Introduction
Liver cancer (LC) is a common and lethal malignancy, but treatment approaches and the clinical management of LC face substantial challenges [
1]. Liver cancer is diagnosed in more than 800,000 people worldwide and accounts for over 700,000 deaths annually [
2]. Hepatocellular carcinoma (HCC), which accounts for 90% of all occurrences of LC [
3], is the most common kind of preinvasive liver cancer. Clinical HCC treatment has made notable strides in recent years [
4‐
6]. In the clinic, surgery paired with chemoradiation is an essential part of treating HCC; nonetheless, some patients’ 5-year survival rates are still low [
7]. In addition, due to the atypical clinical symptoms of early HCC and the low prevalence rate of HCC early diagnosis [
8], most patients miss their best chance for undergoing surgery. Therefore, exploration of potential therapeutic targets and their specific mechanisms in HCC is urgently needed.
More recently, effective tumour immunotherapy, which targets immune checkpoints, has attracted great attention. Immune checkpoints include coinhibitory and costimulatory molecules that are expressed by effector lymphocytes and prevent the development of excessive immune responses. The production of the corresponding ligands by tumor and stromal cells is a biological mechanism used by liver tumors and other malignancies to evade antitumor immune responses [
9]. PD-1, T cell immunoglobulin and mucin domain containing-3 (TIM-3), cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), lymphocyte-activation gene-3 (LAG-3) and others are examples of co-receptors [
10]. Most Treg cells express CTLA-4, which acts as an effector molecule to prevent the proper activation of effector T cells [
11]. In contrast to its ligand, PD-L1, which is expressed by a variety of stromal and tumor cells as well as myeloid cells, including DCs, PD-1 is expressed by activated T cells, natural killer (NK) cells, Treg cells, MDSCs, monocytes, and dendritic cells (DCs). Effector T cells become exhausted or become dysfunctional as a result of PD-1, which prevents effector functions. Immune checkpoint inhibitors (ICIs) work to stop the interaction between checkpoint proteins and their ligands, thereby preventing T cells from becoming inactive. The discovery that an effective immune response can partially or completely eradicate tumor cells across a variety of malignancies has transformed cancer therapy [
12].
Midazolam (MDZ), which is a derivative of benzodiazepine, is extensively used for procedural sedation and the induction of general anaesthesia in clinical practice. Animal experiments have demonstrated that MDZ can inhibit the ability of animals to exercise and induce muscle relaxation [
13]. In addition, many original studies have recently shown that MDZ demonstrates an extraordinary ability to promote apoptosis in several human cancer cells and hinder tumour growth in xenotransplantation mouse models [
14]. However, studies on the relationship between MDZ and the tumour immune microenvironment have not been carried out. In our study, we explored the effect exerted by MDZ on HCC progression and revealed that MDZ reduces the expression of PD-L1 in HCC cell lines by suppressing the NF-κB pathway. The combination of MDZ and the PD-1 mAb is beneficial for increasing antitumor activity, which may provide novel clinical immunotherapeutic approaches for treating HCC and provide new evidence of the relationship between perioperative anaesthesia and HCC tumour progression.
Materials and methods
Cell culture and drug treatments
The Cell Bank of Type Culture Collection provided the human HCC-LM3 and Hep-3B HCC cell lines as well as the mouse Hepa1-6 HCC cell line (Chinese Academy of Sciences, China). HCC-LM3 and Hep-3B cells were grown in DMEM (Gibco, USA) supplemented with 10% FBS and 1% penicillin/streptomycin, whereas Hepa1-6 cells were grown in RIPA 1640 medium (Gibco, USA) with 10% FBS and 1% penicillin/streptomycin. All of the cell lines were kept in a single incubator with a constant temperature of 37 °C and 5% CO2. For the pertinent investigations, the HCC-LM3 and Hep-3B HCC cell lines were treated with MDZ (Nhwa Pharmaceutical, China) diluted with normal saline (0.9%) when they had reached 80–90% confluence.
Cell proliferation assay
For the CCK8 proliferation experiments, HCC-LM3 and Hep-3B cells were meticulously seeded in 96 wells at a density of 2000 cells per well. After the cells were exposed to MDZ (0, 50, 75, 100, 150, 200 μM) for 0 h, 24 h, 48 h, and 72 h, they were then treated with 10 μl of CCK8 solution (RiboBio, China). Following that, a microplate reader was used to measure the cells’ absorbance at 450 nm in accordance with the manufacturer's instructions (Synergy, USA).
Using a Cell-Light EdU DNA Cell Proliferation Kit, the EdU assay was carried out to assess cell proliferation (RiboBio, China). In a 24-well plate, 10 × 104 HCC cells were seeded in each well. After 80–90% of the cells had formed monolayers, we added MDZ (75 and 150 μM) and incubated the mixture for 24 h. The two cell lines were then fixed with 4% paraformaldehyde following a 24-h incubation with a 10 mmol/l EdU solution. Following that, we gave the cells the prescribed amounts of DAPI and Apollo Dye Solution. Using an Olympus FSX100 microscope (Olympus, Japan), photographs of the EdU-incorporated cells were taken, and the quantity of EdU-positive cells was counted.
Transwell invasion assay
In the upper chamber of Transwell chambers (Corning, USA) containing matrix mixes (BD Biosciences, USA) and 300 μl serum-free media, HCC-LM3 and Hep-3B cells (3 × 104 cells) were plated and pretreated with MDZ (75 and 150 μM) for 24 h. 600 μl of 10% FBS-supplemented media, which functioned as the HCC cells’ chemoattractant, were placed in the bottom compartment. The non-invading cells were eliminated after the 24-h incubation period, and the invaded cells were then fixed with 4% paraformaldehyde for 15 min before being stained for 20 min using Crystal Violet Staining Solution (Beyotime, China). We next used a fluorescence microscope to take pictures of the labeled cells and to count them.
Wound healing assay
In 6-well plates, HCC-LM3 and Hep-3B cells were plated. The monolayer was then scratched using a common 20-μl pipette tip to create an artificial homogenous wound after the cells had grown into 90–100% confluent cell monolayers. The plate was then mixed with serum-free medium supplemented with 75 and 150 μM MDZ, and the thermomixer was used to incubate it at 37 °C after the free-floating cells and debris were removed using phosphate-buffered saline (PBS, Gibco, USA). After 0 h, 24 h, and 48 h, images of the wound widths were taken using an inverted microscope for additional analysis.
Western blotting
Using a Mammalian Total Protein Extraction Kit and a protease inhibitor cocktail, the cells' total proteins were extracted (Transgen, Beijing, China). In a nutshell, 10% SDS–polyacrylamide gel electrophoresis was used to separate the proteins. Following that, the proteins were transferred to PVDF membranes. Specific primary antibodies (Abcam, UK) were incubated with the PVDF membranes for an extended period of time at 4 °C in order to identify PD-L1, P65, p-P65, and GAPDH. The membranes were then exposed to peroxidase-conjugated secondary antibodies for two hours at room temperature. Finally, an enhanced chemiluminescence assay was used to see the Western blotting bands (ECL; Thermo Fisher, USA).
Chromatin immunoprecipitation-seq (ChIP-seq) analysis and site prediction
The JASPAR database (
http://jaspar.genereg.net) was used to predict the binding sites for P65 in the promoter of the PD-L1 gene. The human P65-PD-L1 binding sites' CHIP data were obtained from GSE2360959.
Tumour mouse models and treatment
Twenty male C57BL/6 mice, aged 5 to 6 weeks, were acquired from the Laboratory Animal Center of Nanjing Medical University and fed under specified pathogen-free (SPF) conditions. 2 × 106 murine Hepa1-6 cells were subcutaneously implanted into the right axillary areas of the mice to create the subcutaneous tumor mouse model. Subsequently, 20 subcutaneous tumour-bearing mice were randomly divided to four groups: the PBS, MDZ, α-PD-1, and α-PD-1 + MDZ groups. MDZ (1 mg kg−1, every other day) was intraperitoneally injected into the mice in the MDZ and α-PD-1 + MDZ groups when the tumours reached approximately 50–100 mm3 in volume, while the PBS and α-PD-1 groups were injected with an equal dose of PBS. After 7 days, 200 µg anti-mouse-PD-1 (α-PD-1; BE0273; Bio X Cell, USA) was injected intraperitoneally into the mice in the α-PD-1 and α-PD-1 + MDZ groups twice a week. On Day 21, the tumour-bearing mice were sacrificed, and the volume of the tumours was calculated according to the generic formula: volume (mm3) = width2 × length/2. Subsequently, the tumour samples were harvested and weighed for further analysis.
Haematoxylin–eosin (HE) and Immunohistochemistry
We fixed the samples that were collected from the mouse models in paraffin and then sectioned them to 4 mm thickness for HE staining and immunohistochemistry according to the protocols. After using the DAB (SA-HRP) TUNEL Cell Apoptosis Detection Kit, the samples were stained using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) (Servicebio, China). Pressure cooking was used to extract the antigen for three minutes in a 0.01 mol L-1 citrate buffer (pH 6.0; Wobixin Inc., China). At 4 °C, the samples were incubated overnight with specific antibodies against CD3 (ab16669, 1:150 dilution; Abcam, UK), CD8 (ab217344, 1:2000 dilution; Abcam, Cambridge, UK), Ki-67 (ab16667, 1:200 dilution; Abcam, Cambridge, UK), and PD-L1 (ab213524, 1:250 dilution; Abcam, UK). The following day, we carried out immunodetection utilizing 3'-diaminobenzidine, in accordance with the instruction booklet.
Mass cytometry
Following the death of the tumor-bearing mice, full tumor samples from the PBS and MDZ groups were collected and further processed using the Miltenyi Mouse Tumour Dissociation Kit (Miltenyi Biotec, Germany). Following are the phases in the CyTOF staining process: 194Pt staining → Fc block → surface antibody staining → overnight DNA staining (191/193Ir) → intracellular antibody staining → data collection. Two procedures make up the data analysis: FlowJo preprocessing (circle and choose individual, fully functional CD45 + immune cells), and bioinformation analysis (the X-shift algorithm performs cell subpopulation clustering, manual annotation, TSNE dimensionality reduction visual display, and statistical analysis). Most of the CyTOF staining and data processing was done by the PLTTECH Company (Plttech, China).
Statistical analysis
The averages and standard deviations (SD) of at least three different biological replicates are used to represent all of the results. GraphPad Prism 9.0 (GraphPad software Inc., San Diego, USA) was used for the statistical analysis, and a two-tailed P value < 0.05 was regarded as statistically significant. The continuous variables between the two groups were compared using a two-tailed unpaired Student's t test.
Discussion
HCC is the most common tumour type of liver cancer, and its morbidity and mortality are increasing year by year in the United States and Europe [
16]. ICIs are currently promising therapeutic agents for treating patients with advanced HCC and research on these agents has rapidly increased over the past decade. Unfortunately, the available evidence suggests that most HCC patients do not benefit from treatment with ICIs. In HCC patients treated with sorafenib, the overall response rate (ORR) of nivolumab treatment has been reported to be only 16–23% [
17]. Therefore, finding new, potential and effective combinations of immunotherapy drugs has become one of the approaches to overcome the current lack of progress in immunotherapy.
Surprisingly, several recent studies have demonstrated that anaesthetics may have anticancer effects. MDZ belongs to the same class of benzodiazepines. Zhang et al. found that MDZ inhibited the proliferation and metastasis of hepatoma cells through the USP14/PI3K/Akt signalling pathway [
18]. In addition to its sedative, hypnotic, and muscle relaxant effects, Shen et al. found that MDZ also appears to exert inhibitory effects on hepatocellular carcinoma [
19]. In this study, by constructing an HCC xenograft C57BL/6 mouse model, the anti-HCC effect of MDZ in vivo was also confirmed, and this effect was significantly increased when MDZ was combined with a PD1 monoclonal antibody. However, the impact of MDZ on the tumour microenvironment and immune checkpoint blockade therapy have been less well studied. Previous studies indicate that various anaesthetics can affect the expression of PD-L1 and thereby alter the tumour microenvironment [
20,
21]. Therefore, in addition to verifying the anti-HCC effect of MDZ, our study further elucidated the effect of MDZ on the HCC microenvironment and PD-L1 expression and explained the possible mechanism.
PD-L1 (B7-H1) is a ligand of PD1, and PD-L1 can be expressed in different cell types, including macrophages, dendritic cells, vascular endothelial cells, and tumour cells. Tumour cells use PD-L1 to bind to the PD1 receptor expressed by T cells, inhibiting T cell-mediated cytotoxicity in the tumour microenvironment and exerting an immunosuppressive effect [
22]. Notably, in this study, MDZ was found to significantly reduce PD-L1 protein expression in HCC, as demonstrated by our experimental Western blotting results. Considering the significant effect of PD-L1 in inhibiting T-cell immune activity and immunotherapy, this study further explored the possible mechanism or pathway by which MDZ reduces PD-L1 expression. NF-κB is a group of dimeric transcription factors that play vital roles in the occurrence and development of liver inflammation and cancer. The NF-κB transcription factor family mainly includes c-Rel, RelB, p50, p52 and p65 (RelA), which usually exist as dimers [
23]. Multiple studies have demonstrated that the NF-κB signalling pathway regulates PD-L1 expression and that NF-κB affects the transcription of PD-L1 by binding to the PD-L1 promoter. Mo et al. found that icaritin interacts with IKK-α to interfere with the NF-κB pathway and inhibit the expression of PD-L1 in liver cancer cells [
24]. Chrysin downregulates PD-L1 expression in HCC by blocking the STAT3 and NF-κB pathways in vitro and in vivo [
25]. In addition, the CHIP-seq analysis in this study revealed that in HCC, NF-κB can bind to multiple sites in the promoter region of PD-L1 and participates in regulating the expression level of PD-L1. Previous studies have examined whether MDZ can directly or indirectly affect the activation of NF-κB. For example, studies have demonstrated that MDZ can inhibit the activation of NF-κB in macrophages, thereby reducing the release of proinflammatory factors such as iNOS and COX-2 [
26,
27]. According to the Western blotting results in this study, we found that the protein expression levels of P65 and p-P65 were significantly decreased in the MDZ-treated HCC cell lines. Therefore, we infer that MDZ enhances the efficacy of PD-1/PD-L1 blockade therapy by downregulating PD-L1 expression in HCC cells by inhibiting the NF-κB/p-NF-κB pathway.
The tumour microenvironment includes a complicated and changeable relational network composed of cancer cells, immune cells, stromal cells, cytokines, and extracellular matrix components, and this network is extremely important for the clinical treatment of HCC. By mass flow cytometry, we found that MDZ had a significant effect on the HCC microenvironment. Changes in the tumour microenvironment after MDZ treatment may be another critical factor that inhibits PD-L1 expression and HCC growth. In the tumour microenvironment, CD8
+ T cells are the predominant forces that exert antitumor effects and release TNF, perforin and granzyme to kill cancer cells after activation. In addition, CD8
+ T cells are also a significant prognostic marker of HCC [
28]. By mass spectrometry measurements of common exhaustion markers of CD8
+ T cells (TIM3, ICOS and TIGIT), we found that MDZ significantly reduced CD8
+ T-cell exhaustion. The binding of PD-L1 and PD-1 has been demonstrated to inhibit T-cell proliferation and activation, and anti-PD-1/PD-L1 therapy can reactivate the immune response in HCC patients [
29]. Therefore, the present study infers that MDZ increases the efficiency of the PD-1 mAb by inhibiting the exhaustion of CD8
+ T cells, thereby delaying HCC progression.
Taken together, we demonstrate that MDZ shapes an immune-enhancing tumour microenvironment by restricting PD-L1 expression and reducing the depletion of CD8+ T cells in HCC, suggesting that MDZ is a potential antitumor drug. In the in vivo experiments in mouse models, the combination of MDZ and the PD1 monoclonal antibody achieved a better therapeutic effect than monotherapy in the treatment of HCC. MDZ-mediated inhibition of NF-κB reduced PD-L1 expression and ultimately restored the function of cytotoxic T lymphocytes (CTLs), and these effects are expected to be useful in cancer immunotherapy, providing a new direction for solving clinical problems such as PD1 resistance. However, our study also has some limitations. First, as mentioned earlier, although our study showed that MDZ prevented HCC progression and enhanced the effects of PD-1/PD-L1 blockade therapy, this still needs further clinical experimental confirmation. Second, after the injection of MDZ, the number of CD8+ T cells in the HCC tumour immune microenvironment decreased, and the reasons behind this need to be further explored and identified. The cytotoxicity of MDZ may affect not only tumour cells but also other immune cells, and the mechanism remains to be further studied. Furthermore, in this study, the injected doses of MDZ may be higher than those commonly used in clinical systemic administration. Long-term use of MDZ at a high dose could cause a series of side effects, such as excessive sedation, nausea and vomiting, hepatotoxicity and so on. In addition, MDZ has potential neuronal cytotoxicity. Therefore, the efficacy and safety of MDZ as an anticancer drug in clinical practice and its appropriate dose deserve further clinical study. Moreover, it is necessary to conduct more studies to evaluate the advantages and disadvantages of MDZ compared to traditional chemotherapeutic drugs in cancers, especially PD-L1-negative cancer cells.
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