Propofol regulates METTL3-mediated PARP-1 mA modification to promote Parthanatos to improve NSCLC chemotherapy resistance.

Introduction
Globally, lung cancer ranks as the most common cancer and stands as a leading cause of cancer-related deaths1. According to the most recent global cancer statistics, lung cancer remains the leading cause of cancer-related mortality worldwide, with persistently poor long-term survival outcomes2,3. NSCLC accounts for 85% of all lung cancer instances. Presently, NSCLC treatment protocols encompass surgical procedures, radiotherapy, chemotherapy, specialized targeted therapies, or a mix of these methods4. NSCLC patients exhibit the most severe morbidity and mortality rates compared to other malignant tumors, with their five-year overall survival rate falling below 20%5,6. Advancements in clinical diagnosis and treatment methods have eased the management of NSCLC, but patient survival rates are still less than ideal, owing to distinct physiological states and common drug resistance. Lung cancer chemoresistance represents a significant challenge in clinical treatment failure and recurrence, and the emergence of drug resistance is a complex process involving multiple factors and genetic alterations and signaling pathways. Therefore, clarifying the precise molecular underpinnings of NSCLC drug resistance, identifying novel action targets, and creating potent, low-toxicity counteracting agents along with suitable chemotherapy approaches will lead to fresh concepts and tactics for NSCLC therapy.
In the treatment of NSCLC, propofol is a commonly used intravenous anesthetic. Recent studies have revealed its possible anti-tumor effect. Especially in inhibiting tumor growth and metastasis, propofol has shown potential therapeutic value. For example, a study found that propofol inhibited the proliferation and promoted the apoptosis of NSCLC cells in vitro and in vivo by regulating the microribonucleic acid-21 (miR-21)/chromosome 10 (PTEN) /protein kinase B (AKT) signaling pathway7. In addition, another study pointed out that the combination of propofol and AS-IV (astragaloside IV) could enhance the anti-tumor effect of propofol in inhibiting NSCLC cells, which was achieved by inhibiting autophagy8. Propofol can also enhance DDP sensitivity in DDP-resistant NSCLC cells. This effect is achieved by increasing the expression level of miR-486-5p in NSCLC cells by regulating m6A modification and affecting the inactivation of downstream Ras-associated protein1 (RAP1)-NF-kappaB (NF-κB) pathway9. These studies not only provide a scientific basis for the potential application of propofol in the treatment of NSCLC, but also point out the direction for future clinical research. Through these studies, we can see the multifaceted role of propofol in the treatment of NSCLC, including inhibiting tumor cell proliferation, promoting apoptosis, and regulating key signaling pathways. These findings provide new strategies for the comprehensive treatment of NSCLC.
Recent studies have shown that PARP-1 mediates cell death through Parthanatos, i.e. Cell death reliant on PARP-1, where excessive activation of PARP-1 due to DNA damage leads to a depletion of nicotinamide adenine dinucleotide (NAD+) substrate, causing an accumulation of Poly (ADP-ribose) (PAR) in the cytoplasm. PAR binds to apoptosis-inducing factor (AIF) located in the mitochondria, recruits and carries macrophage migration inhibitory factor (MIF) towards the nucleus, leading to extensive DNA fragmentation and chromatin lysis, which mediates cell death10–12. A number of studies have reported that drug-induced Parthanatos can kill a number of different tumour cells, including melanoma, colorectal cancer, prostate cancer, and so forth. This implies that induction of Parthanatos could be used as a therapeutic modality capable of promoting chemo-sensitivity and improving NSCLC resistance12–14. A previous study by our group demonstrated that propofol significantly inhibited NSCLC cell activity and promoted cisplatin sensitivity15. Another study indicated that propofol is involved in the regulation of PARP-1 mediated Parthanatos16. However, it is not clear whether propofol affects chemosensitivity by regulating NSCLC cell Parthanatos.
The methylation of N6 adenosine (m6A), a prevalent RNA alteration, is vital in cancer’s emergence and advancement, and controlling the genetic modification of m6A could be an effective strategy to enhance drug resistance in NSCLC17,18. The alteration of m6A is controlled by enzymes such as methyltransferases, demethylases, and RNA-binding proteins19. Methyltransferase-like 3 (METTL3) is a core constituent subunit of the methyltransferase complex. Aberrant expression of this gene affects the m6A level of intracellular RNA, which in turn affects the expression of target genes. It is notable that METTL3 expression is regulated by propofol20. PARP-1 is regulated by m6A methylation of METTL3, which promotes PARP-1 mRNA stabilisation21. However, the underlying interaction between m6A methylation, PARP-1, and NSCLC chemoresistance have not been fully elucidated yet.
Therefore, this study is aimed to investigate the molecular mechanism of NSCLC drug resistance leading to chemotherapy resistance, and to verify the effect of propofol on NSCLC chemotherapy resistance from the perspective of Parthanatos.

Materials and methods

Reagents
A549, A549/DDP cell lines were purchased from Procell Life Science&Technology Co., Ltd. ( China). Balb/c Nude nude mice (SPF, 4–6 weeks, 20 ± 2 g) (Beijing Vital River Laboratory Animal Technology Co., Ltd., China). DMEM/F12 (11320033) (Gibco, China). Trypsin (R001100) (Gibco, China). Cell Freezing Solution (C0210B-50 ml), PBS Buffer (C0221A) (Beyotime, China). Primers (Sangon, China). OE-METTL3, KD-METTL3, and NC plasmid (Suzhou GenePharma Co., Ltd, China). LipofectamineTM 3000 (L3000015) (Thermo Fisher, USA). APS (ST005), RIPA (P0013B), PMSF (ST506), TEMED (ST728) (Beyotime, China). 30% Acr/Bic (BL513A), Tris-Base (BS083), TBS buffer powder (BL602A), BSA protein standard (BL673A), Tween-20 (BS100) (Biosharp, China). SDS (3250), Glycine (1275), Skimmed Milk (1172) (BioFroxx, China). BCA Protein Content Determination Kit (WB6501), 5xSDS-PAGE Sampling Buffer (WB2001), ECL luminescent solution AB solution (P2100) (NCM Biotech, China). Western antibody diluent (C05-07001), IL-6 ELISA kit (PI325), NAD+/NADH assay kit (WST-8 method) (S0175), mitochondrial reactive oxygen species detection kit (MitoSOX™) (Beyotime, China). RNA immunoprecipitation (RIP) kit (Bes5101 (N)), Co-IP kit (Bes5001 (N)), RNA pulldown: RNA pulldown Kit (Bes5102 (N)), m6A MeRIP kit (Bes5203-1 (N)) (Guangzhou Beyotime Biotechnology Co., Ltd, China). CCK8 cell proliferation detection kit (BA00208) (Bioss, China). TUNEL detection kit (Green Fluorescence) (C1086) (Beyotime, China). Recombinant anti-AIF antibody (ab288370), Recombinant anti-MIF antibody (ab187064) (Abcam, UK). PARP Antibody #9542, Histone H3 Antibody #9715 (CST, US). Actinomycin D (HY-17559, 5 mg; MedChemExpress, USA) and the PARP-1 inhibitor AG14361 (HY-12032; MedChemExpress, USA) were used in this study.

Experimental apparatus
RT-qPCR instrument CFX96 Touch 1,855,195, Western Blot system (Bio-Rad, USA). 4-20R High-low speed universal freezer centrifuge (Hunan Hengnuo Instrument Equipment Co., Ltd., China). JP-K6000 chemiluminescence analyzer (Shanghai Jiapeng, China). BIOER-TC-EA-B-48DA gene amplifier (Hangzhou Bioer Technology Co.,Ltd, China). Ultra-low temperature refrigerator (Haier, China). Attune NxT Flow Cytometer (Themo Fisher, USA). 4-20R High-low speed universal freezer centrifuge (Hunan Hengnuo Instrument Equipment Co., Ltd., China). Attune NxT Flow Cytometer (Themo Fisher, USA).

Cell culture and treatment
The human non-small cell lung cancer cell line A549 (FH0045) and the cisplatin-resistant cell line A549/DDP (FH0091) were obtained from Shanghai Fuheng Biotechnology Co., Ltd. (Shanghai, China). Cell line authentication was performed by short tandem repeat (STR) profiling. Both A549 and A549/DDP cells were cultured in Ham’s F-12 K complete medium supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (50 U/mL). Cells were maintained in a humidified incubator at 37 °C with 5% CO₂. When cell confluence reached approximately 70–80%, cells were harvested for subsequent experiments. To determine the optimal concentration of propofol, A549 and A549/DDP cells were treated with propofol at concentrations of 10, 20, 50, and 100 µM for 24 h. Based on the screening results, 50 µM propofol was selected for subsequent experiments. For mechanistic studies, A549/DDP cells were divided into four groups: Control, DDP, propofol + DDP, and propofol + DDP+AG14361. Cells in the Control group were cultured in complete medium for 24 h. The DDP group was treated with 20 µM cisplatin for 24 h. The propofol + DDP group was treated with 50 µM propofol in combination with 20 µM cisplatin for 24 h. In the propofol + DDP+AG14361 group, cells were additionally treated with the PARP-1 inhibitor AG14361 (15 µM) for 24 h. For METTL3 gain- and loss-of-function studies, A549/DDP cells were transfected with METTL3 overexpression plasmids or METTL3-targeting shRNA constructs, with corresponding negative controls. Transfections were performed using Lipofectamine 3000, and cells were incubated for 24–48 h to establish stable METTL3 overexpression or knockdown before subsequent drug treatments. For METTL3 functional validation experiments, cells were assigned to control, METTL3 knockdown, or METTL3 overexpression groups and cultured for 24 h prior to downstream analyses. For mRNA stability analysis, cells were treated with actinomycin D to inhibit transcription, and METTL3 mRNA levels were quantified by RT-qPCR at the indicated time points.

Cell transfection
The METTL3 overexpression lentiviral vector (pc-METTL3) and the METTL3 knockdown lentiviral vector (sh-METTL3) were synthesized by Suzhou Gemma Genetics Co., Ltd. A549/DDP cells were transfected with the METTL3 overexpression or knockdown constructs to generate OE-METTL3 and KD-METTL3 cell models, respectively.
Transfections were performed using Lipofectamine 3000 reagent according to the manufacturer’s instructions. Briefly, plasmids were introduced into A549/DDP cells under optimized conditions, and cells were incubated for 48 h. Transfection efficiency was subsequently evaluated by RT-qPCR analysis.

NSCLC nude mouse model construction
Our study has been approved by laboratory animal care and ethics committee of Guangdong Laidi Biomedical Research lnstitute Co, LTD (Approval No. 2024027-3). All experiments were performed in accordance with relevant guidelines and regulations. The study is reported in accordance with ARRIVE guidelines. BALB/c nude mice (specific pathogen-free animals) were purchased from Saiye Model Biological Research Center. SPF grade 4–6 weeks old male BALB/c nude nude mice, weighing 20 ± 2 g, 3 mice in each group. The cell suspension concentration was adjusted to 5 × 106 cells/mL, and each mouse was injected subcutaneously with 0.1 mL of cell suspension in the right axilla. The injected cell suspension included A549/DDP cells, A549/DDP cells pretreated with 50µmol/L propofol for 24 h and A549/DDP cells pretreated with 50µmol/L propofol + KD-METTL3 for 24 h. After the tumor grew to 100 mm3 (about 7 days after injection), BALB/c nude mice were divided into 5 groups: model group, DDP+model group, propofol group, propofol + DDP+model group, KD-METTL3 + propofol + DDP+model group. Mice were intraperitoneally injected with 3.5 mg/kg DDP or the same amount of normal saline every three days. After 5 weeks, the nude mice were anesthetized with isoflurane, euthanized via cervical dislocation, and the tumor tissues were collected.

Immunohistochemical (IHC) staining
The protein expression of PARP-1, PAR and γH2AX in tumor tissues was determined by IHC assay. The specific operation refers to the previous research methods22. In short, tumor tissue sections were incubated with primary antibodies (anti-PARP-1, anti-PAR and anti-γH2AX) overnight at 4 ℃ and then incubated with secondary antibodies at room temperature for 1 h. Finally, it was observed under a fluorescence inverted microscope. Hematoxylin-eosin (HE) staining was also performed to confirm tissue oncology.

Cell viability was determined by CCK8
The A549/DDP cells or A549/DDP cells transfected with KD/OE-METTL3 plasmid in the logarithmic growth phase were inoculated in 96-well plates at a density of 2000 cells per well. Cells were cultured in an incubator at 37 °C and 5% CO₂ for 4–8 h. Then, according to the experimental group, cells were added with different concentrations (10, 20, 50 and 100 µmol/L) of propofol or cisplatin. The cells in each group were incubated for 24 h. Two hours prior to the conclusion of the incubation period, 10 µL of CCK8 solution was added to each well. After 1 h of continuous culture, the absorbance of each hole at 450 nm (OD450) wavelength was detected by microplate reader. Six replicate wells were established for each experimental group.

TUNEL staining to observe cell death
Following the treatment of the cells in each group, a single wash with phosphate buffered saline (PBS) was conducted. Subsequently, the cells were fixed with 4% paraformaldehyde for a period of 30 min. Then, 0.3% Triton X-100 was added and incubated at room temperature for 5 min. Prepare TUNEL solution according to manufacturer ‘s instructions: 30 µL of TdT enzyme, 270 µL of fluorescent labelling solution, and 300 µL of TUNEL detection solution. The prepared TUNEL solution was added to the cells at a volume of 100 µL per well and incubated at 37°C for 60 min in dark. The samples were then washed three times with PBS, observed under a fluorescence microscope, and photographed.

Co-IP detection of AIF combined with MIF
The A549/DDP cells were harvested, and the cell lysis buffer was added. The resulting supernatant was then subjected to centrifugation. 2 µL antibody were added to the cell lysate and incubated at 4 °C at a slow rate. Subsequently, 5 µL of Protein A agarose beads and 5 µL of Protein G agarose beads were added and incubated slowly at 4 °C. Following the immunoprecipitation reaction, the Protein A/G beads were subjected to centrifugation at 4 °C. Subsequently, the supernatant was removed and the Protein A/G beads were washed with 1 ml lysis buffer. 20 µL of 2× SDS spiking buffer were added and boiled for 5 min. The eluted protein complexes were denatured by the addition of 1–5% whole cell lysate to SDS loading buffer and subsequent heating to 95 °C for 5 min. The protein samples were separated by SDS-PAGE gel electrophoresis and subsequently detached from the buffer. The samples were then analyzed by SDS-PAGE gel electrophoresis and membrane transfer, capture, primary antibody incubation, secondary antibody incubation and development. The results of the immunoprecipitation were analyzed.

Extraction of nuclear and plasma proteins
The cells were collected and an appropriate volume of PMSF-containing Cytosolic Protein Extraction Reagent A was added according to the manufacturer’s instructions. Subsequently, the mixture was incubated on ice for a period of 10 to 15 min. Subsequently, Cytosolic Protein Extraction Reagent B was added and incubated on ice for 1 min. Subsequently, centrifugation was conducted at 12,000–16,000 g for a period of 5 min at a temperature of 4 °C. The supernatant was collected as the cytoplasmic protein fraction and immediately transferred into a pre-cooled tube. An appropriate volume of PMSF-containing Nucleoprotein Extraction Reagent was added to the pellet, and the mixture was centrifuged at 12,000–16,000 g for 10 min at 4 °C.

Western blot detection of protein expression
The proteins were extracted using radio-immunoprecipitation assay (RIPA) lysate and quantified using the BCA method. Subsequently, the samples were subjected to SDS-PAGE gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. Subsequently, the PVDF membrane was incubated for two hours at room temperature with 5% skimmed milk. Thereafter, the appropriate primary antibodies were applied overnight at 4 °C. Subsequently, secondary antibodies were added, and the membranes were developed using a chemiluminescence imaging system. Primary antibodies against AIF (1:50000), MIF (1:50000), Cytochrome C (1:5000), Bcl-2 (1:2000), Bax (1:1000), Cleaved-Caspase-3 (1:500), PAR (1:1000), γH2AX (1:1000), GAPDH (1:10000), Goat Anti-Rabbit IgG H&L(HRP) (1:20000), and Goat Anti-Mouse IgG H&L(HRP) (1:20000). The grey scale analysis was conducted using the ImageJ software.

Detection of mitochondrial membrane potential by TMRE staining
The requisite quantity of TMRE (1000X) was administered, and the TMRE and assay buffer were diluted in a proportionate manner. A total of 1 × 10⁶ cells were inoculated into each of the six wells of a 6-well plate and incubated at 37 °C for 24 h. Following this incubation period, the culture medium was aspirated and the cells were washed with PBS. Subsequently, 1 ml of the TMRE staining working solution was added, and the cells were incubated at 37 °C for 30 min. The supernatant was aspirated, and the cells were washed twice with pre-warmed culture medium. Next, 2 ml of pre-warmed culture solution containing serum and phenol red was added. The maximum excitation wavelength of TMRE was observed to be 550 nm, while the maximum emission wavelength was 575 nm.

Detection of intracellular NAD⁺/NADH ratio by the WST-8 method
A549/DDP cells were plated at 6,000 in a 6-well plate and 200 µL of NAD+/NADH extract was added to promote cell lysis. Prepare the G6PDH working solution and standard concentration gradient according to the instructions. Pipette 100–200 µL of the sample to be tested into a centrifuge tube and heat at 60 °C for 30 min in a water bath to degrade NAD+. Pipette 50 µL of the supernatant into a 96-well plate as the sample to be tested and the result is the concentration of NADH. The concentration of total NAD⁺ and NADH in the samples was calculated without heat treatment at 60 °C. Add 10 µl of colour developing solution and incubate at 37 °C for 10–20 min in the dark and read the absorbance at 450 nm.

Fluorescent probes to detect cellular ROS levels
The cells were subjected to two washes of 200 µL of PBS buffer. The MitoSOX™ Green Reagent stock solution and working solution were prepared in accordance with the instructions provided. This entails the dissolution of the vial of MSR reagent in 13 µL of DMSO, thereby creating a 5 mM MSR reagent stock solution. Subsequently, 5 µL of the 5 mM MSR stock solution was combined with 50 mL of PBS buffer, resulting in a 500 nM working solution. The cells were stained with 2 mL of 500 nM MSR reagent by incubation at 37 °C and 5% CO₂ for 30 min. Thereafter, the cells were washed with PBS buffer three times and photographed with a fluorescence microscope within 2 h of staining. The absorption and emission wavelengths were selected as 488 nm and 510 nm, respectively.

ELISA for IL-6 expression levels
A total of 1 × 106 cells were inoculated into 6-well plates and incubated for 24 h at 37 °C with 5% CO₂ to allow for cell growth. Subsequently, the samples were subjected to centrifugation for a period of 20 min, after which the resulting supernatant was utilized for the assay. The standards were diluted in accordance with the instructions provided by the ELISA kit. A volume of 50 µL was added to the enzyme-labelled coated plates, comprising the standards and samples to be tested. Subsequently, the plate was sealed with a sealing film and incubated at 37 °C for 30 min. The wash solution was diluted and added, repeating the process five times. A volume of 50 µL of enzyme reagent was added to each well. Following the sealing of the plate with a plate sealing membrane, the plate was incubated at 37 °C for 30 min and then washed. Colour Developer A and Colour Developer B were mixed and added to each well, and the colour was developed for 15 min at 37 °C, protected from light. Termination solution was added to stop the reaction. The absorbance (OD) of each well was then measured at 450 nm.

MeRIP-qPCR detection of m6A-modified PARP-1 levels
The A549/DDP cells were collected, and cell lysis buffer was added. The resulting supernatant was subsequently centrifuged. The cell lysate was combined with 4.5 µL of DNase salt stock, 10 µL of DNase (10 U) in an ice bath, and then mixed with 4.5 µL of 0.5 M EDTA, 1.8 µL of 0.5 M EGTA, and 9 µL of DTT. Subsequently, the mixture was subjected to centrifugation for a period of 10 min at a speed of 16,100 g at a temperature of 4 °C. 20 µL protein A/G magnetic beads were prepared and washed with polysome lysis buffer. Twenty microlitres of protein A/G magnetic beads were added to the IP samples, which were also treated with an IgG antibody. The magnetic beads were then added to the IP samples, which had been previously prepared with 200 µL polysome elution buffer, 2 µL DTT and µL proteinase K. The samples were resuspended and incubated at 55 °C for 1 h. Following this, the RNA was eluted, and the transfer supernatant was collected into RNase-free centrifuge tubes.
The RNA was purified through a series of steps, including chloroform separation, isopropanol precipitation, and 75% ethanol washing. The cDNA was obtained by preparing a reverse transcription system and reacting at 42 °C for 2 min, 37 °C for 15 min, and 85 °C for 5 s. A real-time quantitative polymerase chain reaction (PCR) reaction system was prepared, and the reaction was performed. The results were calculated according to the formula: ΔCt[normalised IP] = Ct [IP] - (Ct [Input] - Log2 (Input Dilution Factor)), % Input = 2 ^(-ΔCt[normalised IP])*100%.

Data analysis
The data were analyzed and graphed using GraphPad Prism 9 (Version 9.4.0). All data were expressed as means ± SD, and statistical differences between groups were tested using either the t-Test or One-way test, with P-values less than 0.05 considered significant.

Results

Propofol promotes the chemosensitivity of NSCLC cells in vitro
In order to determine the optimal concentration of propofol for enhancing the sensitivity of NSCLC cells to DDP, a CCK-8 assay was first performed to evaluate the effects of different concentrations of propofol on cell viability in both A549 and A549/DDP cells under DDP treatment. As shown in Fig. 1a, propofol at concentrations ranging from 10 to 50 µM had no significant effect on the viability of A549 cells, whereas a slight reduction was observed at 100 µM. In contrast, A549/DDP cells exhibited a concentration-dependent decrease in cell viability with increasing concentrations of propofol. Based on these results, 50 µM propofol was selected for subsequent experiments.

To further assess whether propofol enhanced DDP-induced cell death, TUNEL staining was performed in A549/DDP cells. As shown in Fig. 1b, TUNEL-positive cells are indicated by green fluorescence, while nuclei were counterstained with DAPI (blue). Compared with the control group, the proportion of apoptotic cells was significantly increased in the DDP group (P < 0.05). Moreover, combined treatment with propofol and DDP further significantly increased the apoptotic rate compared with DDP treatment alone (P < 0.05). These results indicate that propofol enhances the sensitivity of NSCLC cells to DDP.

Propofol promotes Parthanatos in NSCLC cells
To elucidate the mechanism by which propofol enhances DDP sensitivity in lung cancer cells, we investigated whether Parthanatos was involved in this process. Co-immunoprecipitation analysis revealed an increased interaction between MIF and AIF in A549/DDP cells following propofol treatment (Fig. 2a). Subcellular fractionation was subsequently performed to assess the intracellular distribution of AIF and MIF. As shown in Fig. 2b, propofol treatment significantly increased AIF levels in the cytoplasm while reducing cytoplasmic MIF levels (P < 0.05). In contrast, both AIF and MIF were markedly increased in the nuclear fraction (P < 0.01).

Intracellular ROS levels were then assessed using fluorescent probes. Compared with the DDP group, ROS production was significantly elevated in the propofol + DDP group (P < 0.05), whereas this increase was abolished by treatment with the PARP-1 inhibitor AG14361 (Fig. 2c). Consistently, assessment of mitochondrial membrane potential using TMRE staining showed that propofol + DDP treatment significantly reduced mitochondrial membrane potential (P < 0.01), an effect that was reversed by AG14361 administration (Fig. 2d, P < 0.05). In addition, intracellular NAD⁺ levels were markedly decreased in the propofol + DDP group compared with the DDP group (P < 0.001), while AG14361 treatment significantly restored NAD⁺ levels (Fig. 2e, P < 0.001).
We further measured IL-6 levels in the cell culture supernatants and observed a significant increase following propofol treatment (Fig. 2f, P < 0.001). In contrast, Western blot analysis demonstrated that the expression of apoptosis-related proteins, including cytochrome c, Bax, cleaved caspase-3, and Bcl-2, was not significantly altered by propofol treatment, either alone or in combination with DDP (Fig. 2g). Notably, the expression levels of PAR and γH2AX were significantly increased in the propofol-treated groups (P < 0.001), whereas AG14361 treatment markedly attenuated these increases (Fig. 2h, P < 0.01). Collectively, these results indicate that propofol activates PARP-1–dependent Parthanatos in lung cancer cells.

METTL3-mediated m6A modification of PARP-1 promotes Parthanatos
RT-qPCR analysis revealed that propofol treatment significantly increased METTL3 mRNA expression in DDP-treated A549/DDP cells (Fig. 3a). To further investigate the role of METTL3, METTL3 overexpression and knockdown models were established in A549/DDP cells using a pcDNA3.1-METTL3 expression vector and a sh-METTL3 lentiviral vector, respectively23(Fig. 3b). Western blot analysis confirmed the corresponding changes in METTL3 protein expression, indicating efficient manipulation of METTL3 at the protein level (Fig. 3c). To determine whether propofol regulates METTL3 at the level of mRNA stability, an actinomycin D (ActD)-based RNA decay assay was performed. After transcriptional blockade with ActD, METTL3 mRNA levels were quantified by RT-qPCR at the indicated time points. As shown in Fig. 3d, METTL3 transcripts decayed over time in both the DDP and propofol + DDP groups, while propofol treatment did not markedly alter the decay kinetics compared with DDP alone. These data suggest that propofol-induced upregulation of METTL3 is unlikely to be mediated by enhanced METTL3 mRNA stability, supporting a post-transcriptional/translational mode of regulation.

TUNEL staining demonstrated that METTL3 knockdown significantly increased the proportion of cell death, whereas METTL3 overexpression markedly reduced cell death in A549/DDP cells (Fig. 3e, P < 0.05 and P < 0.01, respectively). MeRIP-qPCR analysis further showed that, compared with the DDP group, the m6A modification level of PARP-1 was significantly decreased in the KD-METTL3 + DDP group (P < 0.01) and significantly increased in the OE-METTL3 + DDP group (P < 0.001) (Fig. 3f). Consistently, RIP-qPCR analysis using an anti-METTL3 antibody revealed that METTL3 knockdown significantly reduced, whereas METTL3 overexpression increased, the enrichment of PARP-1 transcripts (Fig. 3g, P < 0.05 and P < 0.001, respectively).
Intracellular ROS levels and mitochondrial membrane potential were subsequently assessed using fluorescent probes. As shown in Fig. 3h–i, no significant changes in ROS production or mitochondrial membrane potential were observed in the KD-METTL3 + DDP group compared with the DDP group. In contrast, METTL3 overexpression resulted in a significant increase in ROS levels and a marked reduction in mitochondrial membrane potential (P < 0.05). In addition, intracellular NAD⁺ levels were significantly increased following METTL3 knockdown, whereas METTL3 overexpression led to a pronounced decrease in NAD⁺ levels (Fig. 3j, P < 0.001). Western blot analysis further showed that METTL3 knockdown did not significantly affect the expression of PAR or γH2AX, while METTL3 overexpression markedly increased the expression of these Parthanatos-associated proteins (Fig. 3k, P < 0.001). Collectively, these results indicate that METTL3 promotes PARP-1 m6A modification and activates Parthanatos in A549/DDP cells.

Propofol modulates METTL3-mediated PARP-1 m6A modification to promote Parthanatos and promotes chemosensitivity in NSCLC cells
To further investigate whether propofol activates PARP-1–dependent Parthanatos through regulation of METTL3, A549/DDP cells were treated with propofol in combination with DDP. Western blot analysis showed that propofol treatment markedly increased METTL3 protein expression in DDP-treated A549/DDP cells (Fig. 4a). To determine whether the chemosensitizing effect of propofol was dependent on METTL3, propofol and DDP were subsequently applied in a METTL3 knockdown model.

As shown in Fig. 4b-c, METTL3 knockdown significantly attenuated the propofol-induced increase in PARP-1 mRNA expression and reversed the decrease in intracellular NAD⁺ levels (P < 0.05). Consistently, TMRE staining demonstrated that mitochondrial membrane potential was significantly restored in the KD-METTL3 + propofol + DDP group compared with the propofol + DDP group (Fig. 4d, P < 0.01). In addition, Western blot analysis revealed that knockdown of METTL3 markedly attenuated the propofol-induced upregulation of γH2AX and PAR (Fig. 4e, P < 0.05).
Collectively, these results indicate that the chemosensitizing effect of propofol in A549/DDP cells is dependent on METTL3 and is associated with activation of PARP-1-mediated Parthanatos.

In vivo level validation of propofol modulation of METTL3 to promote tumour cell Parthanatos and enhance NSCLC chemosensitivity
A subcutaneous xenograft tumor model was established in BALB/c nude mice to evaluate the in vivo effects of propofol and METTL3 knockdown on DDP sensitivity in NSCLC. As shown in Fig. 5a-c, treatment with propofol significantly enhanced the tumor-suppressive effect of DDP, as evidenced by reduced tumor volume and tumor weight compared with the DDP-treated group. Notably, knockdown of METTL3 markedly attenuated the antitumor efficacy of propofol combined with DDP, indicating that METTL3 is required for propofol-mediated chemosensitization in vivo. Histopathological examination by hematoxylin and eosin (H&E) staining revealed intact tumor architecture with high cellular density in the Model group. In contrast, varying degrees of tumor cell loss and necrotic areas were observed in the DDP- and propofol-treated groups. The most extensive tumor necrosis and structural disruption were detected in the propofol + DDP group, whereas METTL3 knockdown substantially alleviated these pathological changes (Fig. 5d). Furthermore, immunohistochemical analysis demonstrated that propofol synergistically enhanced DDP-induced expression of the Parthanatos-associated markers PARP-1, γH2AX, and PAR in tumor tissues. In contrast, silencing of METTL3 significantly attenuated the upregulation of PARP-1, γH2AX, and PAR induced by propofol and DDP (Fig. 5e-g). Collectively, these in vivo findings demonstrate that propofol enhances DDP chemosensitivity by promoting METTL3-dependent activation of tumor cell Parthanatos, thereby corroborating the mechanistic observations obtained from in vitro experiments.

Discussion
In the present study, we identified a previously unrecognized mechanism by which propofol enhances cisplatin (DDP) sensitivity in non-small cell lung cancer (NSCLC). Our results demonstrate that propofol upregulates METTL3 expression and promotes METTL3-mediated m6A modification of PARP-1, leading to PARP-1 hyperactivation, NAD⁺ depletion, mitochondrial dysfunction, and activation of Parthanatos. This mechanism provides new insight into how anesthetic agents may directly regulate tumor cell fate and contribute to the reversal of chemotherapy resistance24.
An increasing number of studies indicate that anesthetic agents are not biologically inert in the context of cancer. Both local and general anesthetics have been reported to exert direct effects on tumor cell viability, migration, and survival. A comprehensive review by Votta-Velis et al. summarized that local anesthetics can suppress cancer cell proliferation, induce oxidative stress, impair mitochondrial function, and interfere with intracellular signaling pathways involved in tumor progression25. Similarly, experimental evidence has shown that local anesthetics significantly reduce breast cancer cell viability and migration, supporting a direct anti-tumor effect independent of their anesthetic properties26.
Among local anesthetics, lidocaine has been extensively studied for its anti-tumor potential. Notably, lidocaine was reported to suppress the viability and migration of human breast cancer cells by targeting the TRPM7 ion channel, highlighting ion channel-dependent mechanisms as a critical mediator of anesthetic-induced anti-tumor effects27. Although the molecular targets differ from those identified in our study, these findings collectively suggest that anesthetic agents can regulate tumor cell survival through intracellular stress responses, mitochondrial dysfunction, and signaling pathways—processes that conceptually align with regulated cell death programs including Parthanatos.
Propofol, as a commonly used intravenous general anesthetic, has attracted particular interest due to its potential anti-tumor and immunomodulatory effects. A comprehensive review reported that propofol can influence breast cancer cell behavior, immune function, and even patient outcomes, suggesting that propofol may exert both direct tumor-suppressive and indirect immunological effects28. In lung cancer, previous studies demonstrated that propofol induces apoptosis and enhances cisplatin sensitivity through modulation of signaling pathways such as miR-21/PTEN/AKT and m6A-dependent miRNA regulation7,9. Consistent with these observations, our study further confirms the chemosensitizing effect of propofol in NSCLC, particularly in the DDP-resistant A549/DDP cell line, which has been insufficiently explored in prior work.
Notably, although previous studies have demonstrated that propofol enhances chemosensitivity primarily through the regulation of apoptosis-related signaling pathways or microRNA-mediated networks, such as the miR-21/PTEN/AKT axis or m6A-dependent miR-486-5p/RAP1/NF-κB signaling, these mechanisms mainly converge on classical apoptotic or inflammatory responses. In contrast, the present study identifies a fundamentally distinct form of regulated cell death-Parthanatos-as a key mechanism underlying propofol-mediated chemosensitization in NSCLC. Mechanistically, we demonstrate that propofol directly modulates METTL3-dependent m6A modification of PARP-1, thereby promoting PARP-1 overactivation, NAD⁺ depletion, PAR accumulation, and subsequent AIF–MIF nuclear translocation. This METTL3/PARP-1/Parthanatos axis represents a previously unrecognized epigenetic–metabolic mechanism of propofol action, which is mechanistically distinct from apoptosis- or microRNA-centered pathways reported in earlier studies.
Importantly, our findings extend the current understanding of propofol’s anti-tumor activity by identifying Parthanatos as a key downstream cell death pathway. Parthanatos is characterized by PARP-1 hyperactivation, excessive PAR accumulation, NAD⁺ depletion, mitochondrial membrane potential loss, and AIF/MIF-mediated large-scale DNA fragmentation29,30. In the present study, propofol treatment enhanced AIF–MIF interaction and nuclear translocation, increased intracellular ROS levels, depleted NAD⁺, reduced mitochondrial membrane potential, and upregulated PAR and γH2AX expression. These effects were reversed by the PARP-1 inhibitor AG14361, confirming that PARP-1 activation is essential for propofol-induced Parthanatos. Similar Parthanatos-mediated mechanisms have been reported in other tumor models, such as oxaliplatin-induced oral squamous cell carcinoma and cannabinoid-induced leukemia cell death22,30, supporting the broader relevance of this pathway in cancer therapy.
At the clinical level, accumulating evidence suggests that anesthetic strategies used during cancer surgery may influence postoperative outcomes. A recent meta-analysis reported that general anesthetics can affect postoperative metastasis and inflammatory cytokine profiles, implying that anesthetic choice may modulate tumor biology and systemic inflammation31. Furthermore, a recent review emphasized the potential therapeutic applications of local anesthetics in cancer treatment, proposing that anesthetic drugs could be repurposed as adjuvant anti-tumor agents32. Although these studies do not directly address Parthanatos, they support the concept that anesthetic agents can influence tumor progression and treatment response through direct cellular mechanisms.
At the molecular level, our study highlights the importance of epitranscriptomic regulation in chemoresistance. m6A modification is the most prevalent internal modification of eukaryotic mRNA and plays a crucial role in RNA stability and translation17–19. METTL3, a core m6A methyltransferase, has been implicated in tumor progression and drug resistance across multiple cancer types. Previous studies reported that METTL3-mediated m6A modification of PARP-1 enhances PARP-1 mRNA stability and contributes to platinum resistance21. In line with these findings, we demonstrated that METTL3 overexpression increased PARP-1 m6A levels and activated Parthanatos, whereas METTL3 knockdown attenuated propofol-induced PARP-1 activation and Parthanatos.
In addition to epitranscriptomic control, accumulating evidence indicates that the balance between DNA damage responses, metabolic stress adaptation, and regulated cell death critically shapes platinum sensitivity. Recent work has highlighted that altered metabolic homeostasis and stress adaptation can influence tumor vulnerability under therapeutic stress33. In parallel, emerging RNA regulatory networks, including non-coding RNA programs, have been implicated in shaping cell-cycle control and treatment responsiveness in NSCLC34. Together with our findings, these studies support a model in which chemotherapy response is governed by an integrated regulatory network involving metabolic remodeling, RNA modification/regulation, and regulated cell death pathways. In this context, METTL3-mediated m6A modification of PARP-1 may represent a critical molecular node linking RNA regulation to PARP-driven metabolic collapse and Parthanatos activation in NSCLC.
Recent studies further support the relevance of this axis. A recent report35 demonstrated that dysregulation of PARP-1-associated DNA damage responses contributes to platinum resistance by altering the balance between DNA repair and regulated cell death. Moreover, an emerging study36 highlighted that m6A-related regulatory networks critically influence chemotherapy sensitivity through coordination of DNA damage signaling and cell death pathways. These observations are highly consistent with our findings and further reinforce the clinical relevance of the METTL3–PARP-1–Parthanatos axis. Despite these strengths, this study has several limitations. Although our in vivo experiments support the role of METTL3-dependent Parthanatos in propofol-mediated chemosensitization, further validation in additional animal models and clinical samples is required. In addition, other m6A regulators and reader proteins may also participate in propofol-induced Parthanatos and warrant further investigation. Finally, the interaction between anesthetic agents and emerging treatment modalities, such as targeted therapy and immunotherapy, remains to be explored. A limitation of this study is that PARP-1 enzymatic activity was not directly measured; future studies incorporating PARP activity assays will further strengthen the mechanistic conclusion. Moreover, the in vivo cohort size was relatively small and was not based on an a priori power calculation; future studies using power-based sample size estimation will be important to further validate the robustness of these findings.
In conclusion, our study reveals a novel mechanism by which propofol enhances cisplatin sensitivity in NSCLC through METTL3-mediated m6A modification of PARP-1 and subsequent activation of Parthanatos. These findings provide mechanistic insight into the anti-tumor potential of anesthetic agents and suggest that modulation of epitranscriptomic and regulated cell death pathways may represent a promising strategy for overcoming chemotherapy resistance in NSCLC.

Conclusion
In conclusion, this study demonstrates that propofol enhances cisplatin sensitivity in non-small cell lung cancer by activating Parthanatos in drug-resistant tumor cells. Mechanistically, propofol upregulates METTL3 expression and promotes METTL3-mediated m6A modification of PARP-1, leading to PARP-1 activation, NAD+ depletion, and PAR accumulation, which collectively trigger Parthanatos. These findings elucidate a novel epitranscriptomic mechanism underlying propofol-mediated chemosensitization and provide new insight into the regulation of cisplatin resistance in NSCLC. Targeting the METTL3–PARP-1–Parthanatos axis may represent a promising therapeutic strategy for overcoming cisplatin resistance in NSCLC.

Supplementary Information
Below is the link to the electronic supplementary material.