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
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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
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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
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ul/oai:ulspace.ul.ac.za:10386/937 |
| Date | January 2009 |
| Creators | Mbazima, Vusi G. |
| Contributors | Mampuru, L.J. |
| Publisher | University of Limpopo (Turfloop Campus) |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | xxiii, 143 leaves : ill.(some col.) |
| Relation |
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