dissertation summary cell death mechanism as …

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





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





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





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


4. There is an increase in expression of p53 protein in the cultured HeLa cells, oral


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


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


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



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



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


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


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 -


difference.

In the mesenchymal cell group, the mean expression of Ki-67 in ECCT group is 2.38 (1.94 -


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.

REFERENCES [**]

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