Effects of Antibacterial Agents on Cancerous Cell Proliferation

Abstract

Myelosuppression, a side effect of anticancer drugs, makes people more susceptible to infectious diseases by compromising the immune system. When a cancer patient develops a contagious disease, treatment with an anticancer drug is suspended or postponed to treat the infectious disease. If there was a drug that suppresses the growth of cancer cells among antibacterial agents, it would be possible to treat both infectious diseases and cancer. Therefore, this study investigated the effect of antibacterial agents on cancer cell development. Vancomycin (VAN) had little effect on cell proliferation against the breast cancer cell, MCF-7, prostate cancer cell, PC-3, and gallbladder cancer cell, NOZ C-1. Alternatively, Teicoplanin (TEIC) and Daptomycin (DAP) promoted the growth of some cancer cells. In contrast, Linezolid (LZD) suppressed the proliferation of MCF-7, PC-3, and NOZ C-1 cells. Therefore, we found a drug that affects the growth of cancer cells among antibacterial agents. Next, when we examined the effects of the combined use of existing anticancer and antibacterial agents, we found VAN did not affect the growth suppression by anticancer agents. However, TEIC and DAP attenuated the growth suppression of anticancer agents. In contrast, LZD additively enhanced the growth suppression by Docetaxel in PC-3 cells. Furthermore, we showed that LZD inhibits cancer cell growth by mechanisms that involve phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway suppression. Therefore, LZD might simultaneously treat cancer and infectious diseases.

INTRODUCTION

Cancer is the leading cause of death in developed countries.¹⁾ Breast and prostate cancers are examples of cancers with high morbidity.²⁾ Docetaxel (DTX) is used as a therapeutic against breast cancer and prostate cancer.^3,4) Treatment with these anticancer agents has side effects, such as myelosuppression, and increases the risk of various infectious diseases. Additionally, gallbladder cancer is refractory cancer that is difficult to detect early and has a low five-year survival rate.^2,5) Gallbladder cancer is treated using Gemcitabine (GEM), but side effects (myelosuppression) can develop.⁶⁾ Therefore, in cancer patients, treatment with anticancer agents lowers immunity and increases the risk of contracting infectious diseases. Alternatively, antibacterial agents are used to treat infectious diseases. Still, these agents are temporarily suspended during treatment with anticancer agents, or the regimen is postponed. Therefore, when an infectious disease occurs due to a side effect of an anticancer agent, cancer treatment cannot be performed, which may affect the patient’s prognosis.

Recently, drug discovery by drug repositioning has been promoted to shorten drug development and control development costs. Drug repositioning is the use of existing medicines for diseases different from the currently approved treatments. Due to side effects and safety issues of existing drugs, we have undertaken studies to determine whether it might be feasible to receive approval of a therapeutic drug for a new disease at an early stage.

Previously, we examined changes in Vancomycin (VAN) clearance in cancer and non-cancer patients. Consequentially, VAN clearance increased in cancer patients.^7,8) Elevated VAN clearance reduces VAN concentration in the body, making it difficult to treat the infection because VAN concentration is required to treat the infection, cannot be reached. Similarly, the antibacterial agent’s clearance may change when an infectious disease occurs due to an anticancer drug’s side effect. Suppose the antibacterial agent used for treating infectious diseases can suppress the growth of cancer. In that case, the treatment of infectious diseases and cancer can be compatible, which is also interesting for drug repositioning.

Therefore, this study investigated the effects of antibacterial agents used in treating methicillin-resistant Staphylococcus aureus (MRSA) on the growth of cancer cells since we had previously studied VAN. VAN, Teicoplanin (TEIC), Daptomycin (DAP), and Linezolid (LZD) were selected as anti-MRSA agents. We also examined the effects of combining the use of the antibacterial agent and DTX, which is used clinically to treat breast and prostate cancers together with the antibacterial agent and GEM, which is used clinically to treat gallbladder cancer.

MATERIALS AND METHODS

Chemicals

VAN, DAP, LZD, GEM, and DTX were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). TEIC was purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). VAN was dissolved in phosphate-buffered saline (PBS) to prepare a 50-mg/mL stock solution. TEIC was dissolved in PBS to prepare a 40-mg/mL stock solution. DAP was dissolved in PBS to prepare a 50-mg/mL stock solution. LZD was dissolved in PBS to prepare a 2-mg/mL stock solution. GEM was dissolved in PBS to prepare a 10-mM stock solution. DTX was dissolved in ethanol to prepare a 10-mM stock solution. Fetal bovine serum (FBS) was purchased from Gibco (Grand Island, NY, U.S.A.). For other reagents, we purchased and used commercially available special grade products.

Cell Lines and Culture Conditions

MCF-7 and PC-3 cells were grown in RPMI 1640 medium containing 10% FBS and 1-mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Gibco). NOZ C-1 cells were grown in William’s Medium E containing 10% FBS and 2-mM Glutamine (Gibco). The attached cells were removed with trypsin–ethylenediaminetetraacetic acid (EDTA) (Gibco). Cells were incubated at 37 °C in a humidified atmosphere of 5% CO₂ in the air.

Cell Growth

Cells were trypsinized and suspended in an appropriate medium containing 10% FBS. MCF-7 cells (4 × 10⁴ cells/mL), PC-3 cells (4 × 10⁴ cells/mL) and NOZ C-1 cells (1 × 10⁴ cells/mL) were incubated at 37 °C in a humidified atmosphere of 5% CO₂ in air. After one day, various concentrations of chemicals were added to the cultures. Controls were prepared by treating cells with a culture medium containing vehicles without other chemicals. Cells were incubated for 72 h and were not confluent, and then a viable cell number was estimated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) described previously.⁹⁾ Values for percent NET cell growth were calculated using the following formula: [(absorbance of experimental cell concentration) − (absorbance of initial cell concentration)/(absorbance of control cell concentration) − (absorbance of initial cell concentration)] × 100.

Quantitative Reverse Transcriptase (RT)-PCR (qPCR)

Cells (4 × 10⁴ cells/mL) were incubated at 37 °C in a humidified atmosphere of 5% CO₂ in air. After one day, various concentrations of LZD were added to the cultures. Controls were prepared by treating cells with a culture medium containing vehicles without other chemicals. Cells were incubated for 72 h. Total RNA was extracted from cells treated with LZD using an ISOGEN RNA extraction kit (Nippon Gene, Toyama, Japan) as described previously.¹⁰⁾ cDNA was synthesized by RT reactions from 0.5-µg RNA using SuperScript VILO cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, U.S.A.) according to the manufacturer’s instructions. The cDNA and gene-specific primers were added to PowerUp SYBR Green Master Mix (Applied Biosystems Inc., Foster City, CA, U.S.A.), and subjected to PCR amplification in an Applied Biosystems StepOne (Applied Biosystems). The sequences of the PCR primers were as follows: Bax (forward 5′-GGA GCA GCC CAG AGG C-3′, reverse 5′-CTC GAT CCT GGA TGA AAC CCT-3′), Bcl-xL (forward 5′-CCT AAG GCG GAT TTG AAT CTC T-3′, reverse 5′-AAA GTC AAC CAC CAG CTC CC-3′), Bcl-2 (forward 5′-GGG GTC ATG TGT GTG GAG AG-3′, reverse 5′-TTC CAC AAA GGC ATC CCA GC-3′), and β-actin (forward 5′-GAG CAC AGA GCC TCG CCT TT-3′, reverse 5′-TCA TCA TCC ATG GTG AGC TGG-3′). To estimate the level of transcripts quantitatively, β-actin transcript was used as an internal control for each prepared sample.

Flow Cytometry

Cells (4 × 10⁴ cells/mL) were incubated at 37 °C in a humidified atmosphere of 5% CO₂ in air. After one day, cells were treated with 10 µg/mL LZD or PBS for 72 h. Cells were harvested and fixed in 70% ethanol at 4 °C overnight. Before analysis, cells were washed with PBS twice, treated with 100 µg/mL ribonuclease (RNase) A at 37 °C for 30 min, and then stained with 10 µg/mL propidium iodide (PI).⁹⁾ Cell cycle analysis was performed using a FACSVerse flow cytometer (Becton Dickinson, San Jose, CA, U.S.A.).

Effect of Caspase Inhibitor on Cell Growth Inhibition with LZD

Cells (4 × 10⁴ cells/mL) were incubated at 37 °C in a humidified atmosphere of 5% CO₂ in air. After one day, 10 µg/mL LZD and/or 10 µM z-VAD-FMK (PEPTIDE INSTITUTE, INC., Osaka, Japan) were added to the cultures, and then were incubated for 72 h. Viable cell number was estimated using MTT as described above.

Terminal Deoxynucleotidyl Transferase (TdT)-Mediated Deoxyuridine Triphosphate (dUTP)-Biotin Nick End Labeling (TUNEL) Assay

Cells (4 × 10⁴ cells/mL) were incubated at 37 °C in a humidified atmosphere of 5% CO₂ in air. After one day, 10 µg/mL LZD were added to the cultures, and then were incubated for 72 h. Cells were harvested and stained MEBSTAIN Apoptosis TUNEL Kit Direct (MBL, Tokyo, Japan) according to manufacturer’s instructions. Fluorescence was measured using a FACSVerse flow cytometer.

Immunoblotting Analysis

Whole cell lysates were prepared with NP40 lysis buffer [25 mM Tris–HCl (pH 8), 150 mM NaCl, 0.5% NP40, 4 mM NaF, 0.1 mM Na₃VO₄, and protease inhibitor cocktail]. The protein concentration of whole cell lysates was determined by the Bicinchoninic Acid (BCA) Protein Assay (Pierce, Rockford, IL, U.S.A.). Whole lysates were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and were transferred to Immobilon-P membranes (Millipore, Billerica, MA, U.S.A.). The membranes were probed with polyclonal antibodies against Bcl-2, Bcl-xL, extracellular signal-regulated kinase (ERK), phospho-ERK (p-ERK), Akt, phospho-Akt (p-Akt), and LC3 (Cell Signaling, Danvers, MA, U.S.A.), and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.). Proteins were visualized using ECL-plus (Invitrogen).

Statistical Analyses and Results Presentation

The data are expressed as the mean ± standard deviation (S.D.). Data were analyzed using Graph Pad Prism v.6. Statistical significance was assessed using one-way ANOVA followed by Dunnett’s multiple comparisons test or Bonferroni’s multiple comparisons test.

* p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control. ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 vs. DTX or GEM alone.

RESULTS

The Effects of Antibacterial Agents on Prostate Cancer Cells

First, the effects of antibacterial agents on the proliferation of PC-3 prostate cancer cells were examined. As shown in Fig. 1A, VAN did not affect PC-3 cell proliferation. Also, while TEIC did not affect PC-3 cell proliferation at concentrations up to 40 µg/mL, treatment with 80 µg/mL TEIC significantly promoted cell proliferation by 15.5% (Fig. 1B). DAP, which promoted the proliferation of MCF-7 cells, did not affect the proliferation of PC-3 cells (Fig. 1C). In contrast, LZD, which suppressed the proliferation of MCF-7 cells, also showed a concentration-dependent cell proliferation inhibitory effect on PC-3 cells (Fig. 1D). The growth inhibition rates were 7.3% at 0.63 µg/mL, 18.0% at 1.25 µg/mL, 30.4% at 2.5 µg/mL, 42.9% at 5 µg/mL, and 55.2% at 10 µg/mL. Therefore, while VAN and DAP did not affect cell proliferation for PC-3 cells, TEIC promoted cell proliferation, and LZD suppressed cell proliferation.

Fig. 1. Growth Inhibition with Antibacterial Agents in PC-3 Cells

PC-3 cells were treated with various concentrations of Vancomycin (VAN) (A), Teicoplanin (TEIC) (B), Daptomycin (DAP) (C), and Linezolid (LZD) (D) for 72 h. Cell growth was determined as described in Materials and Methods. Control cell number was defined as 100%. Data shown are mean ± S.D. (n = 4). *** p < 0.001 vs. control (Dunnett’s multiple comparisons test).

The Effects of Antibacterial Agents on Breast Cancer Cells

Next, the effects of antibacterial agents on MCF-7 breast cancer cells were examined. As shown in Fig. 2A, VAN did not affect the proliferation of MCF-7 cells. Also, TEIC did not significantly change the proliferation of MCF-7 cells, similar to VAN (Fig. 2B). Alternatively, while DAP did not affect the proliferation of MCF-7 cells at concentrations up to 100 µg/mL, treatment with 200 µg/mL significantly promoted cell proliferation by 13.4% (Fig. 2C). Furthermore, as shown in Fig. 2D, LZD suppresses the proliferation of MCF-7 cells from low concentration in a concentration-dependent manner, and the suppression rate were 11.1% at 0.63 µg/mL, 19.7% at 1.25 µg/mL, 32.8% at 2.5 µg/mL, 48.6% at 5 µg/mL, and 59.4% at 10 µg/mL. Thus, while VAN and TEIC did not affect cell proliferation in MCF-7 cells, high concentrations of DAP promoted cell proliferation. In contrast, LZD, an antibacterial agent, suppressed the proliferation of MCF-7 cells. The effects of antibacterial agents differ depending on the cell type.

Fig. 2. Growth Inhibition with Antibacterial Agents in MCF-7 Cells

MCF-7 cells were treated with various concentrations of Vancomycin (VAN) (A), Teicoplanin (TEIC) (B), Daptomycin (DAP) (C), and Linezolid (LZD) (D) for 72 h. Cell growth was determined as described in Materials and Methods. Control cell number was defined as 100%. Data shown are mean ± S.D. (n = 4). * p < 0.05, *** p < 0.001 vs. control (Dunnett’s multiple comparisons test).

The Effects of Antibacterial Agents on Gallbladder Cancer Cells

The effects of antibacterial agents on the cell proliferation of NOZ C-1 gallbladder cancer cells, a refractory cancer, were investigated. VAN showed a significant growth inhibitory effect at 31.3 and 125 µg/mL, and the inhibition rate was less than 10% (Fig. 3A). Additionally, TEIC and DAP did not significantly affect the proliferation of NOZ C-1 cells (Figs. 3B, C). In contrast, LZD, which suppressed the proliferation of MCF-7 and PC-3 cells, was shown to significantly suppress the proliferation of NOZ C-1 cells (Fig. 3D). The growth inhibition rates were 9.9% at 1.25 µg/mL, 12.7% at 2.5 µg/mL, 18.3% at 5 µg/mL, and 17.0% at 10 µg/mL. Therefore, while treating NOZ C-1 cells with antibacterial agents resulted in LZD suppressing cell proliferation, VAN, TEIC, and DAP had little effect on proliferation. It was also shown that the growth inhibition rate by LZD for NOZ C-1 cells was lower than the growth inhibition rate for PC-3 and MCF-7 cells.

Fig. 3. Growth Inhibition with Antibacterial Agents in NOZ C-1 Cells

NOZ C-1 cells were treated with various concentrations of Vancomycin (VAN) (A), Teicoplanin (TEIC) (B), Daptomycin (DAP) (C), and Linezolid (LZD) (D) for 72 h. Cell growth was determined as described in Materials and Methods. Control cell number was defined as 100%. Data shown are mean ± S.D. (n = 4). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control (Dunnett's multiple comparisons test).

The Effect of Antibacterial Agents on Cell Proliferation Suppression by DTX

First, we examined antiproliferative effects against PC-3 and MCF-7 cells of DTX, which is used clinically to treat breast and prostate cancers. As shown in Fig. 4A, DTX significantly suppressed the proliferation of PC-3 cells from 0.4 nM in a concentration-dependent manner. The suppression rates were 24.4% at 0.4 nM, 56.4% at 1 nM, and 92.3% at 4 nM. Additionally, DTX significantly suppressed the proliferation of MCF-7 cells from 0.1 nM in a concentration-dependent manner (Fig. 4B). The suppression rates were 12.0% at 0.1 nM, 26.0% at 0.4 nM, 55.2% at 1 nM, and 91.9% at 4 nM.

Fig. 4. Growth Inhibition with Docetaxel

PC-3 cells (A) and MCF-7 cells (B) were treated with various concentrations of Docetaxel (DTX) for 72 h. Cell growth was determined as described in Materials and Methods. Control cell number was defined as 100%. Data shown are mean ± S.D. (n = 4). *** p < 0.001 vs. control (Dunnett’s multiple comparisons test).

Next, the effect of the combined use of antibacterial agents and DTX on cell proliferation suppression was investigated. When the effect of the combined use of DTX and VAN on PC-3 cells was examined, the combined use of VAN did not affect cell proliferation suppression by DTX (Fig. 5A). Additionally, 80 µg/mL TEIC promoted the proliferation of PC-3 cells, and the combined use of TEIC and DTX significantly inhibited the suppression of proliferation by DTX (Fig. 5B). Furthermore, the combined use of DAP and DTX did not affect the growth inhibition of PC-3 cells by DTX (Fig. 5C). In contrast, LZD showed a significant proliferation inhibitory effect on PC-3 cells, and the combined use of LZD and DTX significantly enhanced cell proliferation inhibition by DTX (Fig. 5D).

Fig. 5. Growth Inhibition with Docetaxel without or with Antibacterial Agents in PC-3 Cells

PC-3 cells were treated with 0.4 nM of Docetaxel (DTX) without or with 125 or 500 µg/mL of Vancomycin (VAN) (A), 20 or 80 µg/mL Teicoplanin (TEIC) (B), 50 or 200 µg/mL Daptomycin (DAP) (C), and 1.25 or 2.5 µg/mL Linezolid (LZD) (D) for 72 h. Cell growth was determined as described in Materials and Methods. Control cell number was defined as 100%. Data shown are mean ± S.D. (n = 4). ** p < 0.01, *** p < 0.001 vs. control, ^###p < 0.001 vs. DTX alone (Bonferroni’s multiple comparisons test).

As shown in Fig. 6A, VAN did not affect cell proliferation inhibition by DTX in MCF-7 cells. In contrast, TEIC and DAP inhibited the suppression of cell proliferation by DTX. The cell proliferation was suppressed by 25.1% after treatment with DTX alone. However, the cell proliferation was significantly restored to 7.6% by the combined use of 80 µg/mL TEIC and DTX, which was comparable to the control (Fig. 6B). Additionally, the combined use of 200 µg/mL DAP and DTX restored cell proliferation to the same level as the control (Fig. 6C). As shown in Fig. 6D, while LZD significantly suppressed the proliferation of MCF-7 cells, LZD did not affect the suppression of cell proliferation by DTX. From the above statements, it is clear that in MCF-7 and PC-3 cells, there are TEIC and DAP for MCF-7 and TEIC for PC-3 that suppresses cell proliferation inhibition by DTX, and there is also LZD for PC-3 that promotes cell proliferation inhibition by DTX.

Fig. 6. Growth Inhibition with Docetaxel without or with Antibiotics in MCF-7 Cells

MCF-7 cells were treated with 0.4 nM of Docetaxel (DTX) without or with 125 or 500 µg/mL of Vancomycin (VAN) (A), 20 or 80 µg/mL Teicoplanin (TEIC) (B), 50 or 200 µg/mL Daptomycin (DAP) (C), and 1.25 or 2.5 µg/mL Linezolid (LZD) (D) for 72 h. Cell growth was determined as described in Materials and Methods. Control cell number was defined as 100%. Data shown are mean ± S.D. (n = 4). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Control, ^# p < 0.05, ^### p < 0.001 vs. DTX alone (Bonferroni's multiple comparisons test).

The Effect of Antibacterial Agents on Cell Proliferation Inhibition by GEM

Next, we examined the antiproliferative effects against NOZ C-1 cells of GEM, which is used in the clinical treatment of gallbladder cancer. As shown in Fig. 7A, GEM significantly suppressed the proliferation of NOZ C-1 cells from 4 nM in a concentration-dependent manner. The inhibition rates were 23.4% at 4 nM, 39.3% at 10 nM, 66.7% at 40 nM, and 67.1% at 100 nM.

Fig. 7. Growth Inhibition with Gemcitabine without or with Antibiotics in NOZ C-1 Cells

(A) NOZ C-1 cells were treated with various concentrations of Gemcitabine (GEM) for 72 h. Cell growth was determined as described in Materials and Methods. Control cell number was defined as 100%. Data shown are mean ± S.D. (n = 4). *** p < 0.001 vs. control (Dunnett’s multiple comparisons test). (B, C) NOZ C-1 cells were treated with 10-nM Gemcitabine (GEM) without or with 20 or 80 µg/mL Teicoplanin (TEIC) (B) and 5 or 10 µg/mL Linezolid (LZD) (C) for 72 h. Cell growth was determined as described in Materials and Methods. Control cell number was defined as 100%. Data shown are mean ± S.D. (n = 4). *** p < 0.001 vs. Control (Bonferroni’s multiple comparisons test).

Next, we investigated the combined use of antibacterial agents and GEM on cell proliferation inhibition. As shown in Fig. 7B, TEIC did not inhibit cell proliferation by GEM in NOZ C-1 cells. Also, as shown in Fig. 7C, while LZD significantly suppressed the proliferation of NOZ C-1 cells, LZD did not affect cell proliferation inhibition by DTX. From the above, in MCF-7 and PC-3 cells, while there were antibacterial agents that inhibit or enhance the action of anticancer agents, in NOZ C-1 cells, they did not affect cell proliferation inhibition by GEM.

Analysis of Cell Proliferation Suppression Mechanism by LZD

LZD suppressed the growth of breast, prostate, and gallbladder cancer cells in a concentration-dependent manner. Apoptosis, which induces cell death, is considered a mechanism of suppressing cell proliferation. Therefore, to investigate the possibility of inducing apoptosis, we analyzed the gene and protein expression of the Bcl-2 family using PC-3 cells. First, the anti-apoptotic Bcl-2 subfamily examined Bcl-xL and Bcl-2 mRNA expression. Consequently, Bcl-xL and Bcl-2 mRNA expression was significantly reduced by approximately 31.6 and 31.1% by treatment with 10 µg/mL LZD (Figs. 8A, B). When Bax, an apoptosis-promoting Bcl-2 subfamily, was analyzed, the Bax mRNA expression was not changed by treatment with 5 and 10 µg/mL LZD (Fig. 8C). In contrast, when the protein levels of Bcl-xL, Bcl-2, and Bax were examined, Bcl-xL and Bcl-2, protein levels were significantly reduced by approximately 21.0 and 30.1% by treatment with 10 µg/mL LZD, respectively (Figs. 8D, E), while Bax protein level was not changed (Fig. 8F). Next, when we examined the effects of the cell cycle arrest and sub-G1 population, an indicator of apoptosis, in PC-3 cells, the cell cycle and sub-G1 population were not changed by treatment with 10 µg/mL LZD. The co-existence of 10 µM z-VAD-FMK, the caspase inhibitor, did not restore the proliferation suppressed by 10 µg/mL LZD treatment. In addition, TUNEL assay, a method for detecting DNA fragmentation, was performed on LZD-treated cells, but there was no significant change compared to control cells (data not shown). These data indicate that LZD treatment of PC-3 cells might not induce apoptosis, while anti-apoptotic Bcl-2 and BcL-xL mRNA and protein decreased with LZD treatment.

Fig. 8. The mRNA and Protein Levels of Bcl-xL, Bcl-2, and Bax in PC-3 Cells

PC-3 cells were treated with Linezolid (LZD) at a concentration of 0, 5, or 10 µg/mL for 72 h. The expression levels of Bcl-xL (A, D), Bcl-2 (B, E), and Bax (C, F) mRNAs and proteins were analyzed by qPCR and immunoblotting as described in Materials and Methods. The value of control was defined as 1.0. Each bar represents the mean ± S.D. of each group (n = 3). * p < 0.05, ** p < 0.01, vs. control (Dunnett’s multiple comparisons test).

Therefore, we further investigated the effects of LZD on other pathways (phosphorylation inhibition of ERK, phosphorylation inhibition of Akt, and autophagy) related to cell growth. First, phosphorylated ERK levels involved in cell proliferation were examined in PC-3 cells. We found that phosphorylated ERK levels (p-ERK/ERK) were not affected by LZD treatment (Fig. 9A). These results suggest that LZD did not inhibit ERK phosphorylation. Next, we investigated the effect of LZD on the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway, which is a survival signal. We found that phosphorylated Akt levels (p-Akt/Akt) decreased significantly by approximately 27 and 55% by treatment with 5 and 10 µg/mL LZD, respectively (Fig. 9B). This suggests that LZD inhibits the PI3K/Akt pathway in PC-3 cells. In addition, we examined the involvement of autophagy, a non-apoptotic cause of cell death. Treatment of PC-3 cells with 5 µg/mL LZD, significantly increased by approximately 1.1-fold the expression of LC3-II (a lipid adduct of LC3-I), which is an indicator of autophagy (Fig. 9C). This suggests that LZD slightly induces autophagy in PC-3 cells. These data suggest that LZD suppresses the survival signal by Akt in PC-3 cells.

Fig. 9. Protein Levels of Phosphorylated ERK/ERK, Phosphorylated-Akt/Akt, or LC3 in PC-3 Cells

PC-3 cells were treated with Linezolid (LZD) at a concentration of at a concentration of 0, 5, or 10 µg/mL for 72 h. The protein levels of phosphorylated ERK/ERK (A), phosphorylated-Akt/Akt (B), or LC3 (C), were analyzed by Western blot analysis with specific antibodies as described in Materials and Methods. The value of control was defined as 1.0. Each bar represents the mean ± S.D. of each group (n = 3). * p < 0.05, *** p < 0.001 vs. control (Dunnett’s multiple comparisons test).

Finally, collectively, LZD suppresses the proliferation of PC-3 cells primarily by inhibiting the survival signal by Akt, since apoptosis was not induced.

DISCUSSION

In this study, we investigated the effects of antibacterial agents on the growth of cancer cells. VAN and DAP treatment of prostate cancer cell PC-3 did not affect cell proliferation (Figs. 1A, C). In contrast, TEIC treatment promoted the proliferation of PC-3 cells, while LZD treatment suppressed the proliferation of PC-3 cells in a concentration-dependent manner (Figs. 1B, D). Additionally, VAN and TEIC treatments on breast cancer cell MCF-7 did not affect cell proliferation (Figs. 2A, B). In contrast, DAP treatment promoted the proliferation of MCF-7 cells, while LZD treatment suppressed the proliferation of MCF-7 cells in a concentration-dependent manner (Figs. 2C, D). Furthermore, VAN, TEIC, and DAP treatments on gallbladder cancer cells NOZ C-1 did not affect cell proliferation (Figs. 3A–C). In contrast, LZD treatment suppressed the proliferation of NOZ C-1 cells in a concentration-dependent manner (Fig. 3D). Therefore, we could find LZD that affects the growth of cancer cells among antibacterial agents.

LZD is an oxazolidinone-based synthetic antibacterial agent that binds to bacterial ribosomes, prevents the formation of the 70S-initiated complex during the translation process, and inhibits bacterial protein synthesis.¹¹⁾ Additionally, LZD has been reported to exhibit monoamine oxidase (MAO) inhibitory activity.¹²⁾ It is unlikely that inhibition of bacterial protein synthesis is involved in inhibiting proliferation of cancer cells, and MAO inhibitory activity may be involved in inhibiting cell proliferation, while detailed studies are required. TEIC and DAP, which promote cancer cell growth, are classified into glycopeptide and cyclic lipopeptide antibiotics.^13–17) TEIC exhibits antibacterial activity by inhibiting bacterial cell wall synthesis. Alternatively, DAP exerts an antibacterial effect by binding to the bacterial cell membrane and causing K⁺ ions to flow out from the cell membrane to induce depolarization of the membrane potential. Both drugs act on bacteria, and the mechanism that promoted the growth of cancer cells is unknown. It is interesting to elucidate the cell proliferation suppression mechanism by LZD and the cell proliferation promotion mechanism of TEIC and DAP.

Myelosuppression is a side effect of anticancer drug treatment, and the weakened immune system makes people more susceptible to infections. Since the treatment of infectious diseases takes priority over cancer treatment, the patient’s cancer treatment is suspended or postponed. Assuming the suspension or postponement of cancer treatment, the combined use of 0.4-nM DTX and 4-nM GEM had a growth inhibitory effect of approximately 20% and an antibacterial agent on the growth inhibitory effect was investigated (Figs. 4, 7A). In PC-3 cells, the combined use of DTX with VAN or DAP did not affect the cell proliferation inhibitory effect (Figs. 5A, C). In contrast, the combined use of DTX and TEIC promoted the proliferation of PC-3 cells and abolished the cell proliferation inhibition by DTX (Fig. 5B). However, in PC-3 cells, the combined use of DTX and LZD enhanced the cell proliferation inhibitory effect of DTX, and the enhancing effect was additive (Fig. 5D). In MCF-7 cells, the combined use of DTX with VAN or LZD did not affect the cell proliferation inhibitory effect (Figs. 6A, D). In contrast, DTX and TEIC or DAP’s combined use promoted the proliferation of MCF-7 cells and abolished cell proliferation inhibition by DTX (Figs. 6B, C). In NOZ C-1 cells, the combined use of TEIC and LZD did not affect the growth inhibitory effect of GEM, which treats gallbladder cancer (Figs. 7B, C). The above results clarified that an antibacterial agent promotes or effectively suppresses the growth of cancer cells by treatment with an antibacterial agent after the suspension or postponement of cancer treatment. Notably, in prostate cancer cell PC-3, LZD alone showed a cell proliferation inhibitory effect. The combined use of DTX and LZD could perform additive cancer cell proliferation inhibition. Additionally, LZD reduced the patient’s disadvantage when stopping or postponing cancer treatment since LZD alone suppressed the growth of breast and gallbladder cancer and in combination, did not affect the growth of DTX or GEM.

LZD significantly suppressed the proliferation of PC-3 cells when used alone and in combination with DTX. Since LZD had no effect on the cell cycle, in order to clarify whether induction of apoptosis is involved in its cell growth suppression, we measured the effects of LZD on the mRNA and protein expression of the Bcl-2 family. Consequently, LZD significantly decreased mRNA and protein expressions of anti-apoptotic Bcl-xL and Bcl-2 (Figs. 8A, B, D, F), but the expression of pro-apoptotic Bax was not changed (Figs. 8C, F). Regulation of apoptosis by the Bcl-2 family members is achieved by forming homodimers of apoptosis-promoting Bcl-2 subfamily members (e.g. Bax). These homodimers are formed by converting heterodimers that result when apoptosis-promoting Bcl-2 (e.g. Bax) subfamily members associate with anti-apoptotic Bcl-2 subfamily members (e.g. Bcl-2 and Bcl-xL). These results suggest that LZD might induce apoptosis in PC-3 cells. However, in PC-3 cells; (1) LZD treatment did not increase the Sub-G1 population, (2) the results of the TUNEL assay, which could measure apoptosis, were negative, and (3) apoptosis inhibitors had no effect on the proliferation of cells treated with LZD. These results suggest that cell growth inhibition by LZD is not related to apoptosis induction. In our current study, we investigated the other mechanism of action of LZD. We found that LZD does not inhibit the ERK pathway and that LZD inhibits the Akt pathway (Fig. 9).

Hedaya et al. reported that LZD did not suppress MCF-7 cells’ growth and that the derivatives induce apoptosis in breast cancer cells.¹⁸⁾ This result is different from our result (Fig. 2); however, it may be due to the difference in culture conditions and composition of the culture solution. Additionally, it is essential from the perspectives of drug repositioning that the existing antibacterial agent LZD showed a growth inhibitory effect on cancer cells, it is also interesting to enhance the effect of derivatization. Yadav et al. reported that one of the antibacterial agents, Gatifloxacin, suppresses the growth of pancreatic cancer cells through cell cycle arrest.¹⁹⁾ In this study, we clarified the growth inhibitory effect of LZD on gallbladder cancer, which is one of the refractory cancers as well as pancreatic cancer. In the future, it will be interesting to investigate cell cycle arrest in the growth suppression mechanism by LZD for gallbladder cancer cells.

LZD is a clinically used antibacterial agent that has been tested for toxicity against normal NIH/3T3 cells, but not cancer cells.²⁰⁾ This report showed that the IC₅₀ value for toxicity to NIH/3T3 cells is 465 µg/mL. In our current study, we have shown that the IC₅₀ of LZD for growth inhibitory effects on PC-3 and MCF-7 cells was approximately 5–10 µg/mL. Thus, LZD is effective against cancer cells and non-toxic to non-cancer cell lines at low concentrations. Therefore, LZD is a highly promising compound that is non-toxic in vitro and in vivo.

A previous study²¹⁾ has shown the use of antibiotics as anticancer agents. This work provides information on the anticancer role of antibiotics and how they are used in cancer treatment. However, the four antibiotics (VAN, TEIC, DAP, and LZD) used in the clinic examined in our current study, were not covered in the paper. In our current work we show that the clinical antibiotic LZD shows antiproliferative effects. Base on this we speculate whether LZD could potentially be used as an anticancer agent to treat breast cancer, prostate cancer, and gallbladder cancer patients.

CONCLUSION

This study investigated the effects of antibacterial agents on cancer cells. Since LZD suppressed the growth of cancer cells either by itself or when combined with an anticancer drug, it may be a candidate for future use as an anticancer drug. Discovering antibacterial agents that have anticancer effects could potentially improve patient QOL, because such agents may serve dual roles as both anticancer and antibacterial therapeutics.

Acknowledgments

We thank Dr. Terrence Burke, Jr. for his helpful comments. This investigation was supported in part by ‘Hoshi University Otani Research Grants,’ ‘JSPS KAKENHI Grant No. JP17K15485,’ and ‘JSPS Core-to-Core Program, A. Advanced Research Networks, Japan.’

Conflict of Interest

The authors declare no conflict of interest.

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