Thesis (Ph.D. (Biochemistry)) --University of Limpopo, 2009 / Commelina benghalensis Linn is used in traditional medicine in several Asian and African countries for the treatment of various ailments such as stomach irritations, burns, sore throat and feet, diarrhoea and as an anti-inflammatory agent. Recently, our laboratory showed that the crude methanolic extract of Commelina benghalensis L (CMECB) exhibits growth inhibitory and proapoptotic effects in Jurkat T and Wil-2 NS cancer cell lines. In this study, the precise molecular mechanism(s) associated with CMECB-induced growth inhibitory and apoptosis inducing effects in Jurkat T and Wil-2 NS cell lines were investigated. This was achieved by investigating the effects of the extract on the cell division cycle distribution profile as well as its effects on various cell division cycle and apoptosis regulatory genes. Ground stems of C. benghalensis L were extracted with absolute methanol to obtain a crude extract. To assess the effect of CMECB on cancer cell growth, experimental cell cultures were exposed to various concentrations (0 to 600 μg/ml) of CMECB for up to 72 hours. The results demonstrated a significant reduction in cell viability and inhibition of proliferation of experimental cell cultures as determined by the trypan blue dye exclusion assay and the Coulter counter method, respectively. Analysis of nuclear morphological changes in cells stained with Hoechst 33258 confirmed apoptosis as the mode of cell death that is associated with the growth inhibitory effects of CMECB in both the Jurkat T and Wil-2 NS cell lines. This assertion was based on the observed presence of nuclear morphological changes such as chromatin condensation and fragmentation and apoptotic bodies in cells exposed to CMECB. In order to get an insight on the pro-apoptotic mechanisms of CMECB, Western blot xxi and quantitative real-time PCR (qrt-PCR) were used to investigate the expression profiles of various apoptosis and cell division cycle regulatory genes. Qrt-PCR results showed a lack of a clear up- and/or down-regulatory effects of CMECB on the mRNA expression levels of bax and bcl-2 in both Jurkat T and Wil-2 NS cells. Western blot analyses demonstrated that CMECB induced apoptosis by facilitating Bax protein translocation from the cytosol to the mitochondria in both Jurkat T and Wil-2 NS cells. In addition, CMECB down-regulated Bcl-2 protein expression which, as a result, led to the shift in the Bax/Bcl-2 protein ratio at certain time points and concentration in both Jurkat T and Wil-2 NS cells. The modulation of the Bcl-2 family members led to mitochondrial cytochrome c release into the cytosol and activation of caspases-9 and -3; this was also confirmed by caspase activity assays and eventual degradation of PARP. Furthermore, CMECB induced Jurkat T and Wil-2 NS cell division cycle arrest at the G2/M phase as determined by flow cytometric analysis. Western blot analyses of G2/M phase regulatory proteins demonstrated that the CMECB-induced cell division cycle arrest was associated with the downregulation of cyclin B1 and Cdc2 protein expression levels. Western blot analyses results further revealed that the arrest of Wil-2 NS cells at the G2/M phase was independent of p21 protein activity. However, Jurkat T cell division cycle arrest was found to be mediated, in part, by p21. Quantitative real-time PCR results did not show a clear trend in terms of the down- or up-regulatory effects of the extracts on the G2/M phase regulatory genes. The CMECBinduced apoptosis and G2/M arrest was found to occur in a p53-independent xxii manner due to the lack and down-regulation of p53 protein levels in both Jurkat T and Wil-2 NS cells, respectively. In conclusion, CMECB induces its anticancer activity by inducing G2/M phase arrest and mitochondrial-mediated apoptosis independent of p53 protein activity. Although the study did not perform in vivo experiments to ascertain the efficacy of extracts of CMECB against specific tumour types in animal models, the present findings somehow validate the traditional use of C. benghalensis L as an anticancer agent. A more definitive study needs to be done to ascertain this assertion. / National Research Foundation and the University of Limpopo research office

Submitted by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-02-27T13:30:34Z No. of bitstreams: 5 37.pdf: 3780972 bytes, checksum: 25735fd7c2f94a2a52a7a996adefcb6b (MD5) license_url: 52 bytes, checksum: 3d480ae6c91e310daba2020f8787d6f9 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) license.txt: 2109 bytes, checksum: aa477231e840f304454a16eb85a9235f (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-02-27T18:41:07Z (GMT) No. of bitstreams: 5 37.pdf: 3780972 bytes, checksum: 25735fd7c2f94a2a52a7a996adefcb6b (MD5) license_url: 52 bytes, checksum: 3d480ae6c91e310daba2020f8787d6f9 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) license.txt: 2109 bytes, checksum: aa477231e840f304454a16eb85a9235f (MD5) / Made available in DSpace on 2015-02-27T18:41:07Z (GMT). No. of bitstreams: 5 37.pdf: 3780972 bytes, checksum: 25735fd7c2f94a2a52a7a996adefcb6b (MD5) license_url: 52 bytes, checksum: 3d480ae6c91e310daba2020f8787d6f9 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) license.txt: 2109 bytes, checksum: aa477231e840f304454a16eb85a9235f (MD5) Previous issue date: 2011 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (Capes) / Funda??o de Amparo ? Pesquisa do estado de Minas Gerais (FAPEMIG) / Avaliaram-se neste trabalho os efeitos da competi??o entre cultivares de milho, feij?o e mandioca e seis esp?cies de plantas daninhas no ac?mulo e na aloca??o de mat?ria seca e nutrientes pelas plantas; o potencial de algumas dessas esp?cies em reciclar nutrientes e a efici?ncia nutricional do feijoeiro. Para isso foi realizado um experimento para cada cultura com tr?s gen?tipos de milho, tr?s de feij?o e dois de mandioca, cultivados em competi??o com seis esp?cies de plantas daninhas, al?m dos tratamentos adicionais, correspondentes ?s cultivares de milho, feij?o e mandioca e ?s esp?cies daninhas ausentes de competi??o. O per?odo de conviv?ncia entre as cultivares de milho, feij?o e mandioca e as plantas daninhas foi de 60, 45 e 75 dias ap?s emerg?ncia das culturas, respectivamente. Para a cultura do milho e do feij?o foi avaliada a aloca??o de mat?ria seca e de nutrientes, inclusive entre os diferentes ?rg?os. Ainda para o feij?o foram estimados os coeficientes para efici?ncia de absor??o, transporte e utiliza??o dos nutrientes. Para a cultura da mandioca, foram determinadas, al?m da aloca??o de mat?ria seca, caracter?sticas morfofisiol?gicas ligadas ? ?rea foliar. A folha e o caule do milho foram os principais ?rg?os afetados negativamente pela competi??o, enquanto as ra?zes das esp?cies competidoras foram os ?rg?os mais prejudicados. Em competi??o com o feijoeiro, as folhas e o caule das esp?cies daninhas foram os ?rg?os mais prejudicados. Com mandioca, os componentes vegetativos das plantas daninhas sofreram reduzidas modifica??es, sem preju?zos quanto ? produ??o de mat?ria seca. As ra?zes do feijoeiro e de mandioca foram os principais ?rg?os afetados negativamente pela competi??o. Os gen?tipos AL 25 (milho) e IPR Colibri (feij?o) foram os que menos toleraram a competi??o. De forma contr?ria, as cultivares P?rola (feij?o) e Periquita (mandioca) demonstraram possuir as maiores habilidades competitivas. O milho mostrou-se sens?vel ? competi??o com B. brizantha e C. benghalensis e competitivo com B. plantaginea. Esta esp?cie, entretanto, mostrou-se altamente competitiva com feij?o e mandioca. Al?m disso, A. spinosus, em competi??o com o feijoeiro, demonstrou possuir a maior habilidade competitiva. A capacidade competitiva entre esp?cies vegetais, quando se trata do ac?mulo de mat?ria seca e relativo de nutrientes, n?o representou vantagem para as esp?cies de plantas daninhas, quando elas foram mantidas em conviv?ncia com o milho. O feijoeiro apresentou baixa capacidade de competi??o por nutrientes, particularmente N e P. A ?rea foliar espec?fica e a raz?o de ?rea foliar da mandioca n?o foram afetadas pela competi??o. B. brizantha e C. echinatus, livre da conviv?ncia com o milho, apresentaram elevado potencial em reciclar nutrientes. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Produ??o Vegetal, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2011. / ABSTRACT There were evaluated in this study the effects of competition between maize, beans and cassava, and six weed species in the accumulation and allocation of dry matter and nutrients by plants, the potential of some of these species in recycling nutrients and nutritional efficiency of bean. For this end an experiment was performed to each culture with three genotypes of maize, beans and two of three cassava grown in competition with six weeds species, apart from additional treatment corresponding to the maize, beans and cassava cultivars and weed species absent from the competition. The period of coexistence among maize, beans and cassava cultivars and the weeds was 60, 45 and 75 days after crop emergence, respectively. For the corn and beans there were evaluated allocation of dry matter and nutrients, including between different organs. Even for the beans there were estimated coefficients for uptake, transport and utilization of nutrients. For cassava plants there were determined, besides the allocation of dry matter, morphological and physiological characteristics related to leaf area. The leaf and stalk of corn were the main adversely affected organs by competition, while the roots of competing species were the most affected organs. In competition with the beans, leaves and stems of weeds were the most affected organs. With cassava, the weeds vegetative components suffered reduced modifications without losses in the production of dry matter. The roots of bean and cassava were the main adversely affected organs by competition. Genotypes AL 25 (maize) and IPR Colibri (beans) were the less tolerant in the competition. Conversely, the P?rola (beans) and Periquita (cassava) have showed the greatest competitive abilities. The corn was sensitive to competition with B. brizantha and C. benghalensis and competitive with B. plantaginea. This species, however, proved to be highly competitive with beans and cassava. Furthermore, A. spinosus, in competition with the bean, has demonstrated the greatest competitive ability. The competitive ability of plant species when it comes to the accumulation of dry matter and related nutrients did not represent the advantage to the weeds, where they were kept in close contact with the corn. The bean had low capacity to compete for nutrients, particularly N and P. The specific leaf area and leaf area ratio of cassava were not affected by competition. B. brizantha and C. echinatus, free of coexistence with the corn, had high potential to recycle nutrients.