
==== Front
Medicine (Baltimore)
Medicine (Baltimore)
MD
Medicine
0025-7974
1536-5964
Lippincott Williams & Wilkins Hagerstown, MD

MD-D-24-01262
00090
10.1097/MD.0000000000039450
3
7300
Research Article
Observational Study
Cell death and DNA damage via ROS mechanisms after applied antibiotics and antioxidants doses in prostate hyperplasia primary cell cultures
https://orcid.org/0000-0002-8554-7739
Matei Elena PhD a*
Ionescu Anita Cristina (Mitu) MD cristina-ionescu@365.univ-ovidius.ro
bc
Enciu Manuela PhD, MD manuela.enciu@univ-ovidius.ro
cd
Popovici Violeta PhD violeta.popovici@ce-mont.ro
e
Mitroi Anca Florentina PhD, MD anca.mitroi@365.univ-ovidius.ro
ad
Aschie Mariana PhD, MD mariana.aschie@univ-ovidius.ro
acdf
Deacu Mariana PhD, MD mariana.deacu@univ-ovidius.ro
cd
Băltățescu Gabriela Isabela PhD, MD gabriela.baltatescu@univ-ovidius.ro
ad
Nicolau Antonela-Anca PhD, MD antonela.nicolau@365.univ-ovidius.ro
ad
Roșu Mihai Cătălin a
Cristian Miruna PhD miruna.cristian@365.univ-ovidius.ro
a
Dobrin Nicolae PhD nicu.dobrin@365.univ-ovidius.ro
a
Ștefanov Constanța PhD a
Pundiche Butcaru Mihaela PhD, MD butcaru.pundiche@365.univ-ovidius.ro
c
Cozaru Georgeta Camelia PhD, MD georgiana.cozaru@365.univ-ovidius.ro
ad
a Center for Research and Development of the Morphological and Genetic Studies of Malignant Pathology, “Ovidius” University of Constanta, Constanta, Romania
b Institute of Oncology “Prof. Dr. Alexandru Trestioreanu”, Bucharest, Romania
c Medicine Faculty, “Ovidius” University of Constanta, Constanta, Romania
d Clinical Service of Pathology, “Sf. Apostol Andrei” Emergency County Hospital, Constanta, Romania
e Laboratory of Bacteriology, Microbiology and Pharmacology, Center for Mountain Economics (INCE-CE-MONT), National Institute of Economic Research “Costin C. Kiritescu”, Suceava County, Romania
f Romanian Academy of Scientists, Bucharest, Romania.
* Correspondence: Elena Matei, Center for Research and Development of the Morphological and Genetic Studies of Malignant Pathology, “Ovidius” University of Constanta, 145 Tomis Blvd., Constanta 900591, Romania (e-mail: sogorescuelena@yahoo.com, elena_matei@365.univ-ovidius.ro).
13 9 2024
13 9 2024
103 37 e3945001 2 2024
08 3 2024
05 8 2024
Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial License 4.0 (CCBY-NC), where it is permissible to download, share, remix, transform, and buildup the work provided it is properly cited. The work cannot be used commercially without permission from the journal.

Tumor heterogeneity results in aggressive cancer phenotypes with acquired resistance. However, combining chemical treatment with adjuvant therapies that cause cellular structure and function perturbations may diminish the ability of cancer cells to resist at chemical treatment and lead to a less aggressive cancer phenotype. Applied treatments on prostate hyperplasia primary cell cultures exerted their antitumor activities through mechanisms including cell cycle blockage, oxidative stress, and cell death induction by flow cytometry methods. A 5.37 mM Chloramphenicol dose acts on prostate hyperplasia cells by increasing the pro-oxidant status, inducing apoptosis, autophagy, and DNA damage, but without ROS changes. Adding 6.30 mM vitamin C or 622 µM vitamin E as a supplement to 859.33 µM Chloramphenicol dose in prostate hyperplasia cells determines a significant increase of ROS level for a part of cells. However, other cells remain refractory to initial ROS, with significant changes in apoptosis, autophagy, and cell cycle arrest in G0/G1 or G2/M. When the dose of Chloramphenicol was increased to 5.37 mM for 6.30 mM of vitamin C, prostate hyperplasia cells reacted by ROS level drastically decreased, cell cycle arrest in G2/M, active apoptosis, and autophagy. The pro-oxidant action of 1.51 mM Erythromycin dose in prostate hyperplasia cell cultures induces changes in the apoptosis mechanisms and cell cycle arrest in G0/G1. Addition of 6.30 mM vitamin C to 1.51 mM Erythromycin dose in hyperplasia cell cultures, the pro-oxidant status determines diminished caspase 3/7 mechanism activation, but ROS level presents similar changes as Chloramphenicol dose and cell cycle arrest in G2/M. Flow cytometric analysis of cell death, oxidative stress, and cell cycle are recommended as laboratory techniques in therapeutic and diagnostic fields.

antibiotics
antioxidants
apoptosis
DNA damage
flow cytometry
oxidative stress
OPEN-ACCESSTRUE
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pmc1. Introduction

Apoptosis coordinates and controls all cellular processes in normal physiological and pathological conditions. The mechanisms involved in cell apoptosis are complex, involving several synthesis pathways. Defects in synthesis pathways lead to the malignant transformation of affected cells, the development of tumor metastases, and the acquisition of resistance to anticancer drugs. Targeting the cell death pathways, cell cycle, and oxidative stress in cancer cells remains essential to developing the cancer therapies.[1] Apoptosis is a mechanism that removes abnormal cells and plays a pivotal role in controlling tumor growth by counterbalancing proliferation.[2] The morphological changes are highlighted by nuclear apoptosis through chromatin condensation (pyknosis) and nucleus fragmentation (karyorrhexis).[3] Biochemical changes are caspases activation, fragmentation of deoxyribonucleic acid (DNA) sequences encoding protein synthesis, and plasma membrane changes in recognition by phagocytic cells.[4]

A multicellular organism can develop when all its cell functions follow the rules that govern cell growth and reproduction, but losing cell cycle control represents the critical step in cancer development. Cancer cells continue to divide, even when there is a large amount of DNA damage or when the cells are abnormal. The tumor suppressor gene p53 plays an important role in response to cell damage[5] leading to cell cycle arrest and apoptosis.[6] The over-expression of apoptotic-related genes determines an accumulation of pro-apoptotic proteins on the mitochondrial outer membrane. In turn, this causes the release of cytochrome c into the cytoplasm,[7] where it binds to the cytosolic protein Apaf-1 to facilitate the formation of apoptosomes, leading to the activation of caspase-3 and caspase-9 occurs.[3,8,9] Caspases activity of mitochondrial intrinsic pathways is studied in different human cancer cell lines.[10–13] Genetic damage, hypoxia, an increased concentration of calcium ions in the cytoplasm, and oxidative stress activate the mitochondrial intrinsic pathways.[14]

Oxidative stress is studied from carcinogenesis to the tumor-bearing state, from treatment to prevention. Total reactive oxygen species (ROS) contributes to abnormal gene expression, blockage of cell-to-cell communication, and modification of second messenger systems, thus increasing cell proliferation and decreasing cell death.[15–20] ROS induce DNA damage, leading to genetic lesions that initiate tumorigenicity and tumor progression. On the other hand, ROS induces cellular senescence, cell death, and functions as an anti-tumorigenic agent. Whether ROS promotes tumor cell survival or acts as an anti-tumorigenic agent depends on the phenotype of cells and the level of ROS.[19,20] In animal models and humans, uncontrolled tumor cell proliferation requires the upregulation of multiple intracellular signaling pathways and cell cycle progression.[21]

In different studies, antibiotics are used to target mitochondria biogenesis. Erythromycin and Chloramphenicol selectively bind to the large subunit of the mitochondrial ribosome and inhibit mitochondrial biogenesis. Antibiotics inhibit mitochondrial biogenesis and eradicate cancer stem cells in cancer cell lines (breast, ovarian, prostate, lung, pancreatic, melanoma, and glioblastoma).[22,23] Previous studies shown that mitochondrial oxidative function is required for energetically initiating tumorigenesis in different cancer types.[24,25]

A metabolic shift from oxidative to glycolytic metabolism represents an escape mechanism for cancer cells chronically treated with a mitochondrial stressor. The antibiotic acts as an inhibitor of mitochondrial protein translation in cancer cells.[26] Vitamin C acts as a glycolysis inhibitor by targeting glyceraldehyde-3-phosphate dehydrogenase.[27] The antibiotics induce mitochondrial dysfunction and oxidative damage in mammalian cells.[28–30] Metabolic plasticity is the cell’s ability to pass from nutrient source to another, while metabolic inflexibility represents the reducing cell’s ability to change nutrient sources.[26]

In our study, Erythromycin and Chloramphenicol, were used in prostate hyperplasia primary cell cultures to observe how cells adopt a glycolytic metabolism for survival. Because Vitamin C inhibits glyceraldehyde-3-phosphate dehydrogenase, being a glycolytic enzyme and vitamin E blocks ROS-p53 signaling in cancer cells,[27] vitamins were added as antioxidants in prostate hyperplasia primary cell cultures to explore their proliferative changes. Targeting the cell death, oxidative stress, cell cycle, nuclear shrinkage, and lysosomal activity as investigational tools helps to understand the metabolism plasticity in prostate hyperplasia primary cell cultures with phenotypic heterogeneity, after applied treatments.

2. Materials and methods

2.1. Materials

Tissue samples excised by prostate transurethral resection recovered from patients with benign prostatic hyperplasia (BPH) (who signed an informed consent form, agreeing to participate in this study) from the Clinical Service of Pathology, Sf. Apostol Andrei Clinical Emergency County Hospital in Constanta, Romania, were mechanically homogenized with TissueRuptor II (Qiagen, Hilden, Germany). The study was conducted in accordance with the Declaration of Helsinki, being approved by the Institutional Review Board of CEDMOG, University Ovidius from Constanta (0065/September 25, 2023) for studies involving humans.

2.2. Prostate primary cell cultures

Cells and small tissue fragments are placed in a tissue culture flask with a growth area (25 cm2) and cultured in Dulbecco’s Modified Eagle Medium High Glucose without supplementation of antibiotics mixed in humidity conditions of 5% CO2 at 37°C for 14 days. Prostate cells were distributed in tissue culture test plate 6, growth-enhanced treated (ThermoFisher Scientific Inc.; Whatman, MA). After treatments with antibiotics and antioxidants, for 24 hours, caspase-3/7 activity, cell cycle, oxidative stress, nuclear shrinkage, and lysosomal activity were performed by flow cytometry methods in the Cell Biology Department, CEDMOG, Ovidius University of Constanta, Romania.

2.3. Samples and controls

For experiments were prepared, stock solutions of antibiotics (250 mg/mL Chloramphenicol and 200 mg/mL Erythromycin) and antioxidants (1000 mg/mL vitamin C and 100 mg/mL vitamin E). From work solutions were used 50, 100, and 250 µL of Chloramphenicol, 25, 75, and 200 µL of Erythromycin, 100 µL of vitamin C or vitamin E which are applied on prostate hyperplasia primary cell cultures. The final concentrations of antibiotics and antioxidants which were used in our study were 214.50 µM, 859.33 µM, and 5.37 mM of Chloramphenicol, 18.83 µM, 170 µM, and 1.51 mM of Erythromycin, 6.30 mM vitamin C, 622 µM vitamin E.

Experimental observations were reported in the function of 2 controls: prostate hyperplasia cells (positive control) and normal prostate cells (negative control). Negative control is represented by normal prostate primary cells developed from nonmalignant adjacent tissue samples recovered from patients with BPH. Positive control is represented by prostate hyperplasia primary cells with phenotypic heterogeneity, without applied treatments, developed from benign prostatic hyperplasia tissue samples recovered from patients with BPH.

2.4. Equipment

Analysis was performed on an Attune Acoustic focusing cytometer (Applied Biosystems, Waltham). The flow cytometer was set by using Attune performance tracking beads, labeling, and detection (Life Technologies, Europe BV, Bleiswijk, the Netherlands).[31] More than 10,000 cells per sample for each analysis were gated using the flow cytometer’s Forward Scatter (FSC) and Side Scatter (SSC). Data were collected and interpreted using Attune Cytometric Software v.1.2.5, Applied Biosystems, 2010.

2.5. Methods

2.5.1. Caspase-3/7 activity

Cell apoptosis by activating caspase enzymes using Magic Red Caspase Detection kit (MR-DEVD) methodology (MR-DEVD FAM Caspase-3/7 Assay Kit; Invitrogen, Boston, MA). 200 μL of the prostate primary cells were transferred in tubes, 20 μL of work solution MR-DEVD, and 20 μL of propidium iodide. Cells were mixed and incubated for 30 minutes at room temperature into darkness. After incubation, is added 1 ml flow cytometry stain buffer (FCB, eBioscienceTM, Life Technologies Europe BV, the Netherlands). Viability, apoptosis, and necrosis were analyzed on flow cytometry using a 488 nm excitation, a red emission for MR-DEVD (BL3 channel), and an orange emission for propidium iodide (BL2 channel).[32,33]

2.5.2. Cell cycle analysis

In the darkness, 200 µL of prostate cells were introduced into flow cytometry tubes and fixed with 200 µL ethanol for 30 minutes. After fixation, the cells were treated with 10 µL of PI (20 mg/mL) and incubated for 30 minutes at room temperature into darkness. 1 mL FCB was added, and cell cycle phases were detected at the flow cytometer using a 488 nm excitation and orange emission for PI (BL2 channel).

2.5.3. Total reactive oxygen species (ROS)

Total reactive oxygen species (ROS Assay Kit 520 nm; Invitrogen) are important in cell signaling and homeostasis. Two μL of the 500X ROS Assay Stain stock solution is added for 1 mL of cell culture and mixed well. Cell culture tubes are incubated for 60 minutes in a 37°C incubator with 5% CO2, and the cells are analyzed on a flow cytometer on a BL-1 channel.[32,33]

2.5.4. Nuclear shrinkage and lysosomal activity

300 μL of prostate cells are transferred in tubes, 2 μL of Hoechst 33342 stain, and 50 μL of acridine orange (1.0 μM, AO) are added. After that, primary cells are mixed and incubated for 30 minutes at room temperature into darkness. After incubation, 0.5 mL of FCB is added. Cells are analyzed at flow cytometry, using UV excitation, blue emission for Hoechst (VL2 channel) and 488 nm excitation, green emission for acridine orange (BL1 channel).

2.6. Data analysis

Results were presented as means values ± standard deviation (SD), representing our flow cytometry methods as caspase-3/7 activity (%), cell cycle (%), nuclear shrinkage (%), autophagy (%), and count (×106) of oxidative stress. Kolmogorov-Smirnov test was used to assess normal distribution of the variables. To establish the differences between samples and controls, was used independent t test and P < .05 was considered statistically significant, were made by MedCalc v20.111 Software Ltd. (Ostend, Belgium). Figures 1, 2B–K, 3–6, 7B–F, and 8 were made with Attune Cytometric Software v.1.2.5, Applied Biosystems, 2010 (Bedford) and Figures 2A, 7A, 9–14 with MedCalc v20.111 Software Ltd.

Figure 1. Action of different Chloramphenicol doses (C) supplemented with antioxidants on prostate hyperplasia cell cultures. P1: 214.50 µM; P2: 859.33 µM; P3: 5.37 mM; P7: 859.33 µM C: 6.30 mM vitamin C; P8: 5.37 mM C: 6.30 mM vitamin C; P9: 859.33 µM C: 622 µM vitamin E; P10: 5.37 mM C: 622 µM vitamin E. (A, C, E, G, I, K, M) Results are plotted by dual stain MR-DEVD/PI highlighted the caspases- 3/7 activity to observe the viability (V), early, late apoptosis (EA; LA), and necrosis (N). (B, D, F, H, J, L, N, O, P) Cell apoptosis (P1–P3; P7–P10) is represented by PI stain reported to controls (C1, C2). MR-DEVD = Magic Red Caspase Detection kit.

Figure 2. Oxidative stress after applied treatments with Chloramphenicol (C) doses supplemented with vitamins in prostate hyperplasia cell cultures. (A) Statistical analysis. (B) 859.33 µM (P2); (C) 5.37 mM (P3); (D) 859.33 µM C: 6.30 mM vitamin C (P7); (E) 5.37 mM C: 6.30 mM vitamin C (P8); (F) 859.33 µM C: 622 µM vitamin E (P9); (G) 5.37 mM C: 622 µM vitamin E (P10). (H–K) On graphs are extrapolated positive control as C1-prostate hyperplasia cells to samples (P2, P3, P7, P10). **P < .01 and *P < .05 represent significant statistical differences between samples and control, made by independent t tests of the MedCalc program.

Figure 3. Effects of different concentrations of Chloramphenicol (C) supplemented with vitamins on prostate hyperplasia cell cultures. (A, B) 214.50 µM (P1); (C, D) 859.33 µM (P2); (E, F) 5.37 mM (P3); (G, H) 859.33 µM C: 6.30 mM vitamin C (P7); (I, J) 5.37 mM C: 6.30 mM vitamin C (P8); (K, L) 859.33 µM C: 622 µM vitamin E (P9); (M, N) 5.37 mM C: 622 µM vitamin E (P10). (A, C, E, G, I, and M) Results are plotted by PI stain to highlight cell cycle; (B, D, F, H, J, L, and N) On graphs are extrapolated negative and positive controls as C1-normal prostate cells and C2-prostate hyperplasia cells to samples (P1–P3, P7–P10).

Figure 4. Effects of different concentrations of Chloramphenicol (C) supplemented with vitamins on prostate hyperplasia cell cultures. (A, B) 214.50 µM (P1); (C, D) 859.33 µM (P2); (E, F) 5.37 mM (P3); (G, H) 859.33 µM C: 6.30 mM vitamin C (P7); (I, J) 5.37 mM C: 6.30 mM vitamin C (P8); (K, L) 859.33 µM C: 622 µM vitamin E (P9); (M, N) 5.37 mM C: 622 µM vitamin E (P10). (A, C, E, G, I, K, and M) Results are plotted by Hoechst stain to highlight nuclear shrinkage; (B, D, F, H, J, L, and N) Lysosomal activity represented by acridine orange stain; On graphs are extrapolated positive control as C2-prostate hyperplasia cells.

Figure 5. Effects of Erythromycin doses (E) supplemented with vitamin C in prostate hyperplasia cell cultures: P4: 18.83 µM E: 6.30 mM vitamin C; P5: 170.00 µM; P6: 1.51 mM; P12: 1.51 mM E: 6.30 mM vitamin C. (A, C, E, and G) Results are plotted by dual stain MR-DEVD/PI to highlight the caspases- 3/7 activity to observe the viability (V), early and late apoptosis (EA; LA), and necrosis (N). (B, D, F, H, I, and J) Cell apoptosis (P4–P6, P12) is represented by PI stain reported to control samples (C1, C2).

Figure 6. Influence of Erythromycin concentrations supplemented with vitamin C at prostate hyperplasia cell cultures. (A, B) 18.83 µM E: 6.30 mM vitamin C (P4); (C, D) 170.00 µM (P5); (E, F) 1.51 mM (P6); (G, H) 1.51 mM E: 6.30 mM vitamin C (P12); (A–H) Results are plotted by PI stain to highlight cell cycle; (B, D, F, and H) On graphs are extrapolated negative and positive controls as C1-normal prostate cells and C2-prostate hyperplasia cells.

Figure 7. Oxidative stress after applied Erythromycin doses supplemented with vitamin C in prostate hyperplasia cell cultures. (A) Statistical analysis. (B) 18.83 µM E: 6.30 mM vitamin C (P4); (C) 170.00 µM (P5); (D) 1.51 mM (P6); (E) 1.51 mM E: 6.30 mM vitamin C (P12); (F) On graph is extrapolated positive control as C1-prostate hyperplasia cells to samples (P6 and P12). **P < .01 and *P < .05 represent significant statistical differences between samples and control, made by independent t tests of the MedCalc program.

Figure 8. Effects of different concentrations of Erythromycin (E) supplemented with vitamin C on prostate hyperplasia cell cultures. P4: 18.83 µM E: 6.30 mM vitamin C; P5: 170.00 µM; P6: 1.51 mM; P12: 1.51 mM E: 6.30 mM vitamin C. (A, C, E, and G) Results are plotted by Hoechst stain to highlight nuclear shrinkage; (B, D, F, and H) Lysosomal activity represented by acridine orange stain. On graphs is extrapolated positive control as C2-prostate hyperplasia cells.

Figure 9. Effects of different Chloramphenicol concentrations (C) supplemented with antioxidants on prostate hyperplasia cell cultures to observe caspase-3/7 activity by MR -DEVD/PI stain: P1: 214.50 µM; P2: 859.33 µM; P3: 5.37 mM; P7: 859.33 µM C: 6.30 mM vitamin C; P8: 5.37 mM C: 6.30 mM vitamin C; P9: 859.33 µM C: 622 µM vitamin E; P10: 5.37 mM C: 622 µM vitamin E; C1-normal prostate cells (negative control) and C2-prostate hyperplasia cells (positive control). **P < .01 and *P < .05 represent significant statistical differences between samples and controls, made by independent t tests of the MedCalc program. EA = early apoptosis, LA = late apoptosis, N = necrosis, V = viability.

Figure 10. Cell cycle phases at prostate hyperplasia cell cultures after treatments with different doses of Chloramphenicol (C) supplemented with vitamins: P1: 214.50 µM; P2: 859.33 µM; P3: 5.37 mM; P7: 859.33 µM C: 6.30 mM vitamin C; P8: 5.37 mM C: 6.30 mM vitamin C; P9: 859.33 µM C: 622 µM vitamin E; P10: 5.37 mM C: 622 µM vitamin E; C1-normal prostate cells (negative control) and C2-prostate hyperplasia cells (positive control). **P < .01 and *P < .05 represent significant statistical differences between samples and controls, made by independent t tests of the MedCalc program.

Figure 11. Nuclear shrinkage and autophagy in prostate hyperplasia cell cultures after treatments with different doses of Chloramphenicol (C) supplemented with antioxidants: P1: 214.50 µM; P2: 859.33 µM; P3: 5.37 mM; P7: 859.33 µM C: 6.30 mM vitamin C; P8: 5.37 mM C: 6.30 mM vitamin C; P9: 859.33 µM C: 622 µM vitamin E; P10: 5.37 mM C: 622 µM vitamin E; C2-prostate hyperplasia cells (positive control); (A, B) HP or HN-nuclear shrinkage expressed positive or negative by Hoechst stain; (C, D) AN or AOP-autophagy expressed negative or positive with acridine orange stain; (E) AOHP- cell populations expressed double positive by acridine orange and Hoechst stain. **P < .01 and *P < .05 represent significant statistical differences between samples and control, made by independent t tests of the MedCalc program.

Figure 12. Action of Erythromycin concentrations (E) supplemented with vitamin C on prostate hyperplasia cell cultures to observe caspase-3/7 activity by MR-DEVD/PI stain: P4: 18.83 µM E: 6.30 mM vitamin C; P5: 170.00 µM; P6: 1.51 mM; P12: 1.51 mM E: 6.30 mM vitamin C. C1-normal prostate cells (negative control) and C2-prostate hyperplasia cells (positive control). **P < .01 and *P < .05 represent significant statistical differences between samples and controls, made by independent t tests of the MedCalc program. EA = early apoptosis, LA = late apoptosis, N = necrosis, V = viability.

Figure 13. Cell cycle phases in prostate hyperplasia cell cultures after treatments with different concentrations of Erythromycin (E) supplemented with vitamin C: P4: 18.83 µM E: 6.30 mM vitamin C; P5: 170.00 µM; P6: 1.51 mM; P12: 1.51 mM E: 6.30 mM vitamin C. C1-normal prostate cells (negative control) and C2-prostate hyperplasia cells (positive control). **P < .01 and *P < .05 represent significant statistical differences between samples and controls, made by independent t tests of the MedCalc program.

Figure 14. Nuclear shrinkage and autophagy at prostate hyperplasia cell cultures after treatments with different doses of Erythromycin (E) supplemented with vitamin C: P4: 18.83 µM E: 6.30 mM vitamin C; P5- 170.00 µM; P6- 1.51 mM; P12: 1.51 mM E: 6.30 mM vitamin C. (A, B) HP or HN-nuclear shrinkage expressed positive or negative by Hoechst stain; (C, D) AN or AOP-autophagy expressed negative or positive with acridine orange stain; (E) AOHP- cell populations expressed double positive by acridine orange and Hoechst stain; C2-prostate hyperplasia cells (positive control). **P < .01 and *P < .05 represent significant statistical differences between samples and controls, made by independent t tests of the MedCalc program.

3. Results

3.1. Effects of different doses of Chloramphenicol supplemented with antioxidants on prostate hyperplasia cell cultures

3.1.1. Caspase-3/7 activity

To observe the action of Chloramphenicol (C) and antioxidant doses in prostate primary hyperplasia cells reported to prostate hyperplasia cells (positive control) and normal prostate cells (negative control) was analyzed the effector caspase-3/7 intracellular activity by flow cytometry technique, being presented in Figures 1A–P and 9A–D.

After 24 hours of applied treatments with Chloramphenicol (214.50 µM; 859.33 µM; 5.37 mM) in prostate primary hyperplasia cells, it was observed that caspase-3/7 activation mechanism induces changes in cell viability with significant differences between samples and controls (P1: 15.05 ± 0.03; P2: 16.62 ± 0.83; P3: 17.43 ± 1.56 vs C1: 86.95 ± 5.76, P < .01 vs C2: 10.55 ± 1.26, P < .05, Figs. 1A–F and 9A).

When prostate cells were treated with 859.33 µM Chloramphenicol supplemented with 6.30 mM of vitamin C (P7), they showed the highest cell viability reported to positive control (P7: 24.40 ± 1.40 vs C2: 10.55 ± 1.26, P < .01, Figs. 1G, H, and 9A).

The lowest cell viability was observed in prostate cells treated with the highest concentration of Chloramphenicol (5.37 mM) supplemented with a dose of 622 µM vitamin E (P10: 8.93 ± 0.76 vs C1: 86.95 ± 5.76, P < .01, Figs. 1M, N, and 9A).

A biochemical cascade of reactions implied in the pro-apoptotic signal was observed in prostate hyperplasia cell cultures when was applied 6.30 mM of vitamin C as supplement to Chloramphenicol dose reported to positive control (859.33 µM; P7: 4.07 ± 0.40 vs C2: 3.03 ± 1.36, P > .05, Figs. 1G, H, O, P, and 9B).

In Figure 1G–J, O, and P were highlighted apoptosis mechanism actions in prostate hyperplasia cell cultures after applied treatments with 214.50 µM C: 6.30 mM vitamin C (P7) and 5.37 mM C: 6.30 mM vitamin C (P8). Our results suggest that some prostate hyperplasia cells may restore the functional cell apoptosis. Normal prostate cells present an apoptosis peak of 105 (C1), but the prostate hyperplasia cells adopt modified apoptosis with a peak of 106 (C2). When prostate hyperplasia cells were treated with Chloramphenicol and vitamin C doses (P7, P8), it was observed that they might induce a devancement of apoptosis peak from 106 to 105, like normal cell apoptosis (Fig. 1H, J, O, and P).

3.1.2. Oxidative stress

In prostate primary cell cultures, Chloramphenicol supplemented with antioxidant doses increase or decrease the oxidative cellular stress being analyzed by total ROS flow cytometry method. Our results are presented in Figure 2A–K.

A minimal dose of Chloramphenicol (P1: 214.50 µM) applied on primary cells determines a decrease of oxidative stress reported to positive control (11.00 ± 1.41 × 106 vs 46.00 ± 8.49 × 106, P < .05, Fig. 3A).

An interesting oxidative stress mechanism was observed when the Chloramphenicol dose was increased to 859.33 µM supplemented with 6.30 mM of vitamin C, because were quantified two types of cell populations. A part of prostate primary hyperplasia cells reacted on applied treatments by the highest peak of ROS (P7.2: 81.00 ± 1.41 × 107 vs C1: 46.00 ± 8.49 × 106, P < .01). In the meantime, another part of the cell’s rests to initial ROS (P7.1: 45.00 ± 7.07 × 106), remaining in refractory status being compared with positive control and primary cells treated with 859.33 µM of Chloramphenicol (C1: 46.00 ± 8.49 × 106; P2: 41.50 ± 2.12 × 106, P > .05, Fig. 2A, B, D, and H).

When the dose of Chloramphenicol was increased to 5.37 mM and keeping the same concentration of vitamin C (6.30 mM- P8), prostate primary cells reacted by decreasing the ROS level drastically (P8: 28.50 ± 2.12 × 105 vs P7.2: 81.00 ± 1.41 × 107, P < .01 vs C1: 46.00 ± 8.49 × 106, P < .05, Fig. 2A, D, and E), because the Chloramphenicol acts as pro-oxidant on cells and decrease the vitamin C antioxidant activity. Also, the ROS value remained lowest (P8) reported to prostate hyperplasia cultures treated with a dose of 5.37 mM Chloramphenicol (P3: 43.50 ± 2.12 × 106, P < .05, Fig. 2A, C, E, and I).

A similar mechanism of ROS action was observed when vitamin C was replaced with vitamin E doses. When were tested a lower concentration of antibiotic as 859.33 µM C to 622 µM vitamin E (P9), prostate primary cells reacted to increase the ROS reported to positive control (P9: 45.00 ± 7.07 × 107 vs 46.00 ± 8.49 × 106, P < .05, Fig. 2A, B, F, and J). Antioxidant activity of vitamin E was diminished when Chloramphenicol was increased to 5.37 mM to 622 µM vitamin E (P10), oxidative stress had a significantly lower value reported to control and cells treated only with antibiotic (P3: 5.37 mM; P10: 13.50 ± 2.12 × 106 vs C1: 46.00 ± 8.49 × 106 vs P3: 43.50 ± 2.12 × 106, P < .05, Fig. 2A, C, G, and K).

3.1.3. Cell cycle

The cell cycle was performed by propidium iodide (PI) stain to explore the effects of chloramphenicol doses supplemented with antibiotics on prostate primary hyperplasia cells (Figs. 3A–N and 10A–C).

As shown in Figures 3G, H, and 10A, in prostate primary cell cultures, Chloramphenicol supplemented with antioxidants (P7: 859.33 µM C: 6.30 mM vitamin C) induce a cell cycle arrest in G1/G0 phase (82.17 ± 2.57) than controls (C1: 68.67 ± 1.51, P < .05; C2: 0.41 ± 0.06, P < .01). DNA synthesis phase of the cell cycle presented a significantly lower value than positive control (9.05 ± 0.58 vs C2: 14.83 ± 0.37, P < .01, Figs. 3G, H, and 10B).

To understand whether the cell growth inhibition was due to cell cycle arrest, prostate hyperplasia primary cells after 24 hours treatments with different doses of Chloramphenicol (P1: 214.50 µM; P2: 859.33 µM; P3: 5.37 mM) presented significant increasing of G2/M phase than negative control (P1: 83.23 ± 0.64; P2: 79.61 ± 0.71; P3: 73.95 ± 0.42 vs C1: 14.65 ± 2.99, P < .01). DNA synthesis had significantly highest values reported to negative control (14.23 ± 0.17; 14.72 ± 0.94; 22.55 ± 1.68; vs 10.24 ± 0.43, P < .01, Figs. 3A–F and 10B). The last dose of antibiotic determines an increase in DNA synthesis (22.55 ± 1.68; vs 14.83 ± 0.37, P < .05, Figs. 3F and 10B) reported to positive control.

When the doses of Chloramphenicol (P8: 5.37 mM, P9: 859.33 µM, and P10: 5.37mM) were supplemented with antioxidants (P8: 6.30 mM of vitamin C; P9 and P10: 622 µM vitamin E) were observed changes in cell cycle phases, because the prostate cells were presented a cell cycle arrest in G2/M reported to negative control (P8: 78.80 ± 4.07; P9: 72.95 ± 3.44; P10: 89.91 ± 3.10 vs C1: 14.65 ± 2.99, P < .01, Figs. 3I, K, M, and 10C).

When the concentration of Chloramphenicol was increased to 5.37 mM (P8) and vitamin C dose remained to 6.30 mM, was observed significant changes in DNA synthesis compared to controls (P8: 20.58 ± 1.27 vs C1: 10.24 ± 0.43; P < .01; vs C2: 14.83 ± 0.37, P < .05, Figs. 3I, J, and 10B), because the Chloramphenicol acts as pro-oxidant on cells, decreasing the vitamin C antioxidant activity, leading to increasing of S-phase of cell cycle.

An interesting situation was observed when prostate hyperplasia cell cultures were treated with 859.33 µM Chloramphenicol and 622 µM vitamin E (P9) because it acts on cells as pro-oxidants leading to an increase in DNA synthesis compared with controls (P9: 29.52 ± 2.47 vs C1: 10.24 ± 0.43; P < .01; vs C2:14.83 ± 0.37, P < .05, Figs. 3K, L, and 10B).

Instead, when we tested on cells, a higher dose of Chloramphenicol (5.37 mM) supplemented with 622 µM vitamin E, S-phase of the cell cycle was diminished reported to a positive control (P10: 8.70 ± 0.72 vs C2: 14.83 ± 0.37, P < .01, Figs. 3M, N, and 10B), but the cells remained in cell cycle arrest in G2/M phase.

3.1.4. Nuclear shrinkage and lysosomal activity

The presence of pyknotic nuclei stained with Hoechst 33342 and the lysosomal activity by acridine orange (AO) stain after different treatments with antibiotics and antioxidants applied on prostate hyperplasia cell cultures were highlighted in Figures 4A–N and 11A–E.

Tested prostate cells with different doses of Chloramphenicol and vitamins (P1-P3; P7-P10) reported to a positive control (C2) were divided in nuclear shrinkage expressed positive or negative cells populations by Hoechst stain (HP or HN, Figs. 4A, C, E, G, I, K, M, 11A, B, and E). Similarly, autophagy-expressed negative or positive cell populations were highlighted by acridine orange stain (AN or AOP, Figs. 4B, D, F, H, J, L, N, and 11C–E).

A Chloramphenicol dose of 859.33 µM supplemented with 6.30 mM vitamin C showed significantly higher values of HP- nuclear shrinkage expressed than the control (P7: 79.66 ± 1.36 vs C2: 77.16 ± 4.59, P < .05, Figs. 4G and 11A).

While a high dose of 5.37 mM Chloramphenicol but supplemented with 622 µM vitamin E determined a significant HP-nuclear shrinkage decrease (P10: 44.10 ± 1.10 vs C2: 77.16 ± 4.59, P < .01, Figs. 4M and 11A) and increasing of negative cells populations by Hoechst stain (P10: 57.75 ± 1.94 vs C2: 22.19 ± 4.45, P < .01, Figs. 4M and 11B) reported to controlling values.

Concentrations of Chloramphenicol (214.50 µM; 859.33 µM; 5.37 mM) were diminished significantly the lysosomal activity of treated prostate cells than control (P1: 64.04 ± 0.26; P2: 66.38 ± 1.49; P3: 82.15 ± 0.30 vs C2: 92.08 ± 0.37, P < .01, Figs. 4b, D, F, and 11C). However, when we added in prostate cell cultures the vitamin C to supplement the antibiotic doses (P7: 859.33 µM C: 6.30 mM vitamin C; P8: 5.37 mM C: 6.30 mM vitamin C) were observed a slight increase in autophagy process than control (P7: 93.76 ± 1.99; P8: 94.01 ± 0.52 vs C2: 92.08 ± 0.37, P > .05, Figs. 4H, J, and 11C).

Another antioxidant, such as vitamin E combined with Chloramphenicol doses (P9: 859.33 µM C: 622 µM vitamin E; P10: 5.37 mM C: 622 µM vitamin E) applied on cells, induces changes in autophagy by positive cell populations presence (AOP) remained significantly lower than control (P9: 66.56 ± 0.81; P10: 74.94 ± 2.33 vs C2: 92.08 ± 0.37, P < .01, Figs. 4L, N, and 11C).

Small doses of Chloramphenicol (P1: 214.50 µM; P2: 859.33 µM) or supplemented with vitamin E (P9: 859.33 µM C: 622 µM vitamin E) determined an increase of negative cell populations expressed for autophagy (AN) reported to control cells (P1: 35.23 ± 2.14; P2: 29.02 ± 0.46; P9: 31.73 ± 1.24 vs C2: 7.25 ± 0.47, P < .01, Figs. 4B, D, L, and 11D).

A particular interest represents the cell populations expressed double positive by acridine orange and Hoechst dual stain (AOHP). Most valuable observations were highlighted when prostate primary hyperplasia cells were treated with antibiotic doses (859.33 µM; 5.37 mM) and vitamin C (6.30 mM) because induce changes in apoptosis program of hyperplasia cells by increasing the nuclear shrinkage and lysosomal activity reported to positive control (P7: 90.08 ± 1.80; P8: 92.05 ± 2.02 vs C2: 78.07 ± 4.19, P < .01, Fig. 11E).

3.2. Action of Erythromycin doses supplemented with vitamin C on prostate hyperplasia cell cultures

3.2.1. Caspase-3/7 activity

Effector caspase- 3/7 intracellular activity implied in cell apoptosis was presented in Figures 5A–J and 12A–D after applied treatments with Erythromycin and vitamin C doses in prostate hyperplasia cell cultures.

After 24 hours treatment of cultures, were observed changes in cell apoptosis mechanism in prostate primary hyperplasia cells. Different doses of Erythromycin supplemented with antioxidants (P4: 18.83 µM E: 6.30 mM vitamin C; P12: 1.51 mM E: 6.30 mM vitamin C) or not (P5: 170.00 µM; P6: 1.51 mM) showed significant differences on cell viability reported to a negative control (P4: 7.80 ± 0.78; P12: 13.74 ± 2.24; P5: 38.95 ± 2.45; P6: 30.55 ± 1.62 vs C1: 86.95 ± 5.76, P < .01, Figs. 5A–H and 12A).

Various concentrations of Erythromycin (P5: 170.00 µM; P6: 1.51 mM) induce significantly increased values of cell viability than positive control (P5: 38.95 ± 2.45; P6: 30.55 ± 1.62 vs C2: 10.55 ± 1.26, P < .01, Figs. 5C–F and 12A).

The lowest significant value of cell viability was observed in primary cells after treatment with 18.83 µM E: 6.30 mM vitamin C than positive control (P4: 7.80 ± 0.78 vs C2: 10.55 ± 1.26, P < .01, Figs. 5A, B, and 12A).

Because cell apoptosis is adapted to prostate hyperplasia cells’ necessities, the cells support various changes after treatments with antibiotics and antioxidants in their program. A higher dose of Erythromycin (1.51 mM) determinates a silent increase of early apoptosis but without significant differences reported to positive control cells (P6: 6.71 ± 0.82 vs C2: 3.03 ± 1.36, P > .05, Figs. 5E and 12B).

Concentrations of Erythromycin (18.83 µM; 1.51 mM) supplemented with 6.30 mM vitamin C induce changes in late apoptosis in prostate hyperplasia cell cultures, without significant differences reported to positive control (P4: 76.64 ± 5.23; P12: 77.81 ± 2.61 vs C2: 72.71 ± 5.52, P > .05, Figs. 5C, G, and 12C).

While a lower dose of Erythromycin (170.00 µM) determinates significantly decreasing values of late apoptosis than positive control cells (P5: 45.96 ± 1.26 vs C2: 72.71 ± 5.52, P < .05, Figs. 5C and 12C). Erythromycin doses (P4: 18.83 µM E: 6.30 mM vitamin C; P5: 170.00 µM) determine necrosis in prostate hyperplasia cells but without significant differences reported to controls (P4: 20.13 ± 0.33; P5: 17.01 ± 1.86 vs C1: 13.05 ± 5.76; C2: 13.75 ± 5.56, P > .05, Figs. 5A, C, and 12D).

Figure 5D and F showed an interesting pro-oxidant action of Erythromycin doses (P5: 170.00 µM; P6: 1.51 mM) alone in prostate hyperplasia cell cultures after 24 hours because induce changes in the apoptosis mechanism. Mainly, P6 determines a movement of its peak from 106 to 105, like apoptosis of normal cells. When added antioxidant (6.30 mM vitamin C) to 1.51 mM Erythromycin dose (P12), the activation of caspases-3/7 mechanism was diminished (Fig. 5H–J).

3.2.2. Cell cycle

The action of Erythromycin doses supplemented with vitamin C in prostate primary hyperplasia cells was presented in Figures 6A–H and 13A–C to analyze the cell cycle phases by propidium iodide stain.

In prostate primary hyperplasia cells, a higher dose of Erythromycin (1.51 mM) induces a cell cycle arrest in G1/G0 phase than controls (P6: 90.05 ± 0.25 vs C1: 68.67 ± 1.51; C2: 0.41 ± 0.06, P < .01, Figs. 6E, F, and 13A), being able to determine a significant decrease of the S-phase of cell cycle reported to positive control cells (P6: 9.00 ± 0.35 vs C2: 14.83 ± 0.37, P < .01, Figs. 6E, F, and 13B).

Different doses of Erythromycin (18.83 µM; 1.51 mM) supplemented with antioxidants (6.30 mM vitamin C) or not (P5: 170.00 µM) applied to prostate hyperplasia cell culture determine a cell growth inhibition with cell cycle arrest in G2/M phase reported to negative control (P4: 79.99 ± 0.47; P12: 86.17 ± 1.12; P5: 80.07 ± 0.40 vs C1: 14.65 ± 2.99, P < .01), as shown in Figures 6A–D, G, H, and 13C.

A lower dose of Erythromycin supplemented with antioxidant (P4: 18.83 µM E: 6.30 mM vitamin C) determines significant changes in DNA synthesis compared to controls (P4: 19.03 ± 0.86 vs C1: 10.24 ± 0.43; P < .01; vs C2: 14.83 ± 0.37, P < .05, Figs. 6A, B, and 13B). When the concentration of Erythromycin was increased to 1.51 mM supplemented with 6.30 mM vitamin C, the S-phase of the cell cycle was diminished, reported to controls (P12: 6.97 ± 0.09 vs C1: 10.24 ± 0.43; C2: 14.83 ± 0.37, P < .01), but the prostate cells remained in cell cycle arrest in G2/M phase (Figs. 6G, H, and 13B).

3.2.3. Oxidative stress

Our results about total ROS by flow cytometry technique are presented in Figure 7A–F, observing the effects of the Erythromycin concentrations supplemented with vitamin C in prostate primary hyperplasia cell cultures.

Lower doses of Erythromycin (P4: 18.83 µM E: 6.30 mM vitamin C; P5: 170.00 µM) applied in hyperplasia cells registered a silent decreased or increased oxidative stress reported to positive control (P4: 27.50 ± 3.54 × 106; P5: 57.50 ± 3.54 × 106 vs 46.00 ± 8.49 × 106, P > .05, Fig. 7A–C).

When the dose of Erythromycin was increased to 1.51 mM (P6), prostate primary cells reacted by increasing the ROS level (P6: 88.50 ± 2.12 × 106 vs C1: 46.00 ± 8.49 × 106, P < .05, Fig. 7A, D, and F), because the antibiotic may have pro-oxidant action on cells.

An interesting oxidative stress mechanism was observed when the 1.51 mM Erythromycin dose was supplemented with 6.30 mM vitamin C (P12) because two types of cell populations were quantified. A part of primary hyperplasia cells reacted on applied treatments and showed the highest peak of ROS (P12.2: 130.50 ± 0.71 × 107 vs C1: 46.00 ± 8.49 × 106, P < .01, Fig. 7A, E, and F). In the meantime, another part of the cells remained in refractory status by keeping an initial ROS-like positive control (P12.1: 42.50 ± 3.54 × 106 vs C1: 46.00 ± 8.49 × 106; P > .05, Fig. 12A, E, and F).

3.2.4. Nuclear shrinkage and lysosomal activity

After 24 hours of treatments with different Erythromycin doses and vitamin C applied in prostate hyperplasia cell cultures, the pyknosis and autophagy by dual stain (Hoechst 33342/ acridine orange) were highlighted in Figures 8A–H and 14A–E.

A lower dose of 170.00 µM Erythromycin showed significantly decreased values of HP-nuclear shrinkage expressed than control (P5: 58.97 ± 2.01 vs C2: 77.16 ± 4.59, P < .05, Figs. 8C and 14A). While a higher dose of 1.51 mM Erythromycin supplemented with 6.30 mM vitamin C determines a slighted HP-nuclear shrinkage increase (P12: 86.31 ± 1.10 vs C2: 77.16 ± 4.59, P > .05, Figs. 8G and 14A) and decreasing of negative cells populations by Hoechst stain (P12: 15.41 ± 1.37 vs C2: 22.19 ± 4.45, P > .05, Figs. 8G and 14B) reported to controlling values.

Concentrations of Erythromycin supplemented with vitamin C (P12: 1.51 mM E: 6.30 mM vitamin C) or not (P5: 170.00 µM) presented significantly diminished the lysosomal activity of treated prostate cells (AOP) reported to control (P12: 77.73 ± 1.15; P5: 79.60 ± 2.50 vs C2: 92.08 ± 0.37, P < .01, P < .05, Figs. 8D, H, and 14C). In the meantime, autophagy expressed negative cell populations (AN) after all treatments applied on cells were significantly higher values reported to control (P4: 11.08 ± 0.38; P5: 21.13 ± 0.69; P6: 11.21 ± 0.59; P12: 24.81 ± 2.92 vs C2: 7.25 ± 0.47, P < .05; P < .01; P < .05; Figs. 8B, D, F, H, and 14D).

A particular interest is represented by the cell populations expressed double positive by dual stain with Hoechst and acridine orange (AOHP), when prostate hyperplasia cells were treated with the higher dose of Erythromycin supplemented with vitamin C or not (P12: 1.51 mM E: 6.30 mM vitamin C; P6: 1.51 mM) showed silenced changes in apoptosis program of hyperplasia cells by increasing of nuclear shrinkage and lysosomal activity reported to the positive control (P12: 85.31 ± 2.98; P6: 90.21 ± 7.23 vs C2: 78.07 ± 4.19, P > .05, Figs. 14E).

4. Discussion

Clinical research is focused on chemical-based medicine that targets oncogenes or proteins involved in tumor-promoting pathways.[34,35] Tumor heterogeneity results in aggressive cancer phenotypes with acquired resistance. However, combining chemical treatment with adjuvant therapies that cause cellular structure and function perturbations may diminish the ability of cancer cells to resist at chemical treatment and lead to a less aggressive cancer phenotype.[36–38]

In a complex system like cancer cells, it is difficult to determine the cause-and-effect relationships in tumorigenesis to elucidate the factors that control cell processes in tumor progression. Interestingly, it is the emergent property of the pro-oxidant state of tumor cells because it provides a link between the metabolic and genetic theories of carcinogenesis. Warburg’s metabolic defect theory of carcinogenesis sustains that in cells, when respiration is damaged, glycolysis increases to compensate for it, determining the emergence of a cancerous phenotype.[39]

Cells metabolize oxygen and produce low levels of pro-oxidants as byproducts of the electron transport chain. The cell’s antioxidant capacity balances the pro-oxidants via the nicotinamide adenine dinucleotide phosphate and the glutathione/glutathione peroxidase/glutathione reductase system because the normal cell environment is predominantly reducing. Cancer cells have an increased pro-oxidant production and a diminished cellular antioxidant capacity, leading to an increased level of hydroperoxides which may determine a pro-oxidant environment reported to normal cells. Various studies showed that chemicals increase pro-oxidant production and act as promotors of carcinogenesis, stimulating cell proliferation during mitogenesis, generation of mutations, and genomic instability. The antioxidant enzymes and vitamins inhibit the promotion of carcinogenesis instability.[40–42] Various research highlighted the vitamins, natural extracts, and other types of antioxidant’s effects on pro-oxidant environment of the tumor cell.[33,34] A few studies have explored different natural compounds action in tumoral cell cultures to increase the pro-oxidant state determining cell death.[43–46] The pro-oxidant state of tumor cells due to higher concentrations of active oxygen, peroxides, and radicals determines neoplastic development. Pro-oxidant status can modulate the expression of pro-oxidant genes involved in cell growth and differentiation by epigenetic mechanisms or inducing alterations in DNA. Moderate levels of oxidative stress alter processes such as cell signaling, gene activation, cell growth and division, and cell death.[40,47]

Our study shows the effects of antibiotics (Chloramphenicol and Erythromycin), supplemented with antioxidants (vitamins C and E) in prostate hyperplasia primary cell cultures, exerted its antitumor activities through mechanisms including cell cycle blockage and cell death induction. Was identify cells death activation via ROS mechanisms: at higher levels, oxidative stress initiates cell death by autoschizis or necrosis, while at lower concentrations, it acts by a non-oxidative mechanism involving transcription factors and induces autoschizis and apoptosis.

Autoschizis is caspase-3-independent cell death characterized by exaggerated membrane damage and the progressive loss of cytoplasm through a series of self-excisions until the perikaryon consisted of an apparently intact, round nucleus surrounded by a thin rim of cytoplasm which contained damaged organelles.[40]

Necrosis is a form of cell injury that results in the premature death of cells in living tissue by autolysis. Necrosis is caused by factors external to the cell or tissue, which result in the unregulated digestion of cell components. In contrast, apoptosis is a naturally occurring programmed and targeted cause of cellular death.[48,49]

Flow cytometric analysis of the cell cycle and apoptosis as biomarkers recommend laboratory techniques as the efficient means of measurement for adenocarcinoma and hyperplasia prostate tissue samples,[50] being explored in the present study to establish the cell death, DNA damage, and oxidative stress linked mechanisms implied in prostate carcinogenesis.

In our study, a high concentration of Chloramphenicol (5.37 mM) acts in prostate hyperplasia cell cultures by increasing their pro-oxidants status with effects on apoptosis, nuclear shrinkage, autophagy, and DNA damage, but without ROS changes. A high or low doses of antioxidants (6.30 mM vitamin C, 622 µM vitamin E) added as supplements to 859.33 µM Chloramphenicol doses in hyperplasia cell cultures, determine an increasing ROS level for a part of cells, but another part of cells remained to initial ROS, in refractory status, with significant changes in apoptosis, autophagy, and cell cycle arrest in G0/G1 or G2/M. When the dose of Chloramphenicol was increased to 5.37 mM and keeping the same concentration of vitamin C (6.30 mM), it was observed that prostate primary cells reacted by decreasing the ROS level drastically, cell cycle arrest in G2/M phase, with active apoptosis and autophagy.

Chloramphenicol doses (10 µM–1 mM) inhibit mammo-sphere formation in MCF7 breast adenocarcinoma cell lines.[47] Chloramphenicol (100 μg/mL) inhibits hypoxia-inducible factor 1-alpha pathway and induces autophagy in lung squamous carcinoma cells (NSCLC) by increasing biomarkers as beclin-1, Atg12-Atg5 conjugates, and autophagosome-associated protein LC3-II.[51] Chloramphenicol inhibits mitochondrial protein synthesis. Chloramphenicol represses oxygen-labile transcription factor and hypoxia-inducible factor 1-alpha in hypoxic A549 and H1299 lung adenocarcinoma cell lines.[51–53]

In our study, a high Erythromycin dose (1.51 mM) in prostate hyperplasia cell cultures after 24 hours induces changes in the apoptosis mechanism and cell cycle arrest in G0/G1. When were added 6.30 mM vitamin C to 1.51 mM Erythromycin dose, caspases-3/7 mechanism activation was diminished and ROS level was increased for a part of the cells, but another part of the cells remains to initial ROS, in refractory status, similar with the action of Chloramphenicol, and cell cycle arrest in G2/M phase.

Erythromycin is a macrolide antibiotic produced by actinomycete Streptomyces erythreus with role in binding to bacterial 50S ribosomal subunits. It inhibits RNA-dependent protein synthesis by blockage of transpeptidation and/or translocation reactions without affecting the nucleic acid synthesis.[54] Erythromycin exhibits antitumor and neuroprotective effects in different cells, as reported by another study.[55] Doxycycline and Azithromycin showed therapeutic effects in cancer patients, although their selective effects on eradicating cancer stem cells. These trials were performed on advanced or treatment-resistant patients with B-cell lymphoma (Doxycycline) or lung cancer (Azithromycin).[56–60] In lung cancers, Azithromycin significantly increased 1-year patient survival from 45% to 75%.[59] Tetracyclines inhibit protein synthesis by preventing the binding of activated amino-acyl-tRNAs to the A-site on the 30S subunit of bacterial ribosomes. Molecular disruption of mitochondrial biogenesis represents a novel therapeutic strategy for eradicating CSCs-purposing the antibiotics for various cancer types, including pre-malignant and advanced metastatic disease.[47] Authors reported that a 400 µg/mL concentration of Tetracycline inhibits 50% of HeLa cell lines proliferation. Reduced mitochondrial activity in cells results by decreasing the changes of pyruvate to lactate via glycolysis. Mitochondria are responsible for oxidative phosphorylation and cell death activation.[61]

Other authors reported that antibiotics induce mitochondrial dysfunction and oxidative damage in mammary cell lines. They exposed the MCF-10A mammary epithelial cell line to Ampicillin (aβ-lactam), Ciprofloxacin (a fluoroquinolone), and Kanamycin (an aminoglycoside). All three antibiotics caused a dose-time-dependent increase in intracellular reactive oxygen species production.[28]

Antibiotics in MCF-12A non-tumorigenic epithelial cell line, MCF-7, and MDA-MB-231 mammary adenocarcinoma cell lines induce mitochondrial reactive oxygen species and DNA damage. The metabolic effects of antibiotics on mitochondrial function in non-tumorigenic MCF-12A, MCF-7, and MB-MDA-231 human mammary cancer cell lines, were different when were used antibiotic-free media versus antibiotic-containing media. Authors reported that tumorigenesis is caused by mitochondrial dysfunction,[62] and cancer cells presented defective respiration with increased glycolysis and lactate production even in oxygen presence.[30]

Researchers showed that Doxycycline enhances the sensitivity of glioblastoma to chemotherapy by mitochondrial dysfunction and oxidative damage induction.[25] Other studies have presented that Doxycycline decreased the tumor burden of human breast cancer in a bone metastasis model, while others showed that antibiotics could eradicate cancer stem cells across multiple tumor types by targeting mitochondria.[30,47]

On other hand, vitamin C is usually perceived as an antioxidant, but it may also act as a pro-oxidant and increase DNA damage.[63] If vitamin C is added to cells before oxidative stress, it may function as an antioxidant and reduce cytotoxicity and mutagenicity because of its ability to scavenge free radicals and induce G2/M cell cycle arrest, which allows DNA repair to occur. However, if vitamin C is added during oxidative stress, it may act as a pro-oxidant and increase the cytotoxic and mutagenic effects via two mechanisms: increased intracellular dehydroascorbic acid (DHA) levels and DNA damage. For a long time, vitamin C was proposed to prevent and treat cancer because of its antioxidant properties. The mechanisms responsible for the antitumor activity of vitamin C is related to the pro-oxidant properties of ascorbic acid and dehydroascorbic acid. The oxidative product of ascorbic acid which generates reactive oxygen species (ROS) may deplete cellular thiol levels and initiate membrane lipid peroxidation. Antitumor activity of ascorbic acid leads to cell death activation. Vitamin C administered in androgen-dependent prostate cancer cell line (LNCaP) and androgen-independent prostate cancer cell line (DU145), determined in function of dose and time decreasing in cell viability and thymidine incorporation into DNA.[40]

Other studies reported that vitamin C is an endogenous antioxidant agent that inhibits Ca channels selectively, exhibits anti-cancer effects through the generation of ROS, and selective damage to cancer cells.[64,65] Vitamin C (0.1 μM–2 mM) exhibits anti-cancer effects according to SVCT-2 expression and their uptake on human colorectal cancer cell lines.[66] Oxidative stress influences the cellular processes involved in initiating, promoting, and progressing of prostate cancer. Oxidative stress is a mechanism that triggers the reactions chain involved in the prostatic hyperplasia progression. A high ROS level causes a significant decrease in antioxidant defense mechanisms, leading to DNA damage, disruption of cellular functions, and cell death, but at a lower level, it induces subtle signaling pathway changes.[67] Studies reported that oxidative stress was correlated with benign prostatic hyperplasia and prostate cancer progression, and therapy response.[68–71] Antioxidants protect cells against ROS, with a role in prostate cancer prevention. A decreasing antioxidant plasma levels were observed in prostate cancer patients reported to healthy patients.[72,73] High levels of ROS and DNA damage suggest that oxidative stress plays an important role in prostate tumorigenesis. Oxidative stress promotes castration resistance via an androgen receptor (AR) – dependent pathway, leading to castration-resistant prostate cancer. In androgen deprivation therapy, a lower basal ROS level in prostate cancer may sensitize the prostate tumoral cells to radiation.[74]

Exposure of cells to low level of oxidative stress induced by hydrogen peroxide leading to growth arrest without the loss of metabolic capacity, membrane disruption, DNA oxidation, apoptosis, or necrosis. Flow cytometry revealed that cells in G1 at hydrogen peroxide exposure were arrested in G0/G1, while cells in the S phase completed DNA synthesis and were subsequently arrested in G2/M. Hydrogen peroxide-induced cell cycle arrest has been shown to sensitize tumor cells to chemotherapeutic agents and radiation.[40]

Other researchers highlighted the anti-tumoral effects of the antibiotics in animal labs by in vivo studies. Various studies about Erythromycin (gastric intubation, 0.1–50 mg/kg; 30–120 days) decreases tumor growth and prolongs the survival time of mice from a dose of 5 mg/kg in mice. Erythromycin (gastric intubation; 5 mg/kg) protects mice alive even at 120 days after inoculation but shortens mean survival time in tumor-bearing mice by 4 to 5 days with a dose of 50 mg/kg.[55]

In our study, applied antibiotic-antioxidant doses were used to target autophagy-apoptosis (Chloramphenicol), DNA damage (Erythromycin), and oxidative stress-apoptosis (vitamins). Our obtained results showed in prostate primary hyperplasia cells with phenotypic heterogeneity developed from small tissue fragments in a glycolytic substrate for cell metabolism may be extrapolated and tested in future experiments on references prostate carcinoma cell lines. Because antibiotics were used as mitochondrial stressors and oxidative stress represents support to apoptosis mechanisms linked with DNA damage, future experiments may be supplemented with new flow cytometry techniques such as mitochondrial membrane potential and initial caspase 9.

5. Conclusions

In our study were applied different antibiotic and antioxidant doses on BPH primary cell cultures with phenotypic heterogeneity to observe DNA damage and cell death mechanisms via oxidative stress. Were identify 2 cell death mechanisms linked by ROS: at higher levels, it seems to act via oxidative stress to initiate cell death by autoschizis or necrosis, while at lower concentrations, it acts by a non-oxidative mechanism that appears to involve transcription factors and induces autoschizis and apoptosis. In BHP patients, our experimental observations may have therapeutic value being implied in anti-cancer integrative therapy developing.

Acknowledgments

Experimental observations were made in the Cell Biology Department, Center for Research and Development of the Morphological and Genetic Studies of Malignant Pathology, Ovidius University of Constanta, CEDMOG, 145 Tomis Blvd., 900591, Constanta, Romania. Media and kits used for cell apoptosis, oxidative stress, and cell cycle determinations in prostate hyperplasia cell cultures were assured from the European Social Fund under the contract number 36355/23.05.2019 HRD OP/380/6/13.

Author contributions

Conceptualization: Elena Matei, Anita Cristina Ionescu, Manuela Enciu, Violeta Popovici, Anca Florentina Mitroi, Mariana Aschie, Georgeta Camelia Cozaru.

Data curation: Elena Matei, Manuela Enciu, Miruna Cristian.

Formal analysis: Elena Matei, Manuela Enciu.

Funding acquisition: Elena Matei, Manuela Enciu, Mihai Cătălin Roșu, Georgeta Camelia Cozaru.

Investigation: Elena Matei, Violeta Popovici, Mihai Cătălin Roșu.

Methodology: Elena Matei, Anita Cristina Ionescu, Manuela Enciu, Gabriela Isabela Băltățescu, Antonela-Anca Nicolau, Mihai Cătălin Roșu, Miruna Cristian.

Project administration: Elena Matei, Violeta Popovici, Georgeta Camelia Cozaru.

Resources: Elena Matei, Manuela Enciu, Violeta Popovici, Mariana Aschie, Mariana Deacu.

Software: Elena Matei, Nicolae Dobrin, Constanța Ștefanov.

Supervision: Anca Florentina Mitroi, Mariana Aschie, Mariana Deacu, Georgeta Camelia Cozaru.

Validation: Elena Matei, Mariana Aschie, Georgeta Camelia Cozaru.

Visualization: Manuela Enciu, Anca Florentina Mitroi, Gabriela Isabela Băltățescu, Antonela-Anca Nicolau, Georgeta Camelia Cozaru.

Writing – original draft: Elena Matei, Manuela Enciu, Mihaela Pundiche Butcaru, Georgeta Camelia Cozaru.

Writing – review & editing: Elena Matei, Manuela Enciu, Violeta Popovici, Mariana Aschie, Mihaela Pundiche Butcaru, Georgeta Camelia Cozaru.

Abbreviations:

BPH benign prostatic hyperplasia

DNA deoxyribonucleic acid

MR-DEVD Magic Red Caspase Detection kit

ROS total reactive oxygen species

Informed consent was obtained from all subjects involved in the study.

The authors have no funding and conflicts of interest to disclose.

All data generated or analyzed during this study are included in this published article [and its supplementary information files].

How to cite this article: Matei E, Ionescu AC, Enciu M, Popovici V, Mitroi AF, Aschie M, Deacu M, Băltățescu GI, Nicolau A-A, Roșu MC, Cristian M, Dobrin N, Ștefanov C, Pundiche Butcaru M, Cozaru GC. Cell death and DNA damage via ROS mechanisms after applied antibiotics and antioxidants doses in prostate hyperplasia primary cell cultures. Medicine 2024;103:37(e39450).
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