Diss. Phil. Bern.

Köln, Universiẗat, Diss., 2004.

Magister Pharmaceuticae - MPharm / Artemisia afra (A. afra) is traditionally used for a variety of ailments and contain flavonoids e.g. luteolin which may contribute to some of its activity. It is generally administered as a tea or decoction, and such liquid dosage forms present challenges as far as long term storage and stability are concerned, as well as sub-optimal oral bioavailability of actives they contain. Freeze dried aqueous extracts (FDAE) can alleviate such problems but may be hygroscopic and unstable. The use of modified forms of FDAE can counter the problem of hygroscopicity (e.g. use of alginate) and alleviate the issue of sub-optimal bioavailability of plant actives (e.g. polymethylmethacrylate). The objectives of this study, were to: (1) prepare the freeze dried aqueous extract (FDAE) and modified forms, which include alginate-extract beads (alginate-FDAE) and polymethylmethacrylate coated alginate matrix beads of herbal extract (PMMA-alginate-FDAE) of the FDAE of A. afra, (2) determine and compare the pharmaceutical characteristics of the above mentioned preparations of A. afra,(3) quantify and compare the total flavonoid and specifically luteolin levels of the different forms of A. afra,(4) evaluate and compare the release characteristics of FDAE of A. afra from the alginate-FDAE and PMMA-alginate-FDAE beads in gastrointestinal fluids and (5) determine the intestinal permeability of luteolin contained in selected modified Artemisia afra extract preparations. It was hypothesized that making the alginate beads and the polymethylmethacrylate coated alginate beads would make the FDAE less hygroscopic with a lower moisture content, that the rate of release of luteolin from A. afra FDAE into gastrointestinal fluids would be faster than from the modified forms, and that the effective gastrointestinal permeability of luteolin in the alginate-FDAE and PMMA-alginate-FDAE beads of A. afra is equal to that in FDAE. To realize these objectives, the FDAE was prepared by freeze drying the aqueous extract of the A. afra dried leaves, alginate-FDAE prepared by dispersing FDAE into 4% sodium alginate solution, then adding the resulting stock solution into a 2% calcium chloride solution and drying resulting beads and PMMA-alginate-FDAE prepared by a modified water-in-oil-in-water emulsion solvent evaporation method using water as an internal aqueous phase. Using pharmacopoeial methods and methods adapted from other workers the organoleptic and pharmaceutical characteristics were determined to compare the pharmaceutical quality of these preparations of A. afra. To identify and determine the levels of luteolin in the plant preparations, a validated HPLC assay was developed. Finally, the in situ perfused rat intestine model was used to determine the in vitro bioavailability, i.e. gastrointestinal permeability, of luteolin from solutions containing luteolin in pure form, FDAE, alginate-FDAE and PMMA-alginate-FDAE. The A. afra forms were obtained in moderate to good yields and FDAE was brown and hygroscopic in nature, the alginate beads dark brown free flowing and spherical in shape and the PMMA-alginate beads light brown in colour with rough edges. The A. afra plant forms on average contained 0.185 ± 0.24, 0.067 ± 0.014, 0.012 ± 0.071 μg/mg of free luteolin (n=3) in FDAE, alginate-FDAE and PMMA-alginate-FDAE respectively and 0.235 ± 0.026, 0.079 ± 0.093, 0.058 ± 0.082 μg/mg of total luteolin (n=3) in FDAE, alginate-FDAE and PMMAalginate- FDAE respectively. The Plumen values for intestinal uptake of luteolin were significantly higher from solutions of A. afra forms than the pure luteolin solution (i.e. Plumen values in the range of 0.02 - 0.035 cm/s for all plant forms vs Plumen values in the range of 0.010 - 0.014 cm/s for pure luteolin, t-test p = 0.0252). The permeability of luteolin in FDAE appeared to be slighter greater than that of the modified forms (Plumen values >0.03 cm/s for FDAE and Plumen values <0.03 cm/s for both modified forms). In summary, the results showed that, the modified A. afra forms; alginate-FDAE and PMMAalginate- FDAE were of acceptable pharmaceutical quality with luteolin better taken up in the plant forms than in its pure form. The A. afra forms prepared had similar rates of uptake (permeability) of free and total luteolin with the rates being highest for the FDAE. Collectively, these results indicate that alginate-FDAE and PMMA-alginate-FDAE bead forms should be suitable for use in a solid dosage form (e.g. tablet or capsule) of A. afra.

Alzheimer disease (AD) is a progressive neurodegenerative disorder pathologically characterized by deposition of ß-amyloid (Aß) peptides as plaques in the brain. Central to this AD pathology is mismetabolism of the amyloid precursor protein (APP). Recent studies suggest that flavonoids, a class of secondary plant metabolites, may be useful for the prevention and treatment of a variety of neurodegenerative diseases. The studies detailed herein, investigate the ability of two such classes of flavonoids, green tea derived catechins and 5,7-dihydroxyflavones, to modulate APP metabolism in "Swedish" mutant APP (APPsw) models of AD. Studies showed that green tea derived (-)-epigallocatechin-3-gallate (EGCG) effectively reduced Aß generation and resultant amyloidosis both in vitro and in vivo. In concert with these findings, EGCG markedly promoted non-amyloidogenic APP proteolysis via activation of the putative a-secretase, a-disintegrin-and-metalloprotease-10 (ADAM10). Furthermore, luteolin and various related 5,7-dihydroxyflavones, effectively reduced Aß generation and resultant amyloidosis both in vitro and in vivo, as well. Data revealed that luteolin decreased amyloidogenic γ-secretase APP proteolysis via presenilin-1 (PS1) carboxyl-terminal fragment (CTF) phosphorylation. Elucidation of these flavonoids' cellular/molecular mechanisms also revealed their potential for opposing neurofibrillary tangle (NFT) pathology, another hallmark of AD. These data raise the possibility that flavonoid administration to AD patients may prove to be viable and effective prophylactic strategy.

For bone resorption, vacuolar-type H+-ATPases (V-ATPases) on the plasma membranes of osteoclasts acidifies the extracellular millieu adjacent to the bone surface. The V-ATPase a3 and d2 subunits are enriched in osteoclasts. B2 subunit is also expressed on the osteoclast plasma membrane. Disruption of genes encoding subunits a3 and d2 impairs bone resorption. In this study, we have shown an interaction between the a3-B2 and a3-d2 subunits. Luteolin and KM91104 were found to be effective inhibitors of the a3-d2 and a3-B2 interactions respectively. Secondary assays revealed luteolin and KM91104 were not toxic to cells, did not affect osteoclastogenesis yet inhibited bone resorption. Furthermore luteolin did not affect V-ATPase subunit formation or assembly. Inhibitors of osteoclast resorption that do not affect osteoclast viability, preserve osteoclast–osteoblast signalling are desirable than existing anti-resorptives. Therefore, V-ATPase a3–d2 and a3-B2 interactions are viable targets for anti-resorptive therapeutics for osteoporosis.

For bone resorption, vacuolar-type H+-ATPases (V-ATPases) on the plasma membranes of osteoclasts acidifies the extracellular millieu adjacent to the bone surface. The V-ATPase a3 and d2 subunits are enriched in osteoclasts. B2 subunit is also expressed on the osteoclast plasma membrane. Disruption of genes encoding subunits a3 and d2 impairs bone resorption. In this study, we have shown an interaction between the a3-B2 and a3-d2 subunits. Luteolin and KM91104 were found to be effective inhibitors of the a3-d2 and a3-B2 interactions respectively. Secondary assays revealed luteolin and KM91104 were not toxic to cells, did not affect osteoclastogenesis yet inhibited bone resorption. Furthermore luteolin did not affect V-ATPase subunit formation or assembly. Inhibitors of osteoclast resorption that do not affect osteoclast viability, preserve osteoclast–osteoblast signalling are desirable than existing anti-resorptives. Therefore, V-ATPase a3–d2 and a3-B2 interactions are viable targets for anti-resorptive therapeutics for osteoporosis.

No / Potential protective effects of the flavonoids quercetin and luteolin have been examined against the oxidative stress of MC3T3-E1 osteoblast-like cells. Although hydrogen peroxide and menadione reduced cell viability, the toxicity was prevented by desferrioxamine or catalase but not superoxide dismutase, suggesting the involvement of hydrogen peroxide in both cases. Quercetin and luteolin reduced the oxidative damage, especially that caused by hydrogen peroxide. When cultures were pre-incubated with quercetin or luteolin, protection was reduced or lost. Protection was also reduced when a 24 h pre-incubation with the flavonoids was followed by exposure to menadione alone. Pretreating cultures with luteolin impaired protection by quercetin, whereas quercetin pretreatment did not affect protection by luteolin. It is concluded that quercetin and luteolin suppress oxidative damage to MC3T3-E1 cells, especially caused by peroxide. The reduction in protection by pretreatment implies a down-regulation of part of the toxic transduction pathway.

乳腺癌是女性最常見的腫瘤之一，多發生在女性絶經後，並具有雌激素依賴性。芳香化酶(CYP19)是雌激素生物合成過程中的關鍵酶，而芳香化酶抑製劑(AI)則被用於替代治療雌激素依賴性的乳腺癌。然而，AI在降低雌激素水平的同時能夠引起骨質酥鬆。此項研究的目的是找尋AI替代物。 / 黃酮類化合物是一種多酚化合物，廣泛分佈于植物中。我們先前的研究發現二氢黄酮陈皮素能夠抑制芳香化酶的生物活性，并且抑制芳香化酶高表達的乳腺癌生長。在本研究中，我們發現陳皮素在抑制腫瘤生長的同時能夠降低来曲唑引起的骨質流失。木犀草素是另外一種黄酮类化合物，它同樣能夠抑制芳香化酶的活性并減少骨流失。而與陳皮素不同的是，它能夠抑制芳香化酶的表達。在芳香化酶高表達的乳腺癌細胞(MCF-7 aro)中，木犀草素抑制芳香化酶活性的IC50是3 μM。在MCF-7 細胞中，5 μM的木犀草素能夠抑制CYP19 mRNA 的表達，螢光素酶報告實驗顯示木犀草素是通過作用于啟動子I.3和II來抑制CYP19的表達。蛋白印跡實驗表明木犀草素抑制CYP19表達的分子機制可能通過調節JNK信號通路進而減少AP-1的活性來實現。動物實驗結果顯示木犀草素能夠抑制MCF-7aro腫瘤的生長并改善來曲唑引起的骨流失。 / 環氧化酶(COX)是花生四烯酸轉化為前列腺素途徑中的一種關鍵酶。研究發現COX-2在乳腺癌組織中廣泛表達。本實驗研究了COX抑製劑在裸鼠動物模型中對乳腺癌腫瘤的作用機制。研究結果表明塞來昔布和阿司匹林在不影響血液中雌激素水平的情況下抑制乳腺癌腫瘤的生長。蛋白印迹實驗顯示這兩種藥物能夠降低腫瘤中COX-2，Cyclin A和Bcl-xL的表達。miR-98, miR-222和miR-145也能夠被塞來昔布和阿司匹林影響。 / 本研究表明陳皮素，木犀草素及COX抑制劑有潛力成為替代AI的化學治療藥物或共同治療藥物。 / Breast cancer is one of the most prevalent cancers affecting women. The majority of breast tumor growth occurred in the post-menopausal period are estrogen dependent. Aromatase (CYP19) catalyzes the rate-limiting step in the synthetic reaction of estrogen and aromatase inhibitors (AIs) are contemporary treatment for estrogen-positive breast cancer. However, estrogen-lowering drugs may promote osteoporosis. Our objective of this study further identified some alternatives for AIs. / Flavonoids are polyphenolic compounds that are ubiquitously distributed in plants. We have previously found that the flavanone hesperetin can inhibit the activity of aromatase and suppress aromatase-expressing breast tumor growth. In this project, we investigated the potential interaction between hesperetin and the AI letrozole in a mouse model. Our results showed that hesperetin could inhibit the tumor growth and reduce bone loss induced by letrozole. Similarly, another flavonoid luteolin also inhibited aromatase and prevented bone deterioration as observed in this project. In cells stably transfected with CYP19 (MCF-7aro), luteolin inhibited the aromatase activity with an IC50 value of 3μM. In addition, 5μM luteolin significantly reduced CYP19 mRNA expression in MCF-7 cells. Luciferase reporter assay revealed that luteolin could suppress CYP19 transcription at promoter regions I.3 and II. Western analysis illustrated that JNK signaling pathway was involved and deactivation of AP-1 could be the underlying molecular mechanism. Subsequently, we examined the effect in vivo. Our results showed that luteolin could inhibit the MCF-7aro tumor growth and improved bone loss induced by letrozole. / Cyclooxygenase (COX) is an enzyme responsible for the conversion of arachidonic acid into prostaglandins. It is over-expressed in breast cancer tissue and an increased expression of COX-2 was also observed in the xenograft model employed in this project. In the last study we evaluated the importance of COX-2 in breast tumor growth in this model. Our data showed that celecoxib and aspirin could significantly suppress the tumor growth without changing the plasma estrogen level. Western analysis illustrated that COX-2, Cyclin A, Bcl-xL and ER were reduced in celecoxib- and aspirin- treated tumor samples and miR-98, miR-222 and miR-145 were altered by celecoxib or aspirin. / After all, this project demonstrated that hesperetin, luteolin and COX-inhibitors could be potential chemopreventive or co-therapeutic agents. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Li, Fengjuan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 131-148). / Abstract also in Chinese. / ACKNOWLEDGEMENTS --- p.I / ABSTRACT --- p.II / 摘要 --- p.IV / LIST OF ABBREVIATIONS --- p.V / TABLE OF CONTENTS --- p.VII / CHAPTER 1 --- p.1 / GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Types of Breast Cancer --- p.3 / Chapter 1.2 --- Nuclear Receptor Signaling Pathways in Breast Cancer --- p.5 / Chapter 1.3 --- Estrogen and Breast Cancer --- p.7 / Chapter 1.4 --- Estrogen and Bone Health --- p.8 / Chapter 1.5 --- Estrogen Biosynthesis and Aromatase --- p.10 / Chapter 1.6 --- Tissue Specific Promoter for Aromatase Expression --- p.13 / Chapter 1.7 --- Nuclear Receptors and Aromatase Promoter Regulation --- p.15 / Chapter 1.8 --- Signaling Pathway and Aromatase Expression --- p.17 / Chapter 1.9 --- Cell Cycle in Breast Cancer --- p.20 / Chapter 1.10 --- Cell Apoptosis --- p.23 / Chapter 1.11 --- Treatment of breast cancer --- p.25 / Chapter 1.12 --- Phytoestrogens --- p.29 / Chapter 1.13 --- Aim of My Study --- p.32 / CHAPTER 2 --- p.33 / MATERIALS AND METHODS --- p.33 / Chapter 2.1 --- Chemicals and Materials --- p.33 / Chapter 2.1.1 --- Chemicals --- p.33 / Chapter 2.1.2 --- Plasmids --- p.33 / Chapter 2.2 --- Cell Culture --- p.33 / Chapter 2.3 --- Aromatase Activity Assay --- p.34 / Chapter 2.4 --- Quantitative Real Time PCR --- p.36 / Chapter 2.4.1 --- RNA Isolation and cDNA Synthesis --- p.36 / Chapter 2.4.2 --- Quantitative Real Time PCR Assay --- p.37 / Chapter 2.4.3 --- MiRNA Quantitative Real Time PCR Assay --- p.38 / Chapter 2.5 --- Western Blot --- p.39 / Chapter 2.6 --- Measurement of Promoter Activity --- p.41 / Chapter 2.6.1 --- Plasmid Preparation --- p.41 / Chapter 2.6.2 --- Transient Transfection and Dual-Luciferase Assay --- p.42 / Chapter 2.7 --- Electrophoretic Mobility Shift Assay (EMSA) --- p.43 / Chapter 2.7.1 --- Nuclear protein extraction --- p.43 / Chapter 2.7.2 --- Electrophorectic Mobility Shift Assay --- p.44 / Chapter 2.8 --- Animal Experiment Design --- p.45 / Chapter 2.8.1 --- Animal Model for Hesperetin Study --- p.45 / Chapter 2.8.2 --- Animal Model for Luteolin Study --- p.46 / Chapter 2.8.3 --- Animal Model for Cycooxygenase Inhibitors Study --- p.48 / Chapter 2.8.4 --- Serum Estradiol Determination --- p.49 / Chapter 2.8.5 --- Analysis of serum lipoproteins --- p.49 / Chapter 2.8.6 --- Bone Image Acquisition and Region of Interest Selection --- p.50 / Chapter 2.9 --- Statistical Analysis --- p.50 / CHAPTER 3 --- p.51 / The citrus flavonone hesperetin prevents letrozole- induced bone loss in a mouse model of breast cancer --- p.51 / Chapter 3.1 --- Introduction --- p.51 / Chapter 3.2 --- Results --- p.54 / Chapter 3.2.1 --- Murine Body Weight and Liver Weight --- p.54 / Chapter 3.2.2 --- Effect of Hesperetin and Letrozole on Xenograft Growth in Ovariectomized Mice --- p.55 / Chapter 3.2.3 --- Hesperetin Reduced Plasma Estradiol Concentration --- p.58 / Chapter 3.2.4 --- PS2 mRNA Expression in Tumor --- p.59 / Chapter 3.2.5 --- Uterine Wet Weight --- p.60 / Chapter 3.2.6 --- Hesperetin Prevent Bone Deterioration Induced by Letrozole --- p.61 / Chapter 3.3 --- DISCUSSION --- p.63 / CHAPTER 4 --- p.66 / dIETARY FLAVONOID LUTEOLIN ON cyp19 transcription in the breast cancer cells mcf-7 --- p.66 / Chapter 4.1 --- Introduction --- p.66 / Chapter 4.2 --- Results --- p.68 / Chapter 4.2.1 --- Inhibitory Effect of Luteolin on Aromatase Activity --- p.68 / Chapter 4.2.2 --- Luteolin Reduced Aromatase mRNA Expression in MCF-7 Cells --- p.70 / Chapter 4.2.3 --- Effect of Luteolin on Promoter I.3/II Activity of CYP19 in MCF-7 Cells --- p.71 / Chapter 4.2.4 --- The Effect of Luteolin on Truncation CYP19 Gene Reporter Assay --- p.72 / Chapter 4.2.5 --- Luteolin Reduced AP-1 Binding in Promoter I.3/II DNA Fragment --- p.74 / Chapter 4.2.6 --- Inhibitory Effect of Luteolin on Protein Kinase Signaling --- p.76 / Chapter 4.3 --- Discussion --- p.78 / CHAPTER 5 --- p.83 / interaction OF LUTEOLIN and letrozole in a postmenopausal breast cancer model --- p.83 / Chapter 5.1 --- Introduction --- p.83 / Chapter 5.2 --- Results --- p.86 / Chapter 5.2.1 --- Luteolin and letrozole treatment had no effect on mouse body weight and liver weight --- p.86 / Chapter 5.2.2 --- Effect of luteolin and Letrozole on Xenograft Growth in Ovariectomized Mice --- p.88 / Chapter 5.2.3 --- Luteolin reduced plasma estradiol concentration --- p.91 / Chapter 5.2.4 --- Luteolin Counteracted Uterine Weight Reduction under Letrozole Treatment --- p.92 / Chapter 5.2.5 --- Luteolin Prevented Bone Deterioration Induced by Letrozole --- p.93 / Chapter 5.2.6 --- The Effect of Luteolin on Plasma TC and TG --- p.95 / Chapter 5.2.7 --- Luteolin Increased HDL Level and Reduced the Ratio of LDL/HDL --- p.97 / Chapter 5.2.8 --- Effect of Luteolin on Cell Cycle and Apoptotic Protein Expression --- p.99 / Chapter 5.3 --- DISCUSSION --- p.104 / CHAPTER 6 --- p.107 / cyclooxygenase inhibitors suppresse breast tumor growth in NUDE MICE --- p.107 / Chapter 6.1 --- Introduction --- p.107 / Chapter 6.2 --- Results --- p.109 / Chapter 6.2.1 --- Celecoxib and aspirin treatment had no effect on mouse body weight and liver weight --- p.109 / Chapter 6.2.2 --- Effect of celecoxib and aspirin on Xenograft Growth in Ovariectomized Mice --- p.111 / Chapter 6.2.3 --- Celecoxib and aspirin had no effect on plasma estradiol concentration --- p.113 / Chapter 6.2.4 --- Celecoxib and Aspirin Had no Effect on Uterine Weight --- p.114 / Chapter 6.2.5 --- Protein expression of COX-2, Cell cycle-related and cell Apoptotic Genes --- p.115 / Chapter 6.2.6 --- Detection of Related miRNA Expression Level in Tumors --- p.118 / Chapter 6.2.7 --- c-Myc mRNA Expression Level were Regulated in Tumors --- p.121 / Chapter 6.3 --- DISCUSSION --- p.124 / CHAPTER 7 --- p.127 / SUMMARY --- p.127 / REFERENCE --- p.131

The aim of this study was to investigate the effect the plant matrix and the structure of the flavonoid (i.e. whether aglycone or glycoside) may have on the gastrointestinal uptake and metabolism of luteolin derivatives from Artemisia afra traditional plant medicine. Specifically, how these two factors influenced the intestinal uptake and disposition of luteolin derivatives in pure and in Artemisia afra plant extract forms were to be assessed by investigating the uptake and metabolism of the luteolin derivatives in human intestinal epithelial Caco-2 cells and the perfused rat intestinal loop. To realize this aim, the following were determined: (1) identification and characterization of major luteolin derivatives found in Artemisia afra, (2) the effect of the plant matrix on the uptake of luteolin derivatives in Artemisia afra aqueous-extract forms across the Caco-2 cell monolayer, (3) the effect of the plant matrix on the absorption and metabolism of luteolin derivatives in Artemisia afra aqueous-extract forms in the perfused rat small intestine, (4) the effect of gut contents on the uptake and metabolism of luteolin derivatives in intestinal loop and (5) the metabolic profiles of luteolin derivatives obtained for the pure solutions versus plant aqueous extract solutions in Caco-2 cells and the rat intestine. / Philosophiae Doctor - PhD

Objetivos: O objetivo deste estudo foi o de identificar compostos seletivamente tóxicos ao carcinoma espinocelular de boca in vitro por meio do reposicionamento de fármacos. Material e Métodos: Por meio de um escaneamento baseado na viabilidade celular de 1.280 fármacos, nós selecionamos três princípios ativos (luteolin, metixene hydrochloride e nitazoxanide) letais às células de câncer de boca SCC-25 e pouco tóxicos às células de queratinócitos cutâneos imortalizados HaCaT. Os fármacos candidatos foram investigados quanto à sua dose- e tempo-resposta bem como comparados e combinados à agentes quimioterápicos padrão por meio do ensaio por colorimetria com brometo de tiazolil azul de tetrazolio (MTT). O impacto dos fármacos na motilidade do SCC-25 e do HaCaT foi verificado pelo ensaio de migração celular e seus mecanismos de ação também foram explorados por meio da verificação dos níveis das proteínas fosforiladas pelo western blotting. Todos os experimentos foram realizados em triplicata e, pelo menos, três vezes independentes. O teste t de student foi utilizado para confrontar as variáveis e nível de significância de 5% foi estabelecido para todos os testes. Resultados: O flavonoide natural luteolin exerceu citotoxicidade potente contra as células de câncer de boca in vitro, apresentando baixa toxicidade ao HaCaT e alta eficiência quando comparado a quimioterápicos como a cisplatina e o AG1478. Do ponto de vista molecular, a luteolin ativou a via de sinalização do dano do DNA e, combinada com um inibidor do Chk, apresentou efeitos potencializados. Além disso, nós demonstramos que a nitazoxanide e o metixene hydrochloride são capazes de destruir células SCC-25 porém não as HaCaT de maneira proporcional à dose e ao tempo de tratamento. As combinações entre os três fármacos hit e com a cisplatina ou o AG1478 potencializaram seus efeitos contra as células malignas. Conclusões: O presente estudo traz a luteolin, o metixene hydrochloride e a nitazoxanide como fortes candidatos a agentes terapêuticos contra o câncer de boca uma vez que estes compostos apresentam maior eficácia contra células de câncer de boca e menor toxicidade contra células não tumorais in vitro do que agentes quimioterápicos convencionais. / Objectives: Here we aimed at identifying and reposition approved drugs that could be selectively toxic towards oral squamous cell carcinoma cells. Materials and Methods: Through a cell-based drug screening of 1,280 chemical molecules, we selected 3 compounds (luteolin, metixene hydrochloride, and nitazoxanide) lethal to oral cancer SCC-25 cells, while sparing immortalized keratinocyte HaCaT cells. The drugs were then further challenged for their time- and dose-responses, as well as their comparison and combination to standard chemotherapeutic agents by colorimetric assay 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan, Thiazolyl blue formazan (MTT). The impact on SCC-25 and HaCaT motility as well as the mode of action of the drugs was then further explored by scratching assay and western blotting, respectively. All the experiments were performed in triplicated and, at least, three independent times. Students t test was performed to verify the differences among the variables and the level of significance was set at 5%. Results: The natural flavonoid luteolin was a potent cytotoxic agent against oral cancer cells in vitro, presenting low toxicity against HaCaT cells and high efficiency as compared to standard-of-care, such as cisplatin and AG1478. From a molecular standpoint, luteolin coopted the DNA-damage pathway and could be efficiently combined with Chk pharmacological inhibitor. Moreover, we demonstrated that nitazoxanide and metixene hydrochloride kill the SCC-25 but not the HaCaT cells in a dose- and time-dependent. The combinations among the three drugs hit and with cisplatin and AG1478 improved their effect against the malignant cells. Conclusions: Luteolin, metixene hydrochloride, and nitazoxanide emerge as strong cytotoxic and/or adjuvant therapy in oral cancer, as these compounds present higher efficiency and lower toxicity against oral cancer cells in vitro than conventional chemotherapeutic agents.