A Diet Lacking Selenium, but Not Zinc, Copper or Manganese, Induces Anticancer Activity in Mice with Metastatic Cancers.

Selenium, zinc, copper, and manganese are essential components of antioxidant enzymes involved in the elimination of reactive oxygen species (ROS). Given that cancer cells produce high levels of ROS and the accumulation of ROS can lead to cell death, cancer cells may be susceptible to strategies that reduce ROS elimination. In this work, we prepared several artificial diets that contained normal carbohydrate, protein, and lipid levels but lacked selenium, zinc, copper, or manganese. The anticancer activity of these diets was examined in a metastatic ovarian cancer model, established by injecting ID8 Trp53-/- murine ovarian cancer cells into the peritoneal cavity of C57BL/6JRj mice. Treatments started 15 days later and consisted of replacing a normal diet with one of the artificial diets for several weeks. A significant improvement in mice survival was observed when the normal diet was replaced with the selenium-free diet. Diets lacking zinc, copper, or manganese showed no significant impact on mice survival. All diets were very well tolerated. The anticancer efficacy of a diet lacking selenium was confirmed in mice with metastatic colon cancer and in mice with metastatic triple-negative breast cancer. These results suggest that diets lacking selenium hold potential for the treatment of metastatic cancers.

Repurposing Study of 4-Acyl-1-phenylaminocarbonyl-2-substituted-piperazine Derivatives as Potential Anticancer Agents-In Vitro Evaluation against Breast Cancer Cells.

Breast cancer is the most common type of cancer in women. Although current treatments can increase patient survival, they are rarely curative when the disease is advanced (metastasis). Therefore, there is an urgent need to develop new cytotoxic drugs with a high selectivity toward cancer cells. Since repurposing approved drugs for cancer therapy has been a successful strategy in recent years, in this study, we screened a library of antiviral piperazine-derived compounds as anticancer agents. The compounds included a piperazine ring and aryl urea functions, which are privileged structures present in several anti-breast cancer drugs. The selective cytotoxic activity of a set of thirty-four 4-acyl-2-substituted piperazine urea derivatives against MCF7 breast cancer cells and MCF 10A normal breast cells was determined. Compounds 31, 32, 35, and 37 showed high selective anticancer activity against breast cancer cells and were also tested against another common type of cancer, non-small cell lung cancer (A549 lung cancer cells versus MRC-5 lung normal cells). Compounds 35 and 37 also showed selectivity against lung cancer cells. These results suggest that compounds 35 and 37 may be promising hit compounds for the development of new anticancer agents.

Evaluation of Anticancer Activity of 76 Plant Species Collected in Andalusia (Spain) against Lung Cancer Cells.

Every year, cancer kills millions of people around the world. Finding more selective anticancer agents is essential to improve the low survival rates of patients with metastatic cancers. Since the research of natural products is a valuable approach to the discovery of new compounds and the Iberian flora offers a rich source of unstudied plants, we have carried out a random screening of 76 plant species from 43 families collected in Andalusia (South of Spain). Using non-malignant cells (HaCaT) and lung cancer cells (A549), we found that the extract from Arum italicum Mill. subsp. italicum (Araceae), Mandragora autumnalis Bertol. (Solanaceae), Rhamnus alaternus L. (Rhamnaceae), and Lomelosia simplex (Desf.) Raf. subsp. dentata (Jord. & Fourr.) Greuter & Burdet (Dipsacaceae) showed selective cytotoxicity against lung cancer cells. Extracts of plant species belonging to the Iridaceae family showed high selective activity against cancer cells, highlighting that the Xiphion xiphium (L.) M.B. Crespo, Mart.-Azorín & Mavrodiev flower extract was more selective against lung cancer cells than the standard anticancer drugs, cisplatin and 5-fluorouracil. This extract also showed modest selective cytotoxicity against bladder carcinoma cells (T24). The number of cells in the G1 phase increased after treatment with the extract from Xiphion xiphium. Our research indicates that various plants are potential sources for the isolation and development of new anticancer drugs.

Artificial Diets with Altered Levels of Sulfur Amino Acids Induce Anticancer Activity in Mice with Metastatic Colon Cancer, Ovarian Cancer and Renal Cell Carcinoma.

Sulfur-containing amino acids methionine (Met), cysteine (Cys) and taurine (Tau) are common dietary constituents with important cellular roles. Met restriction is already known to exert in vivo anticancer activity. However, since Met is a precursor of Cys and Cys produces Tau, the role of Cys and Tau in the anticancer activity of Met-restricted diets is poorly understood. In this work, we screened the in vivo anticancer activity of several Met-deficient artificial diets supplemented with Cys, Tau or both. Diet B1 (6% casein, 2.5% leucine, 0.2% Cys and 1% lipids) and diet B2B (6% casein, 5% glutamine, 2.5% leucine, 0.2% Tau and 1% lipids) showed the highest activity and were selected for further studies. Both diets induced marked anticancer activity in two animal models of metastatic colon cancer, which were established by injecting CT26.WT murine colon cancer cells in the tail vein or peritoneum of immunocompetent BALB/cAnNRj mice. Diets B1 and B2B also increased survival of mice with disseminated ovarian cancer (intraperitoneal ID8 Tp53-/- cells in C57BL/6JRj mice) and renal cell carcinoma (intraperitoneal Renca cells in BALB/cAnNRj mice). The high activity of diet B1 in mice with metastatic colon cancer may be useful in colon cancer therapy.

Artificial Diets with Selective Restriction of Amino Acids and Very Low Levels of Lipids Induce Anticancer Activity in Mice with Metastatic Triple-Negative Breast Cancer.

Patients with metastatic triple negative breast cancer (TNBC) need new therapies to improve the low survival rates achieved with standard treatments. In this work, we show for the first time that the survival of mice with metastatic TNBC can be markedly increased by replacing their normal diet with artificial diets in which the levels of amino acids (AAs) and lipids are strongly manipulated. After observing selective anticancer activity in vitro, we prepared five artificial diets and evaluated their anticancer activity in a challenging model of metastatic TNBC. The model was established by injecting 4T1 murine TNBC cells into the tail vein of immunocompetent BALB/cAnNRj mice. First-line drugs doxorubicin and capecitabine were also tested in this model. AA manipulation led to modest improvements in mice survival when the levels of lipids were normal. Reducing lipid levels to 1% markedly improved the activity of several diets with different AA content. Some mice fed the artificial diets as monotherapy lived much longer than mice treated with doxorubicin and capecitabine. An artificial diet without 10 non-essential AAs, with reduced levels of essential AAs, and with 1% lipids improved the survival not only of mice with TNBC but also of mice with other types of metastatic cancers.

Manipulation of Amino Acid Levels with Artificial Diets Induces a Marked Anticancer Activity in Mice with Renal Cell Carcinoma.

Targeted therapies with antiangiogenic drugs (e.g., sunitinib) and immune checkpoint inhibitors (e.g., anti-PD-1 antibodies) are the standard of care for patients with metastatic renal cell carcinoma. Although these treatments improve patient survival, they are rarely curative. We previously hypothesized that advanced cancers might be treated without drugs by using artificial diets in which the levels of specific amino acids (AAs) are manipulated. In this work, after showing that AA manipulation induces selective anticancer activity in renal cell carcinoma cells in vitro, we screened 18 artificial diets for anticancer activity in a challenging animal model of renal cell carcinoma. The model was established by injecting murine renal cell carcinoma (Renca) cells into the peritoneum of immunocompetent BALB/cAnNRj mice. Mice survival was markedly improved when their normal diet was replaced with our artificial diets. Mice fed a diet lacking six AAs (diet T2) lived longer than mice treated with sunitinib or anti-PD-1 immunotherapy; several animals lived very long or were cured. Controlling the levels of several AAs (e.g., cysteine, methionine, and leucine) and lipids was important for the anticancer activity of the diets. Additional studies are needed to further evaluate the therapeutic potential and mechanism of action of this simple and inexpensive anticancer strategy.

Overexpressed c-Myc Sensitizes Cells to TH1579, a Mitotic Arrest and Oxidative DNA Damage Inducer.

Previously, we reported that MTH1 inhibitors TH588 and TH1579 selectively induce oxidative damage and kill Ras-expressing or -transforming cancer cells, as compared to non-transforming immortalized or primary cells. While this explains the impressive anti-cancer properties of the compounds, the molecular mechanism remains elusive. Several oncogenes induce replication stress, resulting in under replicated DNA and replication continuing into mitosis, where TH588 and TH1579 treatment causes toxicity and incorporation of oxidative damage. Hence, we hypothesized that oncogene-induced replication stress explains the cancer selectivity. To test this, we overexpressed c-Myc in human epithelial kidney cells (HA1EB), resulting in increased proliferation, polyploidy and replication stress. TH588 and TH1579 selectively kill c-Myc overexpressing clones, enforcing the cancer cell selective killing of these compounds. Moreover, the toxicity of TH588 and TH1579 in c-Myc overexpressing cells is rescued by transcription, proteasome or CDK1 inhibitors, but not by nucleoside supplementation. We conclude that the molecular toxicological mechanisms of how TH588 and TH1579 kill c-Myc overexpressing cells have several components and involve MTH1-independent proteasomal degradation of c-Myc itself, c-Myc-driven transcription and CDK activation.

Artificial Diets Based on Selective Amino Acid Restriction versus Capecitabine in Mice with Metastatic Colon Cancer.

New therapies are needed to improve the low survival rates of patients with metastatic colon cancer. Evidence suggests that amino acid (AA) restriction can be used to target the altered metabolism of cancer cells. In this work, we evaluated the therapeutic potential of selective AA restriction in colon cancer. After observing anticancer activity in vitro, we prepared several artificial diets and evaluated their anticancer activity in two challenging animal models of metastatic colon cancer. These models were established by injecting CT26.WT murine colon cancer cells in the peritoneum (peritoneal dissemination) or in the tail vein (pulmonary metastases) of immunocompetent BALB/cAnNRj mice. Capecitabine, which is a first-line treatment for patients with metastatic colon cancer, was also evaluated in these models. Mice fed diet TC1 (a diet lacking 10 AAs) and diet TC5 (a diet with 6% casein, 5% glutamine, and 2.5% leucine) lived longer than untreated mice in both models; several mice survived the treatment. Diet TC5 was better than several cycles of capecitabine in both cancer models. Cysteine supplementation blocked the activity of diets TC1 and TC5, but cysteine restriction was not sufficient for activity. Our results indicated that artificial diets based on selective AA restriction have therapeutic potential for colon cancer.

Chirality influence on the cytotoxic properties of anionic chiral bis(N-heterocyclic carbene)silver complexes.

Complexes Na3[Ag(NHCR)2], 2a-e and 2b'-c', where NHCR is a N-heterocyclic carbene of the 2,2'-(1H-2λ3,3λ4-imidazole-1,3-diyl)dicarboxylate type, were prepared by treatment of compounds HLR, 1a-e and 1b'-c' (2-(1-(carboxyalkyl)-1H-imidazol-3-ium-3-yl)carboxylate), with silver oxide in the presence of aqueous sodium hydroxide. They were characterized by analytical, spectroscopic (infrared, IR, 1H and 13C nuclear magnetic resonance, NMR, and circular dichroism) and X-ray methods (2a). In the solid state, the anionic part of complex 2a, [Ag(NHCH)2]3-, shows a linear disposition of Ccarbene-Ag-Ccarbene atoms and an eclipsed conformation of the two NHC ligands. The proposed bis(NHC) nature of the silver complexes was maintained in solution according to NMR and density functional theory (DFT) calculations. The cytotoxic activity of compounds 2 was evaluated against four cancer cell lines and one non-cancerous cell line and several structure-activity correlations were found for these complexes. For instance, the activity decreased when the bulkiness of the R alkyl group in Na3[Ag(NHCR)2] increased. More interesting is the detected chirality-anticancer relationship, where complexes Na3[Ag{(S,S)-NHCR}2] (R = Me, 2b; iPr, 2c) showed better anticancer activity than those of their enantiomeric derivatives Na3[Ag{(R,R)-NHCR}2] (R = Me, 2b'; iPr, 2c').

pH-temperature dual-sensitive nucleolipid-containing stealth liposomes anchored with PEGylated AuNPs for triggering delivery of doxorubicin.

Liposomes (Lip) are useful nanocarriers for drug delivery and cancer nanomedicine because of their ability to efficiently encapsulate drugs with different physical and chemical properties. The pH gradient between normal and tumoral tissues, and their rapid metabolism that induces hyperthermia encourage the development of pH- and thermo-sensitive Lip for delivering anticancer drugs. Nucleolipids have been studied as scaffolding material to prepare Lip, mainly for cancer therapy. Herein, we report for the first time the use of 1,2-dipalmitoyl-sn-glycero-3-(cytidine diphosphate) (DG-CDP) to develop pH/thermo-sensitive nucleolipid-containing stealth Lip stabilized by combination with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol, anchored with NH2-PEGylated gold nanoparticles (PEG-AuNPs, 15 nm) for triggering delivery of doxorubicin (Dox). The optimal composition of DPPC, DG-CDP and cholesterol (94:3:3) was established by Langmuir isotherms. Unloaded and Dox-loaded Lip and AuNPs-Lip exhibited nano-scale sizes (415-650 nm), acceptable polydispersity indexes (<0.33), spherical shapes, and negative Z-potential (-23 to -6.6 mV) due to the phosphate groups of DG-CDP, which allowed the anchoring with positively charged AuNPs. High EE% were achieved (>78%) and although efficient control in the Dox release towards different receptor media was observed, the release of Dox from PEG-AuNPs-Lip-Dox was significantly triggered at acidic pH and hyperthermia conditions, demonstrating its responsiveness to both stimuli. Dox-loaded Lip showed high cytotoxic activity against MDA-MB-231 breast cancer cells and SK-OV-3 ovarian cancer cells, suggesting that Dox was released from these nanocarriers over time. Overall, the liposomal formulations showed promising properties as stimuli-responsive nanocarriers for cancer nanomedicine, with prospects for hyperthermia therapy.

Screening for Selective Anticancer Activity of 65 Extracts of Plants Collected in Western Andalusia, Spain.

Finding cytotoxic drugs with a high selectivity towards cancer cells is crucial to improve the low survival rates of patients diagnosed with metastatic cancers. Since plants are an important source of anticancer drugs, we have screened 65 extracts from 45 plants collected in several areas of Western Andalusia (Spain) for cytotoxic activity on lung cancer cells versus lung normal cells. An extract from the leaves of Tetraclinis articulata (Vahl) Mast. (Cupressaceae) showed a marked cytotoxicity (IC50 = 0.37 ± 0.03 µg/mL) and selectivity (selectivity index = 378.3) against the lung cancer cells; cisplatin, 5-fluorouracil, and an extract from the leaves of Taxus baccata L. (Taxaceae) were less cytotoxic and selective. Extracts from Cascabela thevetia (L.) Lippold (Apocynaceae), Frangula alnus Mill. (Rhamnaceae), Iberis ciliata subsp. contracta (Pers.) Moreno (Brassicaceae), Juniperus macrocarpa Sm (Cupressaceae), and Pancratium maritimum L. (Amaryllidaceae) also showed selective cytotoxicity (selectivity index > 10). Active extracts were also tested against a panel of cancer cell lines from a variety tissues. The plants identified in this work are potential sources of natural compounds with selective toxicity towards cancer cells.

MTH1 Inhibitor TH1579 Induces Oxidative DNA Damage and Mitotic Arrest in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy, exhibiting high levels of reactive oxygen species (ROS). ROS levels have been suggested to drive leukemogenesis and is thus a potential novel target for treating AML. MTH1 prevents incorporation of oxidized nucleotides into the DNA to maintain genome integrity and is upregulated in many cancers. Here we demonstrate that hematologic cancers are highly sensitive to MTH1 inhibitor TH1579 (karonudib). A functional precision medicine ex vivo screen in primary AML bone marrow samples demonstrated a broad response profile of TH1579, independent of the genomic alteration of AML, resembling the response profile of the standard-of-care treatments cytarabine and doxorubicin. Furthermore, TH1579 killed primary human AML blast cells (CD45+) as well as chemotherapy resistance leukemic stem cells (CD45+Lin-CD34+CD38-), which are often responsible for AML progression. TH1579 killed AML cells by causing mitotic arrest, elevating intracellular ROS levels, and enhancing oxidative DNA damage. TH1579 showed a significant therapeutic window, was well tolerated in animals, and could be combined with standard-of-care treatments to further improve efficacy. TH1579 significantly improved survival in two different AML disease models in vivo. In conclusion, the preclinical data presented here support that TH1579 is a promising novel anticancer agent for AML, providing a rationale to investigate the clinical usefulness of TH1579 in AML in an ongoing clinical phase I trial. SIGNIFICANCE: The MTH1 inhibitor TH1579 is a potential novel AML treatment, targeting both blasts and the pivotal leukemic stem cells while sparing normal bone marrow cells.

Cholesterol Levels Affect the Performance of AuNPs-Decorated Thermo-Sensitive Liposomes as Nanocarriers for Controlled Doxorubicin Delivery.

Stimulus-responsive liposomes (L) for triggering drug release to the target site are particularly useful in cancer therapy. This research was focused on the evaluation of the effects of cholesterol levels in the performance of gold nanoparticles (AuNPs)-functionalized L for controlled doxorubicin (D) delivery. Their interfacial and morphological properties, drug release behavior against temperature changes and cytotoxic activity against breast and ovarian cancer cells were studied. Langmuir isotherms were performed to identify the most stable combination of lipid components. Two mole fractions of cholesterol (3.35 mol% and 40 mol%, L1 and L2 series, respectively) were evaluated. Thin-film hydration and transmembrane pH-gradient methods were used for preparing the L and for D loading, respectively. The cationic surface of L allowed the anchoring of negatively charged AuNPs by electrostatic interactions, even inducing a shift in the zeta potential of the L2 series. L exhibited nanometric sizes and spherical shape. The higher the proportion of cholesterol, the higher the drug loading. D was released in a controlled manner by diffusion-controlled mechanisms, and the proportions of cholesterol and temperature of release media influenced its release profiles. D-encapsulated L preserved its antiproliferative activity against cancer cells. The developed liposomal formulations exhibit promising properties for cancer treatment and potential for hyperthermia therapy.

Carbohydrate-Based NK1R Antagonists with Broad-Spectrum Anticancer Activity.

NK1R antagonists, investigated for the treatment of several pathologies, have shown encouraging results in the treatment of several cancers. In the present study, we report on the synthesis of carbohydrate-based NK1R antagonists and their evaluation as anticancer agents against a wide range of cancer cells. All of the prepared compounds, derived from either d-galactose or l-arabinose, have shown high affinity and NK1R antagonistic activity with a broad-spectrum anticancer activity and an important selectivity, comparable to Cisplatin. This strategy has allowed us to identify the galactosyl derivative 14α, as an interesting hit exhibiting significant NK1R antagonist effect (kinact 0.209 ± 0.103 µM) and high binding affinity for NK1R (IC50 = 50.4 nM, Ki = 22.4 nM by measuring the displacement of [125I] SP from NK1R). Interestingly, this galactosyl derivative has shown marked selective cytotoxic activity against 12 different types of cancer cell lines.

In Vitro Anticancer Activity and Mechanism of Action of an Aziridinyl Galactopyranoside.

We recently screened a series of new aziridines ß-D-galactopyranoside derivatives for selective anticancer activity and identified 2-methyl-2,3-[N-(4-methylbenzenesulfonyl)imino]propyl 2,3-di-O-benzyl-4,6-O-(S)-benzylidene-ß-D-galactopyranoside (AzGalp) as the most promising compound. In this article, we explore the possible mechanisms involved in the cytotoxicity of this aziridine and evaluate its selective anticancer activity using cancer cells and normal cells from a variety of tissues. Our data show that AzGalp induces DNA damage (comet assay). Cells deficient in the nucleotide excision repair (NER) pathway were hypersensitive to the cytotoxicity of this compound. These results suggest that AzGalp induces bulky DNA adducts, and that cancer cells lacking a functional NER pathway may be particularly vulnerable to the anticancer effects of this aziridine. Several experiments revealed that neither the generation of oxidative stress nor the inhibition of glycolysis played a significant role in the cytotoxicity of AzGalp. Combinations of AzGalp with oxaliplatin or 5-fluorouracil slightly improved the ability of both anticancer drugs to selectively kill cancer cells. AzGalp also showed selective cytotoxicity against a panel of malignant cells versus normal cells; the highest selectivity was observed for two acute promyelocytic leukemia cell lines. Additional preclinical studies are necessary to evaluate the anticancer potential of AzGalp.

Screening for selective anticancer activity of plants from Grazalema Natural Park, Spain.

Since plants are an important source of anticancer drugs, we have carried out a random screening for selective anticancer activity of 57 extracts from 45 plants collected in Grazalema Natural Park, an area in the South of Spain of high plant diversity and endemism. Using lung cancer cells (A549) and lung non-malignant cells (MRC-5), we found that several extracts were more cytotoxic and selective against the cancer cells than the standard anticancer agent cisplatin. Five active extracts were further tested in cancer and normal cell lines from other tissues, including three skin cell lines with increasing degree of malignancy. An extract from the leaves of Daphne laureola L. (Thymelaeaceae) showed a striking potency and selectivity on lung cancer cells and leukemia cells; the IC50 values against these cancer cells were approximately 10,000-fold lower than against the normal cells. Daphnane-type diterpene orthoesters may be responsible for this highly selective anticancer activity.

The Cockayne syndrome protein B is involved in the repair of 5-AZA-2'-deoxycytidine-induced DNA lesions.

The Cockayne Syndrome Protein B (CSB) plays an essential role in Transcription-Coupled Nucleotide Excision Repair (TC-NER) by recruiting repair proteins once transcription is blocked with a DNA lesion. In fact, CSB-deficient cells are unable to recover from transcription-blocking DNA lesions. 5-Aza-2'-deoxycytidine (5-azadC) is a nucleoside analogue that covalently traps DNA methyltransferases (DNMTs) onto DNA. This anticancer drug has a double mechanism of action: it reverts aberrant hypermethylation in tumour-suppressor genes, and it induces DNA damage. We have recently reported that Homologous Recombination and XRCC1/PARP play an important role in the repair of 5-azadC-induced DNA damage. However, the mechanisms involved in the repair of the DNMT adducts induced by azadC remain poorly understood. In this paper, we show for the first time the importance of CSB in the repair of azadC-induced DNA lesions. We propose a model in which CSB initiates a signalling pathway to repair transcription blocks induced by incorporated 5-azadC. Indeed, CSB-deficient cells treated with 5-azadC show a delay in the repair of trapped DNMT1, increased levels of DNA damage and reduced survival.

Selective cytotoxic activity and DNA damage by an epoxyalkyl galactopyranoside.

Preclinical Research & Development Several clinically useful anticancer drugs selectively kill cancer cells by inducing DNA damage; the genomic instability and DNA repair defects of cancer cells make them more vulnerable than normal cells to the cytotoxicity of DNA-damaging agents. Because epoxide-containing compounds can induce DNA damage, we have used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to evaluate the selective cytotoxicity of three epoxyalkyl galactopyranosides against A549 lung cancer cells and MRC-5 lung normal cells. Compound (2S,3S)-2,3-epoxydecyl 4,6-O-(S)-benzylidene-ß-d-galactopyranoside (EDBGP) showed the highest selective anticancer activity and was selected for mechanistic studies. After observing that EDBGP induced cellular DNA damage (comet assay), we found that cells deficient in nucleotide excision repair were hypersensitive to the cytotoxicity of this compound; this suggests that EDBGP may induce bulky DNA adducts. EDBGP did not inhibit glycolysis (glucose consumption and lactate production). Pretreatment of lung cancer cells with several antioxidants did not reduce the cytotoxicity of EDBGP, thereby indicating that reactive oxygen species do not participate in the anticancer activity of this compound. Finally, EDBGP was screened against a panel of cancer cells and normal cells from several tissues, including three genetically modified skin fibroblasts with increasing degree of malignancy. Our results suggest that epoxyalkyl galactopyranosides are promising lead compounds for the development of new anticancer agents.

A 30-s exposure to ethanol 20% is cytotoxic to human keratinocytes: possible mechanistic link between alcohol-containing mouthwashes and oral cancer.

OBJECTIVES: To provide mechanistic evidence for the epidemiological link between long-term use of alcohol-containing mouthwashes and oral cancer. MATERIAL AND METHODS: Human epithelial keratinocytes were exposed for 30 s to concentrations of ethanol commonly present in mouthwashes. After a recovery period, cell viability was assessed with the MTT assay. RESULTS: A marked cytotoxic effect was observed for ethanol concentrations of 20% and above. CONCLUSIONS: The cytotoxicity of ethanol may explain the epidemiological association between mouthwash use and oral cancer. Evidence suggests that the risk of developing cancer in a tissue is strongly determined by the number of stem cell divisions accumulated by the tissue during a person's lifetime; cell division is a major source of mutations and other cancer-promoting errors. Since cell death activates the division of stem cells, the possible cytotoxicity of ethanol on the cells lining the oral mucosa will promote the division of the stem cells located in deeper layers to produce new cells to regenerate the damaged epithelium. If we regularly use mouthwashes containing cytotoxic concentrations of ethanol, the stem cells of the oral cavity may need to divide more often than usual and our risk of developing oral cancer may increase. CLINICAL RELEVANCE: Many mouthwashes contain percentages of ethanol above 20%. Because ethanol is not crucial to prevent and reduce gingivitis and plaque, members of the dental team should consider the potential risk of oral cancer associated with frequent use of alcohol-containing mouthwashes when advising their patients.