Midazolam exhibits antitumour and enhances the efficiency of Anti-PD-1 immunotherapy in hepatocellular carcinoma

Abstract

Background Midazolam (MDZ) is an anaesthetic that is widely used for anxiolysis and sedation. More recently, MDZ has also been described to be related to the outcome of various types of carcinomas. However, how MDZ influences the progression of hepatocellular carcinoma (HCC) and its effects on the biological function and tumour immune microenvironment of this type of tumour remain unknown. Methods The effects of MDZ on the proliferation, invasion, and migration of HCC cell lines were examined in vitro using the Cell Counting Kit 8 (CCK8), 5-ethynyl-2ʹ-deoxyuridine (EdU), Transwell, and wound healing assays. Additionally, western blotting was employed to confirm that PD-L1 was expressed. Chromatin immunoprecipitation-seq (ChIP-seq) analysis was used to pinpoint the transcriptional regulation regions of NF-κB and programmed death-ligand 1 (PD-L1). A C57BL/6 mouse model was used to produce subcutaneous HCC tumors in order to evaluate the in vivo performance of MDZ. Mass spectrometry was also used to assess changes in the tumour immunological microenvironment following MDZ injection. Results The HCC-LM3 and Hep-3B cell lines’ proliferation, invasion, and migration were controlled by MDZ, according to the results of the CCK8, EdU, Transwell, and wound healing assays. PD-L1 expression was shown by ChIP-seq analysis to be boosted by NF-κB, and by Western blotting analysis, it was shown that MDZ downregulated the expression of NF-κB. Additionally, in vivo tests revealed that intraperitoneal MDZ injections reduced HCC tumor development and enhanced the effectiveness of anti-PD-1 therapy. The CD45+ immune cell proportions were higher in the MDZ group than in the PBS group, according to the mass spectrometry results. Injection of MDZ resulted in a decrease in the proportions of CD4+ T cells, CD8+ T cells, natural killer (NK) cells, monocytes, Tregs, and M2 macrophages and a rise in the proportion of dendritic cells. Additionally, the concentrations of the cytokines IFN-g and TNF-a were noticeably raised whereas the concentrations of the CD8+ T-cell fatigue markers ICOS, TIGIT, and TIM3 were noticeably lowered. Conclusion According to this study, MDZ inhibited the progression of HCC by inhibiting the NF-κB pathway and reducing the exhaustion of CD8+ T cells. In clinical practice, MDZ combined with anti-PD-1 therapy might contribute to synergistically improving the antitumor efficacy of HCC treatment.

Role of midazolam on cancer progression/survival - An updated systematic review

“…[ 18 ] Midazolam delayed or inhibited tumour growth compared to controls in six studies. [ 12 , 20 , 21 , 22 , 24 , 25 ] In human leukaemia (K562) and colon cancer (HT29) cell lines, midazolam significantly suppressed the proliferation of K562 cells and HT29 at 100 and 200 μM, respectively. Growth-inhibitory concentrations, the concentration (mM) of midazolam that achieved 50% growth inhibition of cells - presented as % of control of midazolam, were 171.5 and 148.5 μM for K562 and HT29 cells, respectively.…”

“…Four studies noted that they utilised high concentrations of midazolam, which may not be feasible in clinical applications. [ 10 , 11 , 14 , 16 , 25 ] For example, the mean [standard deviation (SD)] of 50% cytotoxic concentration of midazolam in human oral squamous cell carcinoma (OSCC) cell line HSC-2 was 119 (0.8) μM and 232 (59.0) μM in HSC-3;[ 10 ] in FaDu human hypopharyngeal squamous cell carcinoma, cells were incubated with concentrations of up to 100 μM midazolam for 24 and 48 h.[ 11 ] Again, a high concentration of 100 μM of midazolam was applied in human malignant glioma cells, greatly exceeding the clinical range. [ 16 ]…”

“…Limited studies demonstrated midazolam's inhibitory role on cancer progression and survival in vivo . [ 12 , 18 , 19 , 20 , 21 , 22 , 24 , 25 ] This discrepancy between the number of in-vivo and in-vitro studies might be attributable to the potentially toxic effects of high doses of midazolam on animal tissues. In evaluating drug effects on cancer, in-vitro studies are usually prioritised as an initial step before in-vivo studies as they provide a controlled environment.…”

“…In this review, midazolam inhibited cancer cell progression in various cancer cell lines; human non-small cell lung cancer (NSCLC)[ 18 , 19 , 22 , 23 ] and hepatocellular carcinoma[ 20 , 25 , 26 ] were the most studied cancer types. Midazolam also suppressed the progression of human T-cell lymphoma and neuroblastoma,[ 9 ] oral squamous cell carcinoma and glioblastoma,[ 10 ] hypopharyngeal squamous cell carcinoma,[ 11 , 13 ] leukaemia and colon cancer,[ 12 ] mouse Leydig tumour,[ 14 , 17 ] neuroglioma,[ 18 ] melanoma,[ 21 ] pancreatic ductal adenocarcinoma,[ 24 ] breast cancer (oestrogen positive),[ 23 ] and malignant glioma.…”

“…[ 16 ] Midazolam appeared to inhibit cancer cell proliferation in hypopharyngeal squamous cell carcinoma, leukaemia, colon cancer, neuroglioma, NSCLC, breast, pancreatic, glioblastoma, and hepatocellular carcinoma. Midazolam also exerted anti-metastatic properties by inhibiting cancer cell migration,[ 18 , 20 , 23 , 25 , 26 ] invasion,[ 18 , 20 , 23 , 26 ] and epithelial–mesenchymal transition. [ 23 ] Midazolam also decreased lung metastasis of melanoma cells by demonstrating an inhibitory effect on the vascular endothelial cells, with no direct effect on the migration or the proliferation of the melanoma cells.…”