PREFACE
This report was created as part of the criteria for the internship project on the odd semester of the 2021/2022 academic year, aiming to obtain technical ability in laboratory practice of biomedical subjects. This report has been created with the intention of including all details about the study author worked on throughout the credit internship period. This study titled "Investigation of Anti-Cancer Properties on Bryophytes Extract (Pogonatum neesii) Towards HT-29 Cell Line" aimed to look at the potential of natural sourcePogonatum neesiito prevent cancer cell growth.
Thereby, I express my gratitude to God Almighty for His great grace enabling the completion of this internship report and to my supervisors and seniors who assisted me during the internship process. I am grateful to BRIN and i3L for trusting me to participate in this study and also for assisting me in gaining very valuable knowledge for my career. I'd also like to thank my family and colleagues for their help in completing my internship.
ACKNOWLEDGEMENT PAGE
Investigation of Anti-Cancer Properties on Bryophytes Extract (Pogonatum neesii) Towards HT-29 Cell Line
Badan Riset dan Inovasi Nasional
AUTHOR:
SINTIKHE AYUNINGTYAS WENAS 19010128
BIOMEDICINE
ACKNOWLEDGED BY:
FIELD SUPERVISOR SUPERVISOR AT I3L
apt. Pietradewi Hartrianti, S.Farm., M.Farm., Ph.D Richard Sutejo, S.TP, PhD
HEAD OF DEPARTMENT AT I3L
TABLE OF CONTENTS
PREFACE i
ACKNOWLEDGEMENT PAGE ii
TABLE OF CONTENTS iii
LIST OF FIGURES iv
LIST OF ABBREVIATIONS v
ABSTRACT vi
CHAPTER 1: INTRODUCTION 1
1.1 Host Institution 1
1.1.1 Description Of the Institution 1
1.1.2 Description of Departement 2
1.1.3 Product of The Host Institution 2
CHAPTER 2: PROJECT DESCRIPTION 3
2.1 Internship Project 3
2.1.1 Project Background 3
2.1.2 Scope of The Project 4
2.1.3 Objectives 4
2.1.4 Problem Formulation and Proposed Solutions 4
2.1.5 Materials & Method 5
2.1.5.1 Sample Extraction: Maceration Method 5
2.1.5.2 Cell culture 5
2.1.5.2.1 Cell Thawing 5
2.1.5.2.2 Cell Counting and Passaging 5
2.1.5.3 Cytotoxic Test: MTT assays 6
2.1.5.4 Cell Viability Counting 6
2.1.5.5 Statistical Analysis 7
CHAPTER 3: FINDINGS 8
3.1. Results 8
3.1.1 Maceration Process 8
3.1.2 Cytotoxic test: MTT Assay 8
3.1.3 Cell Viability Counting 10
3.2. Discussion 11
CHAPTER 4: CONCLUSION AND RECOMMENDATION 14
CHAPTER 5: SELF REFLECTION 15
APPENDICES 16
REFERENCES 18
LIST OF FIGURES
Figure 3.1Pogonatum neesiiExtract Result 9
Figure 3.2P.neesiiextract cytotoxic test on HEK-293 Cells a. Cytotoxic Test Towards Cells Treated with Different 5FU Concentrations b. Cytotoxic Test Towards Cells Treated with Different Pogonatum neesiiextract Concentration, * p <0.05, ** p<0.005, and “ns” as not significant
10 Figure 3.3P.neesiiextract cytotoxic test on HT-29 Cells a. Cytotoxic Test Towards Cells Treated with
Different 5FU Concentrations b. Cytotoxic Test Towards Cells Treated with Different Pogonatum neesiiextract Concentration, ** p<0.005, **** p<0.0001, while “ns”
implement a non-significant result 10
Figure 3.4P.neesiiextract Cell Viability Counting on HEK-293 Cells a. Cell Viability Counting on Cells Treated with Different 5FU Concentrations b. Cell Viability Counting on Cells Treated with DifferentPogonatum neesiiextract Concentration, ** p<0.005, while “ns” implement a
non-significant result 11
Figure 3.5P.neesiiextract Cell Viability Counting on HT-29 Cells a. Cell Viability Counting on Cells Treated with Different 5FU Concentrations b. Cell Viability Counting on Cells Treated with DifferentPogonatum neesiiextract Concentration, * p<0.05, and “ns” as non-significant 11
LIST OF ABBREVIATIONS
5-FU 5 Fluorouracil ANOVA Analysis of variance
BRIN Badan Riset Inovasi Nasional
cDMEM Complete Dulbecco's Modified Eagle Medium DMEM Dulbecco's Modified Eagle Medium
DMSO Dimethyl sulfoxide HCl Hydrochloric acid
HEK 293 Human embryonic kidney 293
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
RIRN Rencana Induk Riset Nasional RO Research Organization ppm Part per million PRN Prioritas Riset Nasional
ABSTRACT
Cancer is one of the world's most serious health challenges. It was anticipated that more than 19 million recent cancer cases and about 10 million deaths worldwide were recorded in the year of 2020. There are various treatments available, such as chemotherapy and radiotherapy, however, those treatments have severe side effects because they do not precisely aim at the cancerous cell as it is also cytotoxic toward normal cell lines. Nowadays, there has been a lot of research that proves natural ingredients to be potential as cancer treatment such as from the bryophytes class. This research is seeking a targeted and effective alternative treatment using plants as the source that has fewer side effects while effectively preventing cancer cell proliferation. A bryophyte called Pogonatum neesii is one of the natural resources in Indonesia that does not have any study on its cytotoxic activity. In this study, 80%Pogonatum neesiiethanol extract was used to check its ability to inhibit cancer cell growth. By utilizing cell viability analysis through MTT assay and cell counting, it shows that this extract does not have the ability to perform cytotoxic effects towards HT-29 cells as the model of cancerous cells. In addition, further studies are recommended to be performed for Pogonatum neesiispecies.
Keywords: cancer,Pogonatum neesii,HT-29, cell viability assay, MTT assay
CHAPTER 1: INTRODUCTION 1.1 Host Institution
1.1.1 Description Of the Institution
President Joko Widodo established BADAN RISET INOVASI NASIONAL (BRIN) through Presidential Regulation Number 74 of 2019 concerning the National Research and Innovation Agency.
According to this, BRIN aimed to perform research, development, study, and application; and incorporate ideas and inventions. BRIN performs a number of functions in carrying out these responsibilities, including the conceptualization and determination of policies in the field of quality standards for research facilities, protection of intellectual property rights, and their utilization as an outcome of nationwide innovations and inventions in compliance with the rules and guidelines of the laws and policies
BRIN began as a unit of theKementerian Riset dan Teknologi, but along the line, it became independent as Joko Widodo signed Presidential Regulation No. 33 of 2021 on May 5, 2021, thereby making BRIN the primary research institute organization. Furthermore, BRIN is responsible for monitoring, managing, and assessing the performance of the BadanRiset dan Inovasi Daerahduties and powers in compliance with legislative provisions.
The following is the vision and mission of BRIN that underpins its tasks in running this institution.
VISION
The establishment of a National Research and Innovation Agency that is dependable, professional, innovative, and honest in its service to the President and Vice President, in order to realize the President's Vision and Mission: "An Advanced Indonesia that is Sovereign, Independent, and Has a Personality Based on Mutual Cooperation."
MISSION
Provide technical and administrative support, as well as fast, accurate, and responsive analysis, to the President and Vice President in conducting research, development, study, and application, as well as inventions and innovations, nuclear energy implementation, and implementation of integrated national space, as well as monitoring, controlling and evaluating on the implementation of BRIDA's duties and functions. Improving the quality of human resources and infrastructure for integrated research and innovation in the national implementation of nuclear and outer space, as well as fostering the implementation of BRIDA's obligations and functions Organizing effective and efficient services in the areas of oversight, general administration, information, and institutional relations.
1.1.2 Description of Departement
The BRIN Research Organization (RO) conducts technological research, development, assessment, and application, as well as inventions and innovations, nuclear energy implementation, and/or projects. RO's role is similar to that of the campus in the Ministry of Education and Culture. If the campus takes over the education role at the Ministry of Education and Culture, the OR will take over the research function at BRIN. At BRIN, there are 12 Research Organizations, namely Nuclear Energy RO, Earth, and Maritime RO, Social Sciences and Humanities RO, Aviation and Space RO, Archaeological, Language, and Literature RO, RO for Governance, Economics, and Public Welfare, Agricultural and Food RO, Biological and Environmental RO, Health RO, Energy Research and Manufacturing Organization, Electronics and Informatics RO, and lastly Nanotechnology and Materials RO.
In addition to the RO that has been established above, BRIN also opens opportunities to fund researchers through programs called “Prioritas Riset Nasional (PRN)”. This program provides research funding to institutions in order to implement PRN, which is the implementation of theRencana Induk Riset Nasional (RIRN). This financing covers nine research priority areas, which are as follows: food health, transportation, engineering, protection and security, maritime education, social humanities, arts, and culture, cross-sectoral and multidisciplinary with three priorities: screening and diagnostics, vaccinations, and medications and therapy.
1.1.3 Product of The Host Institution
In 2022, BRIN intends to promote 400 innovative products from research-based startup enterprises as part of its performance goals. In addition, more than 25 thousand international scientific journals, more than 50 thousand citations to publications internationally, 150 industry inventions, and 900 registered patents are expected in 2022. Also projected are 15 natural resource technology applications, 35 post-disaster recovery technologies, and 50% of funding originating from outside sources
CHAPTER 2: PROJECT DESCRIPTION
2.1 Internship Project
2.1.1 Project Background
Cancer is a perplexing disease that has affected multiple organisms for more than 200 million years. An indication of cancers among contemporary human ancestors was present over a million years ago. Unlike infectious diseases, cancer is not caused principally by an organism foreign to human systems. Its destroyers are cells from humans that have broken their grip and been attracted to a certain extent, changed into malignant organisms or tumor structural components (Hausman, 2019). As cancer is triggered by the uncontrolled multiplication of numerous types of cells present in our body, there are over a hundred varieties of cancer, each with its own particular behavior and treatment response. In addition, according to Gupta et al (2020), an anticipated 19.3 million new cases and about 10 million deaths worldwide were recorded in 2020. In transitioned countries, the overall incidence was 2 - 3-fold greater than in transitional countries. The worldwide cancer burden is anticipated to reach 28.4 million cases in 2040, a 47% rise over 2020, with a greater increase in transitioning countries compared with transitioned countries, though this could be clarified further.
Efforts to develop cancer prevention and care distribution in transitioning nations are important for worldwide cancer control.
For many years, cancer patients had only limited treatment options, which included surgeries, radiation treatment, and chemotherapy as separate therapies or even in conjunction.
However, several more paths associated with cancer therapeutic development as well as their ability to be targeted have enhanced in recent years, with combinational techniques involving different therapeutic strategies or "traditional" chemotherapeutics, being discovered to have a synergistic impact. Recent ways of treatment, such as medications, biomolecules, and immune-mediated treatments, are being employed even when the expected therapeutic level has not been reached, which opposes mortality and reduces the length of survival for metastatic cancer (Debela et al., 2021). In addition, the most important aspect of creating cancer treatment is determining how to eliminate the cancerous cells without damaging normal cells. As a result, studies have been trying to look for alternative treatments, such as natural medicine, that are more effective with fewer side effects (Kootiet al., 2017).
Through screening of natural compounds from plants, many cancer medicines have been found as their chemicals contain some anti-cancer properties such as vincristine, irinotecan, and paclitaxel (Da Rocha, Lopes & Schwartsmann, 2001). In addition, according to Wang & Jiang (2012).
Natural agents are very likely to be effective in cancer prevention with discernible side effects. There
is evidence that several natural agents, such as green tea and Curcuma longa, have anti-cancer properties. Furthermore, one example available natural source in Indonesia is bryophytes. It has been studied that some bryophytes class such as liverworts and mosses has the ability to have cytotoxic agents toward cancerous cells (Dey & Mukherjee, 2015). Pogonatum neesii is a class of bryophytes mosses that will be used in this research, with the classification of kingdom Plantae, division of Bryophyta mosses, class of Bryopsida, polytrichales order, and family of polytrichaceae.
Where this type of bryophyte has not been studied before, hence this research aim to examine its cytotoxic effects on both cancer and noncancer cells.
2.1.2 Scope of The Project
The scope of this project includes obtaining Pogonatum neesii ethanol extract by implementing the maceration method along, cell culture, also with the investigation of the extract to evaluate their general cytotoxicity towards cancerous cells by performing the cytotoxic assay and cell viability counting.
2.1.3 Objectives
The objective of this study is to:
● ExtractPogonatum neesiiby utilizing the maceration process
● Investigate the cytotoxicity effects of bryophytes (Pogonatum neesii) towards HEK 293 &
HT-29 cell lines through MTT assay
● Investigate the cytotoxicity effects of bryophytes (Pogonatum neesii) towards HEK 293 &
HT-29 cell lines through cell viability counting method
2.1.4 Problem Formulation and Proposed Solutions
Cancer is one of the world's largest health issues. Even though several cancer treatments are available, such as chemotherapy and radiotherapy, those treatments come with severe side effects since they are not specific to target only cancer cells. Hence, a natural source of treatment that is known as having fewer side effects can be the potential to be an alternative for treating cancer. A study that was done in 2015 proves that several types of bryophytes pose cytotoxic properties toward the cancerous cell. For that reason, this study aims to look for a highly targeted and effective treatment using medicinal plants (Pogonatum neesii) as a source that has fewer side effects while efficiently blocking cancer cell growth.
2.1.5 Materials & Method
2.1.5.1 Sample Extraction: Maceration Method
After Pogonatum neesii sample was collected from BRIN, the roots and damaged sections were removed and aboveground plant materials were used after being cleaned, washed, and dried in a 40°C oven for 24 hours. After the powdered samples were prepared, they were dissolved in a conical flask with a 1:5 ethanol (80%) ratio and shaken overnight (24h). Following 24 hours of shaking, the solution was filtered and vacuumed using Whatman filter paper. Following that, the dried extract was mixed with another 1:5 ethanol (80%) ratio, followed by shaking incubation three times in total. Following that, all of the filtered solutions were combined together and evaporated using a rotary evaporator with a water bath (200 rpm; 20-100°C). Lastly, the evaporated sample was placed inside the fumehood until it become viscous. Extract yield was then calculated with the formula below:
𝑦𝑖𝑒𝑙𝑑 (%) = 𝐹𝑖𝑛𝑎𝑙 𝑒𝑥𝑡𝑟𝑎𝑐𝑡 𝑤𝑒𝑖𝑔ℎ𝑡
𝑆𝑎𝑚𝑝𝑙𝑒 𝑢𝑠𝑒𝑑 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 100%
2.1.5.2 Cell culture
2.1.5.2.1 Cell Thawing
After removing the cryovial containing the thawed cells from the -120°C freezer, it was placed directly in a 37°C water bath and gently swirled till there was just a little amount of ice remaining in the vial. It was then placed in a laminar flow hood. A centrifuge tube containing the thawed cells was then filled with pre-warmed complete growth media.
Following that, the cell suspension was centrifuged at 2000 rpm for 5 minutes. Following centrifugation, the clarity of the supernatant and visibility of a cell pellet was examined, and the supernatant was then taken while avoid damaging the cell pellet. Cells were then resuspended in 1 mL cDMEM and transferred to a previously prepared 4 mL cDMEM at T25 flask. The cells were then incubated in a 𝐶𝑂2incubator at 37°C.
2.1.5.2.2 Cell Counting and Passaging
After the cells were healthy and fully grown in the flask, the media from the T25 flask was withdrawn and washed twice with 2 mL DMEM. After that, 1mL Trypsin-EDTA was added to the cells, and they were incubated for 5 minutes at 37°C. Following that, cells were examined to see if they were detached or not. After stopping the reaction with 1mL cDMEM, the cell suspension was transferred to a microcentrifuge tube and centrifuged at 2000 rpm for 5 minutes. The supernatant was then collected from each microcentrifuge tube and resuspended in 1 mL of cDMEM. After that, 10µL of cell suspension was combined with 10 µL
concentration. Finally, the appropriate cell quantity was placed in T25, and cDMEM was added up to 5mL/flask before incubating at 37°C.
.
2.1.5.3 Cytotoxic Test: MTT assays
The cell viability assay was employed to investigate the cytotoxicity ofPogonatum neesii extract toward HEK and HT-29 cells. The cells were seeded in a 96-well plate at a density of5 𝑥 103cells/well in 100 µL cDMEM for HEK cells and 7. 5 𝑥 103cells/well in 100 µL cDMEM for HT-29 cells. For 48 hours, the cells were cultured in an incubator at 37°C and 5% CO. Furthermore, the stock for Pogonatum neesii extract was made in varying concentrations of 12.5, 25, 50, and 100 µg/mL. There was also a negative control of 1%
DMSO, 5% DMEM for blank measurement, and cells treated with 4 different concentrations of 5 Fluorouracil (50,100, 200, 400) ppm as a positive control. When the cells attained confluence, they were treated with varying quantities of Pogonatum neesii extract and cultured for 48 hours within the same incubator setting. Just after incubation time was completed, the media was disposed of and were rinsed with 100 µL of DMEM, which was then discarded, followed by the addition of 100 µL of cDMEM and 10 µL of MTT reagent that was previously prepared into each well (this process was done in a dark room as MTT reagent is light-sensitive), the cells were then incubated for 3 hours followed by the reaction stopping using HCl and isopropanol mixture. The sample cell absorbance was then read using a plate reader at 570 nm wavelength and the formula below was used to calculate cell viability:
𝐶𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 (%) = 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝑏𝑙𝑎𝑛𝑘
𝑠𝑎𝑚𝑝𝑙𝑒 − 𝑏𝑙𝑎𝑛𝑘 𝑥 100%
2.1.5.4 Cell Viability Counting
Following the incubation of the treatment assay, the medium in each well was discarded and rinsed with DMEM. Each well was then filled with 100 µL of trypan blue and allowed to rest for about 3 minutes before being discarded. Following the staining phase, each well was refilled with 100 µL of DMEM to ensure that any leftover surviving cells were not exposed to dry conditions. The dyed wells were then examined using an inverted microscope, and four photographs of different parts of each well were taken. Finally, to assess cell viability, the percent area average of the living cells from each well was determined using an ImageJ program.
2.1.5.5 Statistical Analysis
Graph Pad Prism 9.3.1 for Windows was used to analyze the data. In addition, the normality test and significant differences between the groups were determined using one-way ANOVA.
CHAPTER 3: FINDINGS 3.1. Results
3.1.1 Maceration Process
Figure 3.1Pogonatum neesii Extract Result
This process was aimed to release the phytochemicals amount in the plant sample.
After being mixed with 80% ethanol, going through the maceration process then placed inside the fumehood, the extract results in deep green color with a very viscous consistency solution (figure 3.1) with a total weight of the extract being 28.162 gr. Furthermore, after calculating the percentage yield, it was found that the final concentration of Pogonatum neesiiextract was 1.167% where this extract was used in the following procedure.
3.1.2 Cytotoxic test: MTT Assay
MTT assay was used to determine the cytotoxic activity ofP.neesiiextract towards HT29 cells. The MTT assay was performed using a variety of concentration ranges of treatments. Figure 3.1 shows the outcome of the HEK cell line, which was employed as a normal cell line control. Where Figure 3.1.a shows the result of cells treated with 5FU as positive control and Figure 3.1.b shows the result ofPogonatum neesiitreatment.
Figure 3.2P.neesiiextract cytotoxic test on HEK-293 Cellsa. Cytotoxic Test Towards Cells Treated with Different 5FU Concentrations b. Cytotoxic Test Towards Cells Treated with DifferentPogonatum neesii
extract Concentration, * p <0.05, ** p<0.005, and “ns” as not significant
From the results above, it was shown that HEK-293 cells that were treated with various concentrations of 5-FU have higher cell viability percentages compared with the negative control which is 0.1% DMSO. Where all treatments result in higher than 100% cell viability. Furthermore, the cells that were treated withPogonatum neesiiextract also have higher cell viability percentage than the negative control, where treatment concentrations of 12.5, 25, and 100 ppm has higher result compared with 50 ppm treatment. Also, in comparison with the cells treated with 5-FU, cells treated with Pogonatum neesii extract have a higher percentage of cell viability.
Figure 3.3P.neesiiextract cytotoxic test on HT-29 Cellsa. Cytotoxic Test Towards Cells Treated with Different 5FU Concentrations b. Cytotoxic Test Towards Cells Treated with Different
Pogonatum neesiiextract Concentration, ** p<0.005, **** p<0.0001, while “ns” implement a non-significant result
On the figure above that presents the results of HT-29 cells, demonstrating that cells that were treated with 5FU as the positive control, have lower cell viability percentages compared with the negative control. Where 100,200,400 µM 5-FU treatment concentration
shows a lower than 50% cell viability. On the other hand, cells that were treated with Pogonatum neesii extract does not show any significant result that was identified as “ns”
which means “not significant. This was also can be seen in all of the treatment concentrations that have almost 100% cell viability.
3.1.3 Cell Viability Counting
In order to confirm the MTT results, cell viability counting using trypan blue was performed.
Figure 3.4P.neesiiextract Cell Viability Counting on HEK-293 Cellsa. Cell Viability Counting on Cells Treated with Different 5FU Concentrations b. Cell Viability Counting on Cells Treated with Different Pogonatum neesiiextract Concentration, ** p<0.005, while “ns” implement a non-significant result
From figure 3.4 it was observed that the average number of cells that were treated with 0.1%DMSO, shows higher results up to more than 15000 cells on average, while compared with other treatments that only range from 5000 to less than 10000 cells. In addition for cells that were treated withPogonatum neesiiextract, it was observed that all treatments has a similar average cell number of more than 15000 cells. In addition, when compared with cells treated with 5-FU, cells treated withPogonatum neesiiextract have a much higher cell numbers.
Figure 3.5P.neesiiextract Cell Viability Counting on HT-29 Cells a. Cell Viability Counting on Cells Treated with Different 5FU Concentrations b. Cell Viability Counting on Cells Treated with DifferentPogonatum
neesiiextract Concentration, * p<0.05, and “ns” as non-significant
For HT-29 cells that were treated with 5-FU, the result shows cells treated with 0.1%
DMSO, 50µM have a number of cells that is higher than 500, and for cells that were treated with 100 µM, 200 µM & 400 µM 0.1% DMSO has lower than 500 cells count. Furthermore, the cells that have Pogonatum neesiiextract as their treatment, present that the 25 ppm extract treatment has the highest cell count compared to others.
3.2. Discussion
Maceration is an extraction procedure that involves soaking powdered plant materials in solvents like methanol, ethanol, and hexane. It is a common and inexpensive process for extracting various bioactive chemicals from plant material (Farooq et al, 2022). This method was chosen as there are several things that were considered. First is the consideration of the heat stability of the plant itself, the inexpensive cost of the plant material, a large amount of product, and the intended use for human use consumption (Abubakar & Haque, 2020). Furthermore, several factors affect the percentage yield of the maceration process, such as temperature, duration, and pH (Monton &
Luprasong, 2019). Ethanol (80%) was used in this study due to its high polarity, ethanol has proven to be the most acceptable solvent for extracting chemicals, allowing the extraction of all compounds (Borgeset al.,2020).
HEK 293 (Human Embryonic Kidney) was used as a non-cancerous cell in this study, aiming to
variety of studies ranging from signal transduction to protein expression and biopharmaceutical manufacture. The initial HEK cells were created by transforming the kidney of an aborted human embryo of undetermined parentage with sheared Adenovirus 5 DNA (Linet al,2014). While HT 29 is the cell type that was used as the cancerous cell used in this study. It originated isolated from 44 years old female in 1964 and has been widely employed in human colon cancer studies, where it has been proven to be comparable to in vivo models, implying that it can mimic human colonic tissue (Martinez-Maqueda, Miralles & Recio, 2015). Furthermore, 5 Fluorouracil (5-FU) that was used in this study was aimed as the positive control of its ability to kill the cancerous cell. It has been utilized to treat colon cancer since 1957 and is also used to treat other cancers and is still frequently used as an anticancer treatment today. It acts by converting to fluorodeoxyuridine monophosphate, which creates a complex with thymidylate synthase which suppresses the formation of deoxythymidine monophosphate, which is required for replication reparation of DNA and whose deficiency causes cytotoxicity (Zhanget al., 2018).
Based on the data mentioned above, HEK-293 cells receiving this treatment should not experience cytotoxicity effects, however HT-29 cells should. This is supported by evidence from the results, which demonstrated that HT-29 cells were experiencing cytotoxic effects (figure 3.3.a &
figure 3.5.a), in addition to the result obtained, as HEK-293 cells that were used as the representative of normal cells, it confirms that the treatment of cancer drug which is 5-FU shows a satisfactory result as its only kill HT-29 cell that was indicated by lower than 50% cell viability percentage, but not HEK cell line (figure 3.2.a). However, figure 3.4 shows an opposite result from the cell viability counting method, it presents that HEK-293 cells were shown to have a lower cell number.
There are several possibilities that can lead to this result. First is the major advantage of cell viability counting that selective biased that might happen during photos were taken and processed during imageJ, as there is a lot of adjustment such as pixel, and background that was done during that process creating unprecise result (Bari & Yeasmin, 2022). Another test such as Alamar blue test is recommended to be performed to quantify the amount of living or dead cells. Over the last 50 years, Alamar Blue has been frequently employed in investigations on cell viability and cytotoxicity in a variety of biological and ecological aspects. It is among the most highly referred compounds used for cytotoxicity and viability tests, with over 200 cancer studies and over 1,000 journals in drug screening, development, and discovery (Rampersad, 2012). Another possibility that may lead to this result is the low seeding density. It is stated that cell seeding density is critical for cell proliferation and differentiation. Previous research discovered that a higher cell density improved cell biosynthesis
viability which is more than 100% and HT-29 shows a non-significant result. Where this might be an identification of cell proliferation as the is a higher number of cell viability. To date, there have not been any journals that define what are the chemical compounds contains in this type of moss.
Hence, it can be stated that Pogonatum neesii extract does not have any cytotoxic effects on cancerous cells. Additionally, based on figure 3.2.b indicates that its treatment leads to potential cell proliferation in the HEK-293 cell line. This information is beneficial for further research as the proliferation of normal cells has prospects in the study of antioxidants that has a lot of benefits in treating diseases.
CHAPTER 4: CONCLUSION AND RECOMMENDATION
Pogonatum neesii ethanol extract that was successfully obtained, with a total yield of 1.167% does not demonstrate any cytotoxic effects in both cytotoxic tests using MTT assay and cell viability counting as it does not shows the ability to exert a significant cytotoxic effect on both HEK and HT29 cells. Furthermore, MTT assay is indeed the standard for the viability of cells as cell viability counting might lead to unprecise and biased results.
In addition, since there was limited Pogonatum neesii published literature that was available, it is recommended that further and more studies regardingPogonatum neesiican be done in other fields such as its chemical compounds and anti-oxidant studies, so further studies in many fields can be performed.
CHAPTER 5: SELF REFLECTION
During my internship program, I gained both soft and hard skills. As I was exposed to the world of professional research. When compared to studying in regular teaching-learning classes, the difference is significant. Adaptation to the workplace and decision-making abilities are critical during an internship. Previously, everything was determined and aided by lectures, but now we are the ones who decide and are responsible, also the spirit to never give up and learn from mistakes so that similar incidents do not occur again is as important as the course of this study.
Communication and teamwork are the two most soft skills that I learned during this internship period. As everyone has their own set of responsibilities. I learned how to discuss and express my thoughts on the most exemplary ideas and approaches for conducting this research. I also explore my strength to take the initiative so that everyone can work together to complete it on time and correctly. I also learned the value of honesty. Accepting the outcomes as they are in the right mindset. I also learned that various outcomes do not necessarily indicate the end of things because they can have different meanings or employ different approaches. Moreover, perseverance is another soft skill that was gained during this internship project, as I overcome numerous obstacles such as laboratory equipment malfunctions (unresponsive plate reader machine, incubator malfunction as the cable supply for carbon dioxide into the incubator was not installed properly) also not optimal quality materials such as expired materials.
APPENDICES
Table 1. Cell Viability Percentage on HEK-293 Cells Treated With 5-FU
Treatment Negative Control 50 (µM) 100 (µM) 200 (µM) 400 (µM) Cell
Viability (%)
102.8301887 187.1069182 234.081761 301.8867925 361.0062893 116.6666667 236.4779874 176.7295597 213.2075472 245.9119497 135.2201258 235.5345912 213.2075472 243.081761 294.0251572
Table 2. Cell Viability Percentage on HEK-293 Cells Treated WithPogonatum neesiiExtract Treatment Negative Control 12.5 ppm 25 ppm 50 ppm 100 ppm Cell
Viability (%)
1,352,201,258 341,509,434 4,025,157,233 1,911,949,686 3,874,213,836 1,028,301,887 3,559,748,428 3,455,974,843 2,946,540,881 351,572,327 1,166,666,667 2,028,301,887 1,987,421,384 1,965,408,805 2,393,081,761
Table 3. Cell Viability Percentage on HT-29 Cells Treated With 5-FU
Treatment Negative Control 50 (µM) 100 (µM) 200 (µM) 400 (µM) Cell
Viability (%)
100,070,763 546,055,227 1,710,112,212 1,037,863,523 2,576,959,207 9,918,622,527 8,096,474,763 1,250,152,583 2,576,959,207 1,037,863,523 1,007,783,932 3,143,063,367 1,586,276,928 3,143,063,367 2,559,268,452
Table 4. Cell Viability Percentage on HT-29 Cells Treated WithPogonatum neesiiExtract Treatment Negative Control 12.5 ppm 25 ppm 50 ppm 100 ppm Cell
Viability (%)
100,070,763 1,156,386,274 1,002,476,706 101,662,931 1,092,699,556 9,918,622,527 8,644,888,168 8,786,414,208 8,963,321,758 8,167,237,783 1,007,783,932 8,043,402,498 9,140,229,308 8,485,671,373 8,874,867,983
Table 5. Cell Viability Counting on HEK-293 Cells Treated With 5-FU
Negative Control 50uM 100uM 200uM 400uM
Cell number 21349 10658 9244 8097 6605
Table 6. Cell Viability Counting on HEK-293 Cells Treated WithPogonatum neesiiExtract Negative Control 12.5 ppm 25 ppm 50 ppm 100 ppm
Cell number 21349 22541 19863 24299 23546
19744 21347 19043 21448 22616
18232 19608 17385 19668 18838
Table 7. Cell Viability Counting on HT-29 Cells Treated With 5-FU
Negative Control 50uM 100uM 200uM 400uM
Cell number 1086 951 390 331 345
949 974 356 207 330
721 220 174 180 149
Table 8. Cell Viability Counting on HT-29 Cells Treated WithPogonatum neesiiExtract
Negative Control 12.5 ppm 25 ppm 50 ppm 100 ppm
Cell number 1086 1176 1429 875 673
949 810 1332 659 659
721 382 565 654 568
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