investigate the function of melittin on cisplatin resistance in vitro, A549/DDP cells were incubated with melittin (MEL, 4 μg/mL), cisplatin (DDP, 50 μM), or MEL plus DDP, and then cell proliferation was assessed. Following incubation with melittin in combination with cisplatin, cell proliferation was significantly inhibited in the co-treatment group as compared to that in the DDP-treated group (Fig. 5A). These data indicate that A549/DDP cells presented enhanced cisplatin sensitivity in the presence of melittin.

To investigate the function of melittin in vivo, a mouse xenograft model was constructed by using A549/DDP cells. Tumor growth was significantly decreased by melittin, cisplatin, or melittin plus cisplatin at 12–21 days following treatment, when compared with the control group (p < 0.05, Fig. 5B). At 21 days after treatment, tumor size (Fig. 5C) and tumor weight (Fig. 5D) were decreased in the melittin group, cisplatin group, or melittin plus cisplatin group as compared with the control group (p < 0.01). Co-treatment with melittin plus cisplatin exhibited the best inhibitory effects. Cell apoptosis rate was increased by treatment, and co-treatment also displayed the highest apoptotic rate (Fig. 5E). These results indicate that melittin enhanced cisplatin sensitivity of tumors derived from A549/DDP cells.