Lidocaine inhibited migration of NSCLCA549 cells via the CXCR4 regulation
Abstract
BACKGROUND:
Lidocaine is a local anesthetic that wildly used in surgical treatment and postoperative medical care for lung cancers. We hypothesized that lidocaine at clinical plasma concentration can inhibit CXCL12/CXCR4 axis-regulated cytoskeletal remodeling thereby reduce the migration of Non-small-cell lung cancers (NSCLC) cells.
METHODS:
We determined the effect of lidocaine at clinical plasma concentration on CXCL12-induced cell viability, apoptosis, cell death, monolayer cell wound healing rate, individual cell migration indicators, expression of CXCR4, CD44, and ICAM-1, intracellular Ca
RESULTS:
Lidocaine did not affect cell viability, apoptosis, and cell death but inhibited CXCL12-induced migration, intracellular Ca
CONCLUSION:
Lidocaine at clinical plasma concentrations inhibited CXCL12-induced CXCR4 activation, thereby reduced the intracellular Ca
List of abbreviations
| NSCLC: | Non-small cell lung cancers |
| CXCL12: | C-X-C Motif Chemokine Ligand 12 |
| CXCR4: | C-X-C chemokine receptor type 4 |
1.Introduction
Lung cancer is one of the most fatal cancer types with the most global death number among males and the second most global death number among females [1]. As the medical care for lung cancer develops, the mortality of lung cancer decreased in the USA and UK has decreased in recent years. However, in many industrialized nations, the emerging social smoking culture has resulted in higher lung cancer rates in these areas [2]. Among all lung cancer cases, more than four-fifths of clinical lung cancer diagnosed are non-small cell lung cancer (NSCLC) [3], a lung cancer type that has diverse pathological features and with an undesirable prognosis.
Clinical medical advancements have improved cancer therapy extensively in recent years [4]. As clinical diagnosis is critical for disease treatment [5], in-depth analyses of lung cancer subtypes have been developed for potential targeting therapy and customized treatment according to their genetic and cellular heterogeneity [6]. Surgical treatment is the most applicable intervention for early-stage lung cancer diagnoses and is critical for the further prescription of proper therapeutic options [7]. One of the clinical factors that might affect the outcome of surgical treatments is the anesthesia during the surgery [8, 9]. Many local anesthetics have been found to affect multiple cancer cells [10]. A local anesthetic, lidocaine, has been wildly used in surgical treatment [11] and postoperative medical care [12, 13]. Preclinical studies have revealed that lidocaine can inhibit the proliferation and migration of many cancer types, including lung cancer [14, 15], breast cancer [16, 17], gastric cancer [18], colon cancer [19, 20, 21], etc.
However, the doses of lidocaine used in most previous studies are much higher than the concentration in plasma during clinical use of lidocaine (we mentioned it as “plasma concentration”). In this study, we focused on a lower concentration range of lidocaine that is correspondent to clinical plasma concentration. Lidocaine at this concentration range has been reported to inhibit cytoskeletal remodeling and migration of breast cancer cell MDA-MB-231 [22]. We proposed that a similar effect of lidocaine on cell migration is also present in NSCLC cells.
The metastatic potential of NSCLC has been found to be strongly associated with the chemokine CXCL12 (C-X-C Motif Chemokine Ligand 12) and the activity of the CXCL12 receptor CXCR4 (C-X-C chemokine receptor type 4) [23]. CXCR4/CXCL12 axis controls the immunity of the body and the survival, invasion, and metastasis of cancer cells [24, 25]. CXCR4 has been found to be differently expressed in cancer cells and might contribute to the motility of cancer cells [26]. The CXCR4/CXCL12 axis has been proposed as a potential drug target for NSCLC [27]. In this study, we hypothesized that lidocaine at clinical plasma concentration can inhibit CXCL12/CXCR4 axis-regulated cytoskeletal remodeling, thereby decrease migration of NSCLC cells. This investigation can provide a better understanding of the potential pharmacological effects of lidocaine on the clinical treatment of NSCLC.
2.Methods and materials
2.1Cell line and cells culture
A549 cells were purchased from Biofeng (China). Cells were cultured using Ham’s F12K
2.2Knockdown and overexpression
CXCR4 knockdown was achieved by transfecting the CXCR4 shRNA (TRCN0000256866) plasmid into A549 cells to silence the expression of CXCR4. The plasmid was purchased from Sigma-Aldrich (USA). The Sequences of shRNA oligonucleotides are as follows: 5’-TCCTGTCCTGCTATTGCATTA-3’. CXCR4 overexpression was achieved by transfecting the CXCR4 expressing (hCXCR4-mTFP1) plasmid into A549 cells to overexpress CXCR4. The plasmid was purchased from Addgene (USA). The transfection method was described previously [28] The knowdown and overexpression of CXCR4 were validated by western blotting experiments.
2.3Testing reagents
Lidocaine HCl pre-made parenteral solution was purchased from Hospira Inc. (USA). Human CXCL12 and pertussis toxin were purchased from Sigma-Aldrich (USA). Fura-2 (Fura-2-acetoxymethyl ester) was purchased from Abcam (UK). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc (Kumamoto, Japan).
2.4CCK-8 assay
The CCK-8 assay is used to determine cell overall viability and cytotoxicity. The CCK-8 assay was conducted as previously described [29]. Briefly, cells were seeded in 96 well plates. After the incubation with testing drugs, 10
2.5Apoptosis and cell death detection
Apoptosis and cell death detection were determined using Bcl-2 ELISA kit (Abcam, UK) and Cell Death Detection ELISA plus (Roche, USA) respectively. The method of ELISA was described previously [30, 31]. The positive control was induced by high-temperature culture (55
2.6Western blotting
Membrane protein expression was determined using the western blotting assay Membrane proteins of A549 were extracted using Mem-PER™Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific, USA). The extraction was conducted as previously described [33, 34]. Subsequently, the western blotting assay was conducted as in previous studies [35, 36]. Briefly, SDS-PAGE gel electrophoresis was used to separate proteins and semi-dry protein transfer was conducted as described [37]. The membrane was then incubated with primary and secondary antibodies according to the recommended experimental condition. Na+/K+ ATPase protein was used as an internal reference gene to normalized the data. Antibodies used including CXCR4 Antibody (PA3-305), Anti-Alpha 1 Na+/K+ ATPase Antibody (#ANP-001), Recombinant Anti-CD44 antibody [EPR1013Y] (ab51037), Anti-ICAM1 antibody [EP1442Y] (ab53013), Goat Anti-Mouse IgG H&L (HRP) (ab6789), and Goat Anti-Rabbit IgG H&L (HRP) (ab6721). ECL reagent (Thermo Scientific, USA) was used to visualize the target protein.
2.7QPCR
The CXCR4 mRNA level was determined using a QPCR assay. The extraction was conducted as previously described [38, 39]. Briefly, Trizol buffer (Sigma-Aldrich, USA) was used to isolate cellular RNA. An Agilent Bioanalyzer (Agilent 2100; Agilent Technologies, Inc, USA) was used to assess the concentration and integrity of RNA extracted. QuantiTect Reverse Transcription Kit (QIAGEN, USA) and miRCURY LNA SYBR Green PCR Kit (QIAGEN, USA) were used to conduct RT-PCR. The protocol of PCR reaction was described previously [40]. The GAPDH gene was used as an internal reference gene to normalize the data. CXCR4 Human qPCR Primer Pair (NM_003467) and GAPDH Human qPCR Primer Pair (NM_002046) were purchased from OriGene Technologies (USA).
2.8Wound healing assay
Cells migration was determined using the wound healing assay. The method was described previously [41, 42]. Briefly, cells were grown in six-well plates at over 90% confluency. A scratch wound was created in the cell monolayer using a 200-
2.9Chemotaxis assay
Real-time cell migration of individual A549 cells was recorded using the m-Slide chemotaxis system (ibidi, Germany). The method was described previously [43, 44]. Briefly, A549 cells were cultured on the central channel of the chemotaxis slide at 10% confluency for 8 h to allow adherence. Testing drugs were applied when the assay started and the images of cells were recorded for 15 hours with a time-lapse Micro-Imager. Single-cell tracking was analyzed using the ImageJ software. Spider plots representing the aggregated trajectories of cells. Forward migration indexes and cell velocity were analyzed using the Ibidi software.
2.10Fura-2based intracellular Ca2 +
Fura-2-based fluorescence was used to determine the intracellular Ca
2.11Actin polymerization detection
The cytoskeleton remodeling was indicated by the determination of actin polymerization using Fluorescein Phalloidin staining. The method was described previously [47]. Briefly, cells were fixed in 4% paraformaldehyde for 10 min at 4
2.12Plotting and statistical analysis
Means and standard deviations are displayed in the figures. A T-test or ANOVA was used to analyze the significance of the difference (
Figure 1.
Effect of lidocaine at clinical plasma concentration on A549 viability. A549 were exposed to tested drugs for 24 hours. A. Effect of clinical-plasma-dose lidocaine on A549 viability. B. Effect of lidocaine on A549 apoptosis. C. Effect of lidocaine on A549 cell death. PC: positive control induced by high-temperature culture (55
3.Results
3.1Lidocaine did not affect A549 cell viability at the clinical plasma concentrations
The anti-arrhythmia plasma concentration and approximately equipotent nerve block concentrations of lidocaine are around 10
3.2lidocaine decreased CXCR4 expression
In this study, we focused on the effect of the CXCR4/CXCL12 signal on cancer cell migration. CXCR4/CXCL12 signal has been wildly accepted as a critical signal for the migration of lung cancer cells [49, 50, 51] and CXCR4 has been thought to be a drug target for the inhibition of lung cancer migration [27, 52]. We extracted the membrane expression of CXCR4 in A549 and observed the effect of lidocaine on the surface expression of CXCR4 in A549 cells. Results showed that lidocaine significantly decreased the surface expression of CXCR4 and the total expression of CXCR4 mRNA (Fig. 2). In the CXCR4 knockdown experiments, we knocked down over 80% CXCR4 mRNA (Fig. 2C) and the surface CXCR4 expression was very low that was almost not detectable in the western blotting (Fig. 2A and B). After CXCR4 knockdown, the effect of lidocaine on CXCR4 expression was eliminated (Fig. 2) In
Figure 2.
Effect of lidocaine on CXCR4 expression in A549. AB. membrane expression of CXCR4 protein in A549. C. CXCR4 mRNA expression in A549. (Significant differences compared to both control (CXCL12 and lidocaine are 0) are indicated by
the CXCR4 overexpressing expression, we increased the mRNA of CXCR4 by about 8 times, and the CXCR4 protein expression was increased by about 2 times. At the presence of lidocaine, both the CXCR4 mRNA and protein levels were significantly decreased compared to the control (Fig. 2). Thus, we suggested that lidocaine down-regulated CXCR4 expression when it was highly expressed, but did not affect CXCR4 when it was at a low expression level.
3.3Lidocaine inhibited CXCL12 induced migration
CXCL12 is the agonist of the CXCR4 and has been wildly used for studying chemokinesis or chemotaxis of cells [53]. To tested if lidocaine affected CXCL12 induced migration of A549 cells, we performed both the wound healing assay to observed the migration of monolayer A549 cells as a group and in vitro chemotaxis assay to observe the migration of individual A549 cells. Results showed that CXCL12 significantly increased the A549 wound healing rate. In addition, CXCL12 also increased the migration index and velocity of individual cells. As shown in the aggregated trajectories of individual A549 cells, A549 migrated a longer distance at the presence of CXCL12. At the presence of lidocaine, the A549 wound healing rate was the same as control, but the wound healing rate decreased compared with CXCL12-induced cells (Fig. 3). These results revealed that lidocaine did not affect A549 cell migration but blocked the stimulation of CXCL12.
Figure 3.
Effect of lidocaine on CXCL12-induced A549 migration. A. Effect of lidocaine on CXCL12-induced A549 monolayer wound healing. B. Representative images of wound healing assay. C. Effect of lidocaine on CXCL12-induced A549 forward migration index. D. Effect of lidocaine on CXCL12-induced A549 migration velocity. E. Aggregated trajectories of individual A549 cells at different experimental conditions (10 hours). (Significant differences are indicated by
3.4CXCR4 knockdown block lidocaine effect in migration
To investigate whether lidocaine affects migration through CXCR4, we also performed migration assays in CXCR4 knockdown A549 cells. Results showed that knockdown of CXCR4 eliminated the effects of CXCL12 or lidocaine on A549 migration. The exposure of CXCL12 or lidocaine had no significant effect on the wound healing speed and the migrate index and velocity of individual cells (Fig. 4). This suggested that CXCR4 was essential for the effects of CXCL12 and lidocaine.
Figure 4.
Effect of lidocaine on CXCL12-induced CXCR4 knocked down A549 cells migration. A. Effect of lidocaine on CXCL12-induced CXCR4- knocked down A549 monolayer wound healing. B. Representative images of wound healing assay. C. Effect of lidocaine on CXCL12-induced CXCR4-knocked down A549 forward migration index. D. Effect of lidocaine on CXCL12-induced CXCR4- knocked down A549 migration velocity. E. Aggregated trajectories of individual CXCR4- knocked down A549 cells at different experimental conditions (10 hours). (Significant differences are indicated by
3.5Lidocaine inhibited migration caused by CXCR4 overexpression
To further investigate the effect of lidocaine on CXCR4 mediated migration, we also performed migration assays in CXCR4 overexpressing A549 cells. Results showed that CXCL12 significantly increased CXCR4 overexpressing the A549 wound healing rate. In addition, CXCL12 also increased the migration index and velocity of individual CXCR4 overexpressing A549 cells. As shown in the aggregated trajectories of individual CXCR4 overexpressing A549 cells, these cells migrated a longer distance at the presence of CXCL12. At the presence of lidocaine, CXCR4 overexpressing the A549 wound healing rate was the same as control, but the wound healing rate decreased compared with CXCL12-induced cells (Fig. 5). These results revealed that lidocaine did not affect CXCR4 overexpressing A549 cell migration but blocked the stimulation of CXCL12 (Fig. 6). These results demonstrated that the effect of lidocaine was mediated by CXCR4/CXCL12 signal.
Figure 5.
Effect of lidocaine on CXCL12-induced CXCR4 overexpressing A549 cells migration. A. Effect of lidocaine on CXCL12-induced CXCR4 overexpressing A549 monolayer wound healing. B. Representative images of wound healing assay. C. Effect of lidocaine on CXCL12-induced CXCR4 overexpressing A549 forward migration index D. Effect of lidocaine on CXCL12-induced CXCR4 overexpressing A549 migration velocity. E. Aggregated trajectories of individual CXCR4 overexpressing A549 cells at different experimental conditions (10 hours). (Significant differences are indicated by
Figure 6.
Effect of lidocaine on membrane expression of CD44 and ICAM-1 in A549. A. Representative images of western blotting. B. membrane expression of CD44 protein in A549. C. membrane expression of ICAM-1 protein in A549. (Significant differences are indicated by
3.6Regulations of lidocaine on CD44 and ICAM-1
To further explore the potential mechanisms for lidocaine action on A549 migration, we investigated two critical adhesion molecules on the epithelial cell membrane, CD44 and ICAM-1. CD44 has been wildly used as a key migration-related biomarker for lung cancers [54, 55]. Another migration-related extracellular molecule, ICAM-1, is also thought to be critical in lung cancer cell migration [56, 57, 58]. Both CD44 and ICAM-1 levels were significantly increased by CXCL12. Lidocaine increased CD44 in both wild-type A549 and CXCR4 knockdown A549 cells in the absence or presence of CXCL12, but it did not affect ICAM-1 (Fig. 6). These results suggested that lidocaine can up-regulate CD44 but not ICAM-1. However, the regulation of lidocaine on CD44 was not essential for CXCL12 induced migration.
3.7Lidocaine inhibited CXCL12induced intracellular Ca2 +
Another potential mechanism we hypothesized underlying lidocaine’s effect on migration is the regulation of the cytoskeleton remodeling. To test this hypothesis, we monitored the effect of lidocaine on the intracellular Ca
Figure 7.
Effect of lidocaine on CXCL12-induced intracellular Ca
4.Discussion
The therapeutic effect of surgery in metastatic NSCLC has been controversial [59, 60]. During the surgery, many clinical factors might contribute to the metastasis and recurrence of lung cancer surgery The application of lidocaine during the surgery or preoperational treatment results in a plasma lidocaine micro-environment for lung cancer cell migration and survival. Hence, the potential impact of lidocaine on metastasis and recurrence of lung cancer surgery should be further studied. However, so far, the effect of lidocaine at plasma concentration on cancers was less studied. The doses of lidocaine used in most previous studies are much higher than the clinical plasma concentration. Therefore, although many previous studies demonstrated the effect of lidocaine on lung cancer cells, they fail to convince clinical surgeons that lidocaine exerts a considerable impact on the surgery outcome. Lidocaine is known as a sodium channel blocker. Although the major target of lidocaine, the voltage-gated sodium channels, has been found to play a role in cancer developments [61], lidocaine might also affect cancer independent of sodium channel blockade
Although a previous study suggested that lidocaine at the “mM” concentration range inhibited proliferation [15] and induced apoptosis of A549 [62], our result showed that, at plasma concentration, lidocaine had almost no effect on cell proliferation, apoptosis, and cell death. The previous study also suggested that the migration of A549 was inhibited by lidocaine at 8 mM, but as shown by our data, the effect of lidocaine on migration was not significant when the doses of lidocaine decreased to 100
Different membrane surface proteins expressed on lung cancer cells as adhesion molecules can be critical in the migration of cells [63] CD44 has been reported to play roles in the metastasis of NSCLC cells [54]. In this study, the expression of CD44 was promoted by CXCL12 stimulation. Our results also showed that the surface expression of CD44 on A549 was up-regulated by lidocaine. However, the lidocaine did not further increase the CD44 expression at the presence of CXCL12 and the increase of CD44 in the lidocaine alone group did not affect cell migration. Thus, we suggested that CD44 was directly up-regulated by lidocaine bypassing the CXCL12/CXCR4 axis but the increase of CD44 was not essential for the migration of A549. In addition, we also determined another critical adhesion molecule for migration, the ICAM-1. The expression of ICAM-1 has been associated with lung cancer progression and prognosis [64]. A previous study reported that lidocaine affects the migration of a lung cancer cell line H838 by reducing ICAM-1 [65]. However, in this study, the lidocaine at plasma concentration did not affect ICAM-1 expression. The ICAM-1 expression was up-regulated by CXCR4 activation by CXCL12, hence, we suggested ICAM-1 might be a potential downstream target of lidocaine/CXCR regulation (Fig. 8). Further validation is required in the future.
Figure 8.
Effect of lidocaine on A549 migration.
Another activity that might impact the migration of A549 was the remodeling of the cytoskeleton. The activation of CXCR4 by CXCL12 can trigger intracellular Ca
5.Conclusion
This study demonstrated that lidocaine at clinical plasma concentrations showed a significant inhibition effect on CXCL12-induced CXCR4 activation, thereby reduced the intracellular Ca
Ethics approval and consent to participate
This work was approved and consented by the Ethical Committee of Changzhi Medical College Affiliated Heping hospital.
Funding
This study received funding from the Changzhi Medical College Affiliated Heping hospital (No. is not available).
Availability of data and materials
The raw data of this study are provided from the corresponding author with a reasonable request.
Authors’ contributions
Interpretation or analysis of data: Baichun Xing.
Preparation of the manuscript: Baichun Xing and Linlin Yang.
Revision for important intellectual content: Yanan Cui.
Supervision: Yanan Cui.
Consent for publication
All the authors consent for this publication.
Conflict of interest
The authors claimed that there is no conflict of interest.
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