Plant-derived antimicrobials have similar cytotoxic effects on cancerous and non-cancerous 1 cells 2

Essential oils and their active components, referred here as plant derived antimicrobials (PDAs), 16 have been used for their antimicrobial, anti-inflammatory and antioxidant properties. Many 17 reports also document PDAs’ cytotoxic effects on cancerous cells, raising the hope that they 18 could be used for cancer treatments. Due to the lack of specificity, we hypothesize that PDAs are 19 cytotoxic to both cancerous and non-cancerous cells. Trans -cinnamaldehyde (TCA), carvacrol, 20 and eugenol were assessed for their cytotoxicity on cancerous HeLa cells and normal skin 21 fibroblasts (CCD-1123Sk, CCD) by MTT and LDH assays, flow cytometry, and reverse 22 transcription quantitative PCR (RT-qPCR). After 24 hours of treatment, carvacrol and TCA 23 significantly decreased cell viability (by more than 50%) at 100 µg/ml, whereas eugenol was 24 ineffective up to 400 µg/ml. Cell detachment and significantly increased apoptosis were 25 observed with 100 µg/ml of TCA on both cell types. RT-qPCR for apoptotic genes ( Bcl2, Casp3 26 and Casp8 ) and necrosis genes ( Mlkl, Ripk1 and Ripk3 ) did not show significant differences 27 between control and treated cells of both types, with the exception of eugenol-treated HeLa cells 28 in which expression of Bcl2, Mlkl and Ripk1 was significantly higher than controls. Taken together, we conclude that the three PDAs studied here exhibited similar cytotoxic effects on 30 both cancerous and non-cancerous cells.


Introduction
Essential oils have been used for centuries around the world in foods, cosmetics and as 35 antimicrobials and pharmaceuticals to prevent and treat diseases 1 . The bioactive constituents of 36 essential oils are secondary plant metabolites and their derivatives 2 . An active ingredient of 37 cinnamon oil, for example, is cinnamaldehyde, which predominately occurs in the trans form, Despite extensive reports that PDAs are anti-cancerous, virtually all studies lacked non-57 cancerous cell lines as the control, leading to the speculation that PDAs are specifically anti-58 cancerous. We hypothesize conversely that, due to common mechanisms of cytotoxicity and the 59 lack of a specific targeting pathway, PDAs should have the same effects on non-cancerous cells 60 as on cancerous cells. Here we directly compared cytotoxicity of three PDAs (TCA, carvacrol 61 and eugenol) on cancerous cells (HeLa) and non-cancerous fibroblast cells (CCD-1123Sk; CCD) 62 to support our hypothesis.    Plates were incubated for 10 minutes at 37°C, wells subjected to additional mixing and removal 93 of air bubbles, and absorbance was read at 540 nm using a plate reader (BioTek Synergy 2). with the exception of cell number (5 X 10 3 cells/well) for the optimal functioning of the assay.   60˚C for 1 min and 95˚C for 15s, 60˚C for 15s). The primers (Table S1)   the maximum dose tested at 800 µg/ml, where metabolic activity was reduced to approximately 166 30% of that of untreated controls. 167 Similar to TCA, carvacrol also reduced metabolic activity on both cell types in a dose-dependent 168 manner. Significant differences were observed at 100 µg/ml with viability declining to 40.8% 169 and 59.9% of controls for HeLa and CCD cells, respectively (P<0.05; Figure 1b). Finally, 170 eugenol appeared to be less effective than TCA and carvacrol, and did not exert a significant 171 effect on the metabolic activity up to 400 µg/ml (P<0.05; Figure 1c), when metabolic activity 172 was reduced to 30.8% and 41.34% of controls for HeLa and CCD cells, respectively.

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Interestingly, while not significant, both CAR and EU increased cell viability at low levels (12.5 174 and 25 µg/ml). This is potentially due to the anti-inflammatory effect of these PDAs, while 175 higher levels of PDAs became cytotoxic.   The results on carvacrol and eugenol are consistent with those from the MTT assay.    . We also showed that apoptosis was involved in 255 these processes.

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Out of the three compounds studied, TCA and carvacrol appeared to be more effective than 257 eugenol. TCA and carvacrol significantly induced a reduction in cell viability at a much lower 258 concentration (100 µg/ml) than eugenol (400 µg/ml) as shown by the MTT assays. Carvacrol 259 and eugenol maintained these effective and significant doses in inducing cytotoxicity as 260 determined by the LDH assays. TCA, however, did not cause cytotoxicity until 200 µg/ml. It is 261 possible that TCA was more effective in inhibiting cell viability than inducing cytotoxicity.

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However, from the large variation of the data, we believed that TCA may somehow interfere 263 with the LDH assay. This assumption was supported by previous findings that pre-treatment of 264 cinnamaldehyde decreased LDH activities in rats 25 .

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The MTT assay measures the metabolic activity of mitochondria through the action of succinate cells. We believe that this may be caused by the specific doses of PDAs used in our study.

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Although we chose levels that produced statistical changes in cell viability and cytotoxicity, it was discovered that mitochondrial electron transport was necessary to drive de novo 331 pyrimidine synthesis to overcome cell-cycle arrest 33 . While many cancer cells do indeed employ 332 oxidative phosphorylation, ATP generation was found dispensable for tumorigenesis 33  We conclude that although TCA, carvacrol and eugenol have been reported to be anti-cancerous, 340 they exert similar effects on a non-cancerous fibroblast cell line in regards to viability, 341 cytotoxicity and mechanism of death as they do in the HeLa cell model.