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Molecular and Cellular Biochemistry https://doi.org/10.1007/s11010-018-3490-x

Lidocaine alleviates cytotoxicity-resistance in lung cancer A549/DDP cells via down-regulation of miR-21

Qing Yang1 · Zhi Zhang1 · Haixia Xu1 · Chuangen Ma1

Received: 29 May 2018 / Accepted: 14 December 2018 © Springer Science+Business Media, LLC, part of Springer Nature 2019

AbstractLidocaine (Lido) is a commonly used local anesthetic, which has been reported in various types of cells. However, the effects of Lido on lung cancer cells remain not understood. The study aimed to investigate the underlying mechanisms of Lido in the cisplatin resistance of A549/DDP cells. Different concentrations of cisplatin (0–320 µM) were used to stimulate A549 and A549/DDP cells, and cell viability and apoptosis were examined. To investigate the effect of Lido on A549/DDP cells, the optimum concentration of Lido was selected to treat A549/DDP cells, and cell viability, apoptosis, migration and invasion were then detected. The relative expression of miR-21 in A549/DDP cells or in Lido-treated A549/DDP cells was analyzed by RT-qPCR. MiR-21 mimic, inhibitor and its control were transfected into A549/DDP cells to explore the regulatory effect of miR-21 on the cisplatin resistance in A549 or A549/DDP cells. The effects of miR-21 on PTEN/PI3K/AKT and PDCD4/JNK pathways were detected by western blot. The cisplatin resistance of A549/DDP cells was higher than that of A549 cells. Lido significantly suppressed cell viability, induced apoptosis, and inhibited cell migration and invasion in A549/DDP cells. Additionally, miR-21 expression in A549/DDP was higher than that in A549 cells, and Lido significantly down-regulated miR-21 expression in A549/DDP cells. MmiR-21 inhibition exhibited the same effects as Lido on the cisplatin resistance of A549/DDP cells. Further, miR-21 suppression regulated PTEN/PI3K/AKT and PDCD4/JNK pathways in A549/DDP cells. These findings indicated that Lido alleviated the cytotoxicity resistance of A549/DDP cells via down-regulation of miR-21.

Graphical abstract

Keywords Lung cancer · Lidocaine · Cisplatin · MicroRNA-21 · PTEN/PI3K/AKT · PDCD4/JNK

Introduction

Lung cancer is one of the most malignant tumors with the fastest growth rate of morbidity and mortality [1]. In 2017, Cancer Statistics data demonstrated that 222,500 new cases and 155,870 death cases of lung cancer occur in the United States [2]. A large number of studies show that the vast

Qing Yang and Zhi Zhang contributed equally to this work.

* Haixia Xu yehewei1304rra@163.com

* Chuangen Ma machg522@sina.com

Extended author information available on the last page of the article

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majority (about 85%) of lung cancer cases are closely associ-ated with long-term tobacco smoking [3, 4]. The treatments differ according to the different specific cell types of lung cancer and the patient’s physical condition [5]. Currently, the main treatments of lung cancer include surgery, chemo-therapy, radiation therapy, and palliative care for advanced lung cancer patients [6, 7]. Chemotherapy is an important method for the treatment of lung cancer, while the drug resistance of tumor cells seriously affects the curative effect of chemotherapy [8]. Cisplatin is one of the most commonly used drugs in chemotherapy for lung cancer, but the drug resistance of cisplatin is widespread in clinical treatment [9]. Therefore, to study the mechanism of cisplatin resistance is of great significance for exploring the new chemotherapeutic drugs for the treatment of lung cancer.

It is generally known that local anesthetics are commonly used drugs in surgical operation, which can temporarily block nerve impulses and conduction, thereby causing the disappearance of local pain [10]. Increasing evidences have demonstrated that local anesthetics exhibited the widely pharmacological effects on various cancers, which could suppress cell proliferation, inhibit migration and invasion, as well as induce apoptosis at a range of certain concen-trations [11, 12]. Chan et al. used ropivacaine, Lidocaine (Lido), levobupivacaine and bupivacaine to treat lung cancer A549 cells to explore the effect of local anesthetics on lung cancer, and revealed that these local anesthetics could reduce barrier function in A549 cells [13]. Lido is an aminoamide-type anesthetic, which is a commonly used local anesthetic due to the rapid onset of action, which is generally admin-istered intravenously, and can be used as a general anes-thetic and antiventricular arrhythmic agent. It can also exert antitumor and antitumor drug sensitization effects in vivo [14]. Moreover, Lido participated in mediating the immune system, which could inhibit inflammatory reactions, acute lung injury, inhibit bacteria, protect the brain, and enhance lymphocyte proliferation [15]. Furthermore, evidence from Fu et al. demonstrated that Lido epidural block combined with general anesthesia could alleviate postoperative pain, shorten postoperative recovery time, reduce the risk of postoperative complications [16]. However, the molecular mechanisms of Lido in the cisplatin resistance of lung cancer cells have not been fully investigated.

With the in-depth study of microRNAs (miRNAs), the issue of miRNAs affecting chemo-sensitivity has received considerable critical attention [17]. As an important miRNA, miR-21 has been reported to be involved in regulating vari-ous biological processes in different cancers, including lung cancer. High expression of serum miR-21 has been proven to be associated with poor prognosis in patients with lung cancer [18]. Additionally, study from Liu et al. dem-onstrated that miR-21 could promote non-small cell lung cancer (NSCLC) cells growth, metastasis, and chemo- or

radio-resistance through targeting PTEN [19]. However, whether miR-21 participates in mediating the effect of Lido on lung cancer cell growth is still unclear. Herein, A549 cells and A549/DDP cells were used in the present study. The effect of Lido on cell viability, apoptosis, migration and invasion in these cells were investigated. Moreover, the cor-relation between Lido and miR-21 expression were analyzed. Besides, the relevant signaling pathways were explored in A549/DDP cells. The study might provide more theoretical bases for clinical practice for the treatment of lung cancer.

Materials and methods

Cell culture and treatment

A549 cells and A549/DDP cells were purchased from Academy of Military Medical Science (Beijing, China). These cells were cultured in Roswell Park Memorial Insti-tute (RPMI)-1640 medium (Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS, Hyclone Laboratories, Logan, UT), 100 µg/mL penicillin, and 100 mg/mL streptomycin in a humidified atmosphere containing 5% CO2 at 37 °C. A549 cells and A549/DDP cells were then treated with different concentrations of cis-platin (0, 10, 20, 40, 80, 160 and 320 µM) for 48 h. Lido was obtained from Sigma–Aldrich (St. Louis, MO, USA), and the different concentrations of Lido (0, 1, 10, and 100 µM) were used to treat A549 and A549/DDP cells for 24 h.

Cell counting kit‑8 (CCK‑8) assay

Cell viability was assessed by a CCK-8 assay (Dojindo Molecular Technologies, Gaithersburg, MD). Briefly, A549 and A549/DDP cells were cultured in 96-well plates at a density of 5 × 103 cells/well. After stimulations, the 10 µL CCK-8 solution was added to the culture medium, and the cell plate was incubated for at 37 °C under the conventional culture condition. After incubation for 1 h, the absorbance was measured at 450 nm using a Microplate Reader (Bio-Rad, Hercules, CA, USA).

Apoptosis assay

The percentage of apoptotic cells was assessed using Annexin V-(fluorescein isothiocynate) FITC/propidium iodide (PI) apoptosis detection kit (Beijing Biosea Biotechnology, Bei-jing, China). Briefly, these treated cells were washed twice with cold phosphate-buffered saline (PBS, Gibco, Thermo Fisher Scienti c Inc., Waltham, MA, USA), and re-suspended in buffer. Afterward, the cells were stained by 5 µL PI/FITC-Annexin V according to the manufacturer’s instruction. Finally, the stained cells were measured with flow cytometer

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(Beckman Coulter, USA). These data were analyzed using FlowJo software (Treestar, Ashland, OR, USA).

Migration and invasion assays

Cell migration and invasion of A549 or A549/DDP cells were examined using a modified two-chamber migration assay with a pore size of 8 µm (Millipore, Bedford, Massa-chusetts, USA). In brief, after treatment with 10 µM of Lido, A549 and A549/DDP cells were washed two times with PBS and re-suspended in the serum-free medium. Then, 200 µL DDP cell suspension of serum-free medium were then added to the upper compartment of 24-well Transwell culture chamber, and 600 µL of complete medium was added to the lower compartment. After incubation at 37 °C, the non-migrated cells in the upper compartment were removed by a wet cotton swab. Migrated cells on the lower side of the filter were fixed in methanol for 30 min (Sigma–Aldrich), and stained with 0.5% crystal violet (Merck, Darmstadt, Germany) for 20 min and counted by a Microscope (Olym-pus, Tokyo, Japan). Cell invasion assay was similar with cell migration, but the inserts were coated with BD MatrigelTM Matrix (BD Biosciences, NY, USA).

Cell transfection

The expression plasmids of miR-21 mimic (sense: 5′-UAG CUU AUC AGA CUG AUG UUG A-3′; antisense: 5′-AAC AUC AGU CUG AUA AGC UAU U-3′), miR-21 inhibi-tor (5′-UCA ACA UCA GUC UGA UAA GCU A-3′) and its negative control (NC) (5′-UCA CAA CCU CCU AGA AAG AGU AGA-3′) were synthesized by GenePharma Co. (Shanghai, China). Cell transfections were conducted using Lipofectamine 3000 reagent (Invitrogen) following the man-ufacturer’s protocol. The transfection efficiency of miR-21 mimic and inhibitor was measured using real-time quantita-tive PCR (RT-qPCR). After 48 h of transfection, cells were collected for the following experiments.

RT‑qPCR

Total RNA was isolated from cells using Trizol reagent (Life Technologies Corporation, Carlsbad, CA, USA) according to the manufacturer’s instructions. The cDNA was synthe-sized using a PrimeScript™ 1st Strand cDNA Synthesis kit (TaKaRa, Dalian, China) according to the supplier’s protocol. RT-qPCR assay was performed using the Taqman MicroRNA Reverse Transcription Kit and Taqman Universal Master Mix II with the TaqMan MicroRNA Assay (Applied Biosystems, Foster City, CA, USA). The expression fold of miR-21 was detected by the 2−ΔΔC

t method [20]. U6 was used as the inter-nal control. The specific primer sequence for miR-21 is as Forward: 5′-GTG CAG GGT CCG AGG T-3′ and Reverse:

5′-GCC GCT AGC TTA TCA GAC TGA TGT-3′. U6 primer sequence is as Forward: 5′-CTT CGG CAG CAC ATA TAC T-3′ and Reverse: 5′-AAA ATA TGG AAC GCT TCA CG-3′.

Western blot assay

The protein from treated A549 or A549/DDP cells was lysed in radio immunoprecipitation assay (RIPA) lysis buffer (Bey-otime Biotechnology, Shanghai, China) supplemented with protease inhibitors (Roche, Basle, Switzerland). The concen-trations of proteins were quantified using the BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA). The protein sam-ples were electrophoresed by sodium dodecyl sulfate–poly-acrylamide gel electrophoresis (SDS–PAGE). Afterward, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes, and membranes were blocked in 5% non-fat milk for 1 h at room temperature. Subsequently, these membranes were incubated with primary antibodies of pro-Caspase-3 (ab32150), cleaved-Caspase-3 (ab49822), pro-Caspase-9 (ab32068), cleaved-Caspase-9 (ab2324), matrix metallo-proteinase-9 (MMP-9, ab38898), Vimentin (ab137321), phosphate and tension homology deleted on chromsome ten (PTEN, ab32199), phosphorylated- phosphatidylinositol 3-kinase (p-PI3K, ab182651), t-PI3K (ab133595), phosphoryl-ated-protein Kinase B (p-AKT, ab38449), t-AKT (ab64148), programmed cell death protein 4 (PDCD4, ab80590), phos-phorylated-c-Jun N-terminal kinase (p-JNK, ab47337), t-JNK (ab199380), and β-actin (ab8229, all Abcam, Cambridge, UK) at 4 °C overnight, followed by incubation with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (ab205718, 1:2000, Abcam) for 1 h at room temperature. The ECL West-ern blotting detection reagent (GE Healthcare, Braunschweig, Germany) was performed to visualize the blots. The inten-sity of the bands was quantified using Image Lab™ Software (Bio-Rad).

Statistical analysis

The experiments were repeated three times. These data were presented as the mean ± standard deviation (SD). SPSS 19.0 statistical software (SPSS Inc., Chicago, Illinois) was used to conduct statistical analysis. The P values were calculated using a one-way analysis of variance (ANOVA). A P value of < 0.05 was considered to indicate a statistically significant result.

Results

The cisplatin resistance of A549/DDP cells was higher than A549 cells

In the present study, the different concentrations of cispl-atin (0-320 µM) were used to stimulate A549 and A549/

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DDP cells, and cell viability and apoptosis were then examined. As shown in Fig. 1a, the results revealed that the viability of A549 and A549/DDP cells was signifi-cantly decreased by cisplatin in a dose-dependent manner. However, the inhibitory effect of cisplatin on A549/DDP cells was obviously lower than that on A549 cells at the concentrations of 40 µM (P < 0.001), 80 µM (P < 0.001), 160 µM (P < 0.01) and 320 µM (P < 0.05). Additionally, cisplatin significantly induced cell apoptosis in A549 and A549/DDP cells, and the promoting effect of cisplatin on the percent of apoptotic A549/DDP cells was lower than that on A549 cells (P < 0.05 or P < 0.01, Fig. 1b). Further, western blot results showed that cisplatin nota-bly increased the protein levels of cleaved-caspase-3 and cleaved-caspase-9 in A549 and A549/DDP cells. Whereas, the promoting effect of cisplatin on cleaved-caspase-3/-9 in A549/DDP cells was lower than that in A549 cells (Fig. 1c). All these data suggested that the cisplatin resist-ance of A549/DDP cells was higher than A549 cells.

Lido alleviated the cisplatin resistance of A549/DDP cells

Subsequently, A549/DDP cells were treated with differ-ent concentrations of Lido (0, 1, 10 and 100 µM), and cell viability was detected by CCK-8 assay. The results showed that cell viability was significantly decreased by Lido at the concentration of 100 µM (P < 0.05, Fig. 2a). But, there was no effect of Lido on cell viability at the concentration of 1 and 10 µM. Therefore, 10 µM of Lido was chosen for the treatment of A549/DDP in the following experiments. Then, we observed that Lido significantly declined cell via-bility in cisplatin-treated A549/DDP cells at the concentra-tions of 40 (P < 0.001), 80 (P < 0.001), 160 (P < 0.05) and 320 µM (P < 0.001, Fig. 2b). The percentage of apoptotic cells of A549/DDP cells was further promoted by Lido after treatment of cisplatin (P < 0.05, P < 0.01 or P < 0.001, Fig. 2c). Moreover, the protein levels of cleaved-caspase-3 and cleaved-caspase-9 were further upregulated by Lido in cisplatin-treated A549/DDP cells (Fig. 2d).

Next, the results in Fig. 3a–c revealed that Lido signifi-cantly inhibited cell migration (P < 0.05) and down-reg-ulated MMP-9 expression (P < 0.01 or P < 0.001) in both

Fig. 1 The cisplatin resistance of A549/DDP cells was higher than A549 cells. A549 and A549/DDP cells were stimu-lated with different concentra-tions of cisplatin (0, 10, 20, 40, 80, 160 and 320 µM) for 48 h. a Cell viability, b cell apopto-sis, and c the protein levels of pro-caspase-3/-9 and cleaved-caspase-3/-9 in A549 and A549/DDP cells were examined by CCK-8, flow cytometry and western blot. CCK-8: Cell Counting Kit-8; *P < 0.05, **P < 0.01 and ***P < 0.001

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A549 and A549/DDP cells. Likewise, Lido also suppressed cell invasion (P < 0.05) and down-regulated Vimentin expression (P < 0.01 or P < 0.001) in A549 and A549/DDP cells, (Fig. 3d–f). All above data indicated that Lido alle-viated the cisplatin resistance of A549/DDP, which might through suppressing cell viability, migration, invasion and inducing apoptosis.

Lido down‑regulated miR‑21 expression level in A549/DDP cells

The expression level of miR-21 was then examined using RT-qPCR in A549 and A549/DDP cells. We found that miR-21 expression was upregulated in A549/DDP cells com-pared to in A549 cells (P < 0.05, Fig. 4a). To further confirm whether A549/DDP cells acquires resistance to cisplatin by upregulation of miR-21, the expression vectors of miR-21 mimic and its corresponding control were transfected into A549 cells, and cell viability was then determined. The transfection efficiency of miR-21 mimic was presented in Fig. 4b, the results showed that the expression level of miR-21 was significantly increased in miR-21 mimic-transfected cells (P < 0.001). CCK-8 assay revealed that miR-21 over-expression significantly increased the viability of A549 cells after treatment with cisplatin at the concentrations of 40

(P < 0.01), 80 (P < 0.01), 160 (P < 0.01) and 320 (P < 0.05, Fig. 4c). Further, we observed that the expression level of miR-21 was significantly declined by Lido in A549/DDP cells compared with control group (P < 0.05, Fig. 4d). These data indicated that miR-21 might be involved in mediating the cisplatin resistance of A549/DDP cells.

MiR‑21 suppression declined the cisplatin resistance of A549/DDP cells

Afterward, miR-21 inhibitor and its corresponding control were transfected into A549/DDP cells to regulate miR-21 expression, meanwhile uncover the effect of miR-21 on cisplatin resistance in A549/DDP cells. As expected, we found that the expression level of miR-21 was down-regu-lated in miR-21 inhibitor-transfected cells compared with control group (P < 0.01, Fig. 5a), stating that cell transfec-tion was successful. CCK-8 assay showed that suppression of miR-21 inhibited A549/DDP cell viability after treat-ment with cisplatin at the concentrations of 80 (P < 0.001), 160 (P < 0.05) and 320  µM (P < 0.05, Fig.  5b). More interestingly, we found that the inhibitor effect of Lido on cell viability of A549/DDP cells was further declined by miR-21 inhibition (P < 0.001, Fig. 5c). Flow cytometry results displayed that suppression of miR-21 significantly

Fig. 2 Lido suppressed cell viability and induced apoptosis in A549/DDP cells. A549/DDP cells were stimulated with different concen-trations of Lido (0, 1, 10 and 100 µM) for 24 h. a Cell viability was examined by CCK-8 assay; after co-treated with cisplatin and Lido,

b cell viability, c cell apoptosis, and d the protein levels of pro-cas-pase-3/-9 and cleaved-caspase-3/-9 in A549/DDP cells were exam-ined by CCK-8, flow cytometry and western blot. Lido lidocaine, CCK-8 cell counting kit-8, *P < 0.05, **P < 0.01 and ***P < 0.001

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enhanced cell apoptosis in A549/DDP cells after stimula-tion of cisplatin (P < 0.05, P < 0.01 or P < 0.001, Fig. 5d). Western blot results showed that miR-21 inhibition further

increased the protein levels of cleaved-caspase-3 and cleaved-caspase-9 in A549/DDP cells after stimulation of cisplatin (P < 0.01 or P < 0.001, Fig. 5e, f). These data

Fig. 3 Lido inhibited cell migration and invasion in A549/DDP cells. A549/DDP cells were stimulated with 10  µM Lido for 24  h, a cell migration and b and c MMP-9 expression were detected by transwell and western blot assays; d cell invasion and e and f Vimentin expres-

sion were assessed by transwell and western blot assays. Lido lido-caine, MMP-9 matrix metallopeptidase 9, *P < 0.05, **P < 0.01 and ***P < 0.001

Fig. 4 MiR-21 expression was decreased by Lido in A549/DDP cells. a The expression level of miR-21 in A549 and A549/DDP cells were exam-ined by RT-qPCR assay; b the expression vectors of miR-21 mimic and its corresponding control were transfected into A549 cells, the expression level of miR-21 in these transfected cells was determined by RT-qPCR assay; c cell viability was assessed by CCK-8 assay; after treatment of 10 µM Lido, d the expression level of miR-21 was analyzed by RT-qPCR again. MiR-21 microRNA-21, CCK-8 cell counting kit-8, Lido lidocaine, RT-qPCR real-time quantitative PCR, *P < 0.05, **P < 0.01 and ***P < 0.001

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indicated that miR-21 suppression exerted the same effect as Lido on the cisplatin resistance of A549/DDP cells.

miR‑21 suppression deactivated PTEN/PI3K/AKT and PDCD4/JNK signaling pathways in A549/DDP cells

Finally, the effect of miR-21 on PTEN/PI3K/AKT and PDCD4/JNK signaling pathways in A549/DDP cells were

determined. As results explained in Fig. 6a, b, suppres-sion of miR-21 was notably upregulated PTEN expression (P < 0.001) and down-regulated p-PI3K and p-AKT expres-sion (P < 0.05 or P < 0.01) compared with NC group. Simi-larly, the protein level of PDCD4 was increased by miR-21 suppression, and the protein level of p-JNK was decreased by miR-21 suppression in A549/DDP cells (P < 0.05 or P < 0.001, Fig. 6c, d). These data indicated that miR-21

Fig. 5 MiR-21 suppression alleviated the cisplatin resistance of A549/DDP cells. A549/DDP cells were transfected with miR-21 inhibitor and NC, a the expression level of miR-21 in the trans-fected cells was examined by RT-qPCR; after stimulation of differ-ent concentrations of cisplatin and/or Lido, b and c cell viability, d

cell apoptosis, and e and f the protein levels of pro-caspase-3/-9 and cleaved-caspase-3/-9 in A549/DDP cells were determined by CCK-8, flow cytometry and western blot assays. MiR-21 microRNA-21, NC negative control, RT-qPCR real-time quantitative PCR, *P < 0.05, **P < 0.01 and ***P < 0.001

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could mediate PTEN/PI3K/AKT and PDCD4/JNK signal-ing pathways in A549/DDP cells.

Discussion

The study preliminarily investigated the effect of Lido on A549/DDP cells. The results showed that Lido sig-nificantly alleviated the cisplatin resistance of A549/DDP cells. Moreover, Lido suppressed cell migration and inva-sion in A549/DDP and A549 cells. Additionally, we found that Lido down-regulated the expression level of miR-21 in A549/DDP cells, and miR-21 suppression showed the same regulatory effect as Lido on the cisplatin resistance of A549/DDP cells. Finally, we observed that suppression of miR-21-regulated PTEN/PI3K/AKT and PDCD4/JNK signaling pathways in A549/DDP cells.

Cisplatin is a cell cycle-nonspecific DNA-damaging agent, which exerts anti-cancer effect on variety of solid tumors, such as ovarian cancer, prostate cancer, and also including lung cancer [21–23]. Clinically, cisplatin combi-nations with carboplatin, gemcitabine, paclitaxel, and iri-notecan have been widely used to treat advanced NSCLC [24]. However, the chemo-resistance limits the therapeutic effect of cisplatin in patients with lung cancers [25]. A549/DDP is a multidrug-resistant cell line induced by cisplatin in A549 cells, which has been used to study the mechanism of cisplatin resistance in lung cancer [26]. In our study, we

observed that cisplatin significantly inhibited cell viability and induced apoptosis in A549 cells. However, the regulator effect of cisplatin on cell viability and apoptosis in A549/DDP cells obviously declined, further indicating the cispl-atin resistance of A549/DDP cells was higher than A549 cells.

Lido is a common local anesthetic in clinic, which is mainly used to treat ventricular arrhythmia and acute myo-cardial infarction [27]. Lido has been reported to play an important role in various cancers. For instances, Lido could sensitize the cytotoxicity of cisplatin in breast cancer cells through upregulation of RARβ2 and RASSF1A demethylation [28]. Moreover, Lido could inhibit tumor growth and induce apoptosis in hepatocellular carcinoma (HCC) cells in vivo and in vitro [29]. In lung cancer, Zhang et al. demonstrated that Lido could inhibit the proliferation of lung cancer cells via mediation of GOLT1A expression [30]. Similarly, Wang et al. stated that Lido could inhibit cell viability, induce G0/G1 phase arrest and apoptosis in A549 cells [31]. However, it is still unclear whether Lido affected the cisplatin resist-ance of A549/DDP cells. In this study, we found that Lido significantly suppressed cell viability and induced apopto-sis in cisplatin-treated A549/DDP cells. These data indi-cated that Lido could alleviate the cisplatin resistance of A549/DDP cells. Further, our study also indicated that Lido could inhibit cell migration and invasion through decreas-ing the expression levels of MMP-9 and Vimentin in A549 and A549/DDP cells. It is worth noting that the increase

Fig. 6 miR-21 suppression regulated PTEN/PI3K/AKT and PDCD4/JNK signaling path-ways in A549/DDP cells. A549/DDP cells were transfected with miR-21 inhibitor and NC, the protein levels of a and b PTEN, p/t-PI3K, p/t-AKT and c and d PDCD4 and p/t-JNK in A549/DDP cells were determined by western blot assay. MiR-21 microRNA-21, NC negative control, PTEN phosphate and tension homology deleted on chromsome ten, PI3K phos-phatidylinositol 3-kinase, AKT protein kinase B, PDCD4 programmed cell death protein 4, JNK c-Jun N-terminal kinase, *P < 0.05, **P < 0.01 and ***P < 0.001

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of apoptosis may be an important factor in reducing inva-sion and migration. Thus, further studies are still needed to exclude the effects of apoptosis on migration and invasion in the future.

Mounting evidences indicated that miRNAs may play a vital role in chemo-resistance of lung cancer cells [32, 33]. Several studies focused on investigating the effect of miR-21 on chemo-resistance of lung cancer cells. As Xu et al. showed that suppression of miR-21 could promote the sen-sitivity of A549 cells to cisplatin in vitro and in vivo [34]. Another study from Tian et al. demonstrated that inhibition of miR-21 declined the cisplatin resistance of A549/DDP by regulating cell proliferation and apoptosis [35]. In our study, we found that miR-21 was upregulated in A549/DDP cells, and overexpression of miR-21 increased cell viabil-ity in A549 cells. Further, more important results revealed that Lido down-regulated the expression level of miR-21 in A549/DDP cells. Further, results in the present study were partially similar with above researches that suppression of miR-21 reversed the cisplatin resistance of A549/DDP cells.

To further ascertain the effect of miR-21 on the cisplatin resistance of A549/DDP cells, the drug resistance-related pathways of PTEN/PI3K/AKT and PDCD4/JNK were stud-ied. Recent study showed that miR-21 could repress PTEN and promote lung cancer cell growth [36]. Yang et al. uncov-ered that inhibition of miR-21 and PTEN signaling pathway might decline the cisplatin resistance of lung cancer cells [37]. Additionally, an interesting study from Zhang et al. proved that inhibition of PI3K/AKT signaling pathway could reverse the cisplatin resistance in lung cancer cells [38]. Moreover, Ning et al. reported that miR-182 could mediate chemo-sensitivity of lung cancer cells to cisplatin through down-regulation of PDCD4 [39]. Further, Fu et al. found that inactivation of the JNK signaling pathway could enhance the chemo-sensitivity of A549 cells to cisplatin [40]. Partly similar with these studies, we found that miR-21 suppression could regulate PTEN/PI3K/AKT and PDCD4/JNK signaling pathways in A549/DDP cells. These data indicated that PTEN/PI3K/AKT and PDCD4/JNK signal-ing pathways might be important mediators in regulating the cisplatin resistance of A549/DDP cells.

Taken together, these data suggested that Lido could alleviate the cisplatin resistance of A549/DDP cells through down-regulation of miR-21. Moreover, miR-21 suppression could affect the cisplatin resistance of A549/DDP cells via mediating PTEN/PI3K/AKT and PDCD4/JNK signaling pathways. These findings clarified the effect of Lido on the cisplatin resistance of lung cancer cells, and might provide a novel management for the treatment of lung cancer.

Funding This work was supported by Kaifeng City Social Develop-ment Plan Project (Grant No: 1503113).

References

1. Masters GA, Temin S, Azzoli CG, Giaccone G, Baker S, Brahmer JR, Ellis PM, Gajra A, Rackear N, Schiller JH (2017) Systemic therapy for stage IV non–small-cell lung cancer: American Soci-ety of Clinical Oncology Clinical Practice Guideline update. J Clin Oncol 33:832–837

2. Siegel RL, Miller KD, Jemal A (2017) Cancer statistics, 2017. CA Cancer J Clin 67:7–30. https ://doi.org/10.3322/caac.21387

3. Cheng TD, Cramb SM, Baade PD, Youlden DR, Nwogu C, Reid ME (2016) The international epidemiology of lung cancer: latest trends, disparities, and tumor characteristics. J Thorac Oncol 11:1653–1671

4. Thun MJ, Hannan LM, Adams-Campbell LL, Boffetta P, Buring JE, Feskanich D, Flanders WD, Jee SH, Katanoda K, Kolonel LN, Lee IM, Marugame T, Palmer JR, Riboli E, Sobue T, Avila-Tang E, Wilkens LR, Samet JM (2008) Lung cancer occurrence in never-smokers: an analysis of 13 cohorts and 22 cancer reg-istry studies. PLoS Med 5:e185. https ://doi.org/10.1371/journ al.pmed.00501 85

5. Ebbinghaus S (2015) Lung cancer treatment. Clin Pharm 3:109–114

6. Simone CB 2nd, Jones JA (2013) Palliative care for patients with locally advanced and metastatic non-small cell lung can-cer. Ann Palliat Med 2:178–188. https ://doi.org/10.3978/j.issn.2224-5820.2013.08.02

7. Xue D, Abernethy AP (2010) Management of dyspnea in advanced lung cancer: recent data and emerging concepts. Curr Opin Sup-port Palliat Care 4:85–91. https ://doi.org/10.1097/SPC.0b013 e3283 39920 d

8. Reck M, Rodriguez-Abreu D, Robinson AG, Hui R, Csoszi T, Fulop A, Gottfried M, Peled N, Tafreshi A, Cuffe S, O’Brien M, Rao S, Hotta K, Leiby MA, Lubiniecki GM, Shentu Y, Rang-wala R, Brahmer JR (2016) Pembrolizumab versus chemotherapy for PD-L1-positive non-small-cell lung cancer. N Engl J Med 375:1823–1833. https ://doi.org/10.1056/NEJMo a1606 774

9. Liu MY, Li XQ, Gao TH, Cui Y, Ma N, Zhou Y, Zhang GJ (2016) Elevated HOTAIR expression associated with cisplatin resistance in non-small cell lung cancer patients. J Thorac Dis 8:3314–3322. https ://doi.org/10.21037 /jtd.2016.11.75

10. Bang SU, Kim DJ, Bae JH, Chung K, Kim Y (2016) Minimum effective local anesthetic volume for surgical anesthesia by sub-paraneural, ultrasound-guided popliteal sciatic nerve block: a prospective dose-finding study. Medicine 95:e4652. https ://doi.org/10.1097/md.00000 00000 00465 2

11. Chang YC, Hsu YC, Liu CL, Huang SY, Hu MC, Cheng SP (2014) Local anesthetics induce apoptosis in human thyroid cancer cells through the mitogen-activated protein kinase pathway. PLoS ONE 9:e89563

12. Chang YC, Liu CL, Chen MJ, Hsu YW, Chen SN, Lin CH, Chen CM, Yang FM, Hu MC (2014) Local anesthetics induce apoptosis in human breast tumor cells. Anesth Analg 118:116–124

13. Chan SM, Lin BF, Wong CS, Chuang WT, Chou YT, Wu ZF (2017) Levobuipivacaine-induced dissemination of A549 lung cancer cells. Sci Rep 7:8646. https ://doi.org/10.1038/s4159 8-017-08885 -z

14. Uzun S, Yuce Y, Erden A, Aypar U (2014) Impact of periop-erative lidocaine infusion and bis monitorization on remifentanil dosage in hypotensive anesthesia. Eur Rev Med Pharmacol Sci 18:559–565

15. Yardeni IZ, Beilin B, Mayburd E, Levinson Y, Bessler H (2009) The effect of perioperative intravenous lidocaine on postoperative pain and immune function. Anesth Analg 109:1464–1469. https ://doi.org/10.1213/ANE.0b013 e3181 bab1b d

16. Baochun FU, Wang T, Xinzhou YU, Anesthesiology DO (2017) Effects of lidocaine epidural block combined with general anes-thesia on postoperative related indexes of lung cancer patients. China Pharm 28:1626–1629

Molecular and Cellular Biochemistry

1 3

17. Nordentoft I, Birkenkamp-Demtroder K, Agerbaek M, Theodor-escu D, Ostenfeld MS, Hartmann A, Borre M, Orntoft TF, Dyr-skjot L (2012) miRNAs associated with chemo-sensitivity in cell lines and in advanced bladder cancer. BMC Med Genom 5:40. https ://doi.org/10.1186/1755-8794-5-40

18. Liu XG, Zhu WY, Huang YY, Ma LN, Zhou SQ, Wang YK, Zeng F, Zhou JH, Zhang YK (2012) High expression of serum miR-21 and tumor miR-200c associated with poor prognosis in patients with lung cancer. Med Oncol 29:618–626. https ://doi.org/10.1007/s1203 2-011-9923-y

19. Liu ZL, Wang H, Liu J, Wang ZX (2013) MicroRNA-21 (miR-21) expression promotes growth, metastasis, and chemo- or radiore-sistance in non-small cell lung cancer cells by targeting PTEN. Mol Cell Biochem 372:35–45. https ://doi.org/10.1007/s1101 0-012-1443-3

20. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expres-sion data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25:402–408

21. Ryu SY, Kim K, Lee WS, Kwon HC, Lee KH, Kim CM, Kang S-B (2009) Synergistic growth inhibition by combination of adenovirus mediated p53 transfer and cisplatin in ovarian cancer cell lines. J Gynecol Oncol 20:48–54. https ://doi.org/10.3802/jgo.2009.20.1.48

22. Festuccia C, Gravina GL, D’Alessandro AM, Muzi P, Millimaggi D, Dolo V, Ricevuto E, Vicentini C, Bologna M (2009) Azac-itidine improves antitumor effects of docetaxel and cisplatin in aggressive prostate cancer models. Endocr Relat Cancer 16:401–413. https ://doi.org/10.1677/erc-08-0130

23. Li D, Zhang Y, Xie Y, Xiang J, Zhu Y, Yang J (2013) Enhanced tumor suppression by adenoviral PTEN gene therapy combined with cisplatin chemotherapy in small-cell lung cancer. Cancer Gene Ther 20:251–259

24. Ohe Y, Ohashi Y, Kubota K, Tamura T, Nakagawa K, Negoro S, Nishiwaki Y, Saijo N, Ariyoshi Y, Fukuoka M (2007) Rand-omized phase III study of cisplatin plus irinotecan versus carbopl-atin plus paclitaxel, cisplatin plus gemcitabine, and cisplatin plus vinorelbine for advanced non-small-cell lung cancer: four-arm cooperative study in Japan. Ann Oncol 18:317–323. https ://doi.org/10.1093/annon c/mdl37 7

25. Fennell DA, Summers Y, Cadranel J, Benepal T, Christoph DC, Lal R, Das M, Maxwell F, Visseren-Grul C, Ferry D (2016) Cis-platin in the modern era: the backbone of first-line chemotherapy for non-small cell lung cancer. Cancer Treat Rev 44:42–50. https ://doi.org/10.1016/j.ctrv.2016.01.003

26. Zhang K, Wang X, Wang H (2014) Effect and mechanism of Src tyrosine kinase inhibitor sunitinib on the drug-resistance reversal of human A549/DDP cisplatin-resistant lung cancer cell line. Mol Med Rep 10:2065–2072. https ://doi.org/10.3892/mmr.2014.2440

27. Chaki T, Sugino S, Janicki PK, Ishioka Y, Hatakeyama Y, Hayase T, Kaneuchi-Yamashita M, Kohri N, Yamakage M (2016) Effi-cacy and safety of a lidocaine and ropivacaine mixture for scalp nerve block and local infiltration anesthesia in patients undergoing awake craniotomy. J Neurosurg Anesthesiol 28:1–5. https ://doi.org/10.1097/ana.00000 00000 00014 9

28. Li K, Yang J, Han X (2014) Lidocaine sensitizes the cytotoxicity of cisplatin in breast cancer cells via up-regulation of RARβ2 and RASSF1A demethylation. Int J Mol Sci 15:23519–23536

29. Xing W, Chen DT, Pan JH, Chen YH, Yan Y, Li Q, Xue RF, Yuan YF, Zeng WA (2017) Lidocaine induces apoptosis and suppresses tumor growth in human hepatocellular carcinoma cells in vitro and in a xenograft model in vivo. Anesthesiology 126:868–881. https ://doi.org/10.1097/aln.00000 00000 00152 8

30. Zhang L, Hu R, Cheng Y, Wu X, Xi S, Sun Y, Jiang H (2017) Lidocaine inhibits the proliferation of lung cancer by regu-lating the expression of GOLT1A. Cell Prolif 50. https ://doi.org/10.1111/cpr.12364

31. Wang HW, Wang LY, Jiang L, Tian SM, Zhong TD, Fang XM (2016) Amide-linked local anesthetics induce apoptosis in human non-small cell lung cancer. J Thorac Dis 8:2748–2757. https ://doi.org/10.21037 /jtd.2016.09.66

32. Wang Q, Zhong M, Liu W, Li J, Huang J, Zheng L (2011) Altera-tions of microRNAs in cisplatin-resistant human non-small cell lung cancer cells (A549/DDP). Exp Lung Res 37:427–434. https ://doi.org/10.3109/01902 148.2011.58426 3

33. Ma Y, Li X, Cheng S, Wei W, Li Y (2015) MicroRNA-106a con-fers cisplatin resistance in non-small cell lung cancer A549 cells by targeting adenosine triphosphatase-binding cassette A1. Mol Med Rep 11:625–632. https ://doi.org/10.3892/mmr.2014.2688

34. Xu L, Huang Y, Chen D, He J, Zhu W, Zhang Y, Liu X (2014) Down-regulation of miR-21 increases cisplatin sensitivity of non-small-cell lung cancer. Cancer Genet 207:214–220. https ://doi.org/10.1016/j.cance rgen.2014.04.003

35. Tian M, Wang Y, Miao L, Ding J, Zhang D, Cai H (2014) Effects of miR-21 inhibition on A549/DDP cisplatin resistant cell prolif-eration and apoptosis in non-small cell lung cancer. J Nanjing Med Univ 30:20

36. Zhang JG, Wang JJ, Zhao F, Liu Q, Jiang K, Yang GH (2010) MicroRNA-21 (miR-21) represses tumor suppressor PTEN and promotes growth and invasion in non-small cell lung cancer (NSCLC). Clin Chim Acta 411:846–852

37. Yang Z, Fang S, Di Y, Ying W, Tan Y, Gu W (2015) Modulation of NF-κB/miR-21/PTEN pathway sensitizes non-small cell lung cancer to cisplatin. PLoS ONE 10:e0121547

38. Zhang Y, Bao C, Mu Q, Chen J, Wang J, Mi Y, Sayari AJ, Chen Y, Guo M (2016) Reversal of cisplatin resistance by inhibiting PI3K/Akt signal pathway in human lung cancer cells. Neoplasma 63:362–370. https ://doi.org/10.4149/304_15080 6n433

39. Ning FL, Wang F, Li ML, Yu ZS, Hao YZ, Chen SS (2014) Micro-RNA-182 modulates chemosensitivity of human non-small cell lung cancer to cisplatin by targeting PDCD4. Diagn Pathol 9:143. https ://doi.org/10.1186/1746-1596-9-143

40. Fu Y, Han X, Li Y (2013) Inhibition of the JNK pathway down-regulates lung-resistance protein and enhances the chemosensitiv-ity of A549 cells to cisplatin. Chin J Clin Oncol 40:1518–1522

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Affiliations

Qing Yang1 · Zhi Zhang1 · Haixia Xu1 · Chuangen Ma1

1 Department of Anesthesiology, Huaihe Hospital of Henan University, No. 8, Baobei Road, Gulou District, Kaifeng 475000, China

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