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Title: Cytotoxicity and gene expression of selected apoptotic markers in the human laryngeal carcinoma cell line (HEp-2) by Bulbine spp. fractions
Authors: Singh, Rishan 
Issue Date: 30-Jul-2013
Apoptosis, or programmed cell death, is a process which is pivotal in eliminating damaged,
infected, or unwanted cells from the body. It has been studied in numerous types of cell lines
ranging from normal to infected cell lines, and there have been a wide range of studies on
apoptosis in laryngeal cancer because this type of cancer has become one of the most
common types of head or neck cancer due to the high incidence of alcohol consumption,
tobacco smoking and chewing of betel quid amongst populations. Laryngeal cancer is usually
treated with radiotherapy or is surgically removed, but due to the loss of the function of the
larynx after surgery, it has been suggested that alternative strategies or ways of treating
laryngeal cancer are required. This has prompted the use of, and research in the field of, plant
medicine to combat laryngeal cancer.
Plant medicine has been used for centuries by the Chinese, Indian and Arabian population in
Uhani, Ayurveda and Siddha as a form of replacing conventional medicine with
complementary and alternative medicine, these include many plants from the family
Asphodelaceae, which have become marketable commodities owing to their medicinal values
and traditional uses. Amongst these plants, the genus Bulbine has been used as a form of
natural medicine in rural Africa and they are also exploited for their aloe vera properties as
well as their possession of phytochemical compounds such as isoflavanoids, nor-lignans,
naphthalene derivatives, anthracene and poly prenylated flavonoids. There has been a
compelling amount of literature on the traditional uses of the Bulbine spp. because these are
linked to the Bulbine spp. having secondary metabolites such as pyroles, chromones,
coumarins, bianthraceane, benzene as well as alkaloids. However, for Bulbine natalensis and
B. frutescens, the plants of interest in this study, the location of anticancer compounds in
them are the only amounts of information available. It has been reported, traditionally, that B.
natalensis possesses the anticancer potential in the roots, while the anticancer potential for B.
frutescens is in the leaves. However, this requires scientific clarification. Therefore, this study
was conducted to assess programmed cell death or apoptosis by analysing the responses of
the human laryngeal carcinoma cell line (HEp-2) to crude aqueous and organic (50% and
100% ethanol) fractions of B. natalensis and B. frutescens. In order to have achieved this, the
HEp-2 cell line was exposed to the above mentioned fractions at three different final
concentrations (20, 2 and 1μg/ml) and assessed for cytotoxicity using the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay as an
indicator of cell death after fraction utilisation (3 days) for 5 and 8 days. The differences in
the potency of the Bubline spp. fractions were confirmed using the non-parametric ANOVA
test. Thereafter, selected fractions were screened for apoptotic potential using reverse
transcriptase-polymerase chain reaction (RT-PCR) to determine the expression of bax and
caspase-3 biomarkers, which are the biomarkers that participate in mitochondrial,
endoplasmic reticulum and receptor-ligand mechanism of apoptosis. The fractions of B.
frutescens were selected relative to those of B. natalensis for the RT-PCR procedure (read
section 3.4.1. for the selection procedure) and links between the cytotoxicity and gene
expression results were analysed.
It was found that the B. natalensis fractions had a much greater cytotoxic effect on the HEp-2
cell line compared to fractions of B. frutescens by the fifth day of the MTT assay. On the
eight day of incubation, there was an increase in HEp-2 cell line proliferation by the fractions
of both plant species administered. The fractions selected for bax and caspase-3 gene
expression analysis for B. natalensis were the: 20 μg/ml root and corm aqueous fractions, 20
μg/ml leaf and corm 100% ethanol fractions, 20 μ g/ml corm 50% ethanol fraction, 2 μg/ml
root aqueous fraction, 2 μg/ml leaf 100% ethanol fraction and the corm 1 μg/ml aqueous and
50% ethanol fractions. The fractions that were compared to B. natalensis were the 20 μg/ml
root and leaf aqueous and 100% ethanol fractions respectively, the 2 μg/ml root aqueous
fraction and the 2 μg/ml leaf 100% ethanol fraction. It was found from RT-PCR analysis that
all of the B. natalensis fractions tested induced expression of caspase-3, which indicated that
those fractions were capable of inducing apoptosis in laryngeal carcinoma in vitro, since
caspase-3 is the molecular indicator of apoptosis. The aqueous B. frutescens root fraction, did
not induce expression of caspase-3 gene, although it caused expression of bax. This implied
that the root aqueous B. frutescens fraction, may be involved in some other form of cell
death, other than apoptosis. It was also found that there was variability in the response of the
HEp-2 cell line to the Bulbine spp. fractions because of the variation in bax expression among
fractions of different concentration. It was difficult, from this study, to classify fractions into
categories for their mechanism of action, because not all of the fractions that caused the
expression of capase-3, induced bax gene expression. Hence, proper conclusions were unable
to be made, more so, because all the mechanisms of apoptosis mentioned, involve bax gene
activation in order to proceed to completion. Therefore for those Bulbine spp. fractions to
which the HEp-2 cell line exhibited a variable response to, it was postulated that cell death or
apoptosis occurred through some other unknown mechanism. Overall, the cytotoxicity result
didn’t correlate to the gene expression results because fractions that promoted HEp-2 cell line
growth by day five, expressed apoptotic markers, which highlighted the sensitivity and
accuracy of the cells-to-cDNATM II kit for detecting a few possibly apoptosed cells. This was
confirmed by the fact that the HEp-2 cell line used in the MTT cytotoxicity assay and gene
expression study had the same passage number and were viable, the latter being achieved
because the MTT assay only measures the cytotoxicity of compounds once they have been
taken up by viable cells – measuring mitochondrial activities expressed as absorbances.
Therefore, the deduction that HEp-2 cell death may be due to bax/caspase-3 expression was
valid because the mRNA was isolated from viable HEp-2 cells that had been killed by
Bulbine spp. fractions of different polarity. Furthermore, the lack of correlation between the
cytotoxicity and gene expression results indicated the amount of HEp-2 cell line proliferation
by the fraction out-competes those that died, thereby producing a negative cytotoxicity result.
There was a relationship between the traditional information about the anticancer potential
for B. natalensis and B. frutescens. For example, the aqueous root fractions of B. natalensis
were found to be non-toxic to the HEp-2 cell line, but did express caspase-3, which indicated
the possibility of apoptosis. Similarly, the 100% ethanol leaf B. frutescens fractions were
non-toxic to the HEp-2 cell line, but were able to induce apoptosis as well. This emphasised
that the MTT cytotoxicity assay should be compared with other methods of measuring
cytotoxicity when performing studies like this, because although literature has emphasised
many advantages of using the MTT cytotoxicity assay in apoptotic studies, this study proved
When identical HEp-2 cells were treated with the same extract, only some cells were killed
(apoptosis) whereas others proliferated. This was because although the cells were identical
phenotypically, they were all probably at different phases of the cell cycle resulting in the
HEp-2 cells responding variably to the same fraction at different concentrations. It was also
found that the responses were concentration independent. For example, the 1 μg/ml B.
natalensis corm fraction exhibited the highest toxicity of the three concentrations
administered. The lowest cytotoxicity was achieved for the 20 μg/ml fraction – showing a
proliferative effect on the HEp-2 cell line. Similarly, the 2 μg/ml aqueous B. natalensis leaf
fraction induced the highest cytotoxicity level in the HEp-2 cell line followed by the 1 μg/ml
and then the 20 μg/ml fractions. Apart from the genetic variation in identical HEp-2 cells;
this indicated that the HEp-2 cell line was selective to particular fractions of the Bulbine spp.
for utilisation. Concentration independence and HEp-2 cell preferential selection has been
reported in many other studies involving plant fractions/extracts and natural products.
This study demonstrated that although all the tested B. natalensis fractions were capable of
inducing HEp-2 cell death possibly via. apoptosis (caspase-3 induction), a lack of any link
between apoptosis and the cytotoxicity results (hence the 20 μg/ml corm fraction had a
negative cytotoxicity but expressed both apoptotic markers), indicated the need for
phytochemical screening of both Bulbine spp. in future, to determine the compounds that are
responsible for the cytotoxicity and gene expression result outcomes of both Bulbine spp.
fractions. Furthermore, procaspase genes also have to be analysed since genes are expressed
to form procaspases, which then form active caspases.
Although normal cells also express caspase-3 genes during apoptosis, this study focused
exclusively on the effect of Bulbine natalensis and B. frutescens fractions (selected relative to
the cytotoxicity results of B. natalensis) on the HEp-2 cell line (read cell culture and
cytotoxicity discussion for selection of HEp-2 cell line). The validity of this study is
confirmed by similar experimental designs that assayed the cytotoxicity of plant-derived or
natural compounds on cancer cell lines only, and the detection of apoptosis through caspase-
3 induction and other unrelated methods. This is the first study to report the induction of
apoptosis in cancer cell lines by Bulbine spp. fractions using cytotoxicity and the expression
of bax and caspase-3 apoptotic markers. It provides insight into the interaction between the
HEp-2 cell line and the aqueous and organic fractions of B. natalensis and B. frutescens by
analyzing links between cytotoxicity and bax and caspase-3 gene expression; which could
probably contribute to drug design with selected Bulbine spp. fractions. Further investigations
are required in future, to confirm the possible drug targets of the studied Bulbine spp.
fractions in an attempt of assaying their therapeutic importance.
Dissertation submitted in fulfilment of the requirements for the Degree of Master of Technology: Biotechnology, Durban University of Technology, Durban, South Africa, 2013.
Appears in Collections:Theses and dissertations (Applied Sciences)

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