The Evaluation of Anticancer Properties of Marchantia paleacea Extracts

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STEVE ANTHONIUS LEFRAND MAKALEW 1901034

Field Supervisor

apt. Pietradewi Hartrianti, M.Farm., Ph.D.

i3L Supervisor

Richard Sutejo, S.TP., Ph.D.

The Evaluation of Anticancer Capabilities of

Marchantia paleacea Extracts

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PREFACE

Nelson Mandela said that there is no losing, either you win, or you learn. By this point, I don’t think winning even comes close to the wealth of experience I’ve gained through this Internship.

This internship project, with the title “The Evaluation of Anticancer Properties of Marchantia paleaceaExtracts” was done as a requirement for the “Credit Internship” course in the 7th semester of i3L’s curriculum. The project was a study to investigate whether or not this plant species possesses any cancer-specific cytotoxic effect.

I would first like to thank Jesus Christ, for His eternal love and blessings is what propelled me to finish this internship project. Second, my parents for their ceaseless emotional support. Next, my supervisors Ms. Pietra and Sir Richard for their constant guidance throughout the project. The lab assistants and Ci Erika as my research assistant for their valuable inputs on the project. My fellow internees at anticancer, antiaging, tamanu, and the other PKM projects who shared similar ups and downs. Last but not least I would like to thank i3L and BRIN for this great opportunity to learn, grow, and mature as a scientist.

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ACKNOWLEDGEMENT PAGE

The Evaluation of Anticancer Capabilities ofMarchantia paleaceaExtracts NATIONAL RESEARCH AND INNOVATION AGENCY

AUTHOR:

STEVE ANTHONIUS LEFRAND MAKALEW 19010134

BIOMEDICINE

ACKNOWLEDGED BY

FIELD SUPERVISOR SUPERVISOR AT I3L

apt. Pietradewi Hartrianti, M.Farm., Ph.D. Richard Sutejo, S.TP., Ph.D.

HEAD OF DEPARTMENT AT I3L

Elizabeth Sidhartha, B.Sc., M.Sc.

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LIST OF FIGURES

Figure 3.1.1.1. The dried out M. paleacea extracts after the maceration process. 8 Figure 3.1.2.1. The effect of 5-FU on both HaCaT and HT-29 cells’ viability. 8 Figure 3.1.2.2. The effect of M. paleacea extracts on both HaCaT and HT-29 cells’ viability 9 Figure 3.1.3.1. Cell count of both HaCaT and HT-29 cell lines treated with 5-FU 9 Figure 3.1.3.2. Cell count of both HaCaT and HT-29 cell lines treated with M. paleacea extracts 10

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LIST OF ABBREVIATIONS

5-FU 5-Fluorouracil

cDMEM Complete Dulbecco’s Modified Eagle Media

DMEM Dulbecco’s Modified Eagle Media

DMSO Dimethyl Sulfoxide

FBS Fetal Bovine Serum

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

Pen-Strep Penicillin G-Streptomycin

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ABSTRACT

Cancer is a major burden in the healthcare system, as the leading disease in terms of DALYs (Disability-Adjusted Life Years) afflicted to the patient, and second in terms of mortality rate. Until this day, there is still no permanent universal cure for cancer, with current treatments either capable of afflicting adverse side effects to the patient, or relying on several different mechanisms susceptible to alterations by the cancer cell or its surrounding environment. Alternative compounds would need to be screened to find potentially effective anticancer agents. Phytochemicals have a great potential for anticancer properties, as shown by the presence of plant-based cancer drugs already used in the market. Being one of the 17 megadiverse countries, Indonesia possesses an abundance in potential plants to screen for possible anticancer phytochemicals. Marchantia paleacea, a type of liverwort present in Indonesia, would possibly be one of these anticancer compounds due to the presence of compound families previously already exerting anticancer properties. To evaluate whether or not M. paleacea extracts possess anticancer capabilities, M.paleacea were extracted through an ethanol-based maceration method, then MTT and colony forming assays were performed. Even though the experiment conditions were not optimal, theM.

paleaceaextract did not exert any cytotoxic effects on the normal (HaCaT) and cancerous (HT-29) cell lines, in contrast with the positive control 5-FU.

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CHAPTER 1: INTRODUCTION

1.1. Host Institution/Company

1.1.1. Description about the company

BRIN (Badan Riset Inovasi Nasional) or the National Research and Innovation Agency is an institution made by President Joko Widodo through the President’s Directive Number 74 Year 2019 with the task to conduct research, development, review, and application, as well as an integrated invention and innovation. Initially, BRIN was a part of the ministry of research and technology, however on the 5th of May 2022 President Joko Widodo (through the President’s Directive Number 33 Year 2021) effectively signed BRIN as the only national research institution. The directive in effect merged every one of Indonesia’s national research institutions such as the Indonesian Institute of Science (LIPI) and the National Nuclear Energy Agency of Indonesia (BATAN) into BRIN.

Vision

The realization of a competent, professional, and innovative National Research and Innovation Agency with integrity, in the service of the President and Vice President, to fulfill the President’s Vision and Mission: “A Progressive, Sovereign, and Independent Indonesia based on Mutual Cooperation”.

Mission

● To provide technical and administrative support as well as a rapid, accurate, and responsive analysis to the President and the Vice president on the conduct of research, development, review, and application; invention and innovation; execution of a nationally integrated nuclear energy and space research; as well as conducting monitoring, management, and evaluation actions on the implementation BRIDA’s (Badan Riset Inovasi Daerah or Regional Research and Innovation Agency) tasks and functions

● To improve the quality of human resources as well as research and innovation infrastructure for the realization of a nationally integrated nuclear energy and space research; and to provide guidance on the implementation of BRIDA's tasks and functions

● To carry out an effective and efficient service in terms of monitoring, general administration, information, and inter-institute communication.

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1.1.2. Description of department

The main body responsible for conducting research within BRIN are divided into 12 different Research Organizations (RO), covering numerous fields of studies.

These 12 ROs are: RO for Earth Sciences and Maritime, RO for Life Sciences and Environment, RO for Agriculture and Food, RO for Health, RO for Archaeology, Language, and Letters, RO for Social Sciences and Humanities, RO for Nuclear Energy, RO for Governance, Economy, and Public Welfare, RO for Energy and Manufacture, RO for Nanotechnology and Material, RO for Electronics and Informatics, as well as the RO for Aeronautics and Space.

The RO for Health has its roots as the “National Institute of Health Research and Development” under the Ministry of Health. The RO was functional from the 4th of March 2022, and it comprises several Research Centers (RC). These are: RC for Biomedical Research, RC for Preclinical and Clinical Medicine, RC for Public Health and Nutrition, RC for Pharmaceutical Ingredients and Traditional Medicine, RC for Vaccine and Drug, Eijkman RC of Molecular Biology, and the RC for Veterinary Science. The RO for Health is one of the prioritized RO in the 2022 National Research Priority, which is a government funding initiative to provide research grants to various universities, research institutes, companies, or public organizations. One flagship of the National Research Priority is research on Indonesia’s Endemic Biodiversity, specifically on the utilization of Indonesia’s biological diversity (biological prospecting).

1.1.3. Product of the Host Institution / Company

As Indonesia’s sole national research institution, the research conducted by or funded by BRIN would be made into research papers to either be published internally via the various publishers tied to the research agencies that made up BRIN, or to be published externally in both national or international journals. The research done can also produce novel technologies or inventions that could be patented. BRIN also holds regular conferences that would communicate key findings from their research. Short review articles of the conferences would also be published on the BRIN website, to highlight important information communicated during the conference. BRIN also organizes seminars through meeting apps and makes video podcasts covering specific topics that are made available through their Youtube channel.

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CHAPTER 2: PROJECT DESCRIPTION

2.1. Internship Project 2.1.1. Project Background

Cancer remains one of science’s greatest unsolved mysteries, a disease which remained practically cureless since its discovery over three millennia ago despite vast advances in the field of medicine ("The History of Cancer | First Cancer Diagnosis", n.d.). Cancer is a disease in which regular cells turn rogue, divide uncontrollably, and spread themselves to other parts of the body; monopolizing the body’s share of important nutrients, and wiping out the body’s capacity to perform regular functions, which often would result in death for the patient ("Cancer", 2022).

Cancer is a massive burden on the healthcare system, as it is the highest scoring disease based on how many DALY (Disability-Adjusted Life Years, the total amount of years lost due to premature mortality, or due to being/living in not healthy/disabled conditions) it could afflict on its patients at around 244.6 million DALYs, roughly 9.2% of the world’s total (World Health Organization, 2018). The risk of developing cancer (from 0 to 74 years of age) was estimated at around 20.2%, with males slightly having a larger risk than women ("Cancer today", n.d.). Cancer also sits at number 2 of leading causes of mortality at around 15.8%, with the percentage risk of mortality from any types of cancers sits at around 10.6%, again with males having a slightly larger risk than females (World Health Organization, 2018). Over the course of 16 years, cancer related deaths have increased by a whopping 28%, while WHO have predicted that in 2060, cancer would overtake ischemic heart diseases as the number one cause for mortality in the world with a twice-fold increase in cases within four decades (Mattiuzzi & Lippi, 2019).

Through present, available options to treat cancer each have their own flaws, and there is still yet a universal cure for cancer with a 100% success rate at permanently eradicating cancer cells in a patient, thus alternative compounds which exert anticancer effects are highly sought after. One proven source for these compounds is the various phytochemicals present within or produced by various plant life; as evidenced by vincristine, etoposide, and paclitaxel being popular plant-based anti-cancer drugs (Nobili et al., 2009).

The Marchantia genus possesses antibacterial, antitoxic, and antiseptic, and antidiuretic properties; making it commonly used as traditional medicine throughout the world (Asakawa et al., 2009). These properties came about due to the presence of

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various terpenoids, flavonoids, and simple phenolic species, compounds which previously showed anticancer properties (Xiao et al., 2006; Cragg & Newman, 2009).

2.1.2. Scope of the project

The scope of this internship project revolved around the ethanol-based maceration extraction of Marchantia paleacea, then the assessment of the anti-cancer capabilities of M. paleacea extracts via measuring the cell viability and colony forming capacity of a normal (HaCaT) and a cancerous (HT-29) cell lines treated with either the extract or an anticancer positive control (5-FU) through MTT assay and a trypan blue-based cell counting.

2.1.3. Objectives / Aims

● ExtractMarchantia paleaceavia an Ethanol-based maceration extraction

● Identify the presence of anticancer activity of 5 FU andM. paleaceaextracts by measuring the cell viability via performing an MTT assay on HaCaT and HT-29 cell lines

● Evaluate the colony forming ability of HaCaT and HT-29 cell lines under the treatment of both 5 FU andM. paleaceaextracts by performing a colony forming assay, especially via a trypan blue-assisted cell count

2.1.4. Problem formulation and Proposed Solutions

Cancer therapies currently used are surgery, radiation therapy, chemotherapy, immunotherapy, and targeted therapy. Surgery necessitates early detection and diagnosis of said cancer prior to metastasis, and requires the cancer cells to be in an organ which allows for invasive procedures, both in terms of location and importance, which is not possible in cases such as NSCLC (Non-small Cell Lung Cancer) or pancreatic cancers (Suzuki & Goto, 2020; Vincent et al., 2011). Both radiation therapy and chemotherapy would most often than not lead to adverse side effects to the patient (Mahmood & Nohria, 2016; Airley, 2009). Targeted therapies and Immunotherapies would also both involve other factors in eradicating cancer cells; the cancer cells’

aberrant protein complement or the immune system of the patient, both factors being susceptible to alterations or inhibitions by the cancer cells or by the tumor

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microenvironment (TME) surrounding it (Vinay et al., 2015; Polanczyk et al., 2019;

Buchbinder & Desai, 2016). Targeted therapies could sometimes be inaccurate in their

“targeting”, which leads to nonspecific toxicity towards regular cells, and cancer cells often do not rely on a single mutation which could so easily be targeted by certain inhibitors (Sandoo et al., 2015; Keefe & Bateman, 2019).

Deficiencies in each of cancer’s treatment options prompts the continued search for alternative compounds or treatments that when combined with conventional chemotherapy, would lead to decreased toxicity towards healthy cells through a decreased dose for one specific chemotherapy agent (Lin et al., 2017). These compounds could also provide more interaction with the cancer cells, the TME, or the immune system of the patient by acting as drug sensitizers or by decreasing resistance against cancer therapy through the inhibition of multidrug resistance (MDR) proteins (forcibly fluxes the drugs out of the cancer cell) which leads to an improved antitumor response (Amiri-Kordestani et al., 2012; Turrini et al., 2014; Lin et al., 2020).

Indonesia is one of 17 megadiverse countries; it possesses around 25 thousand plant species (15 thousand being endemic), which results in the second highest number of indigenous medicinal plants just after the Amazon rainforests (Woerdenbag

& Kayser, 2014). This biological prosperity would be a great source to find potentially anti-cancer agents. The marchantia genus exists in Indonesia asMarchantia paleacea.

Though proven to possess compound families which elicited anticancer effects, no research has been conducted to validate this fact. Hence, this study would clarify whether or not M. paleacea extract actually possess anticancer properties.

2.1.5. Methodology

2.1.5.1. Marchantia paleacea Extraction

After the M. paleacea sample was procured from BRIN, its roots and other damaged parts were removed, whilst its shoots were washed and dried in an oven at 40°C for 24 hours. The plant was then powdered and dissolved in a 1:5 ratio of 80% ethanol to then be put in a shaker incubator for 24 hours.

Afterwards, vacuum filtration was performed on the solution. The solution was dissolved again in a 1:5 ratio of 80% ethanol and then was put in a shaker incubator for another 24 hours. The process was repeated one more time, before being evaporated using a rotary evaporator (200 rpm, 20-100°C). The

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evaporated sample was placed in a fume hood to dry out. The yield for the extraction process was then calculated with the formula below.

%𝑌𝑖𝑒𝑙𝑑 = 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑓𝑖𝑛𝑎𝑙 𝑒𝑥𝑡𝑟𝑎𝑐𝑡

𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 𝑢𝑠𝑒𝑑 × 100%

2.1.5.2. Cell Culture

Both cell lines were first thawed before being cultured. Cryovials were taken from the freezer to be put in a water bath until no traces of ice crystals remain. Cells were then mixed with cDMEM (Complete Dulbecco’s Modified Eagle Media) in a 15 mL falcon tube to be centrifuged at 2000 rpm for 5 minutes. Media was removed then the cells were resuspended with cDMEM.

The mixture was transferred to a T-25 flask containing more cDMEM along with 10% FBS (Fetal Bovine Serum) and 1% PenStrep (Penicillin-Streptomycin) to be incubated at 37°C with a 5%𝐶𝑂 concentration. Both the HaCaT and

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HT-29 cell lines were cultured with cDMEM containing 10% FBS and 1%

PenStrep inside T-25 flasks, with periodic passaging/subculturing performed to preserve the cells and prevent cellular death due to overconfluency. The cells were kept in a cell incubator at 37°C with a 5%𝐶𝑂₂concentration.

2.1.5.3. Cytotoxicity Assay

Both cell lines were also seeded into 96-well plates with a seeding density of5 × 10³ 𝑐𝑒𝑙𝑙/𝑤𝑒𝑙𝑙 for HaCaT and 7. 5 × 10³ 𝑐𝑒𝑙𝑙/𝑤𝑒𝑙𝑙 for HT-29.

The cells were incubated at 37°C and 5%𝐶𝑂 concentration overnight and left

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to grow in 100 µL of CDMEM containing 10%FBSand 1%PenStrep. Each cell line was then treated with 100 µL either a blank (5% DMEM), negative control (0.1% DMSO in 5% DMEM), positive control (5-FU in 0.1% DMSO and 5%

DMEM), and the treatment group (M. paleaceaextracts in 0.1% DMSO and 5%

DMEM). 5-FU concentrations used were 50, 100, 200, and 400 µM, while the M. paleaceaextract’s concentrations were 12.5, 25, 50, and 100 ppm. The The plates were then incubated at 37°C with a 5%𝐶𝑂 concentration for 48 hours.

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After 48 hours, media was removed from each well, followed by a wash with 100 µL of DMEM. 100 µL of cDMEM along with 10 µL of MTT reagent. The plates were then incubated at 37°C with a 5%𝐶𝑂₂concentration

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for 3 hours. After incubation, 100 µL of a stop solution containing 0.04 N HCl in isopropanol. Absorbance was then measured at 570 nm with a reference wavelength of 630 nm using a plate reader. Cell viability was then calculated using the formula below based on the sample’s absorbances.

𝐶𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 (%) = 𝑆𝑎𝑚𝑝𝑙𝑒 − 𝐵𝑙𝑎𝑛𝑘

𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐵𝑙𝑎𝑛𝑘 × 100%

2.1.5.4. Colony Forming Assay

Similar to the cytotoxicity assay, both cell lines were seeded into 96-well plates with an identical seeding density for each cell line. The well plates were then incubated at 37°C and 5%𝐶𝑂₂concentration overnight and left to grow in 100 µL of CDMEM containing 10%FBSand 1%PenStrep.Each well was then washed with DMEM, then treated with matching treatment groups with those in the cytotoxic assay. The plates were then incubated again at 37°C and 5%𝐶𝑂 concentration for 48 hours. After incubation, each well

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was washed with DMEM, then 100 µL of trypan blue was added and left there for 3 minutes. The trypan blue was discarded, then 100 µL of DMEM was readded into each well. Photographs of each well were taken using a microscope-mounted camera. four photographs were taken of each well, corresponding to the four quadrants of said well. Each photograph was then analyzed using the ImageJ software that would automatically count the amount of cells present in each well.

2.1.5.5. Statistical Analysis

The data was analyzed using Graph Pad 9.3.1. Differences of cell viability or cell count between each treatment group were evaluated by first analyzing the normality of the data, then performing a one way ANOVA (analysis of variance). p<0.05 was considered as a statistically significant difference. The data was then presented as mean±Standard Error of Means (SEM).

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CHAPTER 3: FINDINGS

3.1. Result

3.1.1.M. paleaceaExtraction

A total of 55.49 g of powderedM. paleaceashoot was processed through the maceration extraction, resulting in a total extracted sample weight of 7.87 g (Figure 3.1.1.1). The %yield of the overall extraction was calculated to be 14.18%.

Figure 3.1.1.1.Dried outM. paleaceaextracts after the maceration process.

3.1.2. Cytotoxicity Assay

Absorbance data of HaCaT and HT-29 cell lines were obtained from the microplate reader, transformed into cell viability, then analyzed through a normality and one way ANOVA test. The 5-FU positive control managed to reduce the cell viability of HT-29 cells, as well as the HaCaT cells, although no statistical significance was displayed in the HaCaT cells due to a rather high standard deviation (Figure 3.1.2.1).

Figure 3.1.2.1The effect of 5-FU on both HaCaT and HT-29 cells’ viability. **p< 0.0021 and ****p<

0.0001, both compared to the control group. Data expressed as mean±SEM (n=3).

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Marchantia paleacea extracts of all concentrations displayed negligible capability to reduce cell viability of either cell lines (Figure 3.1.2.2).

Figure 3.1.2.2.The effect ofM. paleaceaextracts on both HaCaT and HT-29 cells’ viability. Both compared to the control group. Data expressed as mean±SEM (n=3).

3.1.3. Colony forming assay

The photographs of the four quadrants within each of the treated wells were uploaded to the ImageJ software where after some modifications, the software would perform an automated cell counting procedure which would generate roughly the amount of cells present within each well. The 5-FU control managed to reduce the amount of HaCaT and HT-29 cells present in each well dosewise (Figure 3.1.3.1).

Figure 3.1.3.1.Cell count of both HaCaT and HT-29 cell lines treated with 5-FU. *p< 0.0332 and ****p<

0.0001, both compared to the control group. Data expressed as mean±SEM (n=3).

Although the difference of the HT-29 treated group when compared to the negative control is not significant, a trend of decreasing cell count is visible as the 5-FU dose was increased.M. paleaceaextracts did not reduce the cell count of either

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cell lines (Figure 3.1.3.2).

Figure 3.1.3.2.Cell count of both HaCaT and HT-29 cell lines treated withM. paleaceaextracts. Both compared to the control group. Data expressed as mean±SEM (n=3).

3.2. Analysis/Discussion

The %yield of the ethanol-based maceration extraction with this specific method is similar to other studies, highlighting the success of the extraction process (Fadhilla et al., 2012). An increase in the duration of the maceration and a decrease in the plant material/solvent (w/v) ratio would increase the yield of the extraction, however the phytochemical concentration of the extracts would not increase significantly (Chen et al., 2016; Alipieva et al., 2010).

The HaCaT -immortalized human keratinocyte- cell line was used in a comparison to the HT-29 -human colon cancer- cell line to assess the specificity of the potential cytotoxic property of theM. paleaceaextracts, since a good candidate for an anticancer drug would be those that only targets cancer cells. The MTT-based cytotoxicity assay is regarded as an indispensable preliminary step in the pursuit of novel therapeutic agents, especially in screening for cytotoxic abilities (Mosmann, 1983; Verma & Singh, 2020). Meanwhile trypan blue is a commonly used staining dye to assess cellular viability, a process beginning to see semi- or complete- automation due to recent advancements in instruments such as the

“ImageJ” image processing software (Strober, 1997; Louis & Siegel, 2011). Both MTT and a colony forming assay was used concurrently in this experiment due to previous studies identifying the possibility that flavonoid phytochemicals (present in M. paleacea) could react with the MTT reagent, thus increasing the absorbance of a potentially cell deficient sample/well (Talorete et al., 2006; Peng et al., 2005; Grishagin, 2015).

Both the MTT and colony forming assays reveal that 5-FU is cytotoxic to both regular

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and cancerous cell lines, while theM. paleaceaextracts did not significantly alter either cell lines’ viability even at the highest concentration. This nonspecific cytotoxicity that 5-FU exerted was perhaps due to it being an inhibitor of the protein thymidylate synthase; an enzyme that catalyzes the transformation of deoxyuridine monophosphate (dUMP) into deoxythymidine monophosphate (dTMP), of which its inhibition would lead to an imbalance of deoxynucleotides and rise in dUMP levels that causes DNA damage (Peters et al., 2002;

Jackman & Calvert, 1995). This means that 5-FU targets the general cell division mechanism of a cell -something which should be exaggerated in cancer cells albeit also present in other cells-, and not on a specific mutated tumorous protein or a specific mechanism attributed only to cancer cells. 5-FU has also been used topically against skin cancers, which may explain why it was cytotoxic towards the HaCaT cells (Rossi, 2006).

A sizable error bar was also apparent in most of the MTT results, which shows a lack of uniformity in the cell growth. One possible explanation for this is the rather low seeding density of both cell lines into the well; 5 × 10³ 𝑐𝑒𝑙𝑙/𝑤𝑒𝑙𝑙 for HaCaT and for HT-29, which results in a lack of cell-cell adhesion and signaling 7. 5 × 10³ 𝑐𝑒𝑙𝑙/𝑤𝑒𝑙𝑙

that promotes cellular growth (Nelson & Chen, 2002). Regular practice would be to seed at a density of1 × 10⁴ 𝑐𝑒𝑙𝑙/𝑤𝑒𝑙𝑙; treating the wells after 24 hours; then performing the MTT after another 24 hours of incubation, however, previous studies have demonstrated that 5-FU would exert its anticancer properties after 48 hours, thus the methods were adjusted (Jose et al., 2014; Yasumatsu et al., 2012). Another way to reduce this error bar is to increase the amount of biological replicates of which sample data could be collected. An increase in the biological replicate amount would reduce the likelihood of a specific data being a procedural error, resulting in a significantly higher or lower result; an outlier (Vaux et al., 2012).

Difference in seeding density was attributed to the differences in cell doubling time;

HaCaT doubles roughly each 24 hours, while HT-29 doubles around every 4 days (Wilson, 2013; Forgue-Lafitte et al., 1989). HT-29’s doubling time could be reduced to about every 24 hours with the addition of FBS (Forgue-Lafitte et al., 1989). This means that the HT-29 cells greatly depend on the presence of at least 10% of FBS for rapid growth; which is probably the reason why the cell count data clearly shows that the HT-29 cells have vastly lower amounts of cells present in each of the wells, because there were merely 5% FBS content in each of the treatment media used.

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CHAPTER 4: CONCLUSION AND RECOMMENDATION

In this internship project, shoots of M. paleacea were successfully extracted via an ethanol-based maceration extraction, which led to a final extraction yield of 14.18%, which resembles other studies with similar methodology.

M. paleaceaextract’s anticancer capabilities were assessed by performing an MTT assay, of which it elicited no significant cytotoxic activity towards the cancer cell HT-29 and the immortalized regular cell HaCaT, in contrast to the positive control 5-FU which exerted cytotoxic effects to both normal and cancerous cell lines due to its nonspecific mechanism and due to it also being used against keratinocytes. The effects ofM. paleaceaextracts on the cell’s capability to divide was also assessed by performing a colony forming assay, more specifically a trypan blue-based cell count. The assay proved that M. paleaceaextracts once again did not possess any capability to reduce a cell’s proliferative capabilities, unlike the positive control 5-FU.

Abnormal results found in the study were due to the less optimal experimental design of the project; large error bars were due to a lack of enough biological replicates as well as a lower seeding density of both cell lines, of which the lower seeding density was due to the choice of the positive control which exerted its cytotoxic effects after 48 hours. The significantly lower amount of HT-29 cells within a well was also attributed to the rather slow, FBS-dependent growth of the cell line.

For future research, a better study design would have to be made; a well defined normal and cancerous cell lines which would grow regardless of the FBS concentration within the medium, a proper anticancer drug specifically targeting the cancer cells, as well as a larger amount of biological replicates to reduce procedural error.

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CHAPTER 5: SELF REFLECTION

This internship has taught the author many things. First and foremost of course is the author’s laboratory skills, though most visible to observers, the author believes that this is the least important throughout this internship. Second would be on how the author communicates with others; his co-internees, supervisors, lab assistants, even external parties, something the author thought he was capable of until the author was faced with -rather- stressful situations in the lab. The author learned to manage his expectations and emotions, and more importantly on how to compromise with things when they start to diverge from the author’s ideals. Last would be on how the author views his 4th year at campus; of which idyllic scenes of finding something significant in the author’s field of study would be replaced with something (although lacking in theory) more realistic, something that the author is coming to terms with as he is writing this self reflection.

The author would say that he has demonstrated a high degree of standards in the lab, during this internship, both on himself and on his co-internees. The author was also more than willing to learn and improve upon his previous mistakes, and to devise plans to adapt to the situation at hand.

The author however, would still need to improve on how he deals with less-than-ideal results, and on the initiative to carry out certain tasks or reach out to certain parties.

The internship gave the author a glimpse of actual working conditions in the author’s field of study; though filled with complications, it is something that the author would gladly adapt to in order to be excellent in the future. The author would strive so that i3L’s values would be those that the author lives by, and hopefully would be the author’s own values as well.

Basic cell culture laboratory skills; cell culture, MTT assays, ImageJ-based cell culture, as well as data analysis were obtained through i3L’s courses, especially in Basic and Advanced Cellular Oncology Labs, as well as in Principles of Biostatistics. BRIGHT sessions helped me to develop my leadership and communication skills. Both hard- and soft- skills obtained through the courses and the BRIGHT sessions were extremely helpful to the author in finishing this internship process.

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APPENDICES

MTT assay raw data: https://bit.ly/3rGlPIh Cell counting raw data: https://bit.ly/3CEBf5V

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REFERENCES

Alipieva, K., Petreska, J., Gil-Izquierdo, Á., Stefova, M., Evstatieva, L., & Bankova, V. (2010). Influence of the extraction method on the yield of flavonoids and phenolics from Sideritis spp.(Pirin Mountain tea). Natural Product Communications, 5(1), 1934578X1000500113.

Airley, R. (2009). Cancer Chemotherapy: Basic Science to the Clinic. Wiley-Blackwell.

Amiri-Kordestani, L., Basseville, A., Kurdziel, K., Fojo, A. T., & Bates, S. E. (2012). Targeting MDR in breast and lung cancer: discriminating its potential importance from the failure of drug resistance reversal studies. Drug Resistance Updates, 15(1-2), 50-61.

Asakawa, Y., Ludwiczuk, A., & Nagashima, F. (2009). Bryophytes: bio-and chemical diversity, bioactivity and chemosystematics. Heterocycles, 77(1), 99-150.

Buchbinder, E. I., & Desai, A. (2016). CTLA-4 and PD-1 pathways: similarities, differences, and implications of their inhibition. American journal of clinical oncology, 39(1), 98.

Cancer. Who.int. (2022). Retrieved 2 October 2022, from

https://www.who.int/en/news-room/fact-sheets/detail/cancer.

Cancer today. Gco.iarc.fr. Retrieved 3 October 2022, from

http://gco.iarc.fr/today/fact-sheets-cancers.

Chen, Q., Fung, K. Y., Lau, Y. T., Ng, K. M., & Lau, D. T. (2016). Relationship between maceration and extraction yield in the production of Chinese herbal medicine. Food and Bioproducts Processing, 98, 236-243.

Cragg, G. M., & Newman, D. J. (2009). Nature: a vital source of leads for anticancer drug development. Phytochemistry Reviews, 8(2), 313–331. doi:10.1007/s11101-009-9123-y Disability-adjusted life years (DALYs). Who.int. Retrieved 3 October 2022, from

https://www.who.int/data/gho/indicator-metadata-registry/imr-details/158.

Fadhilla, R., Iskandar, E. A., & Kusumaningrum, H. D. (2012). Aktivitas Antibakteri Ekstrak Tumbuhan Lumut Hati (Marchantia paleacea) Terhadap Bakteri Patogen dan Perusak Pangan [Antibacterial Activity of Liverwort (Marchantia paleacea) Extract on Pathogenic and Food Spoilage Bacteria]. Jurnal Teknologi dan Industri Pangan, 23(2), 126-126.

Forgue-Lafitte, M. E., Coudray, A. M., Bréant, B., & Mešter, J. (1989). Proliferation of the human colon carcinoma cell line HT29: autocrine growth and deregulated expression of the c-myc oncogene. Cancer research, 49(23), 6566-6571.

Grishagin, I. V. (2015). Automatic cell counting with ImageJ. Analytical biochemistry, 473, 63-65.

Jackman, A. L., & Calvert, A. H. (1995). Folate-based thymidylate synthase inhibitors as anticancer drugs. Annals of oncology, 6(9), 871-881.

Jose, J. O. B. Y., Sudhakaran, S. U. D. H. E. E. S. H., Kumar, S., Jayaraman, S. O. N. Y., & Variyar, E. J.

(23)

(2014). A comparative evaluation of anticancer activities of flavonoids isolated from Mimosa pudica, Aloe vera and Phyllanthus niruri against human breast carcinoma cell line (MCF-7) using MTT assay. International Journal of Pharmacy and Pharmaceutical Sciences, 6(2), 319-322.

Keefe, D. M., & Bateman, E. H. (2019). Potential successes and challenges of targeted cancer therapies. JNCI Monographs, 2019(53), lgz008.

Lin, S. R., Fu, Y. S., Tsai, M. J., Cheng, H., & Weng, C. F. (2017). Natural compounds from herbs that can potentially execute as autophagy inducers for cancer therapy. International journal of molecular sciences, 18(7), 1412.

Lin, S. R., Chang, C. H., Hsu, C. F., Tsai, M. J., Cheng, H., Leong, M. K., ... & Weng, C. F. (2020). Natural compounds as potential adjuvants to cancer therapy: Preclinical evidence. British journal of pharmacology, 177(6), 1409-1423.

Louis, K. S., & Siegel, A. C. (2011). Cell viability analysis using trypan blue: manual and automated methods. In Mammalian cell viability (pp. 7-12). Humana Press.

Mahmood, S. S., & Nohria, A. (2016). Cardiovascular complications of cranial and neck radiation.

Current treatment options in cardiovascular medicine, 18(7), 1-10.

Mattiuzzi, C., & Lippi, G. (2019). Current cancer epidemiology. Journal of epidemiology and global health, 9(4), 217.

Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of immunological methods, 65(1-2), 55-63.

Nelson, C. M., & Chen, C. S. (2002). Cell-cell signaling by direct contact increases cell proliferation via a PI3K-dependent signal. FEBS letters, 514(2-3), 238-242.

Nobili, S., Lippi, D., Witort, E., Donnini, M., Bausi, L., Mini, E., & Capaccioli, S. (2009). Natural compounds for cancer treatment and prevention. Pharmacological research, 59(6), 365-378.

Peng, L., Wang, B., & Ren, P. (2005). Reduction of MTT by flavonoids in the absence of cells. Colloids and Surfaces B: Biointerfaces, 45(2), 108-111.

Peters, G. J., Backus, H. H. J., Freemantle, S., Van Triest, B., Codacci-Pisanelli, G., Van der Wilt, C. L., ...

& Pinedo, H. M. (2002). Induction of thymidylate synthase as a 5-fluorouracil resistance mechanism. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 1587(2-3), 194-205.

Polanczyk, M. J., Walker, E., Haley, D., Guerrouahen, B. S., & Akporiaye, E. T. (2019). Blockade of TGF-β signaling to enhance the antitumor response is accompanied by dysregulation of the functional activity of CD4+ CD25+ Foxp3+ and CD4+ CD25− Foxp3+ T cells. Journal of translational medicine, 17(1), 1-12.

(24)

Rossi, S. (2006). Australian Medicines Handbook. AMH Pty Ltd.

Sandoo, A., Kitas, G. D., & Carmichael, A. R. (2015). Breast cancer therapy and cardiovascular risk:

focus on trastuzumab. Vascular health and risk management, 11, 223.

Suzuki, S., & Goto, T. (2020). Role of surgical intervention in unresectable non-small cell lung cancer.

Journal of Clinical Medicine, 9(12), 3881.

Strober, W. (1997). Trypan blue exclusion test of cell viability. Current protocols in immunology, 21(1), A-3B.

Talorete, T. P., Bouaziz, M., Sayadi, S., & Isoda, H. (2006). Influence of medium type and serum on MTT reduction by flavonoids in the absence of cells. Cytotechnology, 52(3), 189-198.

The History of Cancer | First Cancer Diagnosis. Cancer.org. Retrieved 2 October 2022, from https://www.cancer.org/treatment/understanding-your-diagnosis/history-of-cancer.html.

Turrini, E., Ferruzzi, L., & Fimognari, C. (2014). Natural compounds to overcome cancer chemoresistance: toxicological and clinical issues. Expert Opinion on Drug Metabolism &

Toxicology, 10(12), 1677-1690.

Vaux, D. L., Fidler, F., & Cumming, G. (2012). Replicates and repeats—what is the difference and is it significant? A brief discussion of statistics and experimental design. EMBO reports, 13(4), 291-296.

Verma, A. K., & Singh, S. (2020). Phytochemical analysis and in vitro cytostatic potential of ethnopharmacological important medicinal plants. Toxicology Reports, 7, 443-452.

Vinay, D. S., Ryan, E. P., Pawelec, G., Talib, W. H., Stagg, J., Elkord, E., ... & Kwon, B. S. (2015, December). Immune evasion in cancer: Mechanistic basis and therapeutic strategies. In Seminars in cancer biology (Vol. 35, pp. S185-S198). Academic Press.

Vincent, A., Herman, J., Schulick, R., Hruban, R. H., & Goggins, M. (2011). Pancreatic cancer. The Lancet, 378(9791), 607-620.

Wilson, V. G. (2013). Growth and differentiation of HaCaT keratinocytes. In Epidermal cells (pp.

33-41). Springer, New York, NY.

Woerdenbag, H. J., & Kayser, O. (2014). Jamu: Indonesian traditional herbal medicine towards rational phytopharmacological use. Journal of herbal medicine, 4(2), 51-73.

World Health Organization. (2018). Global health estimates 2016: deaths by cause, age, sex, by country and by region, 2000–2016. Geneva: World Health Organization.

Xiao, J. B., Chen, X. Q., Zhang, Y. W., Jiang, X. Y., & Xu, M. (2006). Cytotoxicity of Marchantia convoluta leaf extracts to human liver and lung cancer cells. Brazilian Journal of Medical and Biological Research, 39, 731-738.

Yasumatsu, R., Nakashima, T., & Komune, S. (2012). Overexpression of the orotate

(25)

phosphoribosyl-transferase gene enhances the effect of 5-Fluorouracil in head and neck squamous cell carcinoma in vitro. Journal of oncology, 2012.