ANTICANCER RESEARCH 37 : 4371-4378 (2017) doi:10.21873/anticanres.11831 Comparison of Different Muscle-Relaxant Anesthetics on Growth, Migration and Invasion of Gastric Cancer Cells AIHUA JIANG 1* , HUISHAN ZHAO 1* , XIAOFEI LIU 1, MINGWEI YU 1, JIAN CHEN 1 and WEN G. JIANG 2 1Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai, P.R. China; 2Cardiff-China Medical Research Collaborative, Cardiff University School of Medicine, Cardiff, U.K. Abstract. Background/Aim: Muscle relaxants, also known treatment for GC includes radiotherapy, routine surgery, as neuromuscular blocking agents, can block nerve hormone therapy and biotherapy. However, when tumors impulses to the muscles and are always used in surgery for progress there is a risk that not all of the tumor tissue is general anesthesia. However, the effect of muscle-relaxant surgically removed (3, 4). Anesthetics are administered to anesthetics on cell activity in gastric cancer is currently facilitate surgery. Some factors including anesthetic unknown. The present study aimed to examine and techniques and anesthetic drugs may affect the activity of compare the role of three different muscle-relaxant residual tumor cells after surgery and facilitate cancer anesthetics in gastric cancer cells. Materials and Methods: recurrence and/or metastasis (5). Gastric cancer cells (SGC7901 and BGC 823) were Tumor metastasis is the spread of cells from the primary treated with a different dose of muscle-relaxant tumor to new locations in the body, through the circulatory anesthetics, Rocuronium bromide (Rb), Vecuronium and lymphatic system, and is considered the main cause of bromide (Vb) and Cisatracurium Besilate (CB). Using in cancer-related death. Metastatic tumor cells show different vitro models, the effects on gastric cancer cell invasion, genomic and epigenetic profiles generally associated with growth and migration of various anesthetics were aggressive traits. The analgesia technique and the anesthetic subsequently investigated. Results: We found that Rb agents used during surgical tumor resection have been increased the growth, invasion and migration of gastric determined as pathogenic factors for cancer recurrence/ cancer cells SGC7901 and BGC823. However, Vb and CB, metastasis. Thus, anesthesiologists take enormous as relatively mitigative anesthetics, did not significantly responsibility in decreasing metastasis/recurrence rate of affect gastric cancer cell malignant phenotype at their cancer, both during the perioperative period and postoperative regular blood concentration. Conclusion: Our results are analgesia period. There is evidence to suggest that anesthetics important in selecting the type and dose of anesthetic used may potentially have a role in tumor metastasis (6-8). A better for surgery of gastric cancer patients. An understanding of understanding of the mechanisms of metastatic disease and the effect of muscle-relaxant anesthetics and their impact identification of regulators of metastasis, including how on tumor metastasis is critical, since it provides insight metastatic tumor cells invade and grow in the local into the appropriate anesthetic strategy that could improve microenvironment, would enable the treatment and long-term survival in some patients with gastric cancer. development of metastatic prevention strategies. Gastric cancer (GC) is a highly lethal malignancy The effect of anesthetics on cancer metastasis has been worldwide, particular in developing countries, which makes unveiled gradually. Some anesthetics were considered an it a key public health problem (1, 2). The conventional important factor in cancer metastasis. Recently, a review summarized the effect of anesthesia on tumor metastasis, including anesthetic and analgesic techniques (5), suggesting that anesthetics are a non-negligible issue in *These Authors contributed equally to this study. clinical practice. Propofol was investigated in several kinds of cancers and in gastric cancer has been shown to have an Correspondence to: Prof. Wen G. Jiang, Cardiff-China Medical inhibitory role in the growth and survival of gastric cancer Research Collaborative, Cardiff University School of Medicine, cells through up-regulating ING3 (9). In another study Heath Park, Cardiff CF14 4XN, U.K. Tel: +44 2920687065, e-mail: [email protected] propofol inhibited proliferation and apoptosis by regulating microRNA-451 and MMP-2 expression (10). Propofol has Key Words: Anesthetics, migration, breast cancer, invasion, Rb, Vb, also been shown to have an effect on the metastasis of CB. other cancer cells such as ovarian (11), lung (12) and 4371 ANTICANCER RESEARCH 37 : 4371-4378 (2017) Figure 1 . Chemical structures of Rb (A), Vb (B) and CB (C). prostate cancer (13). Increasing numbers of reports on clinical dosages used by the anesthesiologist for which Rb, Vb and procaine have suggested that low dosage of procaine CB were 0.6 mg/kg, 0.1 mg/kg and 0.2 mg/kg respectively. Dilution suppressed the proliferation of lung cancer cells, however ranges were as follows: Rb - 8/80 μg/ml, Vb - .5/15 μg/ml and CB - 2.5/25 μg/ml. this did not appear with higher doses (14). Procaine has also been seen to affect osteosarcoma cells, by inhibiting In vitro cell growth assays. For each cell line 3,000 cells/200 μl/well their migration and growth possibly through inducing cell were added to a 96-well plate (BEAVER, Suzhou, China) and apoptosis (15). Muscle relaxants, including Rocuronium incubated for 1 to 5 days (1, 3, 4 and 5 days respectively). After bromide (Rb), Vecuronium bromide (Vb) and Cisatracurium incubation, culture medium was removed and replaced with 100 μl Besilate (CB), act as effective anesthetics by blocking the of 10% CCK-8 (Cell Counting Kit-8, Dojindo, Japan). Absorbance nerve impulses to the muscles, and thus are generally used of the solution was determined using a spectrophotometer (Thermo in surgery for general anesthesia. Currently there is little Fisher Scientific) at a wavelength of 450 nm. research on the effect of muscle relaxants on tumor In vitro cell scratch assay. Cells were seeded into 12-well plates and metastasis. Last year, our team concentrated on muscle cultured overnight to allow the formation of a confluent monolayer. relaxants’ impact on breast cancer metastasis. Interestingly, Subsequently an artificial wound in the monolayer was created by we found that Rb promoted breast cancer cell growth, using a 200- μl pipette tip to scratch a wound. The cell monolayer migration and invasion, but Vb did not (16). In this study, was washed twice with PBS to remove floating cells. Migration of we investigated how muscle relaxants affect the activity of cells, monitored by the closing of the artificial wound space was gastric cancer cells. recorded every 6 h using an inverted microscope over a period up to 24 h. The sizes of the wounds were subsequently analyzed using Image J software. Materials and Methods In vitro cell adhesion assay. Six-well plates were pre-coated with 5 Cell culture. Gastric cancer cells SGC7901 and BGC 823 were μg/100 μl/well Matrigel (BD Matrigel™ Basement Membrane obtained from China Centre for Type Culture Collection (Shanghai, Matrix, Corning, Tewksbury MA, USA) diluted in serum free media China). These two wild-type cancer cells were routinely cultured in and dried to form an artificial basement membrane. Following RPMI-1640 (Life Technologies Corporation, USA) supplemented rehydration with serum-free media for 40 min, 20,000 cells/200 μl with 10% foetal calf serum (FCS; PAA Laboratories, Somerset, were added into each well. After 4 min incubation culture medium UK), penicillin and streptomycin (Sigma-Aldrich Inc, Poole, Dorset, was removed and the wells were washed with PBS to remove any UK)), in an incubator at 37.0˚C, 5% CO 2 and 95% humidity. non-adherent cells. Adherent cells were fixed with 4% formalin for Muscle relaxants for anesthesia. Three kinds of muscle relaxant 30 min. After washing each well twice with PBS, adherent cells were anesthetics, Rb, Vb and CB, were purchased from Sigma (Sigma- stained with 0.5% crystal violet. Crystal violet staining was dissolved Aldrich, Dorset, UK). They were all dissolved in PBS and stored at with 10% acetic acid and measured using a spectrophotometer 4˚C until use. The doses used during experiments were based on the (Thermo Fisher Scientific) at a wavelength of 540 nm. 4372 Jiang et al : Muscle-relaxant Anesthetics and Gastric Cancer Figure 2. Effects of Rb, Vb and CB on growth of gastric cancer cells in vitro. Rb increased the proliferation of SGC7901 (A) and BGC823(B) in vitro. Vb had no significant effect on the growth of SGC7901 (C) and BGC823 (D) cells. CB hardly stimulated the growth of SGC7901 (E) and BGC823(F) in vitro, but overdose could result in partial cell death (*p<0.05, **p<0.01 vs. control with no treatment). 4373 ANTICANCER RESEARCH 37 : 4371-4378 (2017) Figure 3. The effect of Rb, Vb and CB on migration of gastric cancer cells in vitro. A: Rb promoted the migration of SGC7901 (A) and BGC823 (B). Vb had no effect on the migration of SGC7901(C) and BGC823 (D) at the regular blood concentration in vitro, but facilitated the migration at tenfold blood concentration. CB hardly affected SGC7901 (E) and BGC823 (F) migration in vitro. 4374 Jiang et al : Muscle-relaxant Anesthetics and Gastric Cancer Figure 4. The effect of Rb, Vb and CB on adhesion of gastric cancer cells. Rb increased the adhesion of SGC7901 (A) at tenfold blood concentration in vitro. Rb and Vb affected the adhesion of BGC823 (B) and reached statistical significance. Representative data of adhesion assay for SGC7901 and BGC823 cells treated with different concentration of muscle-relaxant anesthetics for 40 min (C). In vitro cell invasion assay. Transwell chambers (8- μm pore size) Rb promoted gastric cancer cell growth. When compared to polycarbonate filter inserts (6.5 mm diameter) (Becton Dickinson and the untreated control group Rb resulted in a significant increase Company, Oxford, UK), were coated with 50 μg Matrigel/100 μl/insert in gastric cancer cell growth, in SGC7901 ( p< 0.05) (Figure (BD Matrigel™ Basement Membrane Matrix) and air-dried.