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DISSERTATION SUMMARY
CELL DEATH MECHANISM AS RESULT OF THE EXPOSURE TO
LOW-ENERGY ELECTRIC FIELD WITH MEDIUM FREQUENCY
(In Vitro Experimental Research of the ECCT Equipment in the Cultured Hela Cells,
Oral Cavity Cancer Cells and Bone Marrow Mesenchymal Cells)
SAHUDI
STUDY PROGRAM OF MEDICAL SCIENCES
DOCTORAL PROGRAM OF MEDICAL FACULTY
AIRLANGGA UNIVERSITY
SURABAYA
2015
CELL DEATH MECHANISM AS RESULT OF THE EXPOSURE TO
LOW-ENERGY ELECTRIC FIELD WITH MEDIUM FREQUENCY
(In Vitro Experimental Research of the ECCT Equipment in the Cultured Hela Cells,
Oral Cavity Cancer Cells and Bone Marrow Mesenchymal Cells)
DISSERTATION SUMMARY
To obtain a doctoral degree
in Medical Science Study of Doctoral Program
At Medical Faculty of Airlangga University
Tested before the Committee of the Phase II Final Examination (Open)
on 28 September 2015
SAHUDI
Student Registration No. 090970134
STUDY PROGRAM OF MEDICAL SCIENCES
DOCTORAL PROGRAM OF MEDICAL FACULTY
AIRLANGGA UNIVERSITY
SURABAYA
2015
Promotor: Prof. Dr. David S Perdanakusuma, dr. Sp.BP(K)
Copromotor : Prof. Dr. Fedik Abdul Rantam, drh
This dissertation has been tested in the phase-1 final examination (Closed)
Date: 2 September 2015
The Examining Committee:
Chairman: Prof. Dr. I Ketut Sudiana, MS
Members: 1. Prof. Dr. David S Perdanakusuma, dr. Sp.BP (K) 2. Prof. Dr. Fedik Abdul Rantam, DVM 3. Prof Dr. EndangJoewarini, dr.Sp.PA (K) 4. Dr. RusmintoTjaturWidodo, ST 5. Prof. Dr. Subijanto M.S. dr. SpA (K) 6. Prof. Sunarto Reksoprawiro, dr. Spb (K) Onk KL 7. Dr. Hari Basuki Notobroto dr MPH Stipulated by the Decree of the Dean Medical Faculty of Airlangga University Number: 337/UN3.1.1/KD/2015 Date: 1 September 2015
ACKNOWLEDGMENT
Praise be to Allah Almighty for all His blessings, guidance and mercy thereby this research
can be carried out, completed, and made in the form of a dissertation manuscript as one of
the requirements to pursue graduation in Medical Science Study Program in Doctoral
Program in the Faculty of Medicine, Airlangga University.
This dissertation can be completed well thanks to the guidance, direction, advice, assistance
and prayers of many people. Therefore, with all humility and gratitude I extend my high
appreciation to the respectable persons below.
Prof. Dr. David Sontani Perdanakusuma, dr. Sp.BP (K), who has been
willing to be my promotor and provided guidance, direction, instructions and suggestions
while I was pursuing my doctoral education until the completion of the dissertation
manuscript.
Prof. Dr. Fedik Abdul Rantam, DVM, who has been willing to be co-promotor, who sincerely
gave instructions, guidance and suggestions whereby this dissertation manuscript could be
completed well.
Airlangga University Rector, Prof. Dr Moh Nasih SE mT Ak and the former rector Prof. Dr. H.
Fasich, Apt. who have given the great opportunities to me to pursue the doctoral education
in Medical Science Study Program, Faculty of Medicine, Airlangga University.
Prof. Dr. Agung Pranoto, dr., M.Kes., SpPD.,K-EMD.,FINASIM, Dean of the Faculty of
Medicine, Airlangga University who has given me the opportunity to participate in doctoral
education, Medical Science Study Program, Faculty of Medicine, Airlangga University.
Prof. Dr.Teddy Ontoseno, dr., SpA (K) SP JP., FIHA, as Chairman of Doctoral Program of
Medical Science Study Program, Faculty of Medicine, Airlangga University who has helped
me in undergoing the examination of this dissertation manuscript smoothly.
Director of the Graduate School of Airlangga University, Prof. Dr. Hj. Sri
Hajati, SH, MS and a whole range of both lecturers and staff of the graduate school of
Airlangga University Surabaya for the invaluable opportunities and facilities given to me of
being a student of the doctoral program.
Dr. Slamet Riyadi Yuwono, dr. MARS, the former Director of Dr.
Sutomo Hospital Surabaya, Dodo Anondo, dr. MPH, the former director of Dr. Soetomo
hospital, and Harsono, dr., the Acting Director of Dr. Sutomo Hospital
Surabaya, who have given permission and opportunity to continue pursuing
the doctoral education.
Prof. Sunarto Reksoprawiro dr. Spb (K) Onk-KL, the Former Head of
Surgery Department and Head of the Division of Head Neck Surgery in Dr. Soetomo
Hospital, who has given permission, motivation, guidance and opportunities for
continuing doctoral education; Agung Prasmono, dr. SpB TKV,
Head of the Department of Surgery, who has given permission, encouragement, and
an opportunity for me to finish the doctoral education.
Thank to the lecturers at the Doctoral Program in Medical Science
Study Program of Airlangga University, including Prof. Dr. Suhartono Taat Putra, dr, MS.,
Prof. Dr. Harjanto JM, dr., AIFM., Prof. Dr. Zainuddin drs, Apt., Prof. Dr. Juliati Hood A, dr,
MS, Sp. PA (K), FIAC., Prof. Kuntoro, dr, MPH, Dr. PH, Siti Pariani, dr, MSc, PhD., Dr.
Sunarjo, dr, MS, MSc., Dr. F. Sustini, dr., MS., Widodo J. Pudjiharjo, dr, MPH, Dr. PH., Dr.
Hari Basuki Notobroto, dr, M.Kes., Prof.Dr. I Ketut Sudiana, Drs, M.Si., Junaidi Khotib, S Si ,
M.Kes, Ph.D, Apt, Prof. Purnomo Suryohudoyo, dr, Prof. Dr. Fedik Abdul Rantam, drh.
Dr. Imam Susilo, dr., Sp.PA (K). Department of Anatomic Pathology, Airlangga University
Faculty of Medicine, who helped in reading cytologic preparation and provided various useful
inputs in the discussions.
Brothers and sisters, and my colleagues, Urip Murtejo, dr., Sp.B(K)KL, PGD PalMed ECU,
Yoga Wijayahadi dr., Sp.B(K)KL, Dwi Hari Susilo dr., Sp.B (K) KL, Maryono Dwi W dr., Sp.B
(K)KL, who have worked together to develop and promote Division of Head Neck Surgery in
Department of Surgery of Dr. Soetomo Hospital.
Wiwik Ernawati, Dra., Nunung Hardini Ir, Sih Enggar Panglipurwati,
Dra, Tina Marbun, ST, who have helped a management process at the department of
Surgery.
My friends, participants pursuing doctoral education in Medical Science Study Program,
Graduate School of 2009/2010 Class who mutually encourage, support, remind and provide
inputs in completing this study.
My parents whom I love much, my father H. Abdul Mujib (the Late) and mother Chotimah
(the Late), who had been caring for and educating me with affection, and my parent in-laws,
H. Kahono Widyoatmojo, Drs and Hj Partini, whom I love and respect.
Thank to my beloved wife, Hajar Ariyani, dr, Sp Rad (K) who is until
this moment always faithful to accompany me in this life as well as our five children, Nurul
Fitri Shabrina, dr, Izzatun Niswah Ajrina, SSi, Lathifah Nurul Fajri, SKed, Arinal Asma Haq,
and Muhammad Izzudin Syaifullah, who always make me so proud and
encourage me to complete this doctoral education.
I am really aware that this dissertation is far from perfect. Thus, I am acceptable to any
constructive comments and suggestions for improvement and perfection of this dissertation
manuscript. Hopefully the results of research and the writing of this dissertation can
contribute to the advancement of science and useful for colleagues and mankind. Allah SWT
may extend His mercy and blessings to all those who have given helps in any form in the
completion of this dissertation.
Hopefully, this dissertation research and manuscript provide contribution to the betterment of
sciences and also useful for users and foster new researchers who can
complement, sustain and refine this research.
Surabaya, 29 December 2014
Sahudi, dr.Sp.B (K) KL
SUMMARY
CELL DEATH MECHANISM AS RESULT OF THE EXPOSURE TO
LOW-ENERGY ELECTRIC FIELD WITH MEDIUM FREQUENCY
(In Vitro Experimental Research of the ECCT Equipment in the Cultured Hela Cells,
Oral Cavity Cancer Cells and Bone Marrow Mesenchymal Cells)
Cancer has become the main public health issue worldwide. Cancer patients are expected to
grow about 12.7 million people each year, and the cancer may cause death in 7.6 million
people, or 21,000 deaths per day. Modalities of cancer therapy now widely accepted in the
medical world are surgery, chemotherapy, and radiotherapy. Although this is supported by
various researches and scientific findings, but until now the three cancer therapeutic
modalities still have limitations in effectiveness, side effects that are sometimes severe and
expensive. In Indonesia, cancer patients face various kinds of constraints, in which many
patients who came for treatment are in advanced stage, thus the effectiveness of therapy
becomes very low. In this very limited condition, in Indonesia, the medical world was recently
shocked the discovery of cancer therapy device named ECCT (Electro Capacitive Cancer
Treatment) by the doctoral graduate of Japan. Thousands of cancer patients come to the
clinic that provide ECCT therapy, while the majority of the medical community consider that
the findings of ECCT are not scientific, just nonsense, and dangerous for patients.
This study was carried out to bridge the scientific controversy in the field of cancer therapy in
Indonesia by proving the existence of an increase in the percentage of death of cells
exposed to the ECCT cancer therapy equipment and revealing the molecular mechanism of
pathology. This study is an in vitro laboratory experimental research using completely
randomized block design. It aimed at determining the effects of exposure to the low-energy
electric fields with medium frequency of the ECCT cancer therapy equipment, in which
several variables were measured after treatment. Three kinds of cancer cell cultures used in
this research are HeLa cell, oral cavity cancer cells, and bone marrow mesenchymal cells.
All three cell cultures were divided into two groups with 8 replications for each group. They
are the treatment groups exposed to ECCT for 24 hours and the control group. After 24
hours, the number of living cells and died cells were counted using Trypan Blue staining and
examined for their Tubulin A protein, Cyclin B, p53 and Ki-67 expressions.
The results of this research suggested that the group of cells exposed to the ECCT have the
higher amount of cell deaths significantly compared with the control group, occurring in both
cancer cells and non-cancer cells. In line with the viability of the cells, ECCT can reduce the
number of viable cancer cells significantly, while in non-cancer cell cultures, namely bone
marrow mesenchymal cells, the ECCT influences the number of viable cells, but not
statistically significant. In terms of percentage of cell deaths, the ECCT can enhance the
percentage of deaths in all three types of cell cultures.
This research also found that cancer cells exposed to the ECCT for 24 hours will increase
the expressions of Tubulin A, cyclin B1, p53 and Ki-67 significantly compared to the control
group. The author concludes from this study that the low-energy AC electric field with
medium frequency emited by the ECCT can kill cancer cells through mitotic catastrophe
mechanism.
ABSTRACT
CELL DEATH MECHANISM AS RESULT OF THE EXPOSURE TO
LOW-ENERGY ELECTRIC FIELD WITH MEDIUM FREQUENCY
(In Vitro Experimental Research of the ECCT Equipment in the Cultured Hela Cells,
Oral Cavity Cancer Cells and Bone Marrow Mesenchymal Cells)
Background. This study was conducted to answer controversy around the
ECCT cancer therapy device that uses low-energy electric field with medium frequency.
Objective. Proving that there was the increase in the percentage of cell death by expossure
to the ECCT and uncovering the molecular mechanisms of pathology.
Method. This study is an in vitro laboratory experimental research using completely
randomized block design. It aimed at determining the effects of exposure to the low-energy
electric fields with medium frequency of the ECCT cancer therapy equipment. Three kinds of
cancer cell cultures used in this research are HeLa cell, oral cavity cancer cells, and bone
marrow mesenchymal cells. All three cell cultures were divided into two groups with 8
replications for each group. They are the treatment group exposed to ECCT for 24 hours
and the control group. The number of living cells and died cells were counted using Trypan
Blue staining and examined for their Tubulin A protein, Cyclin B, p53 and Ki-67 expressions.
Results. The results of this research suggested that the group of cells exposed to the ECCT
have the higher amount of cell deaths significantly compared with the control group,
occurring in both cancer cells and non-cancer cells. In line with the viability of the cells,
ECCT can reduce the number of viable cancer cells significantly, while in non-cancer cell
cultures, namely bone marrow mesenchymal cells, the ECCT influences the number of
viable cells, but not statistically significant. In terms of percentage of cell deaths, the ECCT
can enhance the percentage of deaths in all three types of cell cultures.
Conclusion. The author concludes from this study that the low-energy AC electric field with
medium frequency emited by the ECCT is able to kill cancer cells through mitotic
catastrophe mechanism.
Key words. ECCT, in vitro, cell culture, Tubulin A, Cyclin B1, p53, Ki-67, mitotic
catastrophe.
GLOSSARY
AC : Alternating Current ACS : American Community Survey (US Census Bureau) AJCC : American Joint Committee on Cancer APC/C : Anaphase Promoting Complex/Cyclosome BAX : BCL2-Associated X Protein CIS : Intracellular fluid CES : Extracellular fluid CDK-1 : Cyclin Dependent Kinase-1 DC : Direct Current ECCT : Electro Capacitive Cancer Treatment Fase M : Mitotic phase FDA : Food & Drug Administration (US) FLIP : FLICE-Like Inhibitory Protein G-1 : Gap-1 HPV : Human Papiloma Virus IARC : International Agency for Research on Cancer MoH : Ministry of Health OCC : Oral Cavity Cancer MAP : Microtubule Associated Proteins MCL-1 : Myeloid Cell Leukemia-1 Mdm-2 : Mouse double minute 2 homolog MTOC : Microtubule Organizing Center PERABOI : Indonesian Society of Surgical Oncology Riskesdas : Basic health research RPCT : Randomized Placebo Controlled Clinical Trial S : Synthesis SEER : Surveillance, Epidemiology, and End Results SKRT : Household Health Survey μT : Micro Teslah nT : Nano Teslah TF-IID : Transcription Factor II-D TTF : Tumor Treating Field UICC : Union for International Cancer Control XIAP : X-Linked Inhibitor of Apoptosis Protein
1. INTRODUCTION
1.1 Background
Cancer has become the major public health issue worldwide. Cancer patients are expected
to grow about 12.7 million people each year, and the cancer may cause death in 7.6 million
people, or 21,000 deaths per day. In advanced countries, the cancer has become the
number one killer, causing 2.8 million deaths per year. In developing countries, the cancer is
the second killer after heart disease and blood vessel disease, with number of 4.8 million
deaths per year. By 2030, new cancer patients worldwide are estimated at 21.4 million, with
number of 13.2 million deaths per year (IARC/Globocan 2008). In Indonesia, the number of
patients and cancer deaths also increase significantly. Data from the Ministry of Health
(MoH) obtained from the basic health research (Riskesdas) state that the prevalence of
cancer reached 4.3 per 1,000 people in 2013, whereas the data of previous five years
mentioned prevalence of 1 per 1,000 people. For now there is an estimated 1 million people
with cancer and this figure will continue to grow each year (MOH, 2013). Household Health
Survey (SKRT) said that cancer deaths in 1992 were 4.8%, in 1995 increased to 5.0% and in
2001 increased to 6.0%. The cancer ranked fifth as a cause of death in Indonesia (MOH,
2001). Regarding the number of patients, cervical and ovarian cancers ranked first with the
number of patients at 19.3, followed by breast cancer at 15.6 per thousand people, whereas
oral cavity cancer (OCC) ranked seventh with number of patients at 5.6 per thousand people
(MoH, 2007). There is a problem in cancer disease in Indonesia, among others, nearly 70%
of people with this disease were found in an advanced stage (Asmino, et al, 1985). OCC
patients who came for treatment at the Dr Sutomo Hospital amounted to 31 patients per year
on average, 89% of them came in advanced stage (Stage III and IV) and only 11% of them
got the curative measures (Sahudi, 2013).
Combating cancer in Indonesia is more therapeutic, and not early detection or prevention.
Most of the cancer patients come for treatment in advanced stage, it is very limiting the
effectiveness of cancer therapy. In this case, there are many problems, such as the
inadequate funds to meet the needs for chemotherapy and radiotherapy, the waiting time to
get radiotherapy which sometimes lasted up to 4-6 months, coupled with phobia about
cancer treatment, either surgery, radiotherapy, or chemotherapy, and the limited access to
get thorough explanation about cancer. As noted by Prof Soehartati, cancer treatment
centers in Indonesia still covers only 22 public hospitals and two private hospitals, and most
of them are located in the island of Java, with only about 15% cancer patients were served
(Willy, 2013). This condition makes alternative treatment for cancer flourish every where,
reflecting the community needs for affordable cancer treatment with minimal side effects.
Indonesian people, especially people with cancer, are recently shocked with news of the
discovery of cancer therapy equipment by the scientist, Japanese doctoral graduate, Dr.
Warsito P Taruno, which is known as a cancer-fighting equipment using Electro Capacitive
Cancer Treatment (ECCT). Soon thousands of cancer patients come to the laboratory which
he founded, hoping cure of cancer, with minimal side effects and affordable price. They are
willing to be registered as volunteers for the equipment that has not passed through a series
of RPCT (Randomized Placebo Controlled Clinical Trial) commonly done for standard
treatment methods in the field of medicine. Meanwhile, the majority of medical practitioners
are very doubt about the effectiveness of the cancer treatment equipment, even the
Indonesian Society of Surgical Oncology (PERABOI) has warned Edwar Tech, the
laboratory which was founded by Dr. Warsito to immediately close and stop services for
cancer patients because it is considered unreasonable and endanger the patients. Such
controversy should immediately be ended, because the creator of the ECCT, through some
media has invited the medical community to do research on this equipment. This ECCT
equipment, if it is proven to kill cancer cells, especially when the cell death mechanism can
be found, could be very valuable for cancer patients and the medical community who is
constantly battling the suffering of cancer patients.
Antimicrotubule drugs such as Paclitaxel, Vincristine, and Nocodazole which have been
accepted and widely used in clinic settings throughout the world, work through a chemical
process, interfere with the microtubule dynamics and causes the cells undergo mitotic arrest,
and finally die by mitotic catastrophe mechanism. There is a big question among experts and
researchers, “are there any ways beyond chemical reactions, which can effectively and
measurably interfere with microtubule dynamics. Butters et al. tested in vitro the effect of a
low-energy electric field on the tubulin polymerization. From this research, they found that
the low-energy electric field with a frequency of 22 KHz works nonthermally, can induce
changes in tubulin subunit interaction, and works like taxanes-class chemotherapy drug.
Low-energy AC electric field supposedly works by affecting the blanket of fluid around the
tubulin poles, changing the density of the fluid around the poles become more viscous, until
the polymerization - depolymerization activities are disturbed. The range of the energy used
in this study is milli Watts, producing a magnetic field with an intensity of 0.1 nT - 10 μT. This
is the first publication of scientific research, which measures directly outside the cellular
system, the interaction between the low-energy AC electric field with medium frequency and
the microtubule polymerization process. From these results we believe that the low-energy
AC electric field can be developed into a medical technology relevant for cancer therapy in
the future (Butters et al.,2014).
ECCT equipment created by CTECH Edwar Technology Lab, using a low-energy electric
field with a frequency of 100 KHz, has been used by thousands of cancer patients, but
brings about controversy in the medical world. This equipment has been investigated and
shown to kill cells in vitro, but no one study uncovers molecular mechanisms of cell death
due to exposure to the ECCT. This study examined several types of cell cultures in vitro to
prove the existence of cell death due to exposure to an electric field of the ECCT equipment,
and uncovered the molecular mechanisms of cell-death process. This study uses OCC cell
cultures and the Hela cells representing a cell line of cervical cancer. The two kinds of
cancers above constitute the top 10 types of cancers suffered by a large part of patients, and
the majority of them come for treatment in advanced stages. We also tested their effects on
bone marrow mesenchymal cell cultures to know the response of non-cancerous cells on the
ECCT equipment.
In associated with the findings of Butters et al. 2014, at the molecular level we can prove
whether an electric field of the ECCT can affect intra-cellular tubulin interaction by examining
changes in expression. The interaction between tubulin A and B, and its polymerization into
microtubules which is disrupted when the cells are undergoing the mitotic process can be
proved by seeing an increase in the expression of one tubulin, i.e. tubulin A, which will be
increased due to hampered microtubule polymerization. The disrupted microtubule
polymerization will trigger a series of subsequent processes. Cyclin B1 expression
increases as the impaired microtubule polymerization will activate spindle checkpoint
complex that continues to keep the Anaphase Promoting Complex/Cyclosome (APC/C)
responsible for doing degradation of the inactive Cyclin. This situation causes the cells are
inhibited while completing mitosis as scheduled, termed as mitotic arrest, where in this
condition the cells have trouble performing protein synthesis. Expression of protein p53
(wild) will increase as the age of Mdm2-sense mRNA is shorter than p53-sense mRNA.
Mdm-2 which serves to perform p53 degradation will be depleted first. These conditions will
drive the cells to die and senesce which ultimately makes the proliferation decrease.
Changes in cell proliferation level can be known by examining changes in the expression of
Ki67.
1.2 Statement of Problem
1. Does the exposure to ECCT equipment increase the percentage of cell death in the
cultured oral cavity cancer cells, HeLa cells, and bone marrow mesenchymal cells?
2. Does the exposure to the ECCT increase the expression of tubulin A in the cultured
Oral Cavity Cancer cells, HeLa cells, and bone marrow mesenchymal cells?
3. Does the exposure to the ECCT increase expression of Cyclin B-1 in the cultured
Oral Cavity Cancer cells, HeLa cells, and bone marrow mesenchymal cells?
4. Does the exposure to the ECCT increase expression of p53 in the cultured Oral
Cavity Cancer cells, HeLa cells, and bone marrow mesenchymal cells?
5. Does the exposure to the ECCT lower the expression of Ki-67 in the cultured Oral
Cavity Cancer cells, HeLa cells, and bone marrow mesenchymal cells?
1.3 Objectives
1.3.1 General Objectives
To prove an increase in the percentage of death of the cells exposed to the ECCT
and uncover the molecular mechanisms of pathology.
1.3.2 Special Objectives
1. To prove the increased percentage of cell death in the cultured Oral Cavity
Cancer cells, HeLa cells, and bone marrow mesenchymal cells exposed to the
ECCT.
2. To prove the increased expression of tubulin A in the cultured Oral Cavity
Cancer Cells, HeLa cells, and bone marrow mesenchymal cells exposed to
ECCT.
3. To prove the increased expression of Cyclin B-1 in the cultured Oral Cavity
Cancer Cells, HeLa cells, and bone marrow mesenchymal cells exposed to
ECCT.
4. To prove the increased expression of p53 in the cultured Oral Cavity Cancer
Cells, HeLa cells, and bone marrow mesenchymal cells exposed to ECCT.
5. To prove the decrease in Ki-67 expression in the cultured Oral Cavity Cancer
Cells, HeLa cells, and bone marrow mesenchymal cells exposed to ECCT.
1.4 Benefits of the Research
1.4.1 Theoretical Benefits
Provide scientific information regarding cytotoxicity of ECCT equipment in cell
cultures and explain the molecular mechanisms underlying the cell death.
1.4.2 Practical Benefits
1. The existence of scientific evidence of the working mechanism of ECCT is expected
to be able to reduce the scientific controversy between CTECH Lab Edwar
Technology and the relevant medical practitioners.
2. It is expected that the existence of scientific evidence of the working mechanism of
the ECCT could become the basis for execution of further in vitro researches in other
cancer cell cultures.
3. It is expected that the existence of scientific evidence of the working mechanism of
ECCT could become the basis for execution of further in vivo researches in test
animals.
4. It is expected that the scientific evidence on the working mechanism of ECCT could
become the basis for the discovery of new cancer therapy modalities other than
surgery, chemotherapy, and radiotherapy.
2. CONCEPTUAL FRAMEWORK & HYPOTHESES
2.2 Explanation of Conceptual Framework
Yoram Palti has performed in vitro study in the cell cultures treated with AC electric field of
voltage 1-2 V/cm with a frequency of 1-3 Khertz, showing a slowdown of the mitotic process
which normally should take place in 1 hour, but does not finish within 3 hours. Besides
slowing down the mitotic process, the disintegration of cells in the final phase of telophase
was also observed. Both of these events are thought to occur because of the effect of an
electric field on the polymerization-depolymerization process of the dimer in microtubule
protein, which has been known to have a high electrical charge in the polar ends of the
molecules.
CDK 1/Cyclin B encourages cells to begin the process of mitosis. Lamin phosphorylation by
Cdk1/Cyclin B causes dissolution of the nuclear membrane. Furthermore, the process of
mitosis enters the main process that is coupling of the kinetochores and microtubules. In the
next process, anaphase-promoting complex/cyclosome (APC/C) degrades Cyclin B marking
the end of mitosis with the lamin dephosphorylation, the formation of the nuclear membrane,
with two daughter cells. When the dynamics of microtubules is interrupted, the spindle
checkpoint continues to keep APC/C inactive, thus preventing damage of Cyclin B and lamin
dephosphorylation. Thus the cells are arrested in the process of mitosis (mitotic arrest). At
the moment of mitotic arrest, synthesis of a variety of proteins will be disrupted. When the
cells enter process of mitosis, the nuclear membrane will be dissolved and the condensed
DNA will form chromosomes. Cell protein synthesis begins with intron splicing in pre-mRNA
in the nucleus of the cell, then is translated and is forming protein in the cytoplasm. Nuclear
membrane has the role of separating transcription from translation processes, which allows
mRNAs to undergo maturation. In the absence of nuclear membrane during mitotic process
underway or cells undergoing mitotic arrest, then the cell protein synthesis will stop. And this
can cause other processes.
Under normal conditions, the cell makes proteins to prevent the cells from apoptosis, for
example, MCL-1 functions to inhibit the release of cytochrome-c from mitochondria, FLIP
which prevents activation of caspase 8, and XIAP which prevents activation process of
caspase 3 by caspase 9. With delays in the translation process for the production of anti-
apoptotic proteins, the cells in mitotic arrest condition will be very susceptible to apoptosis.
Under normal circumstances, Mdm-2 responsible for degradation of the p-53 is in balance
state until the p-53 is in low concentration in the cell. p53 production-coding mRNA is long-
lived until when the transcription process in the cells is hampered, p53 can still continue to
be produced, while production of Mdm-2 will stop. This causes an increase in the amount of
p53, because no Mdm-2 degrades p53. However, the high level of p53 in cells undergoing
mitotic arrest does not always produce apoptosis because the apoptotic process also
requires other protein synthesis where in this state cannot be done.
Degradation of Cyclin B by APC/C is required to escape from mitosis. But in a state of
mitotic arrest, APC/C is not synthesized until when Cyclin B levels decrease slowly due to
natural degradation process, thus the cells not undergoing apoptosis at the time of mitotic
arrest will come out of the process of mitosis known as mitotic slippage in a tetraploidy or
aneuploidy state. In the initial phase of mitotic slippage, cells that previously experienced the
mitotic arrest has high levels of p53. Once coming out of the phase of mitosis, the nuclear
membrane protein has been formed and protein can be synthesized again, then cells with
high levels of p53 will immediately trigger apoptotic process through BAX activation, and
subsequent caspase cascade. In other circumstances, the p53 can activate p21 causing
cells to undergo arrest in G1, then experiencing senessence and death.
The above circumstances, either apoptosis that occurs at the time of mitosis or after the
mitotic slippage, cell senescence, aneuploidy or polyploidy condition, all of which will lead to
cell death which causes the percentage of cell death after exposure to ECCT will increase.
Cell death that occur continuously and lasts a long time eventually will make the proliferation
of cells in the cell culture will decrease. A decrease in the level of proliferation in cell culture
can be seen by the decreased expression of Ki-67 protein.
2.3 Research Hypotheses
Regarding the background of the problem, a literature review, conceptual framework and
objectives to be achieved in this research, several hypotheses can be formulated as follows:
1. There is an increase in the percentage of cell death in the cultured HeLa cells, oral
cavity carcinoma cells and bone marrow mesenchymal cells exposed to ECCT.
2. There is an increase in expression of Tubulin A as a result of microtubule
polymerization disruption in the cultured HeLa cells, Oral cavity carcinoma cells and
bone marrow mesenchymal cells exposed to ECCT.
3. There is an increase in expression of Cyclin B-1 in the cultured HeLa cells, oral
cavity carcinoma cells and bone marrow mesenchymal cells exposed to ECCT.
4. There is an increase in expression of p53 protein in the cultured HeLa cells, oral
cavity carcinoma cells and bone marrow mesenchymal cells exposed to ECCT.
5. There is a decrease in expression of Ki-67 protein in the cultured HeLa cells, oral
cavity carcinoma cells and bone marrow mesenchymal cells exposed to ECCT as a
result of decrease in cell proliferation.
3. RESEARCH METHOD
3.1 Research Type and Design
This study was aimed at analyzing the inhibitory power of ECCT cancer therapy equipment
on the growth of cultured Hela cells, oral cavity carcinoma cells and Bone Marrow
Mesenchymal Cells, as well as their molecular pathobiology. This research is an in vitro
laboratory experimental research using completely randomized block design. It aimed at
determining the effects of exposure to the low-energy electric fields with medium frequency
of the ECCT cancer therapy equipment, in which several variables were measured after
treatment. Experimental unit sampling was conducted randomly and there is control group.
Grouping of the research subjects is showed in figure 3.1.
Figure 3.1 Grouping of the research subjects
Where OCC : Oral Cancer Cell Culture Hela : Hela Cell Culture Mesenchyme: Bone marrow mesenchymal cell culture R : Randomization K : Control group, 1: OCC cells, 2: HeLa cells, 3: Mesenchymal cells P : Treatment group, 1: OCC cells, 2: HeLa cells, 3: Mesenchymal cells Tx : Treatment with ECCT exposure for 24 hours O1-O6 : Determination of protein expression: Tubulin A, Cyclin B, p53, Ki-67
3.2 Experimental Unit, Replication, and Randomization
3.2.1 Experimental Unit
Samples used in the research were the cultured HeLa cells, oral cavity carcinoma cells,
Bone Marrow and mesenchymal cells obtained from the cell bank of the Institut of Tropical
Disease, Airlangga University.
3.2.2 Replication
The amount of replication is determined by the following formula:
Where:
r = number of replication
Z1 - α/2 = value of the standard normal distribution that is equal to the
significance level (for = 0.05 is 1.96)
Z1 - ß = the value of the standard normal distribution that is equal to the desired
power (for ß = 0.10 is 1.28 = standard deviation of the outcome
U1 = mean outcome of the control group, drawn from previous research conducted by Izzatun Niswah = 45500 U2 = mean outcome of the treatment group, drawn from previous research conducted by Izzatun Niswah = 27500 The calculation of the above formula shows n = 4 samples per treatment. The next calculation shows the samples are at least 4 for each treatment group.
3.3. Research Variables
3.3.1 Independent variables
1. Exposure to ECCT cancer therapy equipment
2. Type of Cells
3.3.2 Intervening Variables
1. Expression of Alpha Tubulin.
2. Expression of P53 protein.
3. Expression of Cyclin B-1
4. The expression of Ki67 protein
3.3.3 Dependent variables
Cell death
3.3.4 Operational Definition of Variables
Operational limitations of the variables are as follows:
a. ECCT is an equipment used to generate the electric field. Electric current used is an
alternating electric current with voltage range of -10 volts to +10 volts, with a
frequency of 100 KHz. Based on the research already done by Yolam Palti, the
electric field with a frequency of 100 KHz can be used to inhibit the growth of brain
cancer cells. Electric field generated by the ECCT is static electric field where the
electrodes used in this equipment are not attached to the cancer cells, but they are
in a container (a place to put the plate). Because electrodes used are not attached to
the cells, there is no electric current flowing in the cell. In this experiment, it is the
electric field which affects the cell growth.
b. Alpha tubulin is a part of large family of globular proteins, consisting of alpha-, beta-,
gamma-, delta, epsilon and zeta tubulins. Together with beta tubulin, alpha tubulin
arranges microtubules which are instrumental in the process of mitosis. Alpha tubulin
expression is examined by immunocytochemical staining and the results are
calculated semiquantitatively using Immunoreactive Score method from Remmele
and Stegner.
c. Cyclin-B1 of the human is synthesized by CCNB1 gene code. This protein plays an
important role in the process of mitosis, along with P34 (Cdk1) forming a complex
protein that acts as a switch on to start mitosis, which is characterized by
chromosome condensation and degradation of lamin or nuclear membrane. Similarly,
when mitosis ends, the protein (APC-C) is needed to degrade Cyclin B1. Thus lamin
will be re-formed and chromosomes are in decomposed state. Cyclin B1 expression
is examined with immunocytochemical staining, and the results are calculated
semiquantitatively using Immunoreactive Score method from Remmele and Stegner.
d. Ki-67 in human is synthesized by MKI67 gene code. This nuclear protein plays a role
in cell proliferation by synthesizing ribosomal RNA. Expression of Ki- 67 happens
while cells are proliferating, starting at mid G1 phase, increases at the time of
entering S and G2 phases, and reaches the peak at M phase of the cell cycle. At the
end, Ki-67 will undergo catabolism rapidly in the late phase of M and is not detected
in the phase of G0 or initial G1 phase. Ki-67 expression is determined by
immunocytochemical staining, and the results are calculated semiquantitatively using
Immunoreactive Score method from Remmele and Stegner.
e. p53 is a tumor suppressor where in normal circumstances, its level is low, playing a
role in the cell death process triggered by the gene defect or inhibition in the process
of mitosis. Expression of p53 is examined by immunocytochemical staining, and the
results are calculated semiquantitatively using Immunoreactive Score method from
Remmele and Stegner.
f. Cell death is an event, representing the end of biological phenomenon of cell life. Cell
death is examined by inverted microscope (Nikon TMS, Japan), using trypan blue
staining and counted with a hemocytometer. Viable cells are not colored, clearly
visible in which cells with dead cytoplasms would be stained blue from trypan blue.
Counting is carried out by a competent laboratory personnel.
3.4. Flow chart of the research
Flow chart of the research is showed in Figure 4.2 below.
Figure 3.2. Flow chart of the research
3.5 Research Equipment and Material
3.5.1 Equipment
Some equipment used here are electro field ECCT-17 (C-Tech labs Edwar Technology),
5%CO2 incubator, autoclave, inverted microscope Nikon TMS, Japan, electric centrifuge,
laminar air flow, micro siring Hamilton 1-10 mL, 200 mL adjusted micropipette (Socorex).
Glassware such as 10 ml-, 25 ml- flask, measuring cups Erlen meyer, petri dish, a glass
beaker, vortex, stirrer, aluminum cup, micropipette, microtips, syringe, 2 ml microcentrifuge
tube, eight 24 well-microplate, and Thoma hemocytometer.
3.5.2 Material
The cells used in the research are Hela cells, oral cavity cancer cells and bone marrow
mesenchymal cells obtained from the Cell Bank of Stem Cell Laboratory – Institute of
Tropical Disease (ITD) Airlangga University. Cells stored in Cryo Tube at temperature of -
80C were acclimatized and cultured in α-MEM medium at 37°C with 5% CO2. Culture
medium was changed every 48 hour until the number of cells were sufficient to be
transferred to well cell culture chamber where the cancer cells were treated with ECCT,
while the control well cell culture chamber was not treated with ECCT. After 24 hours, cells
were harvested and examined by immunocytochemistry. The number of cells in each of the
wells is 500,000 cells per cc.
3.6 Procedures for Implementation of the Research The study was carried out with four stages: (1) the cultured OCC cells, HeLa Cells and Bone Marrow Mesenchymal Cells; (2) treatment of OCC cells, Hela Cells and Bone Marrow Mesenchymal Cells with ECCT for 24 hours; (3) fixation and staining of the cell preparation by the immunochemistry method; (4) analysis of the ECCT’s effect based on the expression of variables between treatment and control groups. The research proposal was submitted to the Ethics Commission of Medical Faculty, Airlangga University - Surabaya. Once approved,
the research was carried out at the Stem Cell Laboratory - Institute of Tropical Disease (ITD).
3.7 Data Collection and Analysis
The collected data were processed by using a statistical test of bivariate and multivariate
analysis to test whether the ECCT can interfere with the life of the cell and explain the
mechanism of death.
3.8 Place and Time
The research was conducted at the Stem Cell Laboratory - Institute of Tropical Disease
(ITD) Airlangga University. Fixation process of cytologic preparations and
immunohistochemical staining were also conducted at the Stem Cell Laboratory - Institute of
Tropical Disease (ITD) Airlangga University. Immunohistochemical preparation was
calculated and photographed by competent anatomic pathology experts. The research was
conducted over less than 6 (six) months, including the preparation of materials and
equipment, treatment, examination and preparation of reports.
Table 3.1. Research time
Type of activities Month
1 2 3 4 5 6 7
Proposal preparation x
Material and equipment preparation
X
Culture and determination x x X
Data collection x X
Data analysis x X
Reporting x
4. RESULTS AND DATA ANALYSIS
4.1 Measurement of Voltage and Frequency of ECCT
To ascertain the type and amount of output of the ECCT equipment, the voltage and
frequency generated by the ECCT equipment were measured using an oscilloscope at the
Laboratory of the Surabaya State Electronics Polytechnic. The results of the measurement
showed that electrical energy emitted by ECCT is alternating current with a frequency of 100
KHz and voltage ± 20 Volt. Characteristics of the alternating current is different from direct
current where the size and polarity of the current/voltage is always fixed over time; however,
the size and polarity of the current/voltage in the direct current may change over times
following the shape of the sine function.
Electric current emitted from the ECCT was measured by osciloscope in Electronic Lab
PENS, where the lab showed the alternating electric current with 20-volt voltage and wave
length of 10 micro seconds whereby the frequency of the AC electric current can be
calculated at 100 KHz.
4.2 Preparation and counting of cells
This experimental research was conducted using three different kinds of cell cultures,
including oral cavity cancer cells, Hela cells, and Bone Marrow mesenchymal cells to test the
cytotoxic effect of low-voltage AC electric field with medium frequency produced by ECCT
cancer therapy equipment. The research was conducted at the Stem Cell Laboratory-
Institute of Tropical Disease, Airlangga University. The three kinds of cells stored in Cryo
Tube at temperature of -80 C were acclimatized and cultured in α-MEM medium at
temperature of 37C with 5% CO2. Culture medium was changed every 48 hour until the
number of cells were sufficient to be transferred to well cell culture chamber where the
cancer cells were treated with ECCT, while the control well cell culture chamber was not
treated with ECCT. Each well, either treated with ECCT or control well contains the same
number of cells, ie, 500,000 cells. After 24 hours, the three kinds of cell cultures were
harvested and viable and non-viables cells were then counted using Trypan Blue staining
and immunocytochemistry examination to see the expressions of tubulin A, Cyclin B1, p53
and Ki-67.
Viable and non-viables cells were counted after treatment of ECCT and the control group
were counted by Trypan Blue staining. Integrity of the non-viable cell wall was damaged so
that the material in blue of Trypan Blue penetrated and colored the cytoplasm, while viables
cells had clear cytoplasm, not colored. Viable and non- viable cells were counted by Thoma
hemocytometer.
4.3 An Overview of Research Subjects Results of the research were calculated, collected and tested for normality of the data collected prior to the statistical test. In general, the data of the research results are showed in Table 4.1.
The above data indicates the mean semi-quantitative assessment of Tubulin A, Cyclin-B1,
P53, and KI67 in the three types of samples, showing non-normal data distribution (p <0.05).
While the assessment of the number of living and dead cells indicates normal data
distribution (p> 0.05) in all three cell types.
4.4 Effects ECCT on Cell Death
To determine the effect of the electric field of the ECCT against cell death, the cells are
stained using Trypan Blue and counted by a hemocytometer, and examined with inverted
microscope (Nikon TMS, Japan). Cells with the living cytoplasm are clear, whereas dead
cells as seen in Figure 5.2 will be blue due to being infiltrated by blue from Trypan Blue.
Counting was performed by the competent laboratory personnel. Toxicity effect of ECCT on
the cells was measured by counting the percentage of cell death after treatment for 24
hours, then compared with the control group. Difference in the percentage of cell death
between the treatment groups and control group was tested using independent t-test and the
difference between cell groups was tested using ANOVA test as shown in Table 5.2. The
percentage of cell death in OCC culture group exposed to ECCT is (18.25 ± 3:36)%, which is
different statistically and significantly from the control group (5.72 ± 2:57)%. The percentage
of cell death in HeLa cell culture group exposed to ECCT is (6.66 ± 1.77)%, which is different
statistically and significantly from the control group (2.44 ± 1.05)%. While the percentage of
cell death in groups of mesenchymal cells exposed to ECCT is (33.75 ± 5.80)%, which is
different statistically and significantly from the control group (12.84 ± 4.87)%.
The percentage of cell death is also significantly different when compared among groups of
cells. In the group treated with ECCT exposure, the percentage of cell death is highest in the
mesenchymal cells group (33.75 ± 5.80)%, followed by groups of OCC cells (18.25 ±
3.36)%, and the lowest is the HeLa cells (6.66 ± 1.77)%. Order of the percentage of cell
death is also the same in the control group, where percentage of cell death in the
mesenchymal cells group is (12.84 ± 4.87)%, followed OCC cells at (2.57 ± 5.72)%, and
HeLa cells at (2.44 ± 1.05)%.
Table 4.2. Cell death, Anova test and independent t-test
Treatment
Cells ECCT Control
OCC 18.25 ± 3.36b
5.72 ± 2.57b
0.000
Hela 6.66 ± 1.77a 2.44 ± 1.05a
0.000
Mesenchyme 33.75 ± 5.80c
12.84 ± 4.87c
0.000
P 0.000 0.000
This statistical test using independent t-test shows a significant difference in percentage of
cell death in all three cell groups compared with the control. Anova test also shows
significant difference in the percentage of cell death among all three cell culture groups. The
percentage of cell death caused by exposure to ECCT for 24 hours is illustrated in figure 4.1
depicting comparison in the percentage of cell deaths.
Figure 4.1: The cell death in the ECCT group and treatment group
The average number of cells per well before the treatment is 500,000 cells per cc. Once
treated with exposure to ECCT for 24 hours, the number of living OCC cells are (177,500
±17 728)/cc, which is significantly smaller than the control (255,000 ± 16 035)/cc. Number of
HeLa cells group exposed to ECCT for 24 hours are (778 125 ± 81 017)/cc, significantly
smaller compared with the control at (942,500 ± 28,535)/cc. While in the group of
mesenchymal cells which are not cancer cells, the number of living cells after exposed to
ECCT 24 hours is (115,000 ± 12 535)/cc, smaller yet not statistically significant compared
with the control group of (132 500 ± 17 113)/cc.
4.5 Immunocytochemical examination In OCC cell culture
Results of immunocytochemical determination in cultured OCC cells are showed in the figure
below. Figure 4.2 displays the result of the examination of expression of A tubulin, 4.2 (A) in
cells exposed to ECCT for 24 hours, number of cell expression at 60%, with the medium
expression level, light brown. Figure 4.2 (B) is the control, showing 30% of cell expression,
the weak expression level, pale brown.
Figure 4.3 displays the result of determination of the expression of Cyclin B1, 4.3 (A) in cells
exposed to ECCT for 24 hours, the number of cells expression at 60% with weak expression
levels, pale brown. Figure 4.3 (B) is the control, 10-15 % of cell expression with the weak
expression level.
Figure 4.4 displays the result of examination of the expression of p53, 4.4 (A) in cells
exposed to ECCT for 24 hours, the number of cells expression at 80%, with moderate to
strong expression level, dark brown and light brown. Figure 4.4 (B) is the control, showing
15% of cells expression, weak expression level, pale brown.
Figure 4.5 demonstrates the result of examination of the expression of Ki-67, 4.5 (A) in cells
exposed to ECCT for 24 hours, the number of cells expression at 50%, with moderate to
strong expression levels, dark brown and light brown. Figure 4.5 (B) is the control, showing
10% of the cells expression with weak expression level, brown fade.
Figure 4.6 below shows the results summary of semiquantitative immunohistochemical
examination of OCC cells. Tubulin A expression in the OCC cells exposed to ECCT for 24
hours is (6.75 ±1.04), significantly different from the control (2.00 ± 0.93), Cyclin B is (3.13 ±
0.35) significantly different from the control (1.38 ± 0.52), p53 is (10.50 ± 2.12) significantly
different from the control (1.25 ± 0.71), whereas the expression of Ki-67exposed to ECCT
for 24 hours is (4.88 ±0.83) significantly different from the control (1.25 ± 0.46).
Table 4.3 shows the result of analysis of the independent t-test of immunocytochemical
examination in four parameters using the Mann-Whitney test, suggesting that expressions of
Tubulin A, Cyclin B, p53, and Ki 67 are statistically significantly different between OCC cell
cultures treated with ECCT for 24 hours and the control group with a value of p <0.05.
4.6 Immunohistochemical examination in Hela Cell Culture
Results of immunohistochemical examination in Hela Cell Culture are showed in the figure
below. Figure 4.7 exhibits the result of the examination of tubulin A expression, 4.7 (A) in
cells exposed to ECCT for 24 hours, the number of cells expression is 85% with moderate to
strong expression level, dark brown and light brown. Figure 4.7 (B) is the control, showing
30% of the cells expression, weak to moderate expression, light brown and pale brown.
Figure 4.8 shows the result of examination of the Cyclin B1 expression, 4.8 (A) in cells
exposed to ECCT for 24 hours, the number of cells expression is 85%, with moderate to
strong expression level, dark brown and light brown. Figure 4.8 (B) is the control, showing
30% of cells expression with weak to moderate expression level, light brown and pale brown.
Figure 4.9 represents the result of examination of the expression of p53, 4.9 (A) in cells
exposed to ECCT for 24 hours, the number of cells expression is 85%, with moderate to
strong expression levels, dark brown and light brown. Figure 4.9 (B) is the control, showing
25% of the cells expression, with weak to moderate expressions, light brown and pale
brown.
Figure 4.10 displays the result of examination of the expression of Ki-67, 4:10 (A) in cells
exposed to ECCT for 24 hours, the number of cells expression is 85%, with moderate to
strong expression levels, dark brown and light brown. Figure 4.10 (B) is the control, showing
60% of the cells expression, with weak to moderate expressions, light brown and pale
brown.
Figure 4.11 below exhibits the results summary of semiquantitative immunocytochemical
examination in Hela cells. Tubulin A expression in HeLa cells exposed to ECCT for 24 hours
is (10.25 ± 1.67), significantly different from the control (2.63 ± 0.74), Cyclin B is (11.50 ±
0.93) significantly different from the control (1.88 ± 0.99), p53 is (11.25 ± 1.04) significantly
different from the control (2.88 ±1.25), whereas the expression of Ki-67 exposed to ECCT for
24 hour is (11.75 ± 0.71) significantly different from the control (5.06 ± 0.78).
Table 4.4 displays the result of analysis of the independent t-test of immunocytochemical
examination in four parameters using the Mann-Whitney test, showing that expressions of
Tubulin A, Cyclin B, p53 and Ki-67 are statistically significantly different between groups of
HeLa cell cultures treated with ECCT for 24 hours and the the control with a value of p
<0.05.
4.7 Immunocytochemical examination in mesenchymal cell culture
Results of immunocytochemical examination in mesenchymal cell culture are showed in the
figure below. Figure 4.12 is the result of examination of Tubulin A expression, 4:12 (A) in
cells exposed to ECCT for 24 hours, the number of cells expression is 30%, weak to
moderate expressions, pale brown and light brown. Figure 4.12 (B) is the control, showing
25% of the cells expression, weak expression level, pale brown.
Figure 4.13 exhibits the result of examination of the expression of Cyclin B1, 4:13 (A) in cells
exposed to ECCT for 24 hours, the number of cells expression is 60%, with weak expression
level, pale brown. Figure 4.13 (B) is the control, showing 15% of cell expression, weak
expression level, pale brown.
Figure 4.14 ilustrates the result of examination of the expression of p53, 4:14 (A) in cells
exposed to ECCT for 24 hours, the number of cells expression is 60%, with weak to medium
expression levels, light brown and pale brown. Figure 4.14 (B) is the control, showing 15% of
the cells expression, weak expression level and pale brown.
Figure 4.15 describes the result of examination of the expression of Ki-67, 4:15 (A) in the
cells exposed to ECCT for 24 hours, the number of cells expression is 40%, with weak to
moderate expressions, light brown and pale brown. Figure 4.15 (B) is control, showing 10%
cell expression, with weak expression and pale brown.
Figure 4.16 below shows the results summary of semiquantitative immunocytochemical
examination on mesenchymal cell cultures. Tubulin A expression in the mesenchymal cells
exposed to ECCT 24 hours is (2.38 ± 0.74), not significantly different from the control (2.25
±0.71), Cyclin B is (2.50 ± 0.53) significantly different from the control (0.63 ± 0.52), p53 is
(4.94 ± 1.74) significantly different from the control (0.38 ± 0.52), whereas the expression of
Ki-67 exposed to ECCT 24 hours is (2.38 ± 0.52) significantly different from the control (1.19
± 0.26).
Table 4.5 represents the result of analysis of the independent t-test of immunocytochemical
examination in four parameters using the Mann-Whitney test, suggesting that expression of
Tubulin A between the group exposed to ECCT 24 hours and controls did not differ
significantly with value of p 0.090. While the expressions of Cyclin B, p53, and Ki 67 are
statistically significantly different between groups of mesenchymal cell cultures treated with
ECCT for 24 hours and the control with a value of p <0.05.
5.8 Protein Expression and Path Analysis
To better understand the mechanisms of cell death due to exposure to low-energy current
electric field with medium frequency emitted by ECCT equipment, it is necessary to analyze
the changes in protein expression in each culture, differences between groups of cell
cultures, and perform path analysis to measure the strength of the causal relationship
between the protein expression which is an intervening variable.
Differences in Tubulin A expression between ECCT group and the control in each cell
culture, and the difference between groups of cell cultures are presented in Table 4.6.
Table 4.6 shows that the expression of Tubulin A is statistically significantly different (p
<0.05) in the groups of the cultured HeLa cell and OCC cells, and different but not
statistically significant (p = 0.951) in mesenchymal cell group.
In the groups treated with ECCT, there is statistically significant difference in Tubulin A
expression between cell culture groups, whereas in the control group there is no significant
difference between cell culture groups.
Differences in Cyclin B1 expression between ECCT group and control group in each cell
culture, and the difference between cell culture groups are showed in Table 4.7.
Table 4.7 indicates differences in the strength of Cyclin B1 expression which are statistically
significant (p <0.05) between the ECCT group and the control. There are significant
differences in the strength of a expression of Cyclin B1 between the three cell culture
groups, either in ECCT group or control.
Differences in Ki-67 expression between the ECCT group and the control in each cell
culture, and differences between cell culture groups are illustrated in Table 4.8.
Table 4.8 indicates differences in the strength of Ki-67 expression which are statistically
significant (p <0.05) between the ECCT group and the control. There are significant
differences in the strength of a expression of Ki-67 between the three cell culture groups,
either in ECCT group or control.
Differences in p53 expression between the ECCT group and the control in each cell culture,
and differences between cell culture groups are illustrated in Table 4.9.
Table 4.9 indicates differences in the strength of p53 expression which are statistically
significant (p <0.05) between the ECCT group and the control. There are significant
differences in the strength of a expression of p53 between the three cell culture groups,
either in ECCT group or control.
To determine the relationship between variables, it is necessary to perform path analysis of
all the variables in the study, according to conceptual framework. Relationships between
variables are given in Table 4.10.
Data in table 4.10 above shows the regression analysis required to explain the direction of
the causal relationship of the variables. Exposure to the ECCT can cause a significant
increase (p = 0.000) in tubulin A with the strength of the path coefficient (β) of 0.640. Cell
type also significantly affects the increase in tubulin A with β = 0.520. A significant increase
in tubulin A may cause the increased cyclin B with β = 0.900. Significant increase in Cyclin B
lead to an increase in Ki-67 with β = 0.751. Significant increase in Ki-67 can bring about
reduction in cell death with the path coefficient strength (β) of -0.860. Significant increase of
Ki-67 also causes increase of p53 by β = 0.751. Significant increase in p53 can cause
increased cell death by β = 0.757. Overall relationships between all variables observed in
this study are given Figure 4.18.
Tubulin A expression is influenced by the ECCT treatment and types of cells. Increased
expression of Tubulin A will affect the increase in Cyclin B1 expression, in which the
increased expression of Cyclin B1 will influence increased expression of Ki-67. Increased
expression of Ki-67 will lead to increased expression of p53, and the increased expression of
p53 may cause cell death. This figure also shows that the increase in the expression of Ki-67
results in a decrease in cell death.
5. DISCUSSION
Around the world, more than 10 million patients are diagnosed with cancer each year, not including skin cancer. More than 50%of the cancer patients are living in developing countries, where the cases of cancer continues to increase dramatically from time to time. It is estimated that nearly 15 million people will be diagnosed with cancer in 2015, where nearly all patients from developing countries contribute 85% of the world's population (Anonymous, 2000). The process of cancer formation (carcinogenesis) is an somatic event and caused by an accumulation of genetic and epigenetic changes that result in a change in the regulation of normal control of molecular cell proliferation. The genetic changes may take the form of activation of proto-oncogenes or inactivation of tumor suppressor genes which can trigger tumor formation. A variety of experiments (even millions) have been conducted to investigate the characteristics of cancer using animal models such as mice, mouse, dogs, sheep, cell culture, even single-celled organisms (Kondo, 1993). Biochemical sciences have been used as the main paradigm to explain the function of cells and disease since a century ago. With the biochemical sciences, the pharmaceutical industry has grown rapidly, creating many effective drugs, and becomes a major business in the field of medicine. This success makes the pharmaceutical industry have a major influence in the medical world. Paradigm of biochemistry and a major influence of pharmaceutical industry cause almost all of the researches are directed towards understanding of the chemistry of the body and the effects of drugs to change the chemical reaction (Haltiwenger, 2010). This is why science and applications in the field of biophysics fall behind. Many basic biological questions are not answered by the science of biochemistry, even never questioned, since the molecular biochemistry is demanded to answer many questions that are not in accordance with science. Many physicists believe that living organisms have electrical mechanisms and use electric current to regulate and control the chemical transduction and energy in the process of life (Szent Gyorgyi, 1968). Thus the development of therapeutic equipment or therapeutic methods in the field of biophysics become a strange, lagging, even is rejected a priori by most of the medical community. The emergence of ECCT in Indonesia is one example of the phenomena. The purpose of this research was to determine whether low-voltage AC electric field with a medium frequency generated by ECCT equipment can kill cells, and if it can kill cells, how its death mechanism. To know the mechanism of cell death, it is necessary to examine changes in expressions of Tubulin A, Cyclin B1, p53 and Ki-67. To know whether the ECCT can also kill non-cancerous cells, we examined its effects on cultures of human bone marrow mesenchymal cells and then compare it with both cancer cell cultures. 5.1 Cell Death and Cell Culture Viability This study gives an information about the nature of the growth of three different cell culture groups. This is due to a different genetic trait thereby growth speed and survival may be different. This is observed in the control group, after incubation for 24 hours, if each of the wells is filled with 500,000 cells, then the number of Hela cells group becomes an average of 966,250 cells, number of OCC cells group becomes average of 270,625 cells, whereas number of mesenchymal cell group becomes average of 151,875 cells. Only number of HeLa cell group after 24 hours becomes much larger than initial cell count, that is almost 2-fold. This happens because HeLa cell’s doubling time is only about 23 hours (Jacobson and Ryan, 1982). Number of OCC cells after 24 hours will decrease to just half, while mesenchymal cells decrease to third. It seems that the doubling time of the OCC cells is faster than mesenchymal cells. Naturally cells may be dying due to various causes. Deaths of cell groups not treated with ECCT (natural death) after 24 hours are 2.44% in Hela cells, 5.72% in OCC cells, and 12.84% in the mesenchyme cells. Here, it appears that HeLa cells which are cell line and
having immortal characteristic have the fewest percentage of cell death, while mesenchymal cells which are non-cancerous cells, have the largest percentage of death. Mesenchymal cells in certain culture does have specific age, and will die or suffer from senesence after some time passages. It is associated with the Hayflick-type limited span, where mesenchymal cell telomeres are generally not long, which makes age of mesenchymal cells in culture is not long (Sanford, 1965). In terms of viability, the cell group exposed to ECCT for 24 hours showed a decrease in the number of living cells significantly compared with the control group in both cancer cell cultures (OCC: 255,000/177,500, Hela 942 500/778 125), while group of non-cancer cells (mesenchymal cells) did not show statistically significant differences (132 500/115,000). The number of living cells were not statistically different between the ECCT group and control group in mesenchymal cell culture because mesenchymal cell’s doubling time is the longest. ECCT equipment that works while the cells are in mitotic phase, it will have a major affect on cell mitosis, with most short doubling time or the highest proliferation index. However, the ECCT equipment does not much affect cells which are in non-proliferation state (in G0 phase), or cells whose doubling time is long. The results of this research showed that cell group treated with ECCT has significant higher percentage of cell death compared with the control group in both cancer cells and non-cancerous cells. The highest percentage of cell death occurs in mesenchymal cell group exposed to ECCT at (33.75 ± 5.80)% and the control at (12.84 ± 4.87)%, followed by group of OCC cells at (18.25 ± 3.36)% and the control at (5.72 ± 2.57)%, whereas the percentage of HeLa cell death is the lowest (6.66 ± 1.77)% and the control at (2.44 ± 1.05)%. Mesenchymal cells have the highest percentage of cell death, in both treatment group and the control group, suggesting the existence of other factors besides ECCT, which causes cell death, among others, Hayflick phenomenon that limits the natural lifespan of the mesenchymal cells in culture. 5.2 Expression of Tubulin A Alpha and beta tubulins are a pair of proteins bind to form a heterodimer protein. To form a microtubule polymer, the dimers (pairs of identical molecules) of this protein line up a number of 13 pairs to form a tube. Tubulin A and tubulin protein B are polar and electrically charged proteins, until microtubules they form are also electrically charged. Butters et al published a study, which for the first time investigated the response of microtubule formation from dimers of tubulin A and tubulin B against the weak electric field at medium frequency, in vitro outside the cell. Results of the study found that low-energy electric field with a frequency of 22 KHz, works nonthermally, can induce changes in tubulin-subunit interactions, and works like Taxanes chemotherapy drug class. The low-energy AC electric field is thought to work by affecting the liquid blanket around tubulin poles, changing the density of the fluid around the poles, thereby the polymerization – depolymerization activity is disturbed. In this study, we measured whether the low-energy AC electric field with a frequency of 100 KHz emitted by ECCT equipment can influence the activity of intracellular microtubule polymerization, by measuring changes in the expression of tubulin A in cell cultures exposed to ECCT for 24 hours, and compared it with the control. Tubulin A and tubulin B are a pair of microtubule-forming protein. Inhibition in microtubule polymerization can be proved by an increase in one of the tubulins. In this research, we measured an increase in the expression of tubulin A. In the OCC cell culture group, the mean expression of Tubulin A in ECCT group is 6.75 (5.88 -7.62), higher than the control group of 2.00 (1.23 - 2.77). There is a statistically significant difference.
In the HeLa cell culture group, the mean expression of Tubulin A in the ECCT group is 10.25 (8.86 to 11.65), higher than the control group of 2.63 (2.00 - 3.25). There is a statistically significant difference. In the mesenchymal cell group, the mean expression of Tubulin A in the ECCT group is 2.38 (1.75 - 3.00), higher than the control group of 2.25 (1.66 - 2.84). But there is no a statistically significant difference. In living cells, the microtubule polymerization and depolymerization activities occur most actively and powerfully when cells are in mitotic phase, especially in metaphase and anaphase. Therefore, it is understandable that HeLa cells which are entering the mitotic cycle every 23 hours have the highest increase in expression of Tubulin A (10.25), followed by OCC cells (6.75). While for non-cancerous cells, the mesenchymal cells, the difference in A tubulin expression is not significant. This is related to its very long mitotic cycles, exceeding 2x24 hours, until ECCT treatment for 24 hours only does not produce significant effect on the increased expression of tubulin A. The results are consistent with those obtained by Kirson et al. who examined the influence of a low-voltage electric field with frequency of 100 KHz in melanoma cell culture, which proves that the electric field like this destroys normal process of polymerization-depolymerization of microtubules during the process of mitosis (Kirson ED, Gurvich Z, SchneidermanS, 2004). 5.3 The expression of Cyclin B1 Cyclin B1 is instrumental in the process of mitosis, together with P34 (Cdk1) to form a complex protein that acts as a ON switch to start mitosis, which is characterized by chromosome condensation and degradation of lamin or nuclear membrane. Similarly, when mitosis ends, APC-C protein is required to degrade Cyclin B1. When Cyclin B1 level drops because being degraded by APC-C, then lamin will be re-formed, and chromosome will be in decomposed state again, and phases of mitosis will finish. When the microtubule polymerization is disrupted, causing its binding with Kinetochore not happen perfectly, spindle checkpoint complex will be active and constantly keep APC/C is inactive. This state prevents damage in Cyclin B, and thus the process of mitosis will be stalled for a while (mitotic arrest). In this study we measured the expression of Cyclin B and found the following results: In the OCC cell culture group, the mean expression of Cyclin B in ECCT group is 3.13 (2.83 - 3.42), higher than the control group of 1.38 (0.94 - 1.81). There is a statistically significant difference. In the HeLa cell culture group, the mean expression of Cyclin B in ECCT group is 11.50 (10.73 - 12.27) higher than the control group of 1.88 (1.05 - 2.70). There is a statistically significant difference. While in the mesenchymal cell group, the mean expression of Cyclin B in the ECCT group is 2.50 (2.05 - 2.95) higher than control group of 0.63 (0.19 - 1.06). There is a statistically significant difference. The observation of changes in the expression of Cyclin B indicates the greatest increase in expression of Hela cells at 11.50, followed by OCC cells at 3.13 and the least increase in expression of the mesenchymal cells at 2.5. There are significant differences in increase of Cylin B expression between the three cell culture groups. It is also linked to the cell cycle of the three cell types studied. Cyclin B increases due to the braking mechanism of cells that are undergoing mitosis, which aims to prevent DNA/chromosome division from occurring precisely and properly. The result of this process is the increased Cyclin B at the time cells are in mitotic phase. However, this
condition does not last long, as Cyclin B is naturally also damaged, which eventually forces the cells to terminate its mitosis. Thus it makes sense that cells whose mitotic cycle or doubling time is shortest, i.e., Hela cells, will experience the most substantial increase in expression of Cyclin B. 5.4 The expression of p53 (wild) Protein p53 (wild) is known as proapoptotic protein. In case of the cells undergoing mitotic arrest, many cell proteins cannot be synthesized as mRNA fails to undergo a process of maturation usually occurring in the nuclear lamina. Under normal circumstances, Mdm 2 in charge of the degradation of the p-53 is in balanced state until the p-53 is in low concentration in the cell. p53 production-coding mRNA is more long-lived than Mdm2-sense mRNA until when the transcription process in the cells is hampered, p53 can still continue to be produced, while production of Mdm-2 will stop. This causes an increase in the amount of p53 in the cell in case of mitotic arrest. In this study we measured the expression of p53 (wild) and found following results: In the OCC cell culture group, the mean expression of p53 in ECCT group is 10.50 (8.73 - 12.27), higher than the control group of 1.25 (0.66 - 1.84). There is a statistically significant difference. In the HeLa cell culture group, the mean expression of p53 in ECCT group is 11.25 (10.39 - 12.12) higher than the control group of 2.88 (1.83 - 3.92). There is a statistically significant difference. In the mesenchymal cell group, the mean expression of p53 in ECCT group is 4.94 (3.48 - 6.39) higher than control group of 0.38 (0.6 - 0.8). There is a statistically significant difference. This study found that the expression of p53 (wild) increased significantly in all three groups of cells exposed to ECCT for 24 hours compared with the control. The study also found that p53 expression was significantly different among the three groups of cell cultures. Level of p53 (wild) is high when the cells undergo mitotic arrest. This does not always result in apoptosis because the apoptotic process also requires the synthesis of other proteins, where in these circumstances cannot be done (Chen, et al., 2003). Degradation of Cyclin B by APC/C is required to escape from mitosis. But in a state of
mitotic arrest, APC/C is not synthesized until when Cyclin B levels decrease slowly due to
natural degradation process, thus the cells not undergoing apoptosis at the time of mitotic
arrest will come out of the process of mitosis known as mitotic slippage in a tetraploidy or
aneuploidy state ((Blagosklonny, 2007). In the initial phase of mitotic slippage (initial G1),
cells that previously experienced the mitotic arrest has high levels of p53. Once coming out
of the phase of mitosis, the nuclear membrane has been formed and protein can be
synthesized again, then cells with high levels of p53 will immediately trigger apoptotic
process through BAX activation, and subsequent caspase cascade. In other circumstances,
the p53 can activate p21, causing cells to undergo arrest in G1, then experiencing
senessence and death (Klein, et al., 2005).
5.5 The expression of Ki-67
While undergoing mitosis, the chromosomes observed with a microscope using whole
mount method, would seem covered by a protein layer and RNA, consisting of dense fibrillar
protein, and granular protein. This perichromosomal layer is pre-rRNA, U3 snoRNAs, and
more than 20 ribosomal proteins, including nucleolin and Nopp140, NPM/B23, Bop1, Nop52,
PM-Scl 100, and Ki-67 (Gautier, et al., 1992). This perichromosomal layer is 1.4% of the
chromosome proteome, and their functionalities have not been completely investigated and
are known (Van Hooser, et al., 2005). Ki-67 is a very large protein (about 360 kDa), it is cell
protein that always exists and is involved in the proliferation of eukaryotic cells, always
seems expressed in the active phase of the cell cycle (G1, S, G2, and M), but its expression
will not be visible when cells are inactive (G0 cell cycle). Ki-67 has been widely used and
accepted as an indicator of cell proliferation in a variety of human tissues, including various
types of cancer. Ki-67 has also been routinely used to assess tumor cell proliferation and
measure the aggressiveness of therapy and response to therapy (Yerushalmi, et al., 2010).
In this study we measured expression of Ki-67 to assess the level of proliferation of the cell
culture, and found the following results:
In the OCC cell culture group, the mean expression of Ki-67 in ECCT group is 4.88 (4.18 -
5.57) higher than the control group of 1.25 (0.86 - 1.64). There is a statistically significant
difference.
In the HeLa cell culture group, the mean expression of Ki-67 in ECCT group is 11.75 (11.16 -
12.34) higher than the control group of 5.06 (4.41 - 5.71). There is a statistically significant
difference.
In the mesenchymal cell group, the mean expression of Ki-67 in ECCT group is 2.38 (1.94 -
2.81) higher than the control group of 1.19 (0.97 - 1.40). There is a statistically significant
difference.
This research found the statistically significant increase in expression of Ki-67 in the three cell culture groups, with the highest increase in the group of Hela cells. Thus far, the increased expression of Ki-67 has always been associated with high levels of cell proliferation, poor prognosis, and good response to chemotherapy or radiotherapy. Ki-67 is a prognostic factor which is more superior to the mitotic count in the case of pancreatic tumors and mid gastrointestinal tract which has undergone metastasis (Khan, et al., 2013). Ki-67 is also more superior as prognostic factors than mitotic count and protein marker of PHH3 proliferation, MCM4 and mitosin, in determining prognosis of malignant melanoma (Ladstein, et al., 2010). Despite the death of cells that are statistically significant in the three cell culture groups exposed to electric fields of ECCT for 24 hours, but cell death is not followed by a decrease in cell proliferation indicators, i.e. Ki-67. Ki-67 increases significantly in OCC cell group and mesenchymal cells, even it is overexpressed in HeLa cell group. Some possibilities could be the cause of the increased Ki-67 expression. First, the electric field of the ECCT equipment not only can trigger cell death, but also can stimulate cell proliferation. This can be proven by doing a research with longer exposure duration than 24 hours, up to 72 hours or more to prove whether the ECCT can stimulate cell proliferation. When increased expression of Ki-67 is indeed associated with increased proliferation, then addition of ECCT exposure duration will increase the number of its cell population. Secondly, the increased expression of Ki-67 occurred because many cells experienced mitotic arrest. Manoir et al. have examined the expression level of Ki-67 in each phase of the cell cycle, and found that the expression of Ki-67 decreased after mitosis, but the expression was stably low when cells were in G1 phase, and disappeared when the cells were in the G0 phase. In the G1 phase, the Ki67 concentration was in the nucleolus, i.e. in the intermediate
nucleus. Ki 67 reaches its maximum level when the cells are at profase-metaphase and its concentration is around the chromosomes (Manoir, et al., 1991). The high level of Ki67 in cancer cells treated with ECCT strongly confirms the evidence of mitotic arrest in the cell, where theoretically the cells exposed to ECCT will stop at metaphase, phase wherein the expression of Ki-67 reaches its peak levels. Cells undergoing mitotic arrest may subsequently die through mitotic catasthrophe. Mitotic catasthrophe is defined as a mode of cell death which occurred after a failure of cell in completing mitosis, which is accompanied by some morphological changes such as micronucleation and multinucleation (Rainson, et al., 2001). Rosario et al. have observed the mitotic catasthrophe in epithelial cancer, especially in pleomorphic giant cell carcinoma of the thyroid, lung, and pancreas, which are physically characterized by extensive necrosis in cancer tissue. They argue that the term mitotic catastrophe is a syndrome that is typically characterized by the presence of cells with multinucleation, micronucleation, abnormal mitosis, centrosome aberration, tissue necrosis, and molecularly characterized by overexpression of p53 and Ki-67 (Caruso, et al., 2011). In this research, cell death occurs due to the disrupted microtubule polymerization causing cell death through mitotic catastrophe, which is also characterized by overexpression of p53 and Ki-67. 5.6 Path Analysis Path analysis is the applied form of multiple regression analysis, this analysis uses the path diagram to help making conceptualization of problems or test the complex hypothesis and also to know the direct and indirect influences of the independent variable on the dependent variable (Kerlinger and Pedhazur, 1973). This is a technique for analyzing the causal relationship occurring in multiple regression if the independent variables affect dependent variable not only directly, but also indirectly (Rutherford and Choe, 1993). This relationship test is based on a theory stating that the variables have a relationship. The strength of the theory used in describing the relationship will determine the arrangement of path diagram and affect the results of the analysis and its implementation in science (Widiyanto, 2013). The conceptual framework of this research has described the causal relationship of the variables. Low-energy AC electric field with a frequency of 100 KHz of the ECCT becomes the cause of disruption in microtubule polymerization of tubulin A and B. Disruption of tubulin polymerization can be seen by the increased expressions of tubulin A or B, because it is not polymerized to microtubule. Path analysis proves the strong influence of the ECCT exposure to cause an increase in tubulin A level at significance (p) of 0.000 and the path coefficient (β) of 0.640. This cell type also becomes the cause of the significant increase in expression of tubulin A at (p) of 0.000 and the path coefficient (β) of 0.520. This cell type produces an affect because each cell has a different mitotic cycle, and the electric field of ECCT inhibits microtubules polymerization when cell is in a state of mitosis. When mitotic cell cycle is faster, then disruption of polymerization microtubules will be stronger, and the the increase in the expression of tubulin A is also higher. Increased expression of tubulin A, which reflects the impaired microtubule polymerization will cause disruption in coupling of the microtubules to kinetochore in chromosomes. This disrupted coupling will make the cell do braking mechanism in process of mitosis through the formation of mitotic checkpoint complex which will make CDC-20 become inactive, do not degrade cyclin B, until the process of mitosis is suspended. Result of this process is an increased cyclin B. Path analysis proves the strength of the causal relationship at significance level of (p) 0,000 and the path coefficient (β) of 0.874. Increased cyclin B in cells undergoing mitosis is a condition in which the mitotic process of cells is hampered from metaphase to anaphase. By the time metaphase, cells are in a state without nuclear membrane, and the DNA is condensed in the form of the duplicated
chromososome. Concentration of Ki-67 as a perichromosomal protein reaches the peak while it is at metaphase or stops at metaphase. Path analysis proves that the increased cyclin B may cause significant increase in Ki-67 level at (p) of 0,000 and path coefficient (β) of 0.900. Increased Ki-67 level, reflective of mitotic arrest, may bring about an increase in p53 as the MDM-2 responsible for the degradation of p53 fails to do synthesis and results in an increase in p53. Path analysis proves an increase in Ki-67 as the cause of significant increase in p53 level at (p) of 0.000 and a path coefficient (β) of 0.751. The augmented p53 protein will trigger cell death either through apoptosis or the senescence process of cells. The causal relationship of the increased p53 level and cell death is found significantly at (p) of 0.000 and path coefficient of 0.757. The hiperexpressed Ki-67 reflects number of cells undergoing mitosis. This study also discusses number of cells undergoing mitotic arrest. The state of mitotic arrest can stimulate cell death through apoptosis or mitotic slippage by all the consequences. Path analysis in this study proves significance level at (p) of 0.000 and path coefficient (β) of -0.860, meaning that an increase in Ki-67 generates negative effect, namely decreased cell death. This can be understood as the Ki-67 describes mitosis or cell proliferation, while cells undergoing mitotic arrest not always die. Cells undergoing mitotic slippage after mitotic arrest will become the aneuploid or polyploid cells, and these cells can undergo repair and re-enter the cell cycle. 5.7 New findings Some new findings in this research are:
1. Exposure to a low-energy electric field with a frequency of 100 KHz emitted by the ECCT equipment can increase the percentage of cell death, either cancer or non-cancerous cells.
2. The mechanism of cell death in the low-energy electric field exposure with a frequency of 100 MHz of the ECCT, is through microtubule polymerization inhibition in a cell while undergoing mitosis.
3. Cell death in the electric field exposure of the ECCT is through the phenomenon of mitotic catastrophe.
5.8 Follow-Up Research It has been proven that exposure to low-energy electric field with a frequency of 100 MHz emitted by the ECCT equipment can raise cell death through mitotic catastrophe phenomenon, the further researches need to be conducted by the extended duration of exposure, other types of cancer cell cultures, and deepen in vivo researches in the test animals induced by cancer. 5.9 Limitations This research has several limitations as follows:
1. This research has not involved all the factors that may play a role in increasing protein expression to be examined, for example, MAD2, BUB3, IAP, MCL-1, p21 and Bax.
2. Duration of the observation and the exposure is only one, namely 24 hours, while the process of mitotic catastrophe underlying the cell death due to exposure to the ECCT can take effect until more than 24 hours.
3. This research is in vitro study, so it cannot reflect the real clinical state of the carcinoma patients.
6. CLOSING REMARK 6.1 CONCLUSION 6.1 Conclusion
1. There is an increase in the percentage of cell death in cultured HeLa cells, oral cavity carcinoma cell and bone marrow mesenchymal cells exposed to the electric field of ECCT equipment.
2. There is a disturbance in microtubule dynamics in the cultured Hela cells, oral cavity carcinoma cells and bone marrow mesenchymal cells exposed to ECCT, characterized by increased expression of Tubulin A.
3. There is an increase in expression of cyclin B in the cultured HeLa cell, oral cavity carcinoma cells and bone marrow mesenchymal cells Exposed to ECCT.
4. There is an increase in expression of p53 in the cultured HeLa cells, oral cavity carcinoma cells and bone marrow mesenchymal cells Exposed to ECCT.
5. There is an increase in the expression of Ki-67 in cultured HeLa cells, oral cavity carcinoma cells and bone marrow mesenchymal cells exposed to ECCT.
6. Exposure to low-energy electric field with a frequency of 100 KHz from ECCT equipment can cause cell death through a mechanism of mitotic catastrrophe.
6.2 Suggestions
1. Further researches should be done with a variety of other cell cultures. 2. Further researches should be done with the test animals induced by the cancer cells. 3. Further research with longer duration of ECCT exposure should be done. 4. Further researches on morphological changes in cells after exposure to ECCT to
seek a formation of multinucleation, micronucleation, abnormal mitosis and centrosome aberrations in cells to supports the evidence of mitotic catasthrophe in cells exposed the ECCT-emited electric fields.
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