ATTM and antibodies against METTL3 and METTL14 were purchased from Sigma-Aldrich Co. (St. Louis, MO, US). Antibodies against FTO and YTHDF1 were purchased from Abcam (Plc.Cambridge, MA, US). CuSO₄ and Zn(OAc)₂ were purchased from Meilun Biotechnology Co. (Dalian, China). TRIzol™ Regent (Invitrogen) was provide by Thermo Fisher Scientific Co. (Shanghai, China). Gemcell™ fetal bovine serum (FBS) was supplied by Gemini Company (Woodland, US).

Lung adenocarcinoma cell lines (A549 and HCC827) were purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China), and PC9 was obtained from ATCC. The cells were maintained in RPMI-1640 medium supplemented with 10% FBS at 37°C under an atmosphere of 5% CO₂ and 95% air. They were passaged and harvested with 0.25% trypsin every other day.

Cell number was measured with Cell Counting Kit (CCK)-8 provided by Dojindo Lab (Kyushu, Japan). A549, HCC827, and PC9 cells were plated in 96-well plates at a density of 7, 000 cells/well. When grown to ~60–70% confluence, the cells were treated correspondingly. After the treatments, the CCK-8 solution (100 μL) at a 1:10 dilution with FBS-free medium was added to each well-followed by a 2-h incubation at 37°C. Absorbance (A) was measured at 450 nm with a microplate reader (Molecular Devices, US).

Cell proliferation was tested with BeyoClick™ 5-Ethynyl-2′-deoxyuridine (EdU) kit (Haimen, China). After the treatment of A549 cells with ATTM for 48 h, EdU incorporation assay was performed according to the manufacturer's instructions. TMB-derived color was measured at 630 nm with the microplate reader.

Cell invasion was observed with Transwell Migration Assay, as described (31) with modifications. RPMI-1640 medium (1% FBS) containing ATTM or Na₂S in the absence or presence of Zn(OAc)₂ was added to the lower chambers of the 12-well format transwells (8 μm-pore, BD Biosciences). A549 cells were seeded in the upper chambers at 10⁵ cells per well, following a 48 h-culture. After that, the transwells were fixed in methanol, and stained with Giemsa solution. The unmigrated cells were removed from the top of the membranes using cotton swabs. To quantify the number of migrated cells in the bottom of the membrane, four random images of each group were taken at 10× under a light microscope. Migrated cell number was counted with Image J software.

After treatments of A549 cells with increasing concentrations of ATTM for 24 h, total RNA was extracted using TaKaRa MiniBest kits (Kusatsu, Japan) and quantitated with NanoDrop 1000 spectrophotometer (Thermo Fisher, US). The m⁶A RNA was detected with EpiQuik m⁶A RNA Methylation Colorimetric kit (Farmingdale, US). Briefly, 200 ng of fresh extracted RNA sample was added into strip wells with RNA high binding solution, and incubated for 90 min at 37°C. After three washes, capture antibody, detection antibody, and enhancer solution were applied in turn, and incubated for 1 h at 37°C. After washes, color developing solution was added and incubated for 6 min in the dark. When the solution became blue in the m⁶A positive wells, stop solution was added to turn the color into yellow. Lastly, the absorbance of stable yellow was measured at 450 nm with the microplate reader.

After exposed to 60 μM ATTM for 24 h, A549 cells were collected and split at 4°C. Total proteins in the lysate were quantitated with a BCA kit. Thirty micrograms of total protein sample were loaded in SDS-PAGE and electrophoresed. At the end of electrophoresis, the proteins were transferred to PVDF membranes. The membranes were blocked with 5% fat-free milk in Tris-base buffered saline containing 0.1% Tween-20 (TBS-T) for 1 h at room temperature, and then incubated with the primary antibodies against m⁶A related proteins (METTL3, METTL14, FTO, and YTHDDF1), and H₂S-producing enzymes (CSE, CBS, and MPST), respectively, with gentle agitation overnight at 4°C. After three washes, corresponding HRP-conjugated secondary antibodies were applied and incubated for 1.5 h at room temperature. The signal was visualized using an enhanced chemiluminescence detection system. The intensity of bands was quantified with Image J software.

Transcriptional expressions of m⁶A or H₂S related genes in primary tumor tissues and adjacent tissues of LUAD patients, as well as correlation analysis in tumor tissues, were performed through TCGA cohort research tool (UALCAN). Furthermore, the protein expressions of METTLE3, METTL14, and FTO in LUAD patients were verified with Western blotting assay as above.

Gene expression microarray data were downloaded at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE76367 (29). METTL3 was knocked down through short hairpin (sh) RNA in A549 cells, and gene expression was profiled by high throughput sequencing. Selected genes of eukaryotic translation initiation factors (eIFs) were shown as the maximum vs. minimum of the ratio shMETTL3 to shGFP.

Small interfering RNA (siRNA) against human YTHDF1 (Gene ID: 54915) was synthesized by GenePharma Co., Ltd (Shanghai, China). YTHDF1 siRNA and control random non-targeting siRNA were transfected into the A549 cells using Lipofectamine 2000 (Invitrogen, USA) (32). To raise the transfection efficiency, the cells were incubated with 20 nM YTHDF1 siRNA or Control siRNA for 6 h followed by a 24-h culture. The silencing ability was evaluated by Western blotting assay.

Total RNAs were extracted from A549 cells and quantitated as above. First-strand cDNA was synthesized using TaqMan SYBR® Premix Ex TaqTM II (Tli RNase H Plus) virus reverse transcriptase and 2 μg RNA template in 20 μL reaction volume. The cDNA was used for real-time PCR with Prime Script RT reagent kit with gDNA Eraser (Life Technologies, US). Actin was used to normalize the expression of PRPF6. The results were analyzed using the comparative Ct method (2-ΔΔCt with logarithmic transformation). Primer sequences were displayed as below: PRPF6 forward 5-GTCATGCGTGCCGT GATTG-3 and reverse 5-TCCAGGGCATTGTGGGCTA-3, Actin forward 5-TGGCACCCAGCACAATGAA-3 and reverse 5-CTAAGTCATAGTCCGCCTAGAAGCA-3. PCRs were carried out as follows: initial denaturation at 95°C for 30 s, 40 cycles of 95°C for 5 s, 60°C for 34 s, and 95°C for 15 s, 60°C for 1 h, followed by a final extension at 95°C for 15 s.

ATTM-mediated H₂S generation in cells was determined with a H₂S fluorescent probe WSP-5 (33). A549 cells were inoculated in 24-well plates and grown to 60~70% confluence. After treated with ATTM, the cells were incubated with 10 μM WSP-5 in 1% FBS medium at 37°C for 30 min in the dark. Cell imaging was carried out after a slight wash with PBS. The intracellular H₂S-triggered fluorescence was visualized under AMG fluorescence microscopy (Advanced Microscopy Group, US).

For H₂S scavenging induced by Zn(OAc)₂, CuSO₄, FeCl₃ or VitB₁₂ was observed through a Unisense H₂S micro-sensor (Tueager 1, Denmark) (34). Into 5 mL PBS buffer, fresh Na₂S stock solution was added to produce a 100 μM Na₂S solution. When the curve reached the peak and kept stable, the same dose of above compounds was immediately added, respectively. The curves were recorded correspondingly with the H₂S sensor. For Zn(OAc)₂ or CuSO₄-induced continuous H₂S scavenging from ATTM solution was recorded for 6 h. Into 20 mL of 500 μM ATTM solution (pH 5), fresh Zn(OAc)₂ or CuSO₄ stock solutions was added, respectively, to produce a 250 μM solution. Real-time H₂S content was monitored with the H₂S sensor and quantified against a standard curve.

The experiment data are presented as means ± standard deviation (SD). Significance between groups was evaluated by one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls test using GraphPad Prism 8 software (San Diego, US). A probability <0.05 was considered statistically significant.

As shown in Figure 1A, treatment with high concentrations (≥250 μM) of ATTM for 48 h remarkably reduced cell number, in three types of lung adenocarcinoma cells (A549, HCC827, and PC9). However, at low concentrations (from 60 to 125 μM), the treatment distinctively increased cell number. Notably, another copper chelator TETA, without H₂S releasing activity, was not found to elevate cell number at the same treatment profile (Figure 1B). Furthermore, 60–125 μM of ATTM treatment could also induce A549 cell proliferation as evaluated by EdU assay (Figure 1C). The result indicates that low ATTM concentrations are able to promote lung adenocarcinoma growth.

To understand why ATTM enhanced lung adenocarcinoma growth, A549 cells were selected as a representative in the following experiments. Since mRNA m⁶A methylation is involved in a variety of cancer growth including lung adenocarcinoma, intracellular m⁶A mRNA level was then investigated. As shown in Figure 2A, the m⁶A mRNA content was significantly elevated after the exposure of A549 cells to 60 μM ATTM for 24 h. However, the treatment duration (half of 48 h) did not alter cell number (Figure 2B) or growth status (Figure 2C). Notably, analysis of TCGA cohort shows that LUAD condition significantly upregulates the m⁶A writer METTL3, while downregulates the m⁶A eraser FTO (Figure 2D). The result indicates that ATTM can trigger mRNA m⁶A methylation before cell growth in lung adenocarcinoma cells.

To uncover the mechanisms underlying the increased m⁶A mRNA levels in ATTM-treated cells, m⁶A related proteins were detected with Western blot. As shown in Figures 3A,C, comparing with the adjacent tissues, the writer (METTL3 and METLL14) expressions were upregulated in tumor tissues of LUAD patients, while the eraser FTO expression was downregulated. Importantly, the writer (METTL3 and METLL14) expressions in A549 cells could also be upregulated, while the eraser FTO could be downregulated under ATTM treatment (Figures 3B,D), supporting the finding of the increased intracellular m⁶A content. To confirm the roles of m⁶A methylation, we investigated gene expression profile in METTL3 knockdown A549 cells. As shown in Figure 3E, shRNA-mediated METTL3 knockdown significantly inhibited the expressions of translation initiation factors, including eIFs (2B3, 3B, 3C/CL, 3D, 3IP1, 4A1, and 5/5A). This suggests that m⁶A methylation is necessary to translation process in A549 cells, and that the elevated m⁶A levels may be important for LUAD progression and ATTM-induced tumor growth.

Although the increased m⁶A writer METTL3/14 and the decreased eraser FTO can result in the enhancement of m⁶A content, it is the m⁶A readers that directly determine the outcome of m⁶A methylated mRNA. With TCGA cohort, we examined the four common m⁶A readers (YTHDF1, YTHDF2, YTHDF3, and YTHDC1) in LUAD tumor samples and normal samples, and found YTHDF1 highly expressed in tumor tissues (Figure 4A). The increased YTHDF1 continuously expressed within various stages of LUAD (Figure 4B). Importantly, it was found that treatment of A549 cells with 60 μM ATTM for 24 h remarkably upregulated YTHDF1 protein expression (Figure 4C). After knockdown of YTHDF1 with siRNA in A549 cells (Figure 4D), both basal and ATTM-triggered cell growth were attenuated (Figure 4E).

To explore targets involved in YTHDF1-mediated A549 cell growth, we screened genes that are positively correlated with YTHDF1 in TCGA LUAD cohort. As shown in Figure 5A, PRPF6 was found to be positively expressed with YTHDF1 in LUAD tissues (r = 0.72). Similar to YTHDF1's expression profile, the increased PRPF6 expression lasted various stages of LUAD (Figure 5B). Furthermore, qPCR analysis showed that PRPF6 mRNA level was significantly reduced after YTHDF1 knockdown in A549 cells. Additionally, the knockdown attenuated ATTM-induced PRPF6 mRNA expression (Figure 5C). The result reveals that PRPF6 may be a potential target gene involved in YTHDF1-mediated A549 cell growth.

ATTM was previously demonstrated to generate H₂S on acid conditions in our lab. As shown in Figure 6A, the exposure of A549 cells to ATTM markedly raised intracellular H₂S levels. With TCGA cohort, we studied endogenous H₂S synthetase expressions and, found that CSE/CTH, CBS and MPST expressions were enhanced in tumor tissues comparing with normal samples (Figure 6B). Additionally, the exposure of A549 cells to 60 μM ATTM for 24 h obviously upregulated CBS and MPST expressions (Figures 6D,E), however, remarkable change of CSE was not found (Figure 6C). Notably, direct H₂S donation (Na₂S) could also induce m⁶A methylation (Figure 6F), as well as cell growth (Figure 6G), proliferation (Figure 6H) and invasion (Figures 6I,J), indicating H₂S being an effector in ATTM-induced m⁶A methylation and cell growth.

Since the enhanced H₂S generation was involved in ATTM-induced A549 cell growth and m⁶A methylation, scavenging H₂S might overcome these side-effects of ATTM. Through testing several common H₂S scavengers, it was found that Zn(OAc)₂ and CuSO₄ had powerful ability to remove H₂S, while the ability of VitB₁₂ and FeCl₃ was weak or even undetectable (Figure 7A). Additional cell viability examination showed that both Zn(OAc)₂ and CuSO₄ were non-toxic at concentrations <25 μM (Figures 7B,C).

To make sure H₂S is necessary to ATTM-induced biological process in A549 cells, we observed the scavenging effect of Zn(OAc)₂ or CuSO₄ on ATTM-induced H₂S release within a period of 6 h. As shown in Figure 8A, adding 25 μM Zn(OAc)₂ or CuSO₄ time-dependently attenuated ATTM-induced H₂S release. Notably, application of 25 μM Zn(OAc)₂ inhibited ATTM-induced cell growth (Figure 8B), proliferation (Figure 6H) and invasion (Figures 6I,J). More importantly, Zn(OAc)₂ was also able to attenuate ATTM-induced m⁶A methylation (Figure 8C). Collectively, the result suggests that H₂S is necessary and sufficient to ATTM-induced biological changes, and scavenging H₂S can potentially overcome ATTM's side-effects in lung cancer treatment.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.