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Summer Research Fellowship Programme of India's Science Academies

Effect of Aspirin on proliferation and migration of cervical cancer cells

Ishita Amar

Indian Institute of Science Education and Research, Tirupati, Andhra Pradesh 517507

Dr. Mausumi Bharadwaj

Scientist G (Director Grade and Head), Molecular Biology group, ICMR - National Institute of Cancer Prevention and Research, Noida 201301

Abstract

Cervical cancer (CC) is a major reproductive health problem among women and despite the successful development of a vaccine, the disease is still prevalent. In India, CC ranks as the second most common cancer among women and approximately 90% of deaths from CC occurs in low- and middle-income countries. Current standards of care for CC include surgery, radio- and chemo-therapy. Conventional chemotherapy fails to elicit therapeutic responses and often causes severe systemic toxicity. The major risk-factors for (≥90%) cervical cancers are persistent infections with oncogenic human papillomaviruses (HR-HPVs) that can lead to the development of premalignant lesions and, ultimately, invasive CC. In addition to HPV infection, other cofactors such as cigarette smoking and smoke carcinogen, immunodeficiency, inflammation, multiple sexual partners, long-term use of oral contraceptives etc. are known risk-factors associated with cervical carcinogenesis. Aspirin (acetylsalicylic acid) is an anti-inflammatory drug that is widely used to treat pain and fever. Recently, it has been shown that aspirin reduces inflammation and has anti-cancer effects on several inflammation related cancers, such as colorectal, breast, lung, prostate, esophageal, stomach, ovarian and oral cancers, however, there are still very few data exploring the chemo-preventive role of Aspirin on HPV-related cervical cancers. Therefore, we aimed to study the anti-proliferative and anti-inflammatory effect of aspirin on cervical cancer cells. For this, we have performed MTT assay to check cell viability and migration capacity of cervical cancer cells after exposure to various concentrations of Aspirin at different time intervals.

Keywords: cervical cancer, chemo-prevention, human papillomavirus, Aspirin

Abbreviations

CC Cervical Cancer 
 HR-HPVHigh Risk Human Papillomavirus 
 MTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide 
 FBSFetal Bovine Serum 
 PBSPhosphate Buffer Saline 
VLPs Virus-like Particles 
 NSAIDNon-Steriodal Anti-Inflammatory Drug 
 COX Cyclo-oxygenase

INTRODUCTION

Background

Uncontrolled proliferation of cells of the female cervix gives rise to Cervical Cancer (CC). This disease is a very important health issue for women all over the world, especially for women in developing countries [1]. Worldwide, CC is the fourth most common cancer with high rate of mortality, over 500,000 cases were reported in 2018 with around 300,000 deaths [2]. In India, it ranks as the second most frequent cancer in women. The current estimates indicate approximately 132,000 new cases diagnosed and 74,000 deaths annually in India, accounting to nearly 1/3rd of the global cervical cancer deaths [3].

One of the major risk factors associated with CC is the infection with High-Risk Human Papillomavirus (HR-HPV), with over 90% cases being caused by it. Globally, HR-HPV-16 and 18 contribute over 70% of all cervical cancer cases [4]. Other risk factors for CC include smoking, immunodeficiency, inflammation, multiple sexual partners and long-term use of oral contraceptives. [5] While developed countries with effective screening programmes have lesser cases of the disease, low and middle income countries are the worst hit by this disease. [1]

Current methods for the treatment of CC include radio and chemo therapy, and a vaccine against the major high risk strains of HPV has also been developed successfully. While the vaccine works great for the prevention of the disease, there is a need cheap methods for the treatment if a person contracts the disease, since chemo and radio therapies are costly and often cause systemic toxicity. [6]

Aspirin (Acetylsalicylic acid) is a non-steroidal anti-inflammatory drug (NSAID). Recent research has been able to show that aspirin has a chemopreventive role in several cancers, especially colorectal cancers. [7] Furthermore, it also protects the body against cardiovascular diseases [8] . However, more studies have to be done to define the correct doses that can be administered for treatment in a way that prevents adverse side effects. Furthermore, cancers that have been widely studied are colorectal, breast, prostate, lung, stomach, and oesophageal, but research on the effect of aspirin on cervical cancer is still limited. Through further research, Aspirin has the potential of becoming a cost effective option for treatment of CC.

Objectives of the Research

  • To study the anti-proliferative effect of Aspirin on cervical cancer cells by MTT assay.
  • To evaluate the effect of Aspirin on migratory property of cervical cancer cells

Scope

This study is aimed to provide preliminary data on the effect of Aspirin on SiHa cervical cancer cells through basic experiments: MTT and Migration Assays. Further research needs to be done using more sophisticated animal models to study long term effects, and cell lines taken from different geographical regions to account for genetic differences amongst female populations should also be tested for the same.

LITERATURE REVIEW

Cervical Cancer

In India, there is a population of 453.02 million women older than 15 years of age, and they are at risk of developing cervical cancer. As per current estimates, every year, about 96,000 women are diagnosed with CC and 60,000 die from the disease, indicative of the fact that the disease has a high mortality rate [3].​​ It is now well established that specific types of oncogenic human papillomaviruses (HPVs) are the major etiological agents associated with the development of cervical cancer​​.​ Infection (transmissible) with HR-HPV subtypes results in tumour growth by disrupting cell cycle control through viral oncoproteins E6 and E7. These inactivate p53 and RB tumour suppressor pathways, respectively. This leads to the development of non-invasive cervical dysplasia, but these are not enough to develop cervical cancer, exposure to other risk factors also contributes to the incidence of CC ​[5]​​. About 5% of women of the population harbour cervical HPV-16/18 infection at a given time and 83.2% of invasive cervical cancers are caused due to these strains​​ [4]. ​CC differs from other cancers because it is preventable (due to the successful development of vaccine) and curable if detected early. However, the disease is still not under control in India owing to a lack of effective and organised screening programs, trained people, infrastructure, etc ​[9].​ Furthermore, there is no government sponsored nation-wide public health policy regarding screening or vaccination for CC. 

HPV (Human Papillomavirus)

HPV is a common name for a group of viruses which includes low-risk HPV (LR-HPV) types and high-risk HPV (HR-HPV) types. The most essential risk-factor for genesis of CC is persistent infection with a HR-HPVs [10]. While over 100 serotypes are known, 15-20 are oncogenic. More than 30 HPV genotypes are known to infect cervical epithelium, with a subset of these being associated with lesions that can progress to CC. These cancer-associated HPVs are classified as high-risk HPV types, with HPV types 16, 18, 45 and 31 being the most frequent. The anogenital HPVs have been divided into two groups: the first is associated with a high risk for CC development-the high-risk (HR) HPVs (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82), and the second group with a low carcinogenic potential-the low-risk (LR) HPVs (6, 11, 40, 42, 43, 44, 54, 61, 72 and 81) ​[4]​, [11]

HPVs are a large group of small, non-enveloped, icosahedral, covalent circular, double stranded DNA viruses with a virion size of ~55 nm in diameter which belongs to the family papillomaviridae [12]. Genome size of HPV is about 7200-8000 base pairs which is organized into 3 main regions: 2 protein coding regions (early and late) and a noncoding region known as upstream regulatory region (URR). 

Early proteins of HPV play a key role in its genome maintenance, after HPV infection is established in the basal layer of the epithelium [13]. This infection can be either cutaneous or mucosal, depending on the region. The key early proteins involved in the infection are HPV E6 and HPV E7, they disrupt the host cell regulatory machinery so that replication is altered and incase of persistent infection, without adequate repair or elimination of chromosomes with DNA damage [14]. This is because both E6 and E7 target p53 and retinoblastoma tumour suppression protein (pRB), and this blocks apoptosis evades cell-cycle arrest. Both proteins are expressed at low levels during the infectious process.After the viral cycle goes to various stages, it shifts from early genes to late genes, including L1 and L2, which lead to the assembly of the viral capsid [13]. Infection in the cervix can result in morphological lesions that can be normal, high-grade precancerous lesions and these can subsequently turn into invasive cervical cancer ​[15]​. The development of invasive cancer can take up to 20 years on average, though few cases develop relatively faster [16].​

New Picture.png
    HPV genome and its expression in the epithelium. Adapted from Doorbar.​ [13]

    Two prophylactic HPV vaccines have been successfully developed – a quadrivalent Gerdasil (Merck & Co., Inc.) and a bivalent Cervarix (GlaxoSmithKline). These vaccines use L1 capsid protein of HPV, which is expressed in yeast using recombinant DNA technology. These self-assemble into non-infectious empty shells called Virus-like Particles (VLPs). The vaccine hence contains only an outer L1 protein coat and no genetic material or live virus, so it is not possible to get infected from it. Gerdasil protects against HPV types 6, 11, 16 and 18 and Cervarix protects against types 16 and 18. These vaccines are prophylactic, and prevent most HPV related genital diseases, not just CC [17]. Several studies have shown that these vaccines are efficient in prevention of CC [18], [19]. Furthermore, Gardasil also protects against HPV6‐, HPV11‐, HPV16‐, and HPV18‐related external genital lesions, including genital warts and vulvar and vaginal neoplasia. These vaccines are prophylactic, not therapeutic and therefore, they do not protect against the serotype with which infection has already occurred before vaccination, although protection against other serotypes is conferred. The use and study of vaccines for males is still under way in a few countries like Australia. Despite all of this, there is high incidence of CC in developing countries; hence there is a great need for affordable drugs to combat the disease.

    Aspirin

    Aspirin (Acetylsalicylic acid) is an anti-inflammatory drug (NSAID), and hence, it's regular intake is effective against several diseases like cardiovascular diseases and cancer that develop through inflammation. [7]​​ ​

    A lot is known about the action of Aspirin. It acts as a COX inhibitor, and it inhibits both COX-1 and COX-2 enzymes preferring COX-1 inhibition [20] . In tumor cells, inhibition of COX-2 enzyme results in the reduction of prostaglandin which decreases cell growth, and ultimately results in cell death due to apoptosis. In vitro studies demonstrate overexpression of COX-1 and COX-2 in CC tumour cells [21] . Aspirin is able to acetylate and hence it is significantly different from other NSAIDs. It binds its acetyl group with the cyclooxygenase, bringing an irreversibly inhibiting its function. It can also induce apoptosis in CC cells [22] and inhibit angiogenesis [23].

    Furthermore, aspirin can be used alongside radiation to induce apoptosis in CC cells. Studies done on HeLa cells have shown that treatment with aspirin, followed by irradiation increased the number of apoptotic cells. Hence, sensitivity to radiation was enhanced cells that were treated with aspirin. This has been shown to be mediated by bcl-2 and caspase-3 pathway in cervical cancer cells [24]. Other studies have also indicated that effect of aspirin might be due to its ability to reduce the expression of proto-oncogene ErbB2, which is overexpressed in CC. [25]

    SiHa Cell Line

    SiHa cell line was established from tissue samples of a Japanese patient. The line is HPV16 positive and is reported to contain an integrated HPV16 genome, 1-2 copies per cell [26].​

    METHODOLOGY

    In Vitro Cell Culture experiments

    Cell line used

    • SiHa (HPV16 positive) cervical cancer cell lines was used in the present study.

    Maintenance of cervical cancer cell lines:

    • Dulbecco’s Modified Eagle’s medium, DMEM was used with heat inactivated 10% FBS and 1% penicillin/streptomycin as antibiotics.
    • 1X PBS solution was used for washing.
    • Trypsin-EDTA solution (0.125% Trypsin+0.025% EDTA) was used for detachment.
    • Cells were cultured or treated in 6/96 well plates or T-25 flasks as per the experiment. They were kept in a CO2 incubator with a humidified atmosphere of 95% air and 5% CO2 at 37°C. 

    Subculture

    • Cells were sub-cultured usually in the ratio 1:5 when the confluency of the flask was between 90 and 100%. Briefly, the flasks were first carefully examined for any signs of contamination or deterioration followed by washing with PBS (0.2ml/cm2). Trypsin-EDTA solution was then added (0.1ml/cm2) to the culture and kept in 37°C CO2 incubator for 1-2 min. Upon detachment, fresh DMEM was added (0.1-0.2ml/cm2) and the cells were dispersed carefully by repeated pipetting. After counting of cells with the help of Neubauer’s slide (Paul Marienfeld GmbH & Co., Germany), cells were seeded at a concentration of 0.7 x 106cells/ml. At regular interval of 2-3 weeks, the cells were cryopreserved and stored in liquid nitrogen. Frozen cells were thawed and revived whenever required.

    Cryo-preservation

    • Late log phase cultures with high cell density were cryopreserved. Briefly, single cell suspension was prepared as previously described and re-suspended in freezing medium containing 50% DMEM, 40% FBS and 10% DMSO (Cryoprotectant) in 1:2 ratio. Cells were then distributed into different sterile cryovials in 1ml aliquots and the ampoules were then immediately kept at -70°C for overnight and transferred to ultra-low deep freezer(-150°C) the other day. Regular cryopreservation was done to store the cells. These cells were also revived from time to time to check their viability and any sign of infection.

    Cell revival

    • For revival, stored cryovials were taken out from liquid nitrogen container. The ampoules were rapidly thawed (within 40-60 sec) in 37°C water bath and then cells were transferred quickly to 5ml of culture media in a 25-cm2 flask and kept in a CO2 incubator. When the cells got attached to the surface, the media was changed and fresh media was added to it. The cells were incubated at 37°C and the medium was changed every two days until the culture became confluent and was ready for routine subculturing.

    MTT Assay

    MTT Assay was performed to check SiHa cell viability and dose kinetics of Aspirin. In this assay, MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide], a tetrazolium salt converts to coloured formazan by the mitochondrial dehydrogenase enzymes of live and active cells. This indirectly measures the cell growth and thus gives the extent of cell viability.

    In the present study, the following procedure was followed:

    • Trypsinized SiHa cells were seeded in a 96-well microtitre plate along with media to a cell density of 5000 cells per well.
    • These cells were incubated for 24 hours.
    • As soon as the cells reached log phase, they were treated with different concentrations of Aspirin: 0μM (Untreated), 5μM, 10μM, 15μM, 20μM. The experiment was performed in triplicates.
    • The cells were again incubated at 370C in CO2 incubator for 24-48 hours.
    • After this, 20μl of MTT was added to each well, and the plate was incubated at 370C for 2 hours.
    • 100μl of lysis buffer/DMSO was added.
    • The O.D. was measured at 570nm using a blank solution (only MTT and lysis buffer and no cells) in 96-well plate ELISA reader.

    Migration Assay

    To investigate the healing capacity of SiHa cells, scratch assay was performed using the following protocol:

    • HPV+ve SiHa cells were treated with different concentrations of Aspirin (0μM, 5μM, 10μM, 20μM, 40μM, 80μM) as described above (MTT assay) and incubated under standard conditions to achieve treatment effect.
    • After 48 hours, a scratch was carefully made by scraping through each well using a sterile 200μL pipette tip.
    • The cells were then washed with 1XPBS to remove detached cells.
    • Scratches were monitored with an inverted microscope immediately after wounding and after incubation (370C, 5% CO2) at different time points. Images were taken exactly at the same position before and after the incubation.

    RESULTS

    Analysis of Presence of HPV DNA in Cervical Cancer Cell Lines by PCR

    To confirm the presence of HPV16 in SiHa cells, DNA was extracted from the cultured cells using standard phenol-chloroform method routinely followed in our laboratory. The quality and quantity of DNA was checked by ethidium bromide stained 1% agarose gel. PCR was done to amplify the presence of specific HPV DNA (HPV16) sequences in SiHa cells using specific primers. The presence of HPV16 in SiHa cells was confirmed by amplification of URR region of HPV16 with amplimer size of 217 bp (Figure-2). Primers for β-globin were used as an internal control for checking the quality of DNA as well as authenticity of PCR and its successful amplification.

    New Picture (1).png
    • 1
    • 2
    • 3
    • 4
    PCR amplification of HPV 16 primers in SiHa DNA sample.

    Aspirin Decreases Cell Viability and Induce Growth Inhibition in Cervical Cancer Cells

    Cells were treated with different concentrations of Aspirin for 24 hours and their viability was checked by MTT assay. The cells were plated onto 96-well tissue culture plates, and on the following day, the cells were treated with 0, 5μM, 10μM, 20μM, 40μM, 80μM of Aspirin for 24hrs. As indicated in Figure-3A & B & Table 1, Aspirin resulted in concentration-dependent loss of cell viability in cervical cancer SiHa cells with 50% Inhibitory Dose (ID50) of approximately 33.23 μM of Aspirin and maximal effect was observed at 80 μM. Cells were also checked for their growth kinetics at 24 and 48 hrs in the absence or presence of different concentrations of Aspirin. As shown in Figure-3, Aspirin as low as 10μM could retard the growth of SiHa cells.

    SiHa Cell Lines 5000 cells/Well Seeding
    Drug Treatment 24 hours SiHa Drug Toxicity
    1 2 3
    UT 1.120 0.990 1.010 UT 100
    80μM 0.100 0.090 0.090 5μM 87.97
    40μM 0.300 0.260 0.300 10μM 76.92
    20μM 0.650 0.550 0.680 20μM 60.25
    10μM 0.850 0.800 0.750 40μM 27.54
    5μM 0.920 0.910 0.900 80μM 8.97
    Note
    OD taken for various doses of Aspirin at 24 hours and Drug Toxicity measured with respect to UT
    New Picture (5).png
      A. SiHa cells were treated with indicated doses of Aspirin in triplicates and the viability was measured at different time intervals by MTT assay as described in “Methods”.
      New Picture (4).png
         Fig 3 B. Percent cell viability of cells treated with Aspirin for 48 hrs.

        Aspirin Stimulates Morphological Changes in Cervical Cancer Cell Line; SiHa in a dose dependent manner 

        In order to determine that Aspirin has a cytotoxic effect which leads to any morphological changes in cultured HPV 16 positive SiHa cells, the cells were treated with varying concentrations of Aspirin ranging from 0, 40 μM & 80μM for 48 hrs. The distinct morphological changes in the SiHa cells were observed. We observed significant inhibition in SiHa cell growth at 40μM and 80μM of Aspirin concentrations (Figure-4).

        New Picture (6).png
          A. Effect of Aspirin on the morphology of cells. Photograph of untreated SiHa cells after 48 hours.
          New Picture (7).png
            Fig 4B. Effect of Aspirin on the morphology of cells. Photograph of SiHa cells showing effect of Aspirin treatment with concentrations 40uM after 48 hours of treatment.
            New Picture (8).png
              Fig 4C. Effect of Aspirin on the morphology of cells. Photograph of SiHa cells showing effect of Aspirin treatment with concentration 80uM after 48 hours of treatment.

              Effect of Aspirin on the Migration Ability of SiHa Cells

              A migration assay was performed to check the wound healing capacity of SiHa cells after treatment of Aspirin with varying concentrations (20μM-80μM). Figure 5 illustrates the effects of Aspirin treatment on SiHa cell migration, using gap width at time 0 as a reference. A higher level of migration rate was observed in SiHa untreated cells compared to treated with various doses of Aspirin. Aspirin treatment at 80μM after 48 hour resulted about more than 80% reduction in migration of SiHa cells compared to untreated cells.   

              New Picture (9).png
                Aspirin inhibits the migration ability of SiHa cells.

                Wound healing assay results showing healing capacity of SiHa cells is inhibited as concentration of Aspirin increases in after 48 hours compared to untreated cells. The maximum inhibition of migration rate of SiHa cells was achieved at 80μM of Aspirin concentration. The quantification of cell migration showing percentage wound closure was determined by image J software.

                DISCUSSION

                The prevalence of CC in developing countries like India despite the successful development of vaccines goes to show that there is a need for more research and nationwide programs to combat the disease. In a country like ours, attention should be given to efficient screening programs, mass vaccination, and proper sex education, which are currently either not in place or are not up to mark. The vaccines that have been developed are prophylactic, not therapeutic, so prevention can only occur for serotypes that the person is not previously infected with. Research is still needed towards finding affordable and effective drugs for treatment. The common household drug, aspirin (acetylsalicylic acid) has been around for more than a century. Aspirin, being an easily available and affordable is a promising candidate for such drugs. The mechanism of action of Aspirin as an anti-inflammatory agent is well recorded, yet novel beneficial effects and modes of action keep on adding to its ever-expanding therapeutic repertoire. Accumulating data has shown that regular use of aspirin results in an apparent reduction in the cancer incidence and mortality [27] . Studies done on HeLa cells also show a similar effect, and it is reported that Aspirin induces apoptosis through caspase 3 activation [28]. Not just for CC, Aspirin and other NSAIDs are known to induce apoptosis in different types of cancers [28][29][30].

                Here, we investigated the anti-proliferative and anti-migratory effect Aspirin on SiHa cells. This study highlights the anti-proliferative and anti-migratory properties of aspirin in cervical cancer. The results have indicated that time dependent treatment of aspirin inhibited cell viability and migration rate of SiHa cells. Observational studies have revealed that the regular use of aspirin reduces several cancers incidence and their distant metastases. Scientific evidence supports aspirin as one of the strongest drugs, reducing breast cancer mortality by 50% [31]. Previous research has established that the ability of Aspirin to acetylate lysine and serine residues helps it act as a COX inhibitor; resulting in an irreversible inhibition of its function. Furthermore, it also acetylates tumor suppressor gene p53 which is associated with CC, thereby inducing apoptosis. It was also reported that Acetylsalicylic acid being an effective inhibitor of COX-1 inhibits angiogenesis by COX-1 dependent pathways in CC [20][21] [22][23]. Present study focuses on SiHa cells as they are HPV positive, and HPV is a major causative agent of CC. 

                All of these factors make Aspirin a potential anti-cancer drug. It also has a benefit over other NSAIDs as it is associated with a lowered risk of occlusive cardiovascular diseases. However, there are some side effects associated with NSAIDs like Aspirin, owing to their ability to inhibit COX-1, which prevents aggregation. Therefore, there is risk of bleeding associated with the use of Aspirin. High doses can also increase the risk of gastric ulcers and bleeding. Age-related risks must also be kept in mind before administering the drug to a patient ​[27].​ 

                In summary, the tentative schema is proposed for the possible mechanisms of action of aspirin in SiHa cells in this study, further focused investigations using different cervical cell lines will confirm actual mechanism of action of Aspirin in cervical cancer.​

                CONCLUSION AND RECOMMENDATIONS

                The present study provides sufficient evidence towards Aspirin as an anti-proliferative and cytotoxic agent for cervical cancer cells. A reduction in both proliferation and migration capacity of the cells was observed upon treatment with the drug, and since these two properties are necessary for the development of malignant tumours, Aspirin is a promising therapeutic agents for the treatment of cervical cancer. These findings warrant further study across cell lines and then in animal models for better knowledge of the appropriate dosage required for the desired effect. Based on this study, Aspirin is a cost effective and widely available drug that could act as a chemopreventive agent for CC.

                ACKNOWLEDGEMENTS

                I would like to express my heartfelt gratitude to Dr. Mausumi Bharadwaj, NICPR for giving me the opportunity to be a part of her lab so that I could learn and grow in the field of cancer research.

                I am also deeply indebted to IASc-INSA-NASI for letting me be a part of the summer research fellowship and carry out this project. 

                Furthermore, I am extremely thankful to Dr. Shilpi Gupta for constantly guiding and mentoring me throughout the project and teaching me valuable techniques, despite her busy schedule. 

                Lastly, I would like to thank my seniors in the lab- Heena Ma’am and Mohammad Sir for their constant support and encouragement throughout the two months when I was in the lab. I’m truly grateful for all that I have learnt from them.

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