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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.” References Arai, T., Okomoto, K., Ishiquro, K. &Terao, K. (1976), ‘HeLa cell-tumor in nude mice and its response to anti-tumor agents.’ Gann, (67), pp. 493 – 503. Archana, R., Kanchana, G. &Rubalakshmi, G. (2014), ‘In Vitro Anticancer Activity and DNA Fragmentation Capacity of a Marine Sponge, Spongiatosta’, International Journal of Current Research in Biosciences and Plant Biology, 1(3), pp.87-91. Biba, E. (2010) ‘Henrietta Everlasting: 1950s Cells Still Alive’, Helping Science (Online). 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(2015), ‘The in vitro cytotoxic activity of ethno-pharmacological important plants of Darjeeling district of West Bengal against different human cancer cell lines’, BMC Complementary and Alternative Medicine. Zielinski, S. (2010) ‘Henrietta Lacks’ “Immortal” Cells’, Smith Sonian Magazine. Available at http://www.smithsonianmag. com/science-nature/Henrietta-Lacks-ImmortalCells.html