Apoptosis, Proliferation, and Autophagy Are Involved in Local Anesthetic-Induced Cytotoxicity of Human Breast Cancer Cells

Abstract

Breast cancer accounts for almost one quarter of all female cancers worldwide, and more than 90% of those who are diagnosed with breast cancer undergo mastectomy or breast conservation surgery. Local anesthetics effectively inhibit the invasion of cancer cells at concentrations that are used in surgical procedures. The limited treatment options for triple-negative breast cancer (TNBC) demonstrate unmet clinical needs. In this study, four local anesthetics, lidocaine, levobupivacaine, bupivacaine, and ropivacaine, were applied to two breast tumor cell types, TNBC MDA-MB-231 cells and triple-positive breast cancer BT-474 cells. In addition to the induction of apoptosis and the suppression of the cellular proliferation rate, the four local anesthetics decreased the levels of reactive oxygen species and increased the autophagy elongation indicator in both cell types. Our combination index analysis with doxorubicin showed that ropivacaine had a synergistic effect on the two cell types, and lidocaine had a synergistic effect only in MDA-MB-231 cells; the others had no synergistic effects on doxorubicin. Lidocaine contributed significantly to the formation of autophagolysosomes in a dose-dependent manner in MDA-MB-231 cells but not in BT-474 cells. Our study demonstrated that the four local anesthetics can reduce tumor growth and proliferation and promote apoptosis and autophagy.

Keywords: apoptosis; autophagy; combination index; proliferation; triple-negative breast cancer.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

A diagram of the local anesthetics: lidocaine, levobupivacaine, bupivacaine, and ropivacaine. Compound ID (CID) is the identifier from a database of chemical molecules and their activities of biological assays in PubChem.

Figure 2

The effects of the local anesthetics on cell viability in MDA-MB-231 and BT474 cells. (A,B) MDA-MB-231 and BT-474 (8 × 104) cells that were treated with the indicated concentrations of lidocaine (0.625, 1.25, 2.5, 5, and 10 mM), levobupivacaine (0.125, 0.25, 0.5, 1, and 2 mM), bupivacaine (0.25, 0.5, 1, 2, and 4 mM), and ropivacaine (0.25, 0.5, 1, 2, and 4 mM) for 24 h. Control cells were cultured under identical conditions. Metabolic activity was measured using an MTT colorimetric assay. Data are presented as a percentage of the control. Bars depict the mean ± SD. # p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 3

The effects of the local anesthetics on cell cycle profiles in MDA-MB-231 and BT-474 cells. (A) MDA-MB-231 and (B) BT-474 (3 × 105) cells were treated for 24 h with the indicated concentrations of the local anesthetics. After treatment, the cells were stained with 7-Aminoactinomycin D (7-AAD), which is a dye that can bind to total DNA, coupled with immunofluorescent BrdU staining. Four cell subpopulations (i.e., subG1, G1, S, and G2/M phases) were distinguished based on bivariate BrdU incorporation and DNA content distribution (7-AAD staining): cells in the sub-G1 phase had a lower DNA content (hypodiploid); cells in the G1 and G2/M phases were DNA diploid and tetraploid, respectively; thus, the G2/M phase fluoresced twice as brightly and there was no BrdU incorporation; cells in the S phase had the highest and relatively constant BrdU levels. The 7-AAD staining intensities defined the cell cycle position. The cell cycle profiles were measured using flow cytometry analysis. The results are representative of two independent experiments. Bars depict the mean ± SD. # p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 4

The effects of the local anesthetics on cell DNA synthetic activity in MDA-MB231 and BT-474 cells. (A) MDA-MB-231 and (B) BT-474 (3 × 105) cells were treated for 24 h with the indicated concentrations of the local anesthetics. After treatment, the cells were stained with BrdU, incorporated into newly synthesized DNA, progressed through the S phase (DNA synthesis), and then measured using flow cytometry analysis relative to the BrdU incorporation level. The results are representative of two independent experiments. Bars depict the mean ± SD. # p > 0.05; * p < 0.05; ** p < 0.01; *** p <0.001.

Figure 5

The effects of the local anesthetics on cell ROS status in MDA-MB-231 and BT474 cells. (A) MDA-MB-231 and (B) BT-474 (3 × 105) cells were treated for 24 h with the indicated concentrations of the local anesthetics. The cell ROS status was determined using 20 µM of DCFH-DA and measured using flow cytometry. The cell volume gating strategy involved FSC-H and SSC-H. The median DCFH-DA fluorescence intensity of the vehicle was used as the starting point for M1 gating. The results are representative of two independent experiments. Bars depict the mean ± SD.

Figure 6

The effects of the local anesthetics on autophagosome-related protein expression in MDA-MB-231 and BT-474 cells. (A) MDA-MB-231 and (B) BT-474 (3 × 105) cells were treated with the indicated concentrations of lidocaine (1.25, 2.5, and 5 mM), levobupivacaine (0.5, 1, and 2 mM), bupivacaine (0.5, 1, and 2 mM), and ropivacaine (0.5, 1, and 2 mM) for 24 h; (C) MDA-MB-231 and (D) BT-474 cells were pre-treated for 2 h with 0, 0.1, 0.5, 1, 2, and 5 mM of 3-MA and then combined with 1.25 mM of lidocaine for 24 h. The cell lysates were subjected to Western blotting analysis using antibodies against the indicated proteins. ACTN was used as the protein loading control.

Figure 7

The effects of the local anesthetics on apoptosis-related protein expression in MDA-MB-231 and BT-474 cells. (A) MDA-MB-231 and (C) BT-474 (3 × 105) cells were treated with the indicated concentrations of lidocaine (1.25, 2.5, and 5 mM), levobupivacaine (0.5, 1, and 2 mM), bupivacaine (0.5 and 1 mM), and ropivacaine (0.5, 1, and 2 mM) for 24 h. The cell lysates were subjected to Western blotting analysis using antibodies against the indicated proteins. ACTN was used as the protein loading control. SE: shorter exposure; LE: longer exposure. (B,D) The protein bands (A,C) were quantified through pixel density scanning and evaluated using ImageJ, version 1.44a (http://imagej.nih.gov/ij/) (accessed on 4 December 2022). The ratios of protein/ACTN, including cPARP, cCaspase 3, and BCL-2) were plotted. The molecular weight markers are labeled with kDa.

Figure 8

The combination indices of the local anesthetics with DXR in MDA-MB-231 and BT-474 cells. (A–H) The combination indices of DXR plus lidocaine, levobupivacaine, bupivacaine, and ropivacaine in (A–D) 1.5 × 104 MDA-MB-231 cells and (E–H) 2.5 × 104 BT-474 cells; (A–D) MDA-MB-231 cells were treated with various DXR doses (0, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, and 1 µM) combined with various lidocaine doses (0, 0.156, 0.3125, 0.625, 1.25, 2.5, 5, 10, 20, and 40 mM), levobupivacaine doses (0, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, and 8 mM), bupivacaine doses (0, 0.0156, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2, and 4 mM), and ropivacaine doses (0, 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8, and 10 mM); (E,F, G, H) BT474 cells were treated with various DXR doses (0, 0.156, 0.3125, 0.625, 1.25, 2.5, 5, and 10 µM) combined with various lidocaine doses (0, 0.156, 0.3125, 0.625, 1.25, 2.5, 5, 10, 20, and 40 mM), levobupivacaine doses (0, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, and 8 mM), bupivacaine doses (0, 0.0156, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2, and 4 mM), and ropivacaine doses (0, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, and 8 mM) for 24 h. Metabolic activity was measured using the MTT method. The isobolograms (ED50) of lidocaine, levobupivacaine, bupivacaine, and ropivacaine were calculated using CalcuSyn software.

Figure 9

The effects of lidocaine on autophagy in MDA-MB-231 and BT-474 cells. Acidic vesicular organelles were detected and quantified using acridine orange staining and measured using flow cytometry analysis; (A) MDA-MB-231 and (B) BT-474 (3 × 105) cells were treated with the indicated concentrations of lidocaine (1.25, 2.5, and 5 mM) for 24 h. Acridine orange (1 µg/mL) staining was used to identify autophagic cells via FACS. The intensity of the red fluorescence (y-axis, FL3-H) was proportional to the degree of acidity and the volume of acidic vesicular organelles, including autophagic vacuoles. The values refer to the percentages of cells with a significant proportion of acidic vesicular organelles. The results are representative of two independent experiments. Bars depict the mean ± SD. # p > 0.05; ** p < 0.01; *** p < 0.001.

Figure 10

The detection of lidocaine-induced acidic vesicular organelles using acridine orange staining. MDA-MB-231 (3 × 105) cells were treated with the indicated concentration of lidocaine for 48 h before being stained with 1 µg/mL of acridine orange. The formation of acidic vesicular organelles was examined via fluorescence microscopy. Acridine orange is a weak base that accumulates in acidic spaces and results in bright red fluorescence (punctuation) in the cytoplasm, which can be detected by fluorescence microscopy. Scale bar = 100 µm.

Figure 11

The effects of lidocaine on cell signaling regulation in MDA-MB-231 and BT-474 cells. (A) MDA-MB-231 and (B) BT-474 (3 × 105) cells were treated with 2.5 mM of lidocaine for the indicated time points. The cell lysates were subjected to Western blotting analysis using antibodies against the indicated proteins. ACTN was used as the protein loading control. (C) The protein bands (A,B) were quantified through pixel density scanning and evaluated using ImageJ, version 1.44a (http://imagej.nih.gov/ij/) (accessed on 1 November 2022). The ratios of protein/ACTN and pERK/ERK were plotted.