Receptor subtype-dependent effects of propofol on metalloproteinase activity, NKG2D ligand expression, and NK cell-mediated cytotoxicity in breast cancer: an in vitro study
Article information
Abstract
Background
The effects of propofol, a commonly used intravenous anesthetic, on the breast cancer tumor microenvironment are not well understood. This study examined the influence of propofol on natural killer (NK) group 2, member D (NKG2D) ligand expression, matrix metalloproteinase (MMP)-mediated immune evasion, and NK cell-mediated cytotoxicity in breast cancer cells.
Methods
We studied three human breast cancer cell lines representing distinct receptor subtypes: MCF-7 (estrogen receptor - and progesterone receptor-positive), MDA-MB-453 (human epidermal growth factor receptor 2-positive), and HCC-70 (triple-negative). Cells were treated with propofol at concentrations of 0 μg/ml (control; C), 4 μg/ml (P4), or 8 μg/ml (P8). Assessments included mRNA and protein expression of NKG2D ligands, NK cell cytotoxicity, protein levels of MMP-1 and MMP-2, and concentrations of soluble NKG2D ligands.
Results
In MCF-7 and HCC-70 cell lines, propofol upregulated the mRNA and protein expression of NKG2D ligands in a dose-dependent manner, enhancing NK cell-mediated lysis. In contrast, in MDA-MB-453 cell lines, propofol downregulated the mRNA and protein expression of NKG2D ligands, resulting in diminished NK cell-mediated lysis. Across all receptor subtypes, propofol did not affect the expression of MMP-1 or MMP-2 or the concentration of soluble NKG2D ligands.
Conclusions
Our results demonstrate that propofol exerts receptor subtype-dependent effects on NK cell-mediated immunosurveillance in breast cancer cell lines, potentially mediated by changes in the transcription of NKG2D ligands rather than by alterations in MMP expression or their proteolytic activity.
Introduction
Surgical resection remains the primary curative treatment for breast cancers [1]. However, even if resection is successful, various perioperative factors may lead to cancer cell metastasis, growth, and resistance to postoperative chemotherapy [1–3]. Because the available options for reducing surgical stress are limited, it is critical to identify modifiable factors that reduce cancer progression. By the 2000s, it was suggested that anesthetics and anesthesia techniques could potentially affect cancer metastasis and progression [4,5]. However, clear conclusions have not yet been reached regarding the effects of anesthetics on the prognosis of breast cancers, nor have the mechanisms been clearly explained.
Breast cancer is not a singular disease but rather a heterogeneous group of neoplasms spanning a broad spectrum of molecular subtypes. These subtypes are classified by hormone receptor and human epidermal growth factor receptor 2 (HER2) expression that influence treatment strategies and tumor-immune interactions [6–10]. Despite the importance of receptor-defined differences, their complexity has not been fully addressed in studies on the effects of anesthetics on immune responses and cancer outcomes.
This study explored the effect of propofol, one of the most widely used intravenous anesthetics, on breast cancer immunosurveillance and evasion in a receptor-dependent manner. Specifically, we assessed the impact of propofol on the expression of natural killer (NK) group 2, member D (NKG2D) ligands (major histocompatibility complex class I chain-related molecules [MIC] A/B and UL16-binding proteins [ULBP] 1–3), key mediators of NK cell recognition and activation, and its effect on NK cell-mediated cytotoxicity [11]. We also evaluated propofol’s influence on matrix metalloproteinase (MMP) expression, its role in NKG2D ligand shedding, and the modulation of NK cell responses [12,13].
Materials and Methods
Cell lines, reagents, and propofol treatment
This in vitro study did not involve human participants or materials and did not require institutional review board approval. In this study, three breast cancer cell lines were used, categorized by receptor subtype [7]: MCF-7 (estrogen receptor [ER]- and progesterone receptor [PR]-positive), HCC-70 (triple-negative), and MDA-MB-453 (HER2-positive).
All breast cancer cell lines were obtained from the Korean Cell Line Bank and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin (Welgene, Inc.). The NK92-MI human NK cell line (ATCC) was cultured in α-minimum essential medium (Gibco) supplemented with 12.5% fetal bovine serum (Welgene, Inc.), 12.5% horse serum (HyClone), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich, Inc.), and 2 mM L-glutamine (Gibco). All cell lines were maintained according to their specific requirements and incubated at 37°C in a humidified atmosphere containing 5% CO2.
The use of propofol (Anepol®; Hana Pharm Co., Ltd) was approved by the Ministry of Food and Drug Safety, Republic of Korea. Propofol was administered at 4 μg/ml (P4), reflecting its clinical anesthetic concentration [14,15], and at a higher dose of 8 μg/ml (P8) to evaluate the dose-response relationship. As a control (C), an equivalent volume of distilled water diluted in RPMI-1640 media (0 μg/ml) was used without propofol treatment.
Analysis of mRNA expression was conducted 18 h after treatment. In contrast, other experiments—including cell viability testing, flow cytometry for surface NKG2D ligand expression, Western blotting, enzyme-linked immunosorbent assay (ELISA) for soluble NKG2D ligands, and flow cytometry for NK cell-mediated cytotoxicity—were conducted 24 h later.
The sample size (n) refers to the number of wells analyzed, not the number of multi-well culture plates.
MTT assay for cell viability analysis
As previously described [16], each type of breast cancer cell was plated in 96-well plates at a density of 1 × 104 cells per well and treated with either the C or P8 concentration; the highest propofol concentration (P8) was compared with the C under the assumption that if no cytotoxicity were observed at P8, then P4 would also be non-toxic. After a 24-hour incubation period, MTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Sigma-Aldrich) was added, and the cells were incubated for an additional 4 h. The supernatant was removed, and the formazan crystals were dissolved in dimethyl sulfoxide. Absorbance was measured at 540 nm using a μQuant microplate spectrophotometer (Bio Tek).
Real-time polymerase chain reaction (PCR) for NKG2D ligand mRNA transcription analysis
Real-time PCR was used to analyze mRNA expression of NKG2D ligands, following the procedure outlined by Borchers et al. [17]. Briefly, breast cancer cells were harvested 18 h after treatment. Total RNA was isolated using TRIzol reagent (Invitrogen) and reverse transcribed into cDNA using CellScript All-in-One 5X First Strand cDNA Synthesis Master Mix (CellSafe), according to the manufacturer’s protocol. Real-time PCR was performed on samples with SYBR Green PCR Master Mix (TOPrealTM qPCR 2X PreMIX, Enzynomics) using an ABI 7500 real-time PCR system (Applied Biosystems) in duplicate, following the manufacturer’s guidelines. The primers used in this study are listed in Table 1.
List of Primers Used in Real-time PCR
The PCR protocol consisted of an initial incubation at 50°C for 2 min and initial denaturing at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s and annealing and extension at 60°C for 1 min with a final dissociation step. Relative gene expression was calculated using the Δ cycle threshold (ΔCt) method, normalizing the average Ct value of each sample to its glyceraldehyde-3-phosphate dehydrogenase control. The 2−ΔΔCt formula was then applied to determine the relative expression for each sample.
Flow cytometry for NKG2D ligand expression analysis
Flow cytometry was performed to evaluate the surface expression of NKG2D ligands, as previously described [16]. Briefly, breast cancer cells were collected 24 h after treatment and incubated with 10 μg/ml mouse anti-MICA/B and ULBP1–3 antibodies or their corresponding isotype controls (anti-IgG2a or anti-IgG2b; R&D Systems). The samples were treated with a goat anti-mouse phycoerythrin (PE)-conjugated secondary antibody (BD Biosciences). The mean fluorescence intensity (MFI) was determined using a FACSCantoTM II flow cytometer (BD Biosciences) and analyzed with FlowJo software ver. 10.6.1 (TreeStar, Inc.). Relative expression ratios were calculated by dividing the MFI values of the treated samples by those of the controls.
Western blot analysis for MMP-1 and MMP-2 expression
Samples were collected 24 h after treatment, and Western blot analysis was conducted to evaluate MMP-1 and MMP-2 expression. Cells were washed three times with cold phosphate-buffered saline and lysed with PRO-PREP protein extraction solution (iNtRON Bio). Proteins were resolved on 4–20% sodium dodecyl sulfate-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (MilliporeSigma).
The membranes were blocked at room temperature with 3% bovine serum albumin in Tris-buffered saline containing 0.1% Tween 20. Target proteins were detected by incubating the membranes with primary antibodies specific for MMP-1 and MMP-2 (Cell Signaling Technology), followed by horseradish peroxidase-conjugated secondary antibodies (Enzo Life Sciences), according to the manufacturer’s protocols. Chemiluminescent signals were captured using the AE-9150 Ez-capture II imaging system (Atto Corporation). Since the intensity of chemiluminescence from a peroxidase-mediated reaction is linearly proportional to the concentration of the target molecule [18], we employed this method to measure the quantities of MMP-1 and MMP-2. Band intensities were analyzed using ImageJ software ver. 1.54 (NIH). Protein expression levels of MMPs were normalized to β-actin (Sigma-Aldrich) that served as a loading control to determine the relative expression ratios.
ELISA for soluble NKG2D ligands
Breast cancer cells (4 × 106) were seeded in six-well plates. After 24 h of treatment, cell culture supernatants were collected, centrifuged at 5000 rpm for 5 min at 4°C, and stored at −80°C until analysis. According to the manufacturer’s protocol, the concentration of soluble NKG2D ligands shed into the supernatant was quantified using a human soluble NKG2D ligands ELISA kit (MyBioSource, Inc.). Absorbance at 450 nm was measured with a microplate spectrophotometer (SynergyTM H1, BioTek). Each sample was tested in duplicate, and the mean concentration of soluble NKG2D ligands was analyzed.
Flow cytometry for NK cell-mediated cytotoxicity assay
The NK cell-mediated cytotoxicity assay was performed as described in our previous study [16], and the procedure is outlined in Fig. 1. After 24 h of treatment, breast cancer target cells were harvested, stained with carboxyfluorescein diacetate succinimidyl ester (CFSE; CellTraceTM, Invitrogen), and co-cultured with NK92-MI effector cells for 4 h. Based on recommended protocols from previous studies [16,19,20], effector-to-target (E:T) ratios were established at 1:1 (1 × 105 effector cells to 1 × 105 target cells) and 10:1 (1 × 106 effector cells to 1 × 105 target cells). In this study, the 10:1 ratio was optimal for maximizing NK cell-mediated target cell lysis and yielding the most significant NK cell cytotoxicity effect.
Methodology of NK cell-mediated cytotoxicity assay. NK: natural killer, CFSE: carboxyfluorescein diacetate succinimidyl ester, PI: propidium iodide.
After co-culture, dead cells were labeled with 1 μg/ml propidium iodide (PI; Invitrogen) to distinguish them from live cells. Fluorescence signals from the labeled cells were detected using a FACSCantoTM II flow cytometer and analyzed with BD FACSDivaTM software ver. 7.0 (BD Biosciences).
The percentage of NK cell-mediated lysis was calculated using the formula:
([Dead target cell count] / [Dead target cell count + Live target cell count]) × 100
‘Dead target cells’ are defined as cells positive for both CFSE and PI, whereas ‘live target cells’ are positive for CFSE but negative for PI.
Statistical analysis
MedCalc® ver. 20 (MedCalc Software Ltd.) and IBM SPSS Statistics ver. 25 (IBM) were used for statistical analysis. Variables were summarized as medians with interquartile ranges (Q1, Q3). Group comparisons were conducted using the Mann–Whitney U or Kruskal–Wallis test, with post-hoc analysis by the Bonferroni correction, if significant. The study included a control group (C) and two propofol groups (P4, P8) treated in sequences C, P4, and P8. The Jonckheere–Terpstra trend test was used to evaluate dose-response relationships. Statistical significance was defined as P < 0.05 (two-sided).
Results
Effect of propofol on cell viability: MTT assay
Propofol, at concentrations up to 8 μg/ml, did not significantly affect the viability of MCF-7, HCC-70, and MDA-MB-453 cells. Relative cell viabilities (%) compared to the control (100%) were as follows (median [Q1, Q3]): MCF-7—P8: 107.0 (94.4, 117.3), P = 0.305; HCC-70—P8: 93.5 (91.1, 106.0), P = 0.319; and MDA-MB-453—P8: 76.1 (69.3, 101.7), P = 0.305 (n = 6 per group).
NKG2D ligand mRNA transcription: real-time PCR
Fig. 2 summarizes the results (n = 6 per group; Supplementary Table 1). In MCF-7 cells, the relative mRNA expression levels of ULBP2 at P8 were significantly higher than in the control group (Bonferroni-corrected P = 0.040). A significant increasing trend in relative mRNA expression of ULBP2 was observed with higher propofol concentrations (Jonckheere–Terpstra trend test, P = 0.029).
Effect of propofol on the mRNA expression of NKG2D ligands in (A) MCF-7, (B) HCC-70, and (C) MDA-MB-453 cells. Variables are presented as the median with the first and third quartiles (n = 6 per group). MICA/B: major histocompatibility complex class I chain-related molecules A/B, ULBP: UL16-binding proteins. *P < 0.05 compared to the control (Kruskal–Wallis test with Bonferroni correction).
In HCC-70 cells, the relative mRNA expression levels of ULBP2 at P8 were significantly higher than in the control group (Bonferroni-corrected P = 0.018). The relative mRNA expression of ULBP2 also exhibited a significant increasing trend with rising propofol concentrations (Jonckheere–Terpstra trend test, P = 0.010).
In MDA-MB-453 cells, the relative mRNA expression levels of MICA at P8 were significantly lower than in the control group (Bonferroni-corrected P = 0.007). A significant downregulation in the relative mRNA expression of MICA was observed with increasing propofol concentrations, as demonstrated by the Jonckheere–Terpstra trend test (P = 0.003).
NKG2D ligand expression: flow cytometry analysis
Fig. 3 shows flow cytometry analysis for surface expressions of NKG2D ligands (n = 6 per group; Supplementary Table 2). In MCF-7 cells, MICA and ULBP2 were predominantly expressed. The relative surface expression ratio of MICA at P4 was significantly upregulated compared to the control (Bonferroni-corrected P = 0.001). Similarly, ULBP1 showed increased relative surface expression ratios at P4 and P8 compared to the control (Bonferroni-corrected P = 0.034 and P = 0.002, respectively). ULBP2 and ULBP3 exhibited upregulated relative surface expression ratios at P8 compared to the control (Bonferroni-corrected P < 0.001 and P = 0.009, respectively).
Effect of propofol on surface expressions of NKG2D ligands in (A) MCF-7, (B) HCC-70, and (C) MDA-MB-453 cells. Variables are presented as the median with the first and third quartiles (n = 6 per group). MICA/B: major histocompatibility complex class I chain-related molecules A/B, ULBP: UL16-binding proteins, PE: Phycoerythrin. *P < 0.05 compared to the control (Kruskal–Wallis test with Bonferroni correction).
In HCC-70 cells, the surface expression of MICB, ULBP2, and ULBP3 was prominent. The surface expression ratio of MICA was significantly increased at P4 and P8 relative to the control (Bonferroni-corrected P = 0.018 and P = 0.004, respectively). A significant upregulation of MICB was observed at P8 compared with the control (Bonferroni-corrected P = 0.003). ULBP1 showed higher surface expression ratios at P4 and P8 than the control (Bonferroni-corrected P = 0.013 and P = 0.006, respectively). For ULBP2, surface expression was significantly upregulated at P8 (Bonferroni-corrected P = 0.001). Similarly, ULBP3 exhibited increased surface expression at P4 and P8 relative to the control (Bonferroni-corrected P = 0.040 and P = 0.002, respectively).
In MDA-MB-453 cells, MICA and ULBP2 were the predominant surface-expressed ligands. At P8, the surface expression ratios of MICA and MICB were significantly reduced compared with the control (Bonferroni-corrected P = 0.001 for both). ULBP1 demonstrated lower surface expression ratios at P4 and P8 than the control (Bonferroni-corrected P = 0.040 and P = 0.002, respectively). ULBP2 also showed decreased surface expression ratios at P4 and P8 compared with the control (Bonferroni-corrected P = 0.013 and P = 0.006, respectively). A significant downregulation of ULBP3 was observed at P8 compared with the control (Bonferroni-corrected P = 0.021).
In a dose-response relationship analysis, higher propofol concentrations were associated with significant changes in the relative surface expression ratios of NKG2D ligands across cell lines. In MCF7 cells, the surface expression ratios of ULBP1, ULBP2, and ULBP3 were significantly increased with higher concentrations of propofol (Jonckheere–Terpstra trend test; P < 0.001, P < 0.001, and P = 0.005, respectively). In HCC-70 cells, the surface expression ratios of MICA, MICB, ULBP1, ULBP2, and ULBP3 were also significantly elevated with increasing propofol concentrations (Jonckheere–Terpstra trend test; P = 0.001, P < 0.001, P = 0.002, P < 0.001, and P < 0.001, respectively). In contrast, in MDA-MB-453 cells, higher concentrations of propofol resulted in significantly reduced surface expression ratios of MICA, MICB, ULBP1, ULBP2, and ULBP3 (Jonckheere–Terpstra trend test; P < 0.001, P < 0.001, P < 0.001, P = 0.002, and P = 0.012, respectively).
MMP-1 and MMP-2 expression: Western blot analysis
Fig. 4 shows the Western blot analysis of MMP-1 and MMP-2 expression (n = 6 per group; Supplementary Table 3). No significant differences in protein expression of MMP-1 and MMP-2 were identified between the control and propofol-treated groups in MCF-7, HCC-70, and MDA-MB-453 cells (Kruskal–Wallis test; MMP-1: P = 0.927, 0.587, and 0.932; MMP-2: P = 0.700, 0.977, and 0.960, respectively). Consistently, the Jonckheere–Terpstra trend test indicated no dose-dependent changes in MMP-1 or MMP-2 expression across MCF-7, HCC-70, and MDA-MB-453 cells (MMP-1: P = 0.872, 0.293, and 0.686; MMP-2: P = 0.419, 0.872, and 0.808, respectively).
Effect of propofol on MMPs expression in (A) MCF-7, (B) HCC-70, and (C) MDA-MB-453 cells. Variables are presented as the median with the first and third quartiles (n = 6 per group). MMP: matrix metalloproteinase.
Released soluble NKG2D ligands: ELISA
No significant differences in the concentration of released soluble NKG2D ligands were observed between the control and propofol-treated groups in MCF-7, HCC-70, and MDA-MB-453 cells (n = 9 per group). In MCF-7 cells, the median (Q1, Q3) concentrations of released soluble NKG2D ligands (pg/ml) were: control, 2139.5 (1852.1, 2438.6); P4, 2511.9 (1696.1, 3045.9); and P8, 2702.7 (2204.1, 3208.6). The P values for the Kruskal–Wallis and Jonckheere–Terpstra trend tests were 0.314 and 0.137, respectively.
In HCC-70 cells, the median (Q1, Q3) concentrations of released soluble NKG2D ligands (pg/ml) were: control, 2561.2 (1842.7, 3784.3); P4, 2354.2 (2101.2, 3059.2); and P8, 2106.0 (1986.5, 2622.8). The P values for the Kruskal–Wallis and Jonckheere–Terpstra trend tests were 0.711 and 0.411, respectively.
In MDA-MB-453 cells, the median (Q1, Q3) concentrations of released soluble NKG2D ligands (pg/ml) were: control, 1802.2 (1648.4, 2475.2); P4, 1747.8 (1616.1, 2290.5); and P8, 1659.6 (1531.5, 1906.3). The P values for the Kruskal–Wallis and Jonckheere–Terpstra trend tests were 0.576 and 0.276, respectively.
NK cell-mediated cytotoxicity: flow cytometry analysis
The results summarized in Fig. 5 (Supplementary Table 4) reveal that propofol modulated NK cell-mediated cytotoxicity in a cell line-dependent manner. In MCF-7 and HCC-70 cells, cytotoxicity at P8 was significantly increased compared with the control (Bonferroni-corrected P = 0.013 and P = 0.005, respectively; effector cells:target cells = 10:1, n = 4 per group). Furthermore, the Jonckheere–Terpstra trend test indicated a dose-dependent enhancement of NK cell-mediated cytotoxicity in both MCF-7 and HCC-70 cells with increasing propofol concentrations (P = 0.002 and P < 0.001, respectively).
Effect of propofol on NK cell-mediated cytotoxicity in (A) MCF-7, (B) HCC-70, and (C) MDA-MB-453 cells. Variables are presented as the median with the first and third quartiles (n = 4 per group). NK: natural killer, E: effector cells (NK-92-MI), T: target cells (MCF-7, MDA-MB-453, and HCC-70 cells), CFSE: carboxyfluorescein diacetate succinimidyl ester, PI: propidium iodide. *P < 0.05 compared to the control (Kruskal–Wallis test with Bonferroni correction).
Conversely, in MDA-MB-453 cells, NK cell-mediated cytotoxicity at P8 was significantly reduced compared with the control (Bonferroni-corrected P = 0.005; effector cells:target cells = 10:1, n = 4 per group). Similarly, the Jonckheere–Terpstra trend test demonstrated a dose-dependent reduction in NK cell-mediated cytotoxicity in MDA-MB-453 cells as propofol concentrations increased (P < 0.001).
Discussion
This study demonstrated that propofol exerts receptor subtype-dependent effects on NK cell-mediated tumor immunosurveillance in breast cancer cell lines (Fig. 6). In MCF-7 (ER/PR-positive) and HCC-70 (triple-negative) cell lines, propofol upregulated the mRNA and protein expression of NKG2D ligands in a dose-dependent manner, enhancing NK cell-mediated cancer cell lysis. Conversely, in MDA-MB-453 (HER2-positive) cell lines, propofol downregulated the mRNA and protein expression of NKG2D ligands in a dose-dependent manner, reducing NK cell-mediated cancer cell lysis. Across all receptor subtypes, propofol did not affect the expression of MMP-1 or MMP-2, nor did it influence the concentration of soluble NKG2D ligands generated by proteolytic cleavage of surface NKG2D ligands.
Schematic summary of study findings. ER/PR: estrogen receptor/progesterone receptor, TNBC: triple-negative breast cancer, HER2: human epidermal growth factor receptor 2, NK: natural killer, NKG2D: natural killer group 2, member D, MMP: Matrix metalloproteinase.
Over the past two decades, the potential impact of anesthetic techniques on cancer recurrence and overall survival after surgery has garnered significant attention [1,5]. Numerous prospective and retrospective clinical studies have been conducted on breast cancer, but their conclusions remain inconclusive [21–26]. A single-center retrospective study of 325 patients undergoing modified radical mastectomy reported improved recurrence-free survival with propofol anesthesia compared to sevoflurane (HR: 0.478, 95% CI [0.265–0.862]) [21]. Similarly, Enlund et al.’s multicenter propensity-matched study (n = 418) demonstrated that patients treated with propofol had superior overall survival [22]. In contrast, more extensive retrospective studies by Kim et al. (n = 2645) and Yoo et al. (n = 5331) reported no significant differences in overall or recurrence-free survival between the two anesthetics [23,24]. A randomized controlled trial of 201 breast cancer patients evaluating postoperative circulating tumor cells, a key prognostic marker for metastasis and recurrence, found no significant difference between sevoflurane and propofol anesthesia [25]. Similarly, a recent propensity-matched study reported no significant difference in recurrence-free survival between the two anesthetics in the overall cohort (n = 1026) [26]. However, in receptor-defined subtypes, sevoflurane was associated with a higher risk of recurrence or metastasis in patients with Luminal B (ER+ and/or PR+) and HER2 (+) breast cancer (HR: 5.027, 95% CI [1.369–18.455]) [26].
NKG2D is the primary receptor activating NK cells, facilitating their binding to target cells and triggering cytotoxic signaling [11,27]. NKG2D recognizes multiple human ligands, including MICA/B and ULBPs [28,29]. Under stress, different NKG2D ligand subtypes dominate based on cell type and microenvironment [30,31]. In this study, MICA and ULBP2 were predominant in MCF-7 and MDA-MB-453 cells, while MICB, ULBP2, and ULBP3 were predominant in HCC-70 cells. Importantly, propofol at a clinically relevant concentration significantly altered the expression, with NK cell cytotoxicity exhibiting a dose-dependent sequential trend across the control, P4, and P8 groups.
In our previous study, sevoflurane, an inhalation anesthetic, was observed to downregulate mRNA and protein expression of NKG2D ligands in a dose-dependent manner across hormone receptor-positive, HER2-positive, and triple-negative breast cancer cell lines [16]. This downregulation was accompanied by a reduction in NK-cell-mediated cancer cell lysis, suggesting a potential immunosuppressive effect of sevoflurane in breast cancer [16]. In contrast, the present study found that propofol had a distinct impact on NK cell-mediated tumor immunity, depending on the receptor expression profiles of the breast cancer cell lines. In MCF-7 and HCC-70 cells, propofol upregulated NKG2D ligand expression, enhancing NK cell-mediated lysis. Conversely, in MDA-MB-453 cells, propofol downregulated NKG2D ligand expression, resulting in diminished cancer cell lysis. These findings are presumed to reflect the differential regulation of NKG2D ligand mRNA expression by propofol, with upregulation observed in MCF-7 and HCC-70 cells and downregulation in MDA-MB-453 cells.
There is limited research on the effects of propofol on NK cell-mediated immunity in breast cancer, and the available findings are inconsistent [25,32,33]. In a pilot clinical trial investigating serum from breast cancer patients, propofol combined with paravertebral block anesthesia was associated with increased expression of CD107a on NK cells, a marker of degranulation, and enhanced NK cell-mediated apoptosis in co-culture with hormone receptor-positive breast cancer cells [32]. Similarly, a randomized controlled trial with 50 participants demonstrated that propofol anesthesia with postoperative ketorolac analgesia enhanced NK cell-mediated cytotoxicity 24 h after breast cancer resection [33]. In contrast, a randomized controlled trial on serum from breast cancer surgery patients found no significant difference in the cytotoxicity of circulating NK cells between the propofol and sevoflurane groups (mean apoptosis rate: propofol, 35.7%; sevoflurane, 34.7%; n = 30 per group) [25]. The discrepancies with our findings may stem from the heterogeneity of cancer subtypes, as this study did not account for differences in breast cancer subtypes and from using a chronic myelogenous leukemia cell line (K562) rather than breast cancer cell lines to assess cytotoxicity.
In this study, propofol did not alter the expression of MMP-1 or MMP-2 or the levels of proteolytically cleaved soluble NKG2D ligands across hormone receptor-positive, HER2-positive, and triple-negative breast cancer cell lines. These findings are consistent with our previous study investigating the effects of sevoflurane exposure [16]. These findings suggest that the modulation of NK cell-mediated cytotoxicity by propofol in breast cancer cell lines is primarily driven by changes in the transcription of NKG2D ligands rather than by alterations in ligand shedding mediated through variations in MMP expression. In hormone receptor-positive and triple-negative breast cancer cell lines, propofol directly promotes the transcription of NKG2D ligands, independent of reduced ligand shedding associated with decreased MMP expression. Conversely, in HER2-positive breast cancer cell lines, propofol directly suppresses the transcription of NKG2D ligands rather than increasing ligand shedding through elevated MMP expression.
In this study, propofol modulated the transcriptional regulation of NKG2D ligands without affecting cell viability. The specific molecular pathways by which propofol regulates NKG2D ligand expression remain unknown, but previous research suggests a hypothesis. In response to propofol-induced stress, ER/PR-positive and triple-negative breast cancer cell lines may activate the heat-shock response that inhibits apoptotic and necrotic pathways to promote survival [34]. This response has also been linked to the upregulation of NKG2D ligands [31]. In contrast, in HER2-positive breast cancer cell lines, the Janus kinase-signal transducer and activator of transcription pathway may be preferentially activated under stress conditions [35], promoting cell survival while downregulating NKG2D ligands [36]. Further studies are required to elucidate these regulatory mechanisms.
Our study has certain limitations. First, as an in vitro investigation, the findings cannot be directly extrapolated to in vivo models or clinical practice. Approximately 51% of propofol in circulation binds to erythrocytes and 48% to albumin, leaving only 1.2–1.7% in its free fraction [37]. We used RPMI-1640 medium supplemented with 10% fetal bovine serum that lacks erythrocytes and contains only 0.2–0.3 g/dl of albumin [38–41] compared with ≥ 3.5 g/dl in human serum [42]. However, Mazoit and Samii [37] reported that at low propofol concentrations (< 10 μg/ml), a reduction in erythrocytes has a limited effect on the propofol-free fraction in the presence of albumin, and the impact of albumin itself remains modest. Specifically, a 50% reduction in albumin increased the free fraction by 40% (i.e., the change in the propofol free fraction was approximately from 1.5% to 2.1%) that is attributed to albumin’s high-affinity binding properties that mitigate the effects of reduced binding capacity [37]. Additionally, the propofol concentration required to prevent a response to the incision in 50% of patients ranges from 1.7 to 12.2 μg/ml when co-administered with an opioid [14,15]. This range includes the assumed clinical dose in this study (4 μg/ml). Despite these considerations, the limitations of this in vitro model remain evident, as it does not fully replicate physiological conditions, and the assumed therapeutic concentration of propofol may not fully reflect in vivo conditions.
Second, in this study, cells were exposed to propofol for 18–24 h, a duration exceeding that of typical breast cancer surgery and the terminal half-life of propofol that is approximately 4–7 h [43]. These differences in drug exposure should be carefully considered when assessing the clinical applicability of the findings. Additionally, further studies are needed to investigate the effect of propofol exposure duration on NK cell-mediated cytotoxicity.
In conclusion, propofol exhibited receptor subtype-dependent effects on NK cell-mediated immunosurveillance in breast cancer cell lines, potentially reflecting changes in the transcription of NKG2D ligands rather than alterations in MMP expression or its proteolytic activity. Additional studies are warranted to better understand the impact of propofol on immune modulation and its implications for breast cancer progression.
Notes
Funding
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea Government (MSIT) (No. RS-2022-00167068).
Conflicts of Interest
No potential conflict of interest relevant to this article was reported.
Data Availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
Author Contributions
Hyun-Su Ri (Conceptualization; Formal analysis; Funding acquisition; Methodology; Project administration; Writing – original draft; Writing – review & editing)
Hyeon Jeong Lee (Conceptualization; Methodology; Resources; Supervision; Writing – review & editing)
Jaeho Bae (Methodology; Resources; Supervision; Validation; Writing – review & editing)
Ah-Reum Cho (Formal analysis; Methodology; Resources; Supervision; Validation)
Jae rin Kim (Formal analysis; Investigation; Resources; Software)
Seungbin Park (Investigation; Methodology; Resources; Software; Validation)
Kah Young Lee (Formal analysis; Investigation; Validation)
Soeun Jeon (Conceptualization; Data curation; Formal analysis; Methodology; Project administration; Resources; Writing – original draft; Writing – review & editing)
Supplementary Materials
Effect of propofol on the mRNA expression of NKG2D ligands.
Effect of propofol on surface expressions of NKG2D ligands.
Effect of propofol on MMP expression.
Effect of propofol on NK cell-mediated cytotoxicity.