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. 2020 Sep 28;479(1):180–194. doi: 10.1097/CORR.0000000000001510

Bupivacaine and Lidocaine Induce Apoptosis in Osteosarcoma Tumor Cells

Saied Mirshahidi 1,2,3,4,5,6,7, Troy G Shields 1,2,3,4,5,6,7, Rosalia de Necochea-Campion 1,2,3,4,5,6,7, Xiangpeng Yuan 1,2,3,4,5,6,7, Ata Janjua 1,2,3,4,5,6,7, Nadine L Williams 1,2,3,4,5,6,7, Hamid R Mirshahidi 1,2,3,4,5,6,7, Mark E Reeves 1,2,3,4,5,6,7, Penelope Duerksen-Hughes 1,2,3,4,5,6,7, Lee M Zuckerman 1,2,3,4,5,6,7,✉
PMCID: PMC7899706  PMID: 33009230

Abstract

Background

Osteosarcoma is the most common type of bone cancer in adolescents. There have been no significant improvements in outcomes since chemotherapy was first introduced. Bupivacaine and lidocaine have been shown to be toxic to certain malignancies. This study evaluates the effect of these medications on two osteosarcoma cell lines.

Questions/purposes

(1) Does incubation of osteosarcoma cells with bupivacaine or lidocaine result in cell death? (2) Does this result from an apoptotic mechanism? (3) Is a specific apoptotic pathway implicated?

Methods

Two cell lines were chosen to account for the inherent heterogeneity of osteosarcoma. UMR-108 is a transplantable cell line that has been used in multiple studies as a primary tumor. MNNG/HOS has a high metastatic rate in vivo. Both cell lines were exposed bupivacaine (0.27, 0.54, 1.08, 2.16, 4.33 and 8.66 mM) and lidocaine (0.66, 1.33, 5.33, 10.66, 21.32 and 42.64 mM) for 24 hours, 48 hours, and 72 hours. These concentrations were determined by preliminary experiments that found the median effective dose was 1.4 mM for bupivacaine and 7.0 mM for lidocaine in both cell lines. Microculture tetrazolium and colony formation assay determined whether cell death occurred. Apoptosis induction was evaluated by phase-contrast micrographs, flow cytometry, DNA fragmentation and reactive oxygen species (ROS). The underlying pathways were analyzed by protein electrophoresis and Western blot. All testing was performed in triplicate and compared with pH-adjusted controls. Quantitative results were analyzed without blinding.

Results

Both medications caused cell death in a dose- and time-dependent manner. Exposure to bupivacaine for 24 hours reduced viability of UMR-108 cells by 6 ± 0.75% (95% CI 2.9 to 9.11; p = 0.01) at 1.08 mM and 89.67 ± 1.5% (95% CI 82.2 to 95.5; p < 0.001) at 2.16 mM. Under the same conditions, MNNG/HOS viability was decreased in a similar fashion. After 24 hours, the viability of UMR-108 and MNNG/HOS cells exposed to 5.33 mM of lidocaine decreased by 25.33 ± 8.3% (95% CI 2.1 to 48.49; p = 0.03) and 39.33 ± 3.19% (95% CI 30.46 to 48.21; p < 0.001), respectively, and by 90.67 ± 0.66% (95% CI 88.82 to 92.52; p < 0.001) and 81.6 ± 0.47% (95% CI 79.69 to 82.31; p < 0.001) at 10.66 mM, respectively. After 72 hours, the viability of both cell lines was further reduced. Cell death was consistent with apoptosis based on cell morphology, total number of apoptotic cells and DNA fragmentation. The percentage increase of apoptotic UMR-108 and MNNG/HOS cells confirmed by Annexin-V positivity compared with controls was 21.3 ± 2.82 (95% CI 16.25 to 26.48; p < 0.001) and 21.23 ± 3.23% (95% CI 12.2 to 30.2; p = 0.003) for bupivacaine at 1.08 mM and 25.15 ± 4.38 (95% CI 12.9 to 37.3; p = 0.004) and 9.11 ± 1.74 (95% CI 4.35 to 13.87; p = 0.006) for lidocaine at 5.33 mM. The intrinsic apoptotic pathway was involved as the expression of Bcl-2 and survivin were down-regulated, and Bax, cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase-1 were increased. ROS production increased in the UMR-108 cells but was decreased in the MNNG/HOS cells.

Conclusion

These findings provide a basis for evaluating these medications in the in vivo setting. Studies should be performed in small animals to determine if clinically relevant doses have a similar effect in vivo. In humans, biopsies could be performed with standard doses of these medications to see if there is a difference in biopsy tract contamination on definitive resection.

Clinical Relevance

Bupivacaine and lidocaine could potentially be used for their ability to induce and enhance apoptosis in local osteosarcoma treatment. Outcome data when these medications are used routinely during osteosarcoma treatment can be evaluated compared with controls. Further small animal studies should be performed to determine if injection into the tumor, isolated limb perfusion, or other modalities of treatment are viable.

Introduction

Osteosarcoma is an aggressive primary malignant bone tumor. Osteosarcoma is the most common bone tumor in children and adolescents and typically affects the long tubular bones with an incidence between 3 and 4.4 cases per million [3, 31, 32, 41, 46]. Chemotherapy and surgery are currently standard treatments. Surgery without chemotherapy is associated with an increased risk of recurrence and has a high rate of metastasis, especially to the lung [14, 19, 34]. Currently, the 5-year survival rate of patients with osteosarcoma is 75% for conventional, nonmetastatic disease. However, patients with osteosarcoma treated with surgery alone were found to have metastases with a 5-year survival rate of no more than 20% [4, 16, 17]. Although initial chemotherapeutic agents increased the survival of nonmetastatic disease by 55%, newer agents have not provided a substantial benefit and targeted gene therapies are not currently proven and are expensive [3, 5, 10, 15, 22, 23, 33]. In addition, the severe side effects of chemotherapeutic agents and the development of multidrug resistance are major treatment challenges for patients with osteosarcoma [29]. This highlights the need for the development of new, more effective, and less expensive neoadjuvant and adjuvant therapies for osteosarcoma treatment.

Local anesthetics such as bupivacaine and lidocaine are widely available and inexpensive. In addition to being used clinically in cancer patients to relieve pain and to decrease opioid use, they have been shown to induce apoptosis and arrest cell growth in vitro by both the intrinsic and extrinsic apoptotic signaling pathways in certain malignancies, such as carcinoma of the thyroid, breast, and tongue, neuroblastoma, and lymphoma [8, 9, 30, 39, 44, 47, 48]. Local anesthetics also inhibit axonal transport and prevent the dissemination of cancer cells during biopsies or resection operations [12, 50]. The benefit of local anesthetic administration during the resection of prostate, colon, and breast cancers has been shown to decrease the risk of metastasis, local recurrence, and improve overall survival [6, 11, 13]. Bupivacaine and lidocaine have been shown in vitro to induce cytotoxicity with morphological changes to promote apoptosis via the intrinsic and extrinsic pathways and induce necrosis [2, 18, 27, 37, 38, 40, 47, 48].

However, to our knowledge the effects of local anesthetics have never been investigated on osteosarcoma cell lines. We therefore asked the following questions: (1) Does incubation of osteosarcoma cells with bupivacaine or lidocaine result in cell death? (2) Does this result from an apoptotic mechanism? (3) Is a specific apoptotic pathway implicated? This study allows us to evaluate the treatment effects of medications that are currently available, FDA approved, and cost effective in an in vitro model for osteosarcoma using two representative osteosarcoma cell lines.

Materials and Methods

Our approach to the cell culture and reagents, cell viability, clonogenic survival cell morphology, apoptotic analysis, DNA fragmentation, Western blot analysis, and the statistical analysis noted below has been described previously [35].

Experimental Overview

As bupivacaine and lidocaine have been found to be cytotoxic to certain malignancies, we sought to evaluate if they had a similar effect in osteosarcoma cells. We performed the experiments in two different cell lines that are commonly used in research to provide further validation of the findings and to evaluate whether the same effects occurred in separate cell lines. In preliminary experiments, we established the median effective dose (ED50) of each medication to guide the experimental doses. The ED50 was found to be 1.4 mM for bupivacaine and 7.0 mM for lidocaine in both cell lines. Testing was then performed compared with a pH-adjusted control to determine if the medications were cytotoxic, whether they induced apoptosis, and what mechanisms were involved.

Primary and Secondary Study Endpoints

Our primary study endpoint was to evaluate if bupivacaine and lidocaine was cytotoxic to two separate cell lines in vitro. We assessed this by performing multiple experiments that looked at cell viability and colony formation after cell incubation. Different doses and durations of incubation were used to determine whether the cytotoxicity was affected by increasing dosage and exposure times.

Our secondary study endpoints were to evaluate whether the medications induced apoptosis, and to delineate if it occurred in a similar fashion between cell lines and medications. We assessed these by evaluating photomicrographs, the number of apoptotic cells after exposure, and DNA fragmentation. Induction of apoptosis and the underlying mechanisms were further evaluated by Western blot, testing with a pan-caspase inhibitor and by levels of reactive oxygen species (ROS).

Cell Culture and Reagents

We obtained a rat osteosarcoma cell line (UMR-108) and a human osteosarcoma cell line (MNNG/HOS) (American Type Culture Collections, Rockville, MD, USA Cat# CRL-1663 and CRL-1547, respectively) for this study. UMR-108 was chosen because it is a transplantable cell line that has less hormone reactivity compared with other cell lines and has a rich history of being used as a primary tumor in research. MNNG/HOS is a highly invasive cell line that has been used as a model for metastatic disease. In the clinical setting, osteosarcoma is heterogeneous, whereas cell lines are homogeneous. The study of different cell lines in this case allows for some heterogeneity to determine if the results may be applicable in the clinical setting. The cells were prepared in Dulbecco’s modified Eagle’s medium (Millipore Sigma, Temecula, CA, USA) that was supplemented with 10% fetal bovine serum. Then, we added 100 U/mL penicillin G and streptomycin with 1% nonessential amino acids. The cells were maintained at 37° C with 5% CO2 in a humidified incubator. Preservative-free bupivacaine (Marcaine, 0.5%, 17.34 mM, containing 5 mg/mL bupivacaine HCl with NaCl and pH adjusted with NaOH and HCl, Hopsira, Lake Forest, IL, USA) and preservative-free lidocaine (2%, 85.34 mM, containing 20 mg/mL lidocaine HCl, 6 mg NaCl and pH-adjusted with NaOH and HCl, Hospira) were used during the testing stage of the study.

Evaluation of Cell Viability

To evaluate cell viability, we seeded the osteosarcoma cells at a density of 4000 cells/well in 96-well plates in triplicate. We monitored the cells, and once they reached 80% confluence, we began testing. The cells were treated for 24 hours, 48 hours, and 72 hours at 37° C with various concentrations of bupivacaine (0.27 mM, 0.54 mM, 1.08 mM, 2.16 mM, 4.33 mM and 8.66 mM) and lidocaine (0.66 mM, 1.33 mM, 5.33 mM, 10.66 mM, 21.32 mM and 42.64 mM) and compared with a control. For the control group, phosphate-buffered saline (PBS) with a pH similar to the highest doses of bupivacaine and lidocaine was prepared. Different dilutions of PBS were used as a control for variation in pH and the background was subtracted from the treated groups. The reported data is the difference between treated and control cells. Cell viability was assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide microculture tetrazolium reagent, (Roche Diagnostics, Indianapolis, IN, USA) as per the manufacturer’s protocol. Then, we used a microplate reader (Bio-Rad Model 3550, Hercules, CA, USA) to measure the absorbance at 570 nm. Each experiment was done in triplicate and repeated three times. The results were then expressed as a percentage of the untreated control (% of control). The investigators (SM, AJ, TS, RD) were not blinded to the treatment groups.

Evaluation of Clonogenic Survival

Three concentrations each of bupivacaine (1.08 mM, 2.16 mM, and 4.33 mM) and lidocaine (5.33 mM, 10.66 mM, and 21.32 mM) were used for the clonogenic survival assay. After 24 hours of exposure, fresh media was replaced and after 10 to 12 days the colonies were fixed and stained with crystal violet. We performed the assay three separate times, and we conducted each experiment in triplicate. Colonies containing at least 50 cells were counted. The investigators (SM, TS, XY) were not blinded to the treatment groups.

Assessment of Apoptosis

Evaluation of Cell Morphology

The osteosarcoma cells were exposed to bupivacaine (1.08 mM or 2.16 mM) and lidocaine (5.33 mM or 10.66 mM) for 24 hours, 48 hours, and 72 hours. We performed this portion of the experiment three times. Phase-contrast photomicrographs were taken after each treatment to evaluate any morphological changes in the tumor cells. Cell shrinkage and detachment from the plates were considered markers of apoptosis. The investigators (SM, AJ, RD) were not blinded to the treatment groups.

Apoptotic Analysis

To evaluate the apoptotic cells, we seeded 1x105 of the osteosarcoma cells in 6-well plates. Once the confluence reached 80%, the treated cells were exposed to various concentrations of bupivacaine (1.08 mM, 2.16 mM, and 4.33 mM) and lidocaine (5.33 mM, 10.66 mM, and 21.32 mM). Control groups were treated with PBS with a matching pH to the medications. In some experiments where the cells were exposed to bupivacaine and lidocaine at the ED50, we also treated the cells with Z-VAD-fmk (20 µM) (Cell Signaling Technologies, Beverly, MA, USA) for 1 hour before exposing them to the medications. After 24 to 72 hours, we harvested and washed cells that were both floating and attached. To determine the apoptotic rate, we evaluated apoptotic cells with an Annexin-V and propidium iodide (PI) apoptosis detection kit (BD Biosciences, San Jose, CA, USA). Flow cytometry with FITC-conjugated Annexin-V/PI double staining was used to assess the quantity of apoptotic cells. We used the FlowJo software (TreeStar, Ashland, Oregon, USA) and flow cytometry (MACSQuant, Miltenyi Biotec, Auburn, CA, USA) to analyze the samples. The measurements were then split into four quadrants. The apoptotic rate was then calculated as early apoptosis (Ann+/PI-) and late apoptosis (Ann+/PI+). We performed this experiment three separate times. The investigators (SM, TS, XY) were not blinded to the treatment group.

Evaluation of DNA Fragmentation

MNNG/HOS cells were cultured in 12-well plates for 24 hours. They were then exposed to various concentrations of bupivacaine (1.08 mM, 2.16 mM, and 4.33 mM) and lidocaine (5.33 mM, 10.66 mM, and 21.32 mM) for 48 hours. The UMR-108 cells were also plated in 12-well plates for 24 hours, and then exposed to bupivacaine (1.08 mM) and lidocaine (5.33 mM) for 24 hours, 48 hours, and 72 hours. In some experiments, both cell lines were treated with Z-VAD-fmk (20 µM) (Cell Signaling Technologies) for 1 hour before treatment started at the ED50. Once treatment was completed, the cells were trypsinized. Both floating and attached cells were harvested and washed with Dulbecco`s Phosphate Buffered Saline (Millipore Sigma). We used a DNA extraction kit (Quick-g DNA miniprep kit, Zymo Research, Irvine, CA, USA) to extract the genomic DNA per the manufacturer’s instructions. We used NanoVue (GE Healthcare, Pittsburgh, PA, USA) to measure the DNA from each group, and then we applied 0.4 μg of the DNA to 1.5% agarose gels that contained ethidium bromide. DNA obtained from UMR-108 cells where apoptosis was induced by H2O2 (0.4 mM) was used as a positive control in addition to a DNA molecular marker (1 kb DNA step ladder, Promega, Madison, WI, USA). We performed each experiment three times. We took photographs at the end of each experiment under UV illumination and examined the DNA fragmentation pattern. The investigators (SM, AJ) were not blinded to the treatment groups.

Mechanism of Apoptosis

Analysis by Western Blot

Both cell lines were plated in 6-well plates and left for 24 hours. We then started treatment with either bupivacaine (1.08 mM) or lidocaine (5.33 mM). After 24 hours, we harvested the cells, washed them, and then resuspended them in NP-40 lysis buffer. The whole cell lysates were then homogenized. We used a Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA) to determine the protein concentrations. Thirty micrograms from each sample were then loaded and separated through 12% sodium dodecyl sulfate polyacrylamide gels. The samples were then transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA), and the membranes were blocked with 5% BSA in phosphate-buffered saline with Tween® 20 detergent (PBST) (Millipore) buffer for 1 hour at room temperature. The membranes were then incubated with Bcl-2 (1:1000), Bax (1:1000), caspase-3 (1:1000), cleaved caspase-3 (cCaspase-3) (1:1000), Poly (ADP-ribose) polymerase-1 (PARP) (1:1000), cleaved PARP (cPARP) (1:1000), β-actin (1:10000) (Cell Signaling Technologies) and survivin (1:500) (Novus, Littleton, CO, USA) overnight at 4° C. The membranes were then washed with PBST, and subsequently incubated for 1 hour at room temperature with horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:10000) (Cell Signaling Technologies). We used an enhanced chemiluminescence kit (Thermo Scientific) to visualize the bands as per the manufacturer's instructions. Corresponding values of β-actin densitometry were used to normalize the data. We performed this experiment three separate times. The investigators (SM, XY) were not blinded to the treatment groups.

Determination of ROS

To measure intracellular ROS levels, we used the fluorescent dye H2DCFDA (2’,7’- dichlorodihydrofluoresceindiacetate) (Molecular Probes, Invitrogen, Eugene, Oregon, USA). We seeded 1x105 cells in 6-well plates until confluence reached 80%. The cells were then exposed to bupivacaine (2.16 mM) and lidocaine (10.66 mM) for 24 hours. Cells were then stained with H2DCFDA (20 µM, 30 minutes), harvested by trypsinization and analyzed in PBS buffer by both flow cytometry (MACSQuant, Miltenyi Biotec) and with the FlowJo software (TreeStar). For positive controls, cells were treated with H2O2 (200 µM, 1 hour). The investigators (SM, AJ) were not blinded to the treatment groups.

Statistical Analysis

As noted above, we performed each assay as an independent experiment at least three times. The data was analyzed with ANOVA using Prism 5.01 software (GraphPad Software, San Diego, CA, USA). A t-test was used for comparisons between two groups and ANOVA with Bonferroni was used for comparisons between more than two groups. A threshold value of p < 0.05 was considered statistically significant.

Results

Incubation of Osteosarcoma Cells with Bupivacaine and Lidocaine Results in Cell Death

Both bupivacaine and lidocaine were cytotoxic to both osteosarcoma cell lines in a dose- and time-dependent manner (Fig. 1A-B). Exposure to 1.08 mM of bupivacaine for 24 hours reduced the viability of UMR-108 cells compared with controls (6 ± 0.75% [95% CI 2.9 to 9.11; p = 0.01]). Viability continued to decrease with increasing concentrations of bupivacaine, with a decrease of 89.67 ± 1.5% (95% CI 82.2 to 95.5; p < 0.001) at 2.16 mM. At the same doses and exposure times, MNNG/HOS cell viability decreased by 12.5 ± 4.87% (95% CI 0.04 to 25.04; p = 0.05) and 62.26 ± 3.42% (95% CI 53.44 to 71.06; p < 0.001). When exposed to 1.08 mM of bupivacaine for 72 hours, UMR-108 and MNNG/HOS viability further decreased by 44.67 ± 2.4% (95% CI 37.99 to 51.34; p < 0.001) and 39 ± 3.71% (95% CI 29.44 to 48.56; p < 0.001). After 24 hours of exposure to lidocaine at 5.33 mM, UMR-108 viability decreased by 25.33 ± 8.3% (95% CI 2.1 to 48.49; p = 0.03) and MNNG/HOS viability decreased by 39.33 ± 3.19% (95% CI 30.46 to 47.21; p < 0.001). With exposure to 10.66 mM of lidocaine, UMR-108 and MNNG/HOS cell viability decreased by 90.67 ± 0.66% (95% CI 88.82 to 92.52; p < 0.001) and 81.6 ± 0.47% (95% CI 79.69 to 82.31; p < 0.001), respectively. When the exposure time was increased to 72 hours with exposure to 5.33 mM of lidocaine, UMR-108 and MNNG/HOS viability decreased by 62 ± 6.18% (95% CI 44.84 to 79.16; p < 0.001) and 64.33 ± 2.61% (95% CI 57.05 to 71.62; p < 0.001), respectively. The cytotoxic effects of the medications were further demonstrated in the inhibition of colony formation ability of the tumor cells (Fig. 2A-B). When exposed to 1.08 mM of bupivacaine for 24 hours, the colony formation ability of UMR-108 and MNNG/HOS decreased to 42.33 ± 3.08% (95% CI 33.95 to 50.71; p < 0.001) and 35.17 ± 1.3% (95% CI 31.55 to 38.79; p < 0.001), respectively. When exposed to 4.33 mM, colony formation decreased to 1 ± 0.57% in UMR-108 cells and 0.16 ± 0.16% in MNNG/HOS cells. Exposure to lidocaine also decreased colony formation with increasing concentrations (Fig. 2A-B).

Fig. 1.

Fig. 1

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A-B This figure shows that bupivacaine and lidocaine reduced the viability of osteosarcoma tumor cells. (A) UMR-108 and (B) MNNG/HOS osteosarcoma cells were treated with bupivacaine and lidocaine. An microculture tetrazolium assay was performed at 24 hours, 48 hours, and 72 hours. The bars represent the mean values of three independent experiments with standard error shown as error bars. Each experiment was done in triplicate and compared with a pH-adjusted control. ap < 0.05, bp < 0.01, cp < 0.001.

Fig. 2.

Fig. 2

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A-B This figure demonstrats that bupivacaine and lidocaine inhibited the colony formation ability of osteosarcoma cells. (A) UMR-108 and (B) MNNG/HOS cells were exposed to bupivacaine and lidocaine at various concentrations for 24 hours. The colonies were stained with crystal violet after 12 days to 14 days, and the number of colonies was counted. The bars represent the mean values of three independent experiments with standard error as error bars. The data were derived from three independent experiments and compared with a pH-adjusted control. ap < 0.01.

Bupivacaine and Lidocaine Induced Apoptosis in Osteosarcoma Cells in an In Vitro Setting

Both medications caused morphologic changes in each cell line based on phase-contrast photomicrographs consistent with apoptosis. These changes were characterized by cellular shrinkage, severe blebbing, loss of shape by becoming round and detaching, floating, and eventually death. The morphologic changes corresponded with loss of adherence to the cell culture dishes (Fig. 3A-B). Quantitative analysis of the flow cytometry showed that the total number apoptotic cells (Annexin-V+) rose with increasing doses of each medication (Fig. 4A-B). Bupivacaine at concentrations of 1.08 mM increased the percentage of apoptotic cells by 21.3 ± 2.82% (95% CI 16.25 to 26.48; p < 0.001) for UMR-108 cells and by 21.23 ± 3.23% (95% CI 12.27 to 30.2; p = 0.003) for MNNG/HOS cells. After exposure to 2.16 mM, the percentage increased by 71.29 ± 2.13% (95% CI 66.44 to 76.57; p < 0.001) for UMR-108 cells and 60.23 ± 5.6% (95% CI 44.42 to 76.04; p < 0.001) for MNNG/HOS cells. Lidocaine increased the percentage of apoptotic UMR-108 and MNNG/HOS cells by 25.15 ± 4.38% (95% CI 12.99 to 37.3; p = 0.005) and 9.11 ± 1.74% (95% CI 4.35 to 13.87; p = 0.006) at 5.33 mM, respectively, and by 73.36 ± 4.16% (95% CI 63.17 to 83.55; p < 0.001) and 62.55 ± 4.2% (95% CI 50.88 to 74.21; p < 0.001) at 10.66 mM, respectively. The rate of apoptosis continued to increase after 72 hours of treatment indicating that the medications also induced apoptosis in a time-dependent manner (Fig. 4C). To further confirm that the cell death was caused by apoptosis induction, the integrity of the DNA was evaluated by gel electrophoresis. Both medications increased the fluorescence intensity of ethidium bromide, which indicated that the degree of DNA fragmentation occurred in a time- and dose-dependent manner. After 48 hours of exposure to 1.08 mM of bupivacaine, DNA fragmentation increased by 2.03 ± 0.12-fold (95% CI 1.7 to 2.36; p < 0.001) in UMR-108 cells and by 0.73 ± 0.07-fold (95% CI 0.52 to 0.94; p < 0.001) in MNNG/HOS cells (Fig. 5A-B). After 48 hours of exposure to 5.33 mM of lidocaine, UMR-108 fragmentation increased by 3.54 ± 0.49-fold (95% CI 2.09 to 4.81; p = 0.002). After 48 hours of exposure to 10.66 mM of lidocaine, MNNG/HOS fragmentation increased by 0.93 ± 0.14-fold (95% CI 0.51 to 1.34; p = 0.003). There was no change in the degree of DNA fragmentation in the control groups between 24 and 72 hours (data not shown).

Fig. 3.

Fig. 3

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A-B This figure illustrates that bupivacaine and lidocaine induced abnormal morphologic changes in osteosarcoma tumor cells. (A) UMR-108 cells after 24 hours, 48 hours, and 72 hours of treatment and (B) MNNG/HOS cells 48 hours after treatment with various concentrations of bupivacaine and lidocaine. Morphologic changes in tumor cells were examined by phase-contrast photomicrograph and representative images of each treatment condition are shown. The data represent three independent experiments. Scale bars represent 50 µm.

Fig. 4.

Fig. 4

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A-C This figure shows that bupivacaine and lidocaine induced apoptosis in osteosarcoma tumor cells in a dose- and time-dependent manner. (A) UMR-108 cells and (B) MNNG/HOS cells were exposed to various concentrations of bupivacaine and lidocaine for 48 hours and (C) UMR-108 cells were exposed to 1.08 mM of bupivacaine and 5.3 mM of lidocaine for 24 and 72 hours. Both floating and attached cells were harvested and washed. Flow cytometry with FITC-conjugated Annexin-V (AnxV-FITC)/propidium iodide (PI) double staining was used to assess the number of apoptotic cells. Associated plot graphs are shown in (A) and (B) where the left upper quadrant represents dead cells, lower left is living cells, upper right is late apoptosis, and lower right is early apoptosis. The data were derived from three independent experiments compared to pH-adjusted controls and are presented as the mean ± standard error of the mean. ap < 0.01 and bp < 0.001.

Fig. 5.

Fig. 5

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A-B This figure shows that bupivacaine and lidocaine induced DNA degradation in a dose- and time-dependent manner. (A) UMR-108 cells were exposed to bupivacaine (1.08 mM) and lidocaine (5.33 mM) for 24 hours, 48 hours, and 72 hours. (B) MNNG/HOS cells were exposed to various concentrations of bupivacaine and lidocaine and after 48 hours both floating and attached cells were harvested. The genomic DNA was applied to 1.5% agarose gels containing μg/mL ethidium bromide. A DNA step ladder (1 kb) and the DNA from apoptotic UMR-108 cells induced by H2O2 (0.4 mM) were used as positive control. The DNA fragmentation pattern was examined in photographs taken under UV illumination. The data were derived from three independent experiments and compared to pH-adjusted controls. The data is presented as the mean ± standard error of the mean with the corresponding DNA fragmentation pattern on the right. ap < 0.01 and bp < 0.001.

Apoptosis Occurred by the Intrinsic Pathway in Both Cell Lines but ROS Production Differed

The densitometry analysis that was performed showed that 1.08 mM of bupivacaine and 5.33 mM of lidocaine had an effect on the expression level of anti-apoptotic proteins when normalized to β-actin densitometry (Fig. 6A-B). For both cell lines and medications, Bcl-2, survivin and pro-caspase-3 were reduced. An increase in cPARP, Bax and cCaspase-3 was noted in both cell lines and with both medications. The level of cCaspase-3 was higher in MNNG/HOS cells treated with bupivacaine compared to UMR-108 cells (Fig. 6A-B). To further investigate the role of caspase, both cells were exposed to Z-VAD-fmk, a broad caspase family inhibitor before treatment with bupivacaine and lidocaine. Addition of Z-VAD-fmk had no effect on the apoptosis of treated UMR-108 cells (Fig. 7A) or to the control group (data not shown). A protective effect of Z-VAD-fmk was found against bupivacaine and lidocaine in the MNNG/HOS cell line (Fig. 7B). The percentage of apoptotic cells exposed to bupivacaine decreased by 24.2 ± 2.27 (95% CI 17.89 to 30.51; p < 0.001) and by 36.64 ± 2.44 (95% CI 29.86 to 43.3; p < 0.001) with lidocaine. In addition to this, pre-treatment with Z-VAD-fmk reduced the DNA fragmentation of MNNG/HOS cells after treatment with 1.08 mM of bupivacaine and 5.33 mM of lidocaine (Fig. 7B). Fragmentation decreased by 25.23 ± 2.6% (95% CI 17.9 to 32.57; p = 0.007) for bupivacaine and by 17.82 ± 3.23% (95% CI 8.82 to 26.61; p = 0.053) for lidocaine. This effect was not observed in the UMR-108 cells where the difference for bupivacaine was 3.46 ±1.64% (95% CI -1.08 to 8.02; p = 0.10) and lidocaine was 3.83 ± 2.26% (95% CI -2.44 to 10.11; p = 0.16). Next, we measured ROS levels, which demonstrated an increase in ROS levels in the UMR-108 cells compared with a decrease in the MNNG/HOS cells (Fig. 8A). To confirm this effect, the UMR-108 cells were pre-treated with Trolox (Millipore), an ROS scavenger, for 1 hour, and then exposed to bupivacaine and lidocaine (Fig. 8B). The ROS levels were attenuated by Trolox in the UMR-108 cells after administration of both 1.08 mM of bupivacaine (1.24 ± 0.19-fold [95% CI 0.71 to 1.76; p = 0.003]) and 5.33 mM of lidocaine (0.94 ± 0.11-fold [95% CI 0.6 to 1.2; p = 0.001]).

Fig. 6.

Fig. 6

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A-B This figure shows the expression of apoptotic related proteins in osteosarcoma tumor cells after being exposed to bupivacaine and lidocaine. (A) UMR-108 cells and (B) MNNG/HOS cells were exposed to bupivacaine (1.08 mM) and lidocaine (5.33 mM). After 24 hours, the total protein was isolated. Equal amounts of protein from each sample were loaded and separated through 12% sodium dodecyl sulfate polyacrylamide gels and then transferred to polyvinylidene difluoride membranes. We used the following antibodies: Bcl-2, Bax, survivin, caspase-3, cleaved caspase-3 (cCaspase-3), PARP, cleaved PARP (cPARP), and β-actin. The bands were visualized by enhanced chemiluminescence kit instructions. Data were normalized to corresponding values of β-actin densitometry. The data were derived from three independent experiments and compared to pH-adjusted controls. Photographs of the associated Western blot are shown on the left. ap < 0.05, bp < 0.01, and cp < 0.001.

Fig. 7.

Fig. 7

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A-B This figure shows the effect of the pan-caspase inhibitor, Z-VAD-fmk, on bupivacaine- and lidocaine-induced apoptosis and DNA fragmentation. (A) UMR-108 cells and (B) MNNG/HOS cells were exposed to bupivacaine (1.08 mM) and lidocaine (5.33 mM) for 24 hours. Both floating and attached cells were harvested and washed. Flow cytometry with FITC-conjugated Annexin-V (AnxV-FITC)/propidium iodide (PI) double staining was used to assess the number of apoptotic cells. Associated plot graphs are shown below the bar graphs where the left upper quadrant represents dead cells, lower left is living cells, upper right is late apoptosis and lower right is early apoptosis. The data were derived from three independent experiments compared with pH-adjusted controls and are presented as the mean ± standard error of the mean. Genomic DNA was applied to 1.5% agarose gels containing μg/mL ethidium bromide. A DNA step ladder (1 kb) and the DNA from apoptotic UMR-108 cells induced by H2O2 (0.4 mM) were used as a positive control. The DNA fragmentation pattern was examined in photographs taken under UV illumination and shown at the bottom. The data were derived from three independent experiments. ap < 0.01.

Fig. 8.

Fig. 8

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A-B This figure shows bupivacaine- and lidocaine-mediated ROS production. (A) UMR-108 cells and MNNG/HOS cells were exposed to bupivacaine (1.08 mM) and lidocaine (5.33 mM) for 24 hours. ROS levels were determined by H2DCFDA staining and flow cytometry analysis. Bupivacaine and lidocaine treatment were compared with pH-adjusted controls (untreated). H2O2-treated cells (200 mM; 1 hour) served as positive controls. A shift to higher fluorescence intensity corresponds to increased ROS levels. (B) UMR-108 cells were pretreated for 1 hour with 1 mM Trolox before bupivacaine and lidocaine were administered for 24 hours. Bupivacaine- and lidocaine-treated cells were compared with pH-adjusted controls (untreated). The data were derived from three independent experiments and are presented as the mean ± standard error of the mean. ap < 0.01.

Discussion

Previous studies have shown that local anesthetics are toxic to multiple cancer types [8, 9, 30, 44, 47, 48]. Local anesthetics are commonly used medications and may be used during biopsies of sarcomas and as an adjunct for pain control with resection. The common use of these medications stresses the importance of knowing their effects on cancer cells. Although prior studies have demonstrated that local anesthetics cause apoptosis in several types of cancers, the literature is limited in regard to the effect of these medications on osteosarcoma, and treatment options for osteosarcoma have not improved much since chemotherapy was introduced [8, 9, 30, 44, 47, 48]. The results of this study demonstrated that both bupivacaine and lidocaine increased apoptosis of both rat and human osteosarcoma cells in a time- and dose-dependent manner, decreased the viability of the cells and decreased colony formation. The intrinsic pathway is presumed to be involved, although the mechanisms were found to differ between the two cell lines. This underscores the importance of studying multiple cell lines in regard to treatment modalities and provides a basis for further in vivo studies.

Limitations

Our work is not without limitation. These experiments were only performed in an in vitro setting, and therefore future studies are needed to replicate the results in an in vivo model. The doses used in an in vitro setting may not correlate with the doses required in the in vivo setting due to multiple factors. In this study, the ED50 for both cell lines was 1.4 nmM for bupivacaine and 7.0 mM for lidocaine. Previous in vitro studies investigating the effects of LAs local anesthetics on cancers have used wide ranges of both bupivacaine and lidocaine (1-30 mM) [50]. It is uncertain if these doses would be applicable in certain clinical settings or disease contexts. The concentrations of 0.5% bupivacaine and 2% lidocaine that are commonly used in the clinical setting are 17.34 mM and 85.35 mM, respectively. The concentrations used in this study that resulted in reduced cell viability were well below these concentrations, and therefore the results would hopefully be able to be translated to the in vivo setting. This is particularly important as a lower dosage is important to prevent complications that can be associated with these medications, including seizure and cardiac arrest [20]. In addition to this, the cell lines were incubated with the medications for at least 24 hours. This does not account for the half-life and distribution of the medications that will occur in vivo. Whether a single dose of the medication can affect contamination of a biopsy tract should be considered for further study both with an animal model and in the clinical setting. Contamination of the biopsy tract could be evaluated after definitive resection to see if there is a difference between patients who received a local anesthetic during their biopsy and those who did not. Use of a continuous infusion pain pump that could bathe the tumor bed after surgery as well as the use of liposomal bupivacaine are also options for further study. Another consideration is that cell lines were used. Even though cell lines provide for a control that can be replicated in other studies, osteosarcoma is heterogeneous and has multiple subtypes. We have recently shown that bupivacaine had a similar effect on patient-derived osteosarcoma cells in the in vitro setting; however, this still does not take the multiple subtypes or patient factors into account [52]. The fact that bupivacaine and lidocaine were cytotoxic in different cell lines and due to differing mechanisms provides some promise that the effect may be seen in multiple subtypes. Further in vitro and in vivo studies involving multiple cell lines as well as patient-derived cells should be performed to confirm these findings.

Incubation of Osteosarcoma Cells with Bupivacaine and Lidocaine Results in Cell Death

Both bupivacaine and lidocaine were cytotoxic to both osteosarcoma cell lines, with prolonged incubation, and at higher concentrations. Furthermore, the effects of bupivacaine and lidocaine on tumor cell growth were determined by clonogenic assay and the reduced ability of the cells to form colonies was also observed. These findings agree with other studies that have evaluated the effects of these medications on other malignancies [8, 9, 30, 44, 47, 48]. The findings in this study validate that further in vivo testing should be performed to determine if these results can be translated to the clinical setting.

Bupivacaine and Lidocaine Induced Apoptosis in Osteosarcoma Cells in an In Vitro Setting

We found that cell death was consistent with apoptosis and was based on cell morphology, the total number of apoptotic cells, and DNA fragmentation. There was no cell cycle arrest in cells treated with bupivacaine and lidocaine, but we observed a dose-dependent increase in the sub-G1 fraction following treatment with medications (data not shown). Moreover, apoptotic factors in the mitochondrial intermembrane space are released into the cytoplasm and transfer to the nucleus and bind to DNA, causing nuclear condensation and DNA fragmentation. This is consistent with the increased fluorescence intensity of ethidium bromide seen in our results and indicate that the degree of DNA fragmentation occurred in a time- and dose-dependent manner. Previous studies have shown that bupivacaine and lidocaine cause apoptosis in other forms of cancer [9, 48, 51]. The results of this study support this mechanism of action.

Apoptosis Occurred by the Intrinsic Pathway in Both Cell Lines but ROS Production Differed

We found that apoptosis occurred by the intrinsic pathway, and that inhibitors of apoptosis were downregulated as well. In this study, Bcl-2, survivin and pro-caspase-3 were reduced and cPARP, Bax and cCaspase-3 were increased. The intrinsic, or mitochondrial-mediated pathway of apoptosis is mediated by the release of anti- and pro-apoptotic proteins and regulates apoptotic cascades [21]. Bcl-2, an anti-apoptotic protein, interacts with mitochondrial proteins, which prevents the release of apoptogenic factors, and consequently, protects cell membrane integrity. Our findings of a decrease in the level of Bcl-2 after treatment support the findings that the intrinsic pathway is involved. Kalimuthu and Se-Kwon [24] reported that the intrinsic apoptotic pathway increases the Bax/Bcl-2 ratio leading to the activation of caspase-3. Our experiments confirmed that bupivacaine and lidocaine decreased the expression of Bcl-2 and increased the expression of Bax, leading to a higher ratio between anti-apoptotic and pro-apoptotic proteins. The change in Bcl-2/Bax ratio facilitates the opening of the mitochondrial permeability transition pore and the release of cytochrome c, apoptosis inducing factor, and sustains caspase in the active state that cleaves PARP into inactive fragments. The results of the present study agree with these findings with an increase in expression of cCaspase-3 and cPARP. Survivin can antagonize the apoptotic cascade by targeting the terminal effector caspase-3, and overexpression of survivin is associated with inhibition of apoptosis through the intrinsic or extrinsic apoptotic pathways [1, 28]. As survivin was reduced, involvement of both the intrinsic and extrinsic pathways are possible; however, how survivin inhibits apoptotic pathways is controversial and further studies would be needed to clarify if the extrinsic pathway is involved [49]. We demonstrated that pretreatment with Z-VAD-fmk, a pan-caspase inhibitor, prevented apoptosis and DNA fragmentation in the MNNG/HOS cells but not in the UMR-108 cells. This finding is interesting since we observed increased cCaspase-3 in both cell lines. However, several studies have shown that Z-VAD-fmk does not prevent all types of caspase-dependent apoptosis, and it may not inhibit all caspases to the same degree [43]. In addition, the effect and cross reactivity of this inhibitor in a human cell line and a rat cell line might be different. Prior studies have shown that formation of ROS may be a potential mechanism for bupivacaine and lidocaine cytotoxicity [7, 29, 36, 40, 48]. Increased cellular levels of ROS cause Bax/Bak channels on the mitochondrial membrane to open which release apoptosis-promoting factors into the cytosol and lead to activation of apoptosis and direct damage to nuclear and mitochondrial DNA [26, 42, 51]. The caspase-8/Bid/cytochrome c axis plays a key role in death receptor-mediation in ROS generation and apoptosis [25]. It has been reported that bupivacaine can induce activation of caspase-9 and caspase-8, and that caspase-8 activation can occur independent of the death receptor ligand, leading to activation of caspase-3, which affects ROS generation [45, 51]. Therefore, the increase in ROS production in the UMR-108 cells demonstrates that bupivacaine and lidocaine might cause activation of different initiator caspases in these cells, but further studies would be needed to clarify this (Fig. 9). This heterogeneity is likely due to the difference in the cell lines and is consistent with differing apoptotic pathways observed for both bupivacaine and lidocaine in other malignancies [9, 48, 51].

Fig. 9.

Fig. 9

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The proposed molecular mechanism for the intrinsic pathway in bupivacaine and lidocaine induced tumor cell apoptosis is demonstrated. Addition of a pan-caspase inhibitor prevented apoptosis and DNA fragmentation in the MNNG/HOS cells but not in the UMR-108 cells, while reactive oxygen species (ROS) were increased in UMR-108 cells but decreased in MNNG/HOS cells. These findings are likely due to the medications activating different initiator caspases.

Conclusion

We found that bupivacaine and lidocaine were cytotoxic to both human and rat osteosarcoma cell lines and caused apoptosis in a time- and dose-dependent manner. Apoptosis seems to occur via the intrinsic pathway in both cell lines; however, in contrast to MNNG/HOS cells, the UMR-108 cell line had increased ROS levels with both bupivacaine and lidocaine administration, and apoptosis was possibly activated through a different initiator caspase. As local anesthetics are currently administered in several ways, there is potential for multiple uses as a chemotherapeutic agent pending validation in vivo. Future studies could evaluate local infiltration of these medications during biopsies, use of a continuous infusion pump postoperatively, and whether use with a Bier block or isolated limb perfusion is feasible. The results of this study offer initial optimism that these drugs can be used in an adjuvant or neoadjuvant fashion in osteosarcoma treatment and validate that further studies should be performed to determine if these results can be translated to the clinical setting.

Acknowledgments

We thank Elisabeth Clarke for her assistance in coordination of this study.

Footnotes

The institution of one or more of the authors (LMZ, SM) has received, during the study period, funding from the Loma Linda University School of Medicine Dean’s Office and the Department of Orthopedic Surgery for the Grants to Promote Collaborative and Translational Research (GCAT 2016, #681163).

Each author certifies that neither he or she, nor any member of his or her immediate family, has funding or commercial associations (consultancies, stock ownership, equity interest, patent/licensing arrangements, etc.) that might pose a conflict of interest in connection with the submitted article.

All ICMJE Conflict of Interest Forms for authors and Clinical Orthopaedics and Related Research® editors and board members are on file with the publication and can be viewed on request.

Clinical Orthopaedics and Related Research® neither advocates nor endorses the use of any treatment, drug, or device. Readers are encouraged to always seek additional information, including FDA approval status, of any drug or device before clinical use.

Each author certifies that his or her institution approved the reporting of this investigation and that all investigations were conducted in conformity with ethical principles of research.

This experiment was conducted at the Biospecimen Laboratory, Loma Linda University Cancer Center, Loma Linda University School of Medicine, Loma Linda, CA, USA.

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