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Pharmacology Study Using Hela Cell
Introduction
The HeLa cell line is the oldest, most widely used and permanent human cell line. Its aggressive
growth characteristics make it a difficult contaminant that can overgrow less robust and
temporary cell lines. HeLa contamination is very common in research laboratories and its
detection efficiency is limited by the absence of a rapid, sensitive and robust diagnostic assay.
The history of HeLa cell line is still surrounding controversy. The commonly accepted view is
that the line was derived from cervical cancer cells from Henrietta Lacks, a patient who
eventually died of her cancer. The cell line was found to be remarkably durable and prolific
(Arai, 1976). In spite of being significant as the first continuous and permanent human cancer
cell line, there have been a lot of uncertainties regarding HeLa. It has undergone various strong
adaptations to the culture conditions being the first human cell line to be cultured so widely. The
demand for HeLa cells for research went up quickly and they were put into mass production. The
cell line has been used around the globe by scientists for Cancer and AIDS research,
Chemotherapy tests, Gene Mapping, etc. HeLa cells are used as human sensitivity indicator to
tape, cosmetics, glue and several other products (Biba, 2010).
History of HeLa cell line in Pharmacology Research
HeLa cells improved, improvised and standardized tissue culture. Doctors were able to closely
examine cell division by using frozen HeLa cells. Apart from freezing, HeLa were first human
cells to be cloned. The cloning technology initiated by HeLa led to other advances which utilized
the ability of cells to grow in culture. These include isolating stem cells, cloning entire animal
and most importantly, in vitro fertilization. HeLa cells proved to be a path breaking technology
in human genetics which earlier believed that human gene contains forty-eight chromosomes. It
was discovered later that normal human cells have 46 chromosomes. Knowing the normal
number of chromosomes, it became possible to detect genetic disorders. HeLa cells were
exposed to nuclear radiation to study its effect on normal human cells. Advances in virology,
Polio research, Live cell transport, cloning, genetic hybrids, HPV, HIV, etc. is all due to the
robustness of HeLa cell line(Garner, 2011).
Current Pharmacology Research and HeLa cell Line
The control over diseases requires efficient implementation of research knowledge derived from
more than two decades of study. Most of the diseases are preventable, including cancers and
many of them than be cured if diagnosed in the early stages. Continuous pharmacological
research is going on these days to come up with different cures for the prominent diseases like
HIV and Cancers. In cancer, with limited resources, a major impact can be achieved. HeLa cell
line owing to its versatility in disease research and therapy proves to be very significant.
Medicinal plants have been used for pharmaceutical research for a long time now. They are
considered safer and easily biodegradable than the prevailing synthetic chemicals. They also
reduce the problems of drug resistance. Research for anti-cancer drugs from such natural sources
has increased. Chemical Therapy is effective against a range of tumors, but it does not guarantee
freedom from side effects. The research evaluates some plant products against cancer, presuming
they will cause lesser side effects (Ekwall, 1990).
1. Evaluation of in vitro anticancer activity of hydroalcoholic extract of Tabernaemontana
divaricate
Akhila et al (2012) presents their research on Tabernaemontana divaricate, which is an
ornamental and flowering shrub. It is commonly found in Brazil, Malaysia, India, Vietnam and
Thailand. The author in his study evaluated the anticancer activity of the dried leaves of
Tabernaemontana divaricate. The extract was prepared by drying the plant in shade and
processing the powder with petroleum ether at about 323 K for 18 hours. It was further treated
with Hydro alcoholic solution by the same extraction process. It was followed by the evaporation
of solvent to retain a crude hydro alcoholic extract.
The human cervical adenocarcinoma cell line (HeLa) cells were turned into single cell
suspensions using trypsin-EDTA. A density of 100,000 cells/ml were achieved. Using Serial
dilution method, various concentrations of test drug solutions was prepared and the cells were
treated with it. The medium without sample worked as control. After 48 hours incubation, 15µl
of MTT (5mg/ml) in phosphate buffered saline (PBS) was added and incubated for 4 hours. The
medium now formed formazan crystals. They were solubilized in DMSO and absorbance was
measured at 570 nm.
Cell Inhibition was determined using the formula:
% Cell Inhibition = [100- Abs (sample)/Abs (control)] x100.
The results from the in-vitro studies performed using HeLa cell line proved that the plant has a
moderate anti-cancer activity. Though there was an inhibition in cell growth when the sample
concentration was increased, the IC50 value was around 100 µg/ml for the cell line studies by the
MTT assay method. Therefore, the level of cytotoxicity of the sample can be inferred to be less
effective.
2. Cytotoxicity analysis by MTT assay of isolated Gossypol from Bt and Non-Bt Cotton
Seeds on HeLa Cell Lines
Chandrasekar et al (2014), employed a dose dependent method to evaluate the toxicity of the
isolated gossypol from both Bt and non-Bt cotton seeds on HeLa cell lines at varying
concentrations in this study. Research has shown that cotton plant contain compounds that can
potentially help in treating Cancer and HIV. This paper deals with study of In-vitro cytotoxicity
effect of isolated gossypol from Bt and Non-Bt cotton seeds on HeLa cell lines. Gossypol is a
phenolic compound which enables cotton’s self-defense mechanism against insect pests and
some diseases. Gossypol that reacts with the substances in cotton seeds is called “Bound
gossypol” and is not harmful. The unreacted gossypol called “Free gossypol” is toxic which is an
anti-nutritional factor and anticancer agent that limits the use of cotton seed.
The Bt and Non-Bt cotton seeds were crushed and extracted with diethyl ether. At low
temperature, the solvent was evaporated till gossypol was obtained which was stored for further
use. Cytotoxicity and cell viability assay were used for drug screening and cytotoxicity tests of
chemical compounds. HeLa cells were used for this study. HeLa cells were cultured and subcultured and maintained at 370 degree Celsius at 5% CO2 in CO2 incubator. Cultures were
continuously monitored, sub-cultured and were transferred and incubated for 24 hours. Isolated
gossypol was added at varying concentrations. After incubation, the media containing the drug
was removed and MTT was added to each well. Absorbance was read at 570 nm with a reference
filter at 630 nm. Percentage cytotoxicity was calculated and used for finding the IC50 value of
Gossypol obtained from Bt and Non-Bt cotton seeds.
Results show that a significant cytotoxicity is observed with dose dependent concentrations. The
analysis showed that gossypol is more in Non-Bt seeds. It showed more percentage of cell
viability compared to standard anti-cancer drug Doxorubicin. The study also confirms the mild
toxic effect of gossypol on HeLa cell lines and validates that it can be used as an effective anticancer drug in combination with other similar natural compounds.
3. The in vitro cytotoxic activity of ethno-pharmacological important plants of Darjeeling
district of West Bengal against different human cancer cell lines
RunuGhosh et al (2015) evaluates the in-vitro cytotoxic activity of 30 ethno-pharmacological
plant extracts against three different human cancer cell lines, including HeLa cell lines. It also
characterizes the constituents with the aim of extracting compounds which may help in drug
development against cancer.
The ethanolic leaf extracts of these plants were tested and observed for their cytotoxicity. It was
evaluated using the MTT assay, tryptan blue elusion assay and morphological characterization
under phase contrast inverted microscope. The extracts which gave positive results were
calculated for IC50 i.e. the concentration that inhibited the cell growth by 50%. They were
subjected further to Thin Layer Chromatography to validate their phytochemical nature and
properties. Out of the 30 tested plants, 5 plants displayed a greater than 50% growth inhibition of
cell lines at 50 μg/ml. Phytochemical analysis validated the presence of falvonoids, steroids,
coumarins, tannins and terpenes.
This article claims to be the first screening report of Darjeeling’s traditional medicinal plants.
Darjeeling is a district in West Bengal State in India. The report confirmed the cytotoxic activity
of these plants against MCF7, HepG2 and HeLa cell lines. The extracts of a particular plant,
MaesaMacrophylla inhibited the growth of MCF7 and HeLa significantly. It is also reported to
constitute multiple known biologically active chemical compounds.
4. Invitro and Invivo anticancer activity of Ethanolic extract of CanthiumParviflorum Lam
on DLA and Hela cell lines
Prabhu et al (2011) evaluated some plant products against cancer, presuming they will cause
lesser side effects. Canthiumparviflorum Lam is used alone or in combination with other plants
for therapy. But very few literature was available regarding the scientific evidence to prove the
anti-tumor activity. The research aims to evaluate in vitro and in vivo anti-cancer activity of
Canthiumparviflorum leaves on HeLa cell lines.
Colonies inbreed strains of Swiss albino male mice were kept under standard conditions in 12
hour light/dark cycles. They were acclimatized to the conditions for a week prior to
experimentation and randomly divided into six animals a group. MTT assay was performed by
the standard protocol and absorbance is taken as 570 nm. The growth inhibition was determined
using Growth inhibition = (control O.D – sample O.D/ control O.D) and further IC50 value were
determined. It is based on the reduction of MTT (3-(4, 5- dimethyl thiazolyl)-2, 5-diphenyltetrazolium bromide) by mitochondrial dehydrogenase to purple formazan product.
Tumor growth affects different haematological parameters and the anticancer activity is
generally assessed by restoring the changes in these parameters to square one and most
significantly in increased RBC, lymphocyte and haemoglobin content and decreased WBC, as
compared to tumor control. The acceptance criteria for determining the antitumor activity of a
compound is the determination of circulating WBC and the life span prolongation.
5. The Evidence of HeLa Cell Apoptosis Induced with Tetraethylammonium Using
Proteomics and Various Analytical Methods
Lin Huang et al (2014) work with Tetraethylammonium (TEA), a potassium channel (KCh)
blocker which founds its applications in the functional and pharmacological studies of the KChs.
The MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) colorimetric assay,
which quantitatively measure living cells, demonstrated that TEA reduced the HeLa cell viability
dose-dependently. The optical density was measured at 570 nm using a plate reader named
FLUOstar Omega. This assay is widely used to calculate cell viability and observe cytotoxic
effects of drugs on cell lines in vitro. Cells without treatment were taken as a control for 100%
cellular viability. Each division included triplicate assays to validate the results. Flow cytometry
analysis reflected a steep increase in apoptosis rate of the HeLa cell after getting exposed to
TEA. The patch clamp technique indicated that the K+ current of the HeLa cell was inhibited up
to 80% when exposed to TEA. In addition, quantitative real-time PCR approach set up cross-talk
among the cytotoxicity of TEA, 4-aminopyridine, and anti-cancer drug such as cisplatin.
It is well identified that TEA can block KChs and induce cell death in a clinic study. In this
study, the researchers chose different concentrations and treatment periods of TEA exposed to
HeLa cells. The MTT assay reveal that HeLa cells are very sensitive to TEA, especially for the
concentration of TEA greater than 2 mM, and the growth rates of HeLa cell are inhibited by TEA
dose-dependently. The results obtained from both staining methods are close to each other,
supporting that the methods set up in flow cytometry are reliable.
K+ current in HeLa cells was investigated using the whole-cell configuration of the patch clamp
technique. The results show that the K+ current was obviously blocked up to 80.8% relative to
that in the control group.
6. In-vitro cytotoxicity activity of Solanumnigrum extract against HeLa cell line and Vero
cell line.
Patel et al (2009) aimed at evaluating the anticancer activity of Solanumnigrum fruits on the
HeLa cell line. The methanolic extract of Solanumnigrum was tested for its effects on the HeLa
cell line. The percentage cell line viability was carried out using the Tryptan blue dye exclusion
technique. Trypan Blue is a blue acid dye that has two azo chromophores. Trypan blue doesn’t
enter into the cell wall of plant cells which are grown in culture. Trypan Blue is an essential dye,
use in estimating the number of viable cells present in a population SRB assay and MTT assay
were used to evaluate the cytotoxicity of the plant fruit extract on HeLa cell.
SolanumNigrummethanolic extract has significant cytotoxicity effect on HeLa Cell Line in
concentration range between 10 mg/ml to 0.0196 mg/ml by using SRB assay and study also
showed that inhibitory action on HeLa cell line in concentration range between 10 mg/ml to
0.0196 mg/ml by using MTT assay. IC50 value and R2 value of SolanumNigrum on HeLa cell
and Vero cell were 847.8 and 0.8724, 9088 and 0.1017 respectively by SRB assay. IC50 value
and R2 value of SolanumNigrumon HeLa cell was 265.0 and 0.9496 respectively by MTT assay.
IC50 value of SolanumNigrum on Vero cell was 6.862 by MTT assay. R2 value of
SolanumNigrum was not found by MTT assay. From the performed assay, methanolic extract of
these drug shows greater activity on HeLa cell line and little activity on Vero cell line and that
mean SolanumNigrum can be used as anticancer activity substance.
7. In Vitro Anticancer Activity and DNA Fragmentation Capacity of a Marine Sponge,
Spongiatosta
Archana et al (2014) worked with marine sponges due to the diversity in their secondary
metabolites. Many natural products form marine sources are adorned with potential
immunomodulation, and hence can act as invaluable leads in drug discovery. Although the
molecular biology of the mode of action is still unclear, for a significant number of compounds,
the mechanism by which they interact with the pathogenesis is widely reported. Therefore, the
study is taken up with the aim to study the cytotoxic property from S. tosta which will help in
formulation of doses for therapeutic efficacy.
For cytotoxicity studies, S. tosta was dissolved in distilled DMSO to obtain a stock solution of
1mg/ml concentration and sterilized ultimately by filtration. Two fold serial dilutions were
prepared. The samples were washed with water, air dried and lyophilized to store them for
further use.
Using apoptotic DNA ladder kit [G Bioscience], HeLa cells were seeded in two 6 well plates and
allowed to fix for 24 hours. They were then incubated for 48 hours at 310 K in humid conditions.
After trypsinization, cells were washed with PBS. Apoptotic cells were incubated with lysis/
binding buffer in 15-25 degree Celsius for 10 minutes. After incubation, the base sample was
mixed with isopropanol and pipetted into a filter tube. DNA bound to the filter tube was isolated
by centrifugation of the sample. The bound DNA was washed and the unbound fragments were
disposed. Eluted DNA was collected, mixed with loading buffer , electrophoresed on 0.8%
agarose gels at 90 V for 1.5 hours and then visualized using a UV Trans illuminator (Masters,
2002).
This study validates that methanolic extract of S. tosta possess tremendous anticancer properties.
Also, in the DNA fragmentation assay,effective induction of apoptosis was confirmed by
electrophoretic pattern of separated DNA fragments in HeLa cell line.
8. Induction of apoptosis in the cervical cancer cell line HeLa by a novel metabolite
extracted from the fungus Aspergillusjaponicus Saito
Apoorva et al (2014) presented the research analyzing the secondary metabolite extracted from
Aspergillus japonicas and its potential anti-cancer property. It was tested on Human cervical
cancer cell line HeLa. To evaluate its safety in humans, it was also tested on normal human
peripheral lymphocytes. The research also aims to characterize the compound responsible for
anti-cancer activity by partial purification and subsequent Thin Layer Chromatography (TLC)
and Liquid chromatography-Mass Spectroscopy (LC-MS analysis).
The bioactive compound was extracted by preparing 20 ml of A. japonicas from a 48 hours old
culture. The enriched culture was then transferred as seed into culture medium and incubated for
8 days at 25 °C in stationary condition. The dried hyphae were homogenized and the metabolites
extracted with methanol, applying standard methods. The extract from the mycelia was
evaporated under vacuum at 50 °C till it dried. The obtained solid was weighed and then
dissolved in dimethyl sulfoxide (DMSO) to form the crude extract at 1 mg/mL concentration.
The lymphocytes and HeLa cells were both treated with the crude extract and then exposed to the
MTT assay for observing the cytotoxicity of the extract. After trypsinization, 24 hour old
cultures of HeLa cells and lymphocytes were harvested. Then the MTT assay was performed in
quadruplicate and absorbance was noted at 540 nm. This was followed was detection of active
compound isolated in TLC. The partially purified fragments from TLC were tested against both
HeLa and lymphocytes for cytotoxicity by MTT Assay.
Fluorescence Microscopy was used for apoptosis determination of HeLa cells. HeLa cells were
treated with bioactive fraction for 24 hours. The cells were washed 3 times with PBS, trypsinized
and centrifuged. The supernatant was discarded and cells were resuspended in PBS. Stain
solution of Ethidium bromide: Acridine Orange was mixed with the cell. Slides were observed
under fluorescence microscope using a blue-green filter. Control cells were similarly processed
for fluorescence microscopy.
The bioactive fraction from A. japonicus was found to have high anticancer and antiproliferative
effects with an IC50 value of 10 µg/ mL. Both the crude extract and the bioactive fraction from
this have shown immunostimulatory effects on normal human lymphocytes. A. japonicas was
found to be an excellent source of extracting a secondary metabolite with cytotoxic property
against cervical cancer cell HeLa.
9. Anticancer effect of Moringaoleifera leaf extract on human breast cancer cell
NilanjanaGhosh in her research (2013) reported that the leaf extract of a plant called
Moringaoliferawhen interacts with human cervical cancer cell line, gives an anti-proliferative
effect. The author majorly talks about the impact of the leaf extract on Breast Cancer. Different
fragments were added in various solvents and crude methanolic extract of Moringaoleifera was
prepared, using the standard accepted extraction protocols(Tiloke, 2013).
To observe the anti-proliferative property, all the fractions were screened on the HeLa cell line.
The chosen extract was tested for MCF 7 and MDAA MB 231, two breast cancer cell lines in
different concentrations. MTT assay was used for evaluation of cell viability for 24 hours and 48
hours. The Lethal Dose 50 value was calculated and other morphometric studies were done with
the effective dose of the extract and was compared to cis-Platinum. Normal cell study was also
done to establish a control sample.
The leaf extract displayed a dose and time dependent inhibition on the cell proliferation in the
breast cancer cell lines. It showed low cytotoxicity in the normal cells and in the treated cancer
cells, it healed the wounds and inhibited cellular adhesion. The author suggests that the leaf
extract of M. olifera induces anticancer impact on the HeLa, breast cancer cell line. Further
research might confirm it as an anti-cancer drug.
10. HeLa cell Line Xenograft Tumor as a Suitable Cervical Cancer Model: Growth Kinetic
Characterization and Immunohistochemistry Array
M. Arjomandnejad et al (2014) describes cervical cancer as third most common cancer in
women. Despite significant progress in the therapy, it is not completely curable (Fiebig et al,
2004). Animal models are one of the most significant practical tools in cancer research. The
author’s study is aimed at characterize the surface markers and growth behaviour of HeLa cells
after heterotrophic and systematic inoculation to athymic nude mice.
Ten 6-week old nude mice were used in this study. HeLa cells were inoculated into the tail vein
or the flank. Tumor volume was calculated and representative growth curves were drawn.
Tumor-affected mice were sacrificed and the lesions obtained after harvesting were observed.
One slide per tumor was stained with hematoxylin and eosin (H&E) and other slides were
immunohistochemically stained by cytokeratins (CK), Ki-67, vimentins, CD34 and P53.
Tumor take rate was calculated as 94.4%, whereas mean doubling time and latency period were
5.29 days and 15.27 days respectively. H&E results showed highly malignant hyperchromatin
epithelial cells. Immunohistochemical examination of the heterotopic tumors reflected greater
expression of CK and less expression of vimentin compared to the static ones. 60% of cells were
P53-positive and more than 80% were Ki-67-positive. CD34 expression indicated the intensity of
angiogenesis in tumor (Headley, 2011).
The research comprehensively described the HeLa xenograft model for in-vivo cancer
investigations, enabling researchers to assess latest treatments for cervical cancer.
Alexander Del Carpio from Berkeley Science Review (2014) assess what makes HeLa cells so
wanted and special. They are the first immortal cell line cultured by the scientists. Unlike normal
human cell population which divide about 40-50 times, HeLa can divide indefinitely. HeLa was
one of the jumpstart for research on modern virology. It was able to tell the researchers how the
viruses act and reprogram the cell(Moorthi, 2014). There are other cell lines which are developed
in recent years, but the familiarity and hardy growth, as the author likes to call it, makes HeLa a
popular choice amongst others.
Taking a detailed look into the karyotype of HeLa may answer our questions as to what makes it
so special and robust. Normal human cells have 46 chromosomes, while HeLa has 76 to 80
heavily mutated chromosomes. The source of this deviation from normalcy comes from the
human papilloma virus (HPV), the root cause of nearly all cervical cancers (Qi, 2014). HPV
inserts its DNA into a host, causing it to begin producing a protein that binds to and inactivates
the native p53 protein. p53 is the guardian of the genome as it prevents mutations and suppress
tumors. Non-functional p53 protein have disastrous consequences.
The growth of HeLa cell is unusually fast. Early researchers were amazed to see that within 24
hours of culturing, the number of cells have doubled. This abnormal characteristic can be
credited to HeLa’s telomerase enzyme. During normal cell division, the string of repetitive DNA
at the tips of all chromosomes, known as telomeres, are shortened. This ultimately leads to
apoptosis, or cell death. Normal cells have a maximum number of divisions before these
telomeres are depleted. HeLa cellshave an overactive telomerase enzyme that rebuilds
telomerases after cell division, thus circumventing the aging process and skirting death.
The popularity of HeLa began as soon as it was discovered. Then, it was HeLa’s vigor and
human origin that made it stand out of the box. Today, it is HeLa’s familiarity and robustness
which makes it comfortable to use. Many tools, protocols and techniques were developed using
HeLa, and hence are optimized for it. HeLa has a brilliant transfection efficiency, i.e. when
researchers transfect HeLa with some protein, a large percentage of the protein population will
have this protein of interest (Liu, 2012).
HeLa specific genome database will make things easier for research. National Institute of
Health’s database has now made it possible to gain access to HeLa genome. This bioinformatics
approach will significantly reduce the time required to design efficient therapy models as it will
be a source to tell what genes are expressed and to what extent, hence minimizing the time
required for pre-research activities.
The discovery of HeLa cells has played a crucial role in medicine. HeLa cells were part of
research into the genes that cause cancer and those that suppress it; they helped develop drugs for
treating herpes, leukemia, influenza, hemophilia and Parkinson's disease; and they've been used
to study lactose digestion, sexually transmitted diseases, appendicitis, human longevity,
mosquito mating and the negative cellular effects of working in sewers. Doctors have created the
field of virology which is the study of viruses after infecting her cells with everything from
measles to mumps so that they could see how the virus affects the cell. Because of her cells
people were able to be cured by the advances in medicine.
HeLa cells have come a long way since its discovery and found their applications in a broad
range of diseases and disorders. They have the potential to be more helping towards medicine
and pharmacology, but that would require the publication of the genome database of HeLa cells.
A newly constituted body at NIH will review the scientists’ applications for access to the full
genome sequence data from HeLa cells. This agreement was reached after Lack’s family had
cited issues of their genetic profile being made public and ultimately letting open their history of
genetic disorders, if any. The committee will be composed of Scientists, bioethicists and Lack’s
descendants, and they will decide whether the researcher can be trusted to keep the genomic data
secure and not disclose it for any commercial interests (Zielinski, 2010).
More than 60 years after Lack’s death, HeLa cells still are a source and base for groundbreaking
medical and pharmacological innovations (Nair, 2015). Jay Shendure et al (2013) properly sum it
up by calling it the first successful attempt to immortalize human-derived cells in vitro. The
robust growth and unrestricted distribution of HeLa cells resulted in its broad adoption—both
intentionally and through widespread cross-contamination—and for the past 60 years it has
served a role analogous to that of a model organism. The genomic architecture of HeLa remains
largely unexplored beyond its karyotype, partly because like many cancers, its extensive
aneuploidy renders such analyses challenging. Medicinal and Pharmacological Research, if
allowed to be conducted efficiently, may even further reveal the untapped potentials of HeLa
cells.
Conclusion
Henrietta Lacks may not have contributed directly to any of the advancements in the
pharmacological and medicinal technology, but she indeed left her cells behind. Her cells proved
an invaluable gist that helped the scientists and researchers of 20th and 21st century to progress on
therapy and treatment. Though a mild controversy may have hampered the progress of
technology via HeLa cells, but thankfully, the family and the National Institutes of Health came
to a mutual understanding about future procedures involving HeLa cells, ensuring their
continued utility for medical and biotechnological research. To put it in a single line, we can say
that, “For cells may come and cells may go, but HeLa will go on forever.”
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