Avaliação in vitro do potencial antineoplástico de plantas do Cerrado em carcinoma de cabeça e pescoço

Tese (doutorado)—Universidade de Brasília, Faculdade de Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2014.As moléculas antineoplásicas derivadas de plantas têm ganhado atenção frente aos tratamentos convencionais do câncer. Assim, esse trabalho tem como objetivo avaliar a atividade antineoplásica de extratos de plantas derivadas do Bioma Cerrado em linhagens de carcinoma de cabeça e pescoço (HNSCC). Também, objetiva-se averiguar a ação concomitante do tratamento radioterápico associado aos extratos de plantas do Cerrado e o possível sinergismo entre eles, além de identificar vias de ação e substâncias com potencial biológico nas linhagens de HNSCC. Para os experimentos foram usados extratos de folhas das espécies Erythroxylum daphnites, E. suberosum, E. subrotundum, Pouteria ramiflora e P. torta provenientes do bioma Cerrado. Para cada planta foram produzidos três diferentes extratos divididos de acordo com sua solubilidade e polaridade, são eles: aquoso, etanólico e hexânico. Os extratos com maior atividade citotóxica tiveram sua concentração de IC50 definidas e passaram por novos testes a fim de determinar o mecanismo de ação. Dentre os 15 extratos testados, o extrato hexânico de E. daphnites (EDH) foi o que apresentou maior toxicidade para a linhagem SCC-9 (carcinoma de língua) e extrato hexânico de E. suberosum (ESH) para a linhagem FaDu. De maneira geral os extratos induziram efeito citotóxico supraaditivo quando comparado às linhagens que receberam apenas irradiação. O extrato EDH apresentou ainda atividade antiproliferativa, estabilização do ciclo celular na fase G0/G1, diminuição de expressão das ciclinas D e E e aumento de expressão das proteínas p16 e p21, além de ter atividade apoptótica comprovada pela expressão de caspase 3. O extrato ESH, apesar de citotóxico, não possui efeito antiproliferativo para a linhagem FaDu e o perfil de morte descrito foi apenas o de necrose. Os triterpenos foram os principais constituintes encontrados nos extratos com melhor resposta citotóxica. Diante dos resultados obtidos, esse estudo demonstra a relevância biológica das substâncias advindas de plantas do Cerrado e destaca o extrato EDH como uma opção promissora para o tratamento do carcinoma de língua.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).Antineoplastic effects of molecules derived from plants have recently gained increased attention as an additive to traditional therapies. This study aims to investigate the antineoplastic activity of plant extracts derived from the Cerrado biome in head and neck carcinoma cell lines. It also investigates the concomitant activity of radiotherapic treatment associated with plant extracts and the possible synergism between them, identify action mechanisms and possible active substances with biological activity for HNSCC cells. For the experiments, leaf extracts of plant species Erythroxylum daphnites, E. suberosum, E. subrotundum, Pouteria ramiflora and P. torta from Cerrado Biome were used. Of each plant, three extracts were produced according to their solubility and polarity: aqueous, ethanolic and hexanic. Extracts with increased activity had the IC50 concentration defined. New assays were done to determinate mechanism of action and probable constitution. Among 15 extracts tested, hexanic extract from E. daphnites (EDH) showed the highest toxicity for SCC-9 and hexanic extract from E. suberosum (ESH) for FaDu cells. Generally extracts induced supra-additive cytotoxic effect compared with cells that received only radiation. The extract EDH also showed antiproliferative effect, stabilization of cell cycle on G0/G1 phase, decreased levels of ciclins D and E, and increased labeling of proteins p16 and p21, as well as apoptotic activity proven by expression of caspase 3. Extract ESH, despite cytotoxicity activity, didn’t show antiproliferative result on FaDu cell line and the profile of cell death described is only necrosis. Triterpenes are major constituents found in both extracts with better cytotoxic index. Based on these dates, this study highlights the potential biological relevance of Cerrado plants and EDH emerges as a promising option for the treatment of tongue cancer

Plants from Brazilian cerrado with potent tyrosinase inhibitory activity

The increased amount of melanin leads to skin disorders such as age spots, freckles, melasma and malignant melanoma. Tyrosinase is known to be the key enzyme in melanin production. Plants and their extracts are inexpensive and rich resources of active compounds that can be utilized to inhibit tyrosinase as well as can be used for the treatment of dermatological disorders associated with melanin hyperpigmentation. Using in vitro tyrosinase inhibitory activity assay, extracts from 13 plant species from Brazilian Cerrado were evaluated. The results showed that Pouteria torta and Eugenia dysenterica extracts presented potent in vitro tyrosinase inhibition compared to positive control kojic acid. Ethanol extract of Eugenia dysenterica leaves showed significant (p<0.05) tyrosinase inhibitory activity exhibiting the IC50 value of 11.88 µg/mL, compared to kojic acid (IC50 value of 13.14 µg/mL). Pouteria torta aqueous extract leaves also showed significant inhibitory activity with IC50 value of 30.01 µg/mL. These results indicate that Pouteria torta and Eugenia dysenterica extracts and their isolated constituents are promising agents for skin-whitening or antimelanogenesis formulations

Asparaginase induces selective dose- and time- dependent cytotoxicity, apoptosis, and reduction of NFκB expression in oral cancer cells

Asparaginase is fundamental to the treatment of haematological malignancies. However, little has been studied on the effects that asparaginase could exert on solid tumours. Thus, this study aimed to evaluate the effects of asparaginase on an oral carcinoma cell line. The cytotoxicity of asparaginase in SCC- 9 (tongue squamous cell carcinoma) and HaCaT (human keratinocyte) cell lines was evaluated with MTT cell viability assay. The cells were treated with asparaginase at 0.04, 0.16, 0.63, 1.0, 1.5, 2.5, and 5.0 IU/mL. Dose- response curves and IC50 values were obtained and the Tumour Selectivity Index (TSI) was calculated. The effect of asparaginase on procaspase- 3 and nuclear factor κB (NFκB) expression was evaluated with western blot because it was reported that the overexpression of NFκB has been shown to contribute to tumour cell survival, proliferation, and migration. Caspase 3/7 staining was performed to identify cell death using flow cytometry. Effective asparaginase concentrations were lower for SCC- 9 cells when compared to HaCaT cells. The cytotoxicity results at 48 and 72 hours were significantly different for SCC- 9 cells. The TSI indicated that asparaginase was selective for the tumour cells. A decrease in procaspase- 3 and NFκB protein levels was observed in SCC- 9 cells. Furthermore, asparaginase resulted in significant apoptosis after 48 and 72 hours. Based on these results, asparaginase was cytotoxic in a dose- and time- dependent manner, induces apoptosis, and reduces NFκB expression in oral cancer cells. These results encourage further studies on the effectiveness of this enzyme as a treatment for solid tumours, especially head and neck cancer.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154950/1/cep13256.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154950/2/cep13256_am.pd

Cytotoxic effect of Erythroxylum suberosum combined with radiotherapy in head and neck cancer cell lines

O câncer de boca e de orofaringe emerge como o 6º tipo de câncer mais comum no mundo. O tratamento pode envolver cirurgia, quimioterapia e radioterapia. Mais de 50% das drogas com atividade de combate ao câncer foram isoladas de fontes naturais, tais como a Catharanthus roseus e a epipodofilotoxina, isolada de Podophyllum. O maior desafio é maximizar o controle da doença, enquanto minimiza a morbidade e toxicidade para os tecidos normais circundantes. O Erythroxylum suberosum é uma planta comum no bioma Cerrado brasileiro e é popularmente conhecida como "cabelo-de-negro". O objetivo deste estudo foi avaliar a citotoxicidade dos extratos da planta Erythroxylum suberosum do bioma Cerrado brasileiro, associados à radioterapia em linhagens celulares humanas de carcinomas de língua e de hipofaringe. As células foram tratadas com os extratos aquoso, etanólico e hexânico do Erythroxylum suberosum e irradiadas com 4 Gy, 6 Gy e 8 Gy. A citotoxidade foi avaliada pelo ensaio de MTT e a absorvância foi medida a 570 nm em uma leitora Beckman. A cisplatina, quimioterápico padrão, foi utilizada como controle positivo. O uso de extratos de Erythroxylum suberosum mostrou potencial efeito radiosensibilizante in vitro no câncer de cabeça e pescoço. O efeito da citotoxicidade nas linhagens foi de forma não seletiva e muito semelhante ao efeito da quimioterapia padrão. O extrato aquoso de Erythroxylum suberosum, combinado com radioterapia, foi o extrato mais citotóxico para os carcinomas de língua e hipofaringe, associados à radioterapia.The mouth and oropharynx cancer is the 6th most common type of cancer in the world. The treatment may involve surgery, chemotherapy and radiotherapy. More than 50% of drugs against cancer were isolated from natural sources, such as Catharanthus roseus and epipodophyllotoxin, isolated from Podophyllum. The biggest challenge is to maximize the control of the disease, while minimizing morbidity and toxicity to the surrounding normal tissues. The Erythroxylum suberosum is a common plant in the Brazilian Cerrado biome and is popularly known as "cabelo-de-negro". The objective of this study was to evaluate the cytotoxic activity of Erythroxylum suberosum plant extracts of the Brazilian Cerrado biome associated with radiotherapy in human cell lines of oral and hypopharynx carcinomas. Cells were treated with aqueous, ethanolic and hexanic extracts of Erythroxylum suberosum and irradiated at 4 Gy, 6 Gy and 8 Gy. Cytotoxicity was evaluated by MTT assay and the absorbance was measured at 570 nm in a Beckman Counter reader. Cisplatin, standard chemotherapy, was used as positive control. The use of Erythroxylum suberosum extracts showed a possible radiosensitizing effect in vitro for head and neck cancer. The cytotoxicity effect in the cell lines was not selective and it is very similar to the effect of standard chemotherapy. The aqueous extract of Erythroxylum suberosum, combined with radiotherapy was the most cytotoxic extract to oral and hypopharynx carcinomas

Efeito apoptótico do extrato bruto de tabaco em linhagem de carcinoma oral humano

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ciências da Saúde, 2009.O câncer é um problema mundial de saúde pública. Nas últimas décadas a incidência de câncer de cabeça e pescoço vem aumentando, sendo os países desenvolvidos os mais afetados. As terapias convencionais para este tipo de câncer, cirurgia e radioterapia, são agressivas e não aumentam de maneira considerável a qualidade e expectativa de vida dos pacientes. Nesse cenário, a descoberta de novas terapias é necessária. Esse trabalho tem como objetivo investigar o efeito citotóxico do extrato bruto de tabaco (EBT) e cinco frações provenientes dele (metanólica, neutra, carbônica, ácida e básica) em células de carcinoma oral humano. A exposição das células de câncer oral ao EBT induz a morte celular e diminui a viabilidade celular de maneira dose-dependente. Das frações testadas, apenas a neutra foi capaz de induzir uma significante morte celular (acima de 50%) após 48 horas de tratamento. Ademais, fragmentação do DNA e ativação da caspase-3 indicam que a morte celular induzida por EBT e pela fração neutra tem natureza apoptótica. Os resultados mostram que apesar do tabaco ser um carcinógeno conhecido, possuir inúmeros componentes indutores de tumor, ele também pode conter componentes capazes de induzir morte em células de câncer. Pelo fato da fração neutra ser capaz de também induzir morte celular, pode-se inferir que o componente apoptótico é apolar. Novos estudos são necessários para avaliar a estrutura química envolvida neste processo. _______________________________________________________________________________________ ABSTRACTCancer is a public health problem worldwide. Incidences of head and neck cancer are increasing in the last decades, and the developed countries are the most affected. Current therapeutic options for this type of cancer are aggressive, including surgery and radiotherapy. In addition, they have not yet translated into an improvement of life quality or expectancy to patients. In this scenario, new therapeuticals are urgently needed and actively sought after. The goal of this study was to investigate the cytotoxic effects of tobacco crude extract (TCE) and 5 fractions thereof (methanolic, neutral, carbonic, acid and basic) on a human oral squamous cell carcinoma. Exposure of oral cancer cells to TCE induced cell death and decreased cell viability in a dose-dependent manner. Out of the tested fractions, only the neutral one was able to induce significant cell death (over 50%) over the period of 48h. In addition, DNA laddering and caspase-3 activation indicate that the cell death processes induced by TCE and neutral fraction were apoptotic in nature. Our results indicate that although tobacco is a known carcinogen, possessing many tumorinitiating compounds, it could also contain compounds that are useful to induce apoptosis in cancer cells. Because the neutral fraction was also able to induce apoptosis, it is postulated that this putative compound is non-polar, although further investigation is needed to uncover its true nature and chemical structure

In vitro Anti-Tumor Effects of Statins on Head and Neck Squamous Cell Carcinoma: A Systematic Review

<div><p>Background</p><p>Statins are commonly used against arteriosclerotic disease, but recent retrospective analyses have suggested that statins also prevent cancer. The aim of this systematic review is to verify the vitro anti-tumor effects of statins on head and neck squamous cell carcinoma.</p><p>Methods</p><p>Studies were gathered by searching Cochrane, MEDLINE, EMBASE, LILACS, and PubMed, up until May 9, 2015, with no time or language restrictions. Only in vitro studies that discuss the effect of statins on head and neck carcinoma were selected.</p><p>Results</p><p>Of 153 identified papers, 14 studies met the inclusion criteria. These studies demonstrated that statins had a significant effect on head and neck squamous cell carcinoma cell lines and influenced cell viability, cell cycle, cell death, and protein expression levels involved in pathways of carcinogenesis, which corroborates with the potential in vitro anti-tumor effects. It provides highlights about the biological mechanisms of statins used alone or associated with traditional therapy for cancer.</p><p>Conclusions</p><p>Though there are few studies on the topic, currently available evidence suggests that statins shows that preclinical experiments supports the potentiality of statin as an adjuvant agent in chemotherapy and/or radiotherapy approaches routinely used in the management of HNSCC and should undergo further clinical assessment.</p></div

Curcumin downregulates the PI3K–AKT–mTOR

Curcumin, a polyphenol isolated from the rhizome of Curcuma longa, has been studied because of its antioxidant, antimicrobial, and antiinflammatory properties. This study aimed to evaluate the effects of curcumin on head and neck cancer (HNC) cell lines and how it modulates the PI3K–AKT–mTOR signaling pathway. Dose‐response curves for curcumin were established for hypopharynx carcinoma (FaDu), tongue carcinoma (SCC‐9), and keratinocytes (HaCaT) cell lines and IC50 values were calculated. Cell cycle and cell death were investigated through flow cytometry. Cytoskeleton organization was assessed through phalloidin+FITC staining. qPCR array and western blot were performed to analyze gene and protein expression. Curcumin reduced cell viability in a dose‐dependent and selective manner, induced cell death on SCC‐9 cells (necrosis/late apoptosis: 44% curcumin vs. 16.4% vehicle), and arrested cell cycle at phase G2/M on SCC‐9 and FaDu (G2: SCC‐9—19.1% curcumin vs. 13.4% vehicle; FaDu—37.8% curcumin vs. 12.9% vehicle). Disorganized cytoskeleton and altered cell morphology were observed. Furthermore, curcumin downregulated the PI3K–AKT–mTOR signaling pathway by modifying the expression of key genes and proteins. These findings highlight the promising therapeutic potential of curcumin to inhibit HNC growth and progression and to modulate the PI3K–AKT–mTOR pathway.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/163924/1/ptr6780.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/163924/2/ptr6780_am.pd

Curcumin downregulates the PI3K–AKT–mTOR pathway and inhibits growth and progression in head and neck cancer cells

Curcumin, a polyphenol isolated from the rhizome of Curcuma longa, has been studied because of its antioxidant, antimicrobial, and antiinflammatory properties. This study aimed to evaluate the effects of curcumin on head and neck cancer (HNC) cell lines and how it modulates the PI3K–AKT–mTOR signaling pathway. Dose‐response curves for curcumin were established for hypopharynx carcinoma (FaDu), tongue carcinoma (SCC‐9), and keratinocytes (HaCaT) cell lines and IC50 values were calculated. Cell cycle and cell death were investigated through flow cytometry. Cytoskeleton organization was assessed through phalloidin+FITC staining. qPCR array and western blot were performed to analyze gene and protein expression. Curcumin reduced cell viability in a dose‐dependent and selective manner, induced cell death on SCC‐9 cells (necrosis/late apoptosis: 44% curcumin vs. 16.4% vehicle), and arrested cell cycle at phase G2/M on SCC‐9 and FaDu (G2: SCC‐9—19.1% curcumin vs. 13.4% vehicle; FaDu—37.8% curcumin vs. 12.9% vehicle). Disorganized cytoskeleton and altered cell morphology were observed. Furthermore, curcumin downregulated the PI3K–AKT–mTOR signaling pathway by modifying the expression of key genes and proteins. These findings highlight the promising therapeutic potential of curcumin to inhibit HNC growth and progression and to modulate the PI3K–AKT–mTOR pathway.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/163924/1/ptr6780.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/163924/2/ptr6780_am.pd

Summary of descriptive characteristics of included articles (n = 14).

<p>The statin clinical application was classified as (1) potential effect in HNSCC treatment; (2) inconclusive, and (3) evidence not supportive as a drug to HNSCC treatment. Abbreviations: U87MG—Human primary glioblastoma cell line; Gy–Gray; ISR—Integrated stress response; HNSCC—Head and neck squamous cell carcinomas; CC—Cervical carcinoma; EGFR—Epidermal growth factor receptor; LKB1- The Liver Kinase B1; AMPK- AMP-Activated Protein Kinase; RhoC—a GTPase belonging to the Ras superfamily; SCC—Squamous cell carcinomas; EGF—Epidermal growth factor; ATF3- activation of transcription factor; mRNA- Messenger RNA; 5-FU 5-fluorouracil; NPC–Nasopharyngeal Carcinoma; LMP1 –latent membrane protein 1; NPC.- Nasopharyngeal Cancer; GGPP- Geranyl pyrosphosphate; ERK- Extracellular-signal-regulated kinases; RT PCR- Reverse transcription polymerase chain reaction; UM- SCC-1—Squamous cell carcinoma cell lines derived from floor of the mouth; UM-SCC-47—Squamous cell carcinoma cell lines derived from tongue; DMSO—Dimethyl sulfoxide; SCC9 (Homo sapiens tongue squamous cell carcinoma); SCC25 (Homo sapiens tongue squamous cell carcinoma); HeLa (cervical carcinoma); A549 (lung carcinoma); MEFs/LKB1 -/- (Murine Embryonic Fibroblast); CCL-30 (Nasopharyngeal carcinoma cells); 293T (Human Embryonic Kidney); Tu167 (Squamous cell carcinoma cell lines derived from floor of the mouth); JMAR (Squamous cell carcinoma cell lines derived from floor of the mouth); CAL27 (Oral Squamous Cell Carcinoma); SIHA (Cervical Carcinoma); Cos-7 (monkey Kidney Cell Line); SCC4 (Oral epithelial cell lines); SCC15 (Oral epithelial cell lines); C15 (Nasopharyngeal Carcinoma Cells); C17 (Nasopharyngeal Carcinoma Cells); A431 cell (epidermoid carcinoma); GM-38 (Diploid fibroblasts cell line).</p><p>Summary of descriptive characteristics of included articles (n = 14).</p

Interventions used to test head and neck carcinoma cell lines viability in cell culture.

<p>Cells: Head and Neck Squamous Cell Carcinoma cell lines, (immortalized or primary cell lines). C: Control. O: Outcomes S: Study (RCT or Comparable baselines). Yes- √”, No “–“. Percentage of cell viability: 1 = 0 to 49% of viable cells; 2 = 50 to 100% of viable cells.</p><p>Interventions used to test head and neck carcinoma cell lines viability in cell culture.</p