Holistic Healing for Cancer by Prof. Chris Teo
The Cancer Care Therapy
Realising that cancer is a complex problem we advocate a multiapproach
to healing. We tell cancer patients the following:
Step 1: Give total commitment to your healing. This is your
sickness and only you can heal yourself – others can only help.
If you come to us but do not believe that we can help you, then
go and find someone else whom you have faith in or can trust
so that he can help you better. Finding healing for cancer needs
your whole-hearted commitment. You need to change your ways
– your lifestyle and mental attitude. It goes beyond swallowing
some pills or getting an injection. Our experiences show that
those who actually participate in the healing process benefit
and win but those with a kiasu (only wanting to win) attitude
fail.
Step 2: Seek proper medical help. We do not substitute
medical doctors. We advise you to consult your doctors and
get the best possible treatments, but at the same time be aware
of the limitations of what medical science can do. There may
come a stage when there is only that much a doctor could do
and no more. After this point, to do more may perhaps just
mean mere adventurism. So, learn to know when enough is
enough.
Step 3: Take herbs. Herbs are not magic potions. You need
them to restore the imbalances that exist in the sick body.
Herbs do not kill cancer cells like the cytotoxic drugs used in
chemotherapy. Many educated (presumably scientific) people are
sceptical about taking herbs. Our experiences show that herbs
do no harm but on the contrary, help many cancer patients.
There is a saying: the believer asks no question, while no answer can
satisfy the unbeliever! If you do not believe in herbs, our advice is
not to take them. Find other avenues for your healing.
Step 4: Eat rightly. We believe that diet by itself cannot heal
cancer, just as drugs or herbs alone cannot cure it. However,
diet is important and can determine the progress and direction
of your healing. Of course, our stand is being refuted and even
detested by some patients. They counter: My doctors say I can eat
anything I like – food has nothing to do with my cancer. To the
sceptics, we suggest that they read more widely. Every year,
more and more evidence accumulates and points to the fact
that what we eat determines the progress and direction of our
healing or have a great influence on whether we get sick or not.
Today, we are aware that the causes of 70% of cancers are
related to our food and lifestyle. Studies have shown that as the
Western way of life gains popularity in the developing countries,
more and more people are getting cancers.
From the literature, we know that cancer patients benefit from
a diet that consists of the following:
• Fresh vegetables, either raw or cooked.
• Lots of fresh juices and fruits.
• Whole grains, cereals, seeds and nuts in an as unprocessed
form as possible.
• Avoid taking meat, dairy products, table salt, white sugar
and cooking oils in the diet.
• Avoid types of food that are preserved, canned or processed.
Step 5: Lead a happy, stress-free life. We advocate that you
do proper exercise daily but do not over-strain yourself. Qi
kung, tai qi and deep abdominal breathing exercises are good for
you. Go ahead and practise them.
Cultivate a positive attitude. Avoid anxiety, anger and other
forms of negative emotions. Learn to love and to forgive and
forget. Let go of any emotional baggage that you have been
carrying all along in life!
We also find that those who believe in God or some form of
Superior Being have a better sense of coping with their sickness.
We advise cancer patients to find solace in the Loving God, in
ways that are compatible with their own individual beliefs. We
also find that those who receive full-hearted family support
manage better. The love, care and understanding of the spouse,
children and relatives are important for healing. Those who do
not have such emotional support from their family members
generally get worse.
Why Some People Fail to Find Healing
Many patients come to us with high expectations. They expect
us to dish out cures for their cancers, most often after all medical
treatments have failed them. Surely this is extremely unrealistic.
Some have even reach the point of being absurd – expecting
miraculous results within one or two weeks!
A lady and her two daughters traveled all the way from Kuala
Lumpur by bus to see me. The reason? The lady had colon
cancer that had spread to the liver. She was on oral 5-FU. In
such a condition, her chance of survival for long was almost nil.
As the three visitors sat down by my table, the daughters
requested me not to tell their mother that she had cancer.
That was a fair request and I had no problem with that except
that in some cases, especially like this one, it was a non-starter!
When I turned to the lady, she blurted out that she did not
want to take the prescribed herbs. Then, why come and see me?
I asked. I told the lady that I was not a magician. Healing needed
genuine efforts on her part and if she was not prepared to help
herself, nobody else could. In fact, in our earlier days, we used
to feel very upset, discouraged and saddened by such an attitude.
But with time we have learnt to take things easy and cope with
them. There is a saying: There is no incurable disease, only incurable
people. Since our mission is to help those who seek us, we
continue to help them the best we know how.
From experience, NOT all who come and see us find their
healing. If you are interested in statistics, let me share this with
you: from my calculation I conclude that only 30% of those who
come to seek our help benefit or find their healing in one way
or another. The remaining 70% of them are mostly on a fishing
trip, hoping to find a magic potion and luck. Let me also share
with you some of my observations as to why people can or
cannot find their healing.
• Fear. Many who come are generally lost in fear, and they
panic. They go nuts after being told of their cancer diagnosis.
Unfortunately, things are made worse when they are told
that the treatments: surgery, chemotherapy or radiotherapy,
must be done fast – sometimes, by tomorrow or the day
after, or else the cancer will spread like wild fire. Indeed,
this is sad misinformation! Cancer patients should know that
any decision made in a state of fear and panic is never a good
decision. I have often told patients to relax and take it easy.
Calm down and have some space and peace. Take a bit of
time to think things over, evaluate the strategy and seek a
second or third opinion before you commit your life into
someone else’s hands. Committing your life and believing in
only one doctor is perhaps not the best option.
• Ignorance. There is a Chinese saying: The road to health is
the road of knowledge. Ignoring knowledge is to end up with
sickness. Many people who come to us are basically ignorant
of their own health and are caught off-guard when faced
with cancer! Almost overnight they have to cope with a lifeor-
death problem for which they are not prepared for or
ever expected. There is another Chinese saying: You don’t
sharpen your sword while in the battlefield! Or, start to dig a well
when you are thirsty. But how many people actually practise
this? In fact, many well-to-do, healthy people would consider
that reading about cancer when they are not sick would
bring them soi or bad luck. It is a taboo! They do not wish to
hear anything about such a dreaded disease.
Many cancer patients have the impression that after surgery,
chemotherapy or radiotherapy they will be cured of their cancer.
One cancer patient, Devi told me: I have not read much about
cancer and after I have had my radiotherapy I thought I was cured! I
was sent for a scan and the doctor said there was no evidence of
cancer. That was it, I thought that was the end. I never ever conceived
that the cancer would come back again. I thought I was cured. Exactly
a year later, I had a relapse of the lymphoma. And Devi is not a
village housewife or any person on the street. She is a lawyer!
• Quick fix and instant noodles mentality. As said earlier, a
majority of patients who come to see us expect a quick fix.
This is unfortunate and we tell them in no uncertain terms
that we have no such thing. Healing takes time. Some patients
write to us while others request that we give them herbs
that do not require boiling. They want the herbs in capsules,
which are easier to take. Indeed, we know that too. But
probably that can be done only at the expense of the herbs’
effectiveness – so what choice do we have? If our mission is
to sell as much of our herbs as possible, then we would
probably do things to please our customers. In our case, we
are not running a popularity contest and our bottom line is
never a sale volume, so we stick to the ways which we feel
would bring the most benefits to those whom we help.
This instant noodles mentality is also seen in those who e-mail me
after visiting our website. After reading their mail, I just wonder
how many really read the contents of the website? They write
to ask for information that is already given in the website anyway.
My only conclusion is that many people want things the easy
way – if it is easier to ask and get answers, why bother to read
and spend time looking them up?
• Kiasu – seeking healing on your own terms. Many patients
have the impression that managing cancer is as simple as
taking Panadol for a headache. Unfortunately, it is very much
more complex than that with cancer. We need to change
our mental attitude, our lifestyle and our diet besides
undergoing all the treatments and taking drugs. Even after
they have suffered relapses from their cancers, many patients
do not seem to realise or learn that there is more than the
medical treatments, if they want to regain their health. Yet
these people still insist on playing the game according to their
own rules or seeking to be healed on their own terms. Their
argument is that: My doctors never asked me to do what you
have asked me to do. You say I cannot eat this, I cannot eat that
– then what do I eat? We let all cancer patients know that
with cancer, it is not easy to win all the time nor can you
win on your own terms.
In the book, When Heaven is Silent by Ronald Dunn, the author
wrote: Ten years of … overloaded schedules, …and fast food
restaurants had taken their toll. On my way to saving the world, I had
acquired a stomach disorder that was throwing me into the hospital
with increasing frequency, threatening my ministry and my sanity…To
me a well-balanced meal was a Coke in one hand and …pizza in the
other. I was too busy to get well. I wanted to get well – but without
changing my lifestyle…I wanted a pill. The doctor assured me there
was no such pill, but I’m still looking….I am not alone in my quest for
the quick-fix. The doctors I talk to say that most of their patients
come to them looking for a way to continue living as they like, but
without suffering the consequences… We look for the easy path,
always ripe for a quick-fix scheme.
• Complacence. Those who are really sick very often come
with open ears to our advice. They seem to readily agree to
practise what we advise them to do. I tell them this: When
you are dying you will listen and follow what I advise you, but when
you are a bit better you curse me! As patients become more
energetic and regain their appetite they start to be fussy
and demand to go back to their old lifestyle. So, we always
remind patients not to be complacent even after they feel
well. The cancer may come back. If you think you are done
with cancer, remember, cancer may not be done with you
yet.
Our advice to cancer patients is always to relax and be positive.
We also hope that their healing experience has changed their
lifestyle and attitude towards life positively and permanently.
Often people ask me this question: When can I go back to my old
favourite food? Some people learn easily while some learn things
the hard way. Many others do not seem to want to learn at all.
Our answer to such a question is well known: Never go back to
your old unhealthy habits. But later, if for some reasons, you decide
that you are bored or tired with life – then indulge in whatever you
want, and pray that you go quickly and peacefully.
The second question often asked of me is: When can I stop
taking the herbs? Honestly, I do not know. You should take the
herbs for as long as you wish to remain well. Do whatever you wish. If
you value life more and do not mind that extra chore of cooking and
swallowing the herbs, then continue. There are many patients who
have been on the herbs for the last three to five years and they
are still taking them. While on this, my mind goes back to a
pretty lady – our friend Su, a matron in a hospital. She had
thyroid cancer and was one of our earliest patients. The doctor
told her that she would not be able to get her voice back and
that she was in a bad shape indeed. She took Capsule A and
drank fresh rodent tuber juice. In addition, she underwent
radioiodine treatment. With God’s grace and mercy, she became
well and could even sing with a beautiful voice. She became
whole again. One evening she came to CA Care with a nice cake
as a present to us. The cake was loaded with sugar and it really
disappointed me! A few months later, we learnt that Su was
not well again. We called Su’s husband and asked if we could be
of any help. It was too late as the cancer had spread to the
lungs. She was breathless and died soon afterwards. We went
to her house and talked to her husband, a staff of a health
department. I asked him this frank question: Why did Su stop
taking the herbs? His answer was: We thought that the problem
was over. We were over-confident because she had recovered so well.
So we stopped the herbs and went back to eating our catered food
again.
Indeed, patients do have a choice. So exercise that prerogative
wisely. Reflect on the above true story. For me, if at all there is
a lesson to learn or advice to give, let not Su die in vain – she
has a message for you.
Doubting Thomas. As I am editing this paragraph an e-mail
came from the son of a lady in Kuala Lumpur. This is what the
son wrote: …After her chemo, my mom didn’t look good. I supposed
that is one of the side effects of chemo. She has been vomiting and
her appetite was not encouraging either….my mom is rather reluctant
to explore the alternative therapy of using rodent tuber due to many
negative comments she has been hearing from well-meaning but
misinformed relatives and friends…… Well, I have always said this
to all cancer patients: If you are doubtful then go to someone else
for help. But to trust and believe well-meaning friends and relatives
who only hear stories but know next to nothing about what
they hear would be foolish.
But what baffles me most is why patients who are doing
extremely well suddenly disappear out of the picture totally.
We never see them again. They stop taking the herbs and of
course, they do not inform us. At times these supposedly cured
patients resurface and this time they visit us because they have
suffered a relapse of their cancers. One reason they give us for
abandoning the herbs is that the herbs can be toxic if consumed
for too long!
Melisa is a lady with cervical cancer that had spread to the liver
and lungs. The doctor gave her three to six months to live. She
has been taking the herbs for almost two years now. Then she
decided to take the fresh rodent tuber juice. After a few days,
she came and asked me: Doctor, one Datuk relative of mine told me
that I will die if I take the rodent tuber. I gave Melisa a big smile and
said: Melisa, you were supposed to have died two years ago when you
had that chemotherapy. Your doctor asked you to prepare your will
and gave you only three to six months to live. You took the herbs and
some of them contained the rodent tuber. If the rodent tuber can kill
you wouldn’t be here talking to me. But you are still alive and well! I
have numerous patients coming to tell me that after they have
taken the fresh rodent tuber juice, they feel better and well.
We have many patients taking Capsule A for four to five years
now and none of them has ever dropped dead because of the
herbs. Also, none of them has ever returned with new or strange
diseases or side effects. On the contrary, they have felt better
and become healthier. From literature, I have come to know
that bad mouthing often happens everywhere. At times, the rivalry
between the drug companies and complementary medicine has
become so fierce that the rich would finance scientists to conduct
fake or flawed experiments and churn out dubious data to provide
proof and justification that complementary medicine is harmful.
Such is the world that we live in.
The Exceptional Patients – Those Who Find Healing
Power of the Mind: The main basic difference between medical
science and holistic healing is the perception of what we are.
The holistic view regards man as a trinity, made up of the body,
mind and soul. Indeed, the body is only the physical case that
houses the mind and soul. Medicine, however, is based on the
philosophy proposed by Rene Descartes, a French mathematician
and philosopher in the 17th century. He regarded man as a
machine that obeys only the physical laws. The mind and soul
play no roles in the body.
Given this conflicting view about man, it is therefore up to you
to decide what you want to make out of yourself. If you believe
that you are just a pile of chemicals, bones and flesh that has no
mind and soul, then so be it. Worry not, for like an old car, you
can remove any worn-out parts of your body and replace them
with some newer parts from somewhere, if you can. Hopefully
by doing so, you can function better. Or can you? I am reminded
of an article in Newsweek (Special Issue 1999) about a man
who had undergone a heart transplant. Everything done, the
man recovered very well. Modern technology had saved him.
What surprised the cardiac surgeon was that the man with the
new heart did not behave as he did before. He had the tendency
to become suicidal. So, that much about the mind and soul not
having any influence over the physical body.
From the holistic point of view, the mind is the root cause of
problems. Carolyn Myss (in Creation of Health) says that cancer
is created through excessive fear, guilt feelings, inability to cope
with changes, self-hate and self-denial. Debbie Shapiro wrote
(in The Bodymind Workbook): Cancer appears to be the result of
many years of inner conflict, guilt, hurt, grief, resentment, confusion or
tension surrounding deeply personal issues. It is connected to feelings
of hopelessness, inadequacy and self-rejection.
According to Freud, the mind is an iceberg and only one-third
floats above water. However, Avni Sali, a professor of surgery at
the University of Melbourne, Australia, said: But I’m sure that in
cancer patients it’s probably 10%. Most of it is under water.
Paramahansa Yogonanda said that there is an innate connection
between the mind and the body. Whatever you hold in your mind will
be produced in the physical body…All diseases have their origins in
the mind. The pains that affect the physical body are secondary
diseases. The holistic healers view pain, sickness or the disease
of the body as a signal that there is an imbalance within, perhaps
due to conflicts of emotions and thoughts deep down within
us, and is threatening our survival. This disease represents a
wake-up call for us to do something to heal ourselves. However,
few ever understand this message.
Contrary to medical views, body-mind healers have pointed out
that:
• The healing power of the body is within us. There is a
physician within us and this power heals us absolutely.
• Many of us are ignorant or are unaware of our own body’s
healing potential.
• Many of us block ourselves off from this potential, preferring
to trust others whom we think can cure us rather than
trust our own Infinite Intelligence within.
• Many of us create unhealthy circumstances by thinking
negatively and harbouring self-denying thoughts that
eventually make us sick. We fail to recognise that these are
the root causes of our many illnesses.
• We are unaware that from the day we are born, we have
been bombarded with negative suggestions. Negativity begets
illness.
The seat of our real mind is the subconscious. Joseph Murphy
(in The Power of Your Subconscious Mind) wrote that we
view our world through thoughts in the subconscious mind.
Think good and good will follow, think evil, evil follows. Change your
thought and you change your destiny. Ralph Emerson said that
man is what he thinks all day long.
From the above, I can conclude that the very first step in healing
is for every cancer patient to recognise the influence of his own
mind on his illness and recovery process. Many a time, I ask
patients if they carry any emotional baggage or harbour any
unresolved emotional conflicts within them. Invariably the answer
would be: No, no, I have no emotional problems at all. They simply
do not want to admit or discuss them, perhaps for fear that
people know their secrets or they do not see the relevance or
relationship between unresolved personal emotions and their
cancers. After all, in schools we are taught that illness is caused
by some kind of bug, virus or germ and it has nothing to do with
the mind. Most patients would tell me that whatever problems
they have are all old issues and have long been forgotten. Such
comments and attitudes only go to show sheer ignorance or
arrogance, whichever way you wish to put it. However, as I began
to dig and probe deeper, some of them just break down and
cry. So, my first advice to all cancer patients is to be honest
with your own self, especially your subconscious mind. Think
again, do you carry any baggage? Remember that you are not
just a machine – without any mind or soul. You are sick because
something inside is eating you up.
Let me suggest a list of some active steps that you can take to
heal your mind, your soul and lastly, your body and its cancer.
• Think positively: as you think, so you become! According
to Susan Bannerman, a clinical psychologist working with
cancer patients (in As you think, so you become. Proc. 1st
World Congress on Cancer, Sydney, 1999, pg. 199): Illness
is a reflection of a person’s negative perception and self-defeating
ways of thinking. Hatred, envy, selfishness, jealousy, self-judgement,
self-doubt, self-criticism, lack of self-respect, feeling of unworthiness,
etc. All these are negative and destructive thoughts. Do not
harbour them for they do not help you at all. On the other
hand, cultivate positive attitudes of love, joy, happiness,
sharing, caring, self-esteem and self-confidence.
When you see a glass of carrot juice that is not filled up to the
brim: how would you see it – as half full or half empty? Our
perceptions of many situations can be either negative or positive.
Learn to see things positively. Alex is a colon cancer patient. He
told me: I do not consider myself taking herbs. I just drink tea and it
brings me a lot of good. On the other hand, I have patients telling
me: Yuck! the tea just tastes horrible! Think for yourself, do you
think you will benefit from drinking a yucky drink?
From talking to patients, I can easily pick out those who can
potentially heal themselves and those who cannot. I see the
potential healing right in their faces – by the way they talk and
behave. What messages do you think the following remarks
convey to you?
• Are your herbs hygienic?
• Have I to boil the herbs? I have no time to do so. They are so
bitter!
• My doctor said this…… My doctor said that…..
• I have this problem for such a long time already, my doctor said
there is no more cure….
You may want to tell me: No, I can’t change. Take me for what I
am. I remember one young lady who had breast cancer. She
hated her father very intensely. The sight and thought of her
father worked her up so much so that she felt pains in the lump
of her breast. I told her to go home and hug her father and love
him. She was adamant at sticking to her guns saying: No, I hate
him. I then told her: The problem is it is you who have to suffer and
perhaps die. It is not your father. Again here, patients have a choice,
to embark on the healing journey rightly or to be left in misery.
In our Centre, there is a quotation: when you hate people, the
only person who gets hurt is you, because most people you hate, don’t
know. And the rest of the world don’t care. To cultivate positive
thinking is simple and easy. It is just like letting go of your
baggage. Put it down! This requires no skill at all but you must
have a loving heart. Unfortunately many people insist on hanging
onto their baggage. Again, my advice is: Let go!
Free yourself of negative thoughts. Many negative thoughts
are ingrained into our subconscious mind and they become a
part of us. You and I have similar problems. And more often
than not, you do not even know that these negative thoughts
ever exist or are causing you all these problems. We are the
product of our environment and the experiences of our lives.
So to be able to free yourself from these negative thoughts you
must first and foremost be aware and recognise that these
negative thoughts are in you. If you keep insisting that they are
non-existent, there is no reason for change or wanting to be
free.
If you admit that negative thoughts are your problems, the next
step is to consciously correct them and replace them with
positive thoughts. Keep on doing this correction like cultivating
new habits. With time these good values stick in your mind. All
along tell yourself that you want to change and are capable of
doing so. In time you will see that you do change. Remember,
you need not be trapped in your past. Life is not living in the
past, it is living for the present and future.
Go for Healing!!!!
Most people who come to see us are generally desperate and
lost. They do not know what else to do. Some of them have
never had any experience with cancer before. So they are unsure
of what to do: to go for surgery, chemotherapy or radiotherapy?
Yet many others are told point blank that there is no more
treatment available or there is nothing that can be done. In
simple language it means to go home and wait to die. They
receive a good supply of painkillers or morphine to tide them
against the pains. There are also the more experienced patients.
They know what these treatments are about for they have gone
through them all. Apparently they have been well and thought
that they have been fully cured. Then cancer strikes again. They
come to seek other avenues of help. Whatever category you
are in, I have the following advice for you: Go for healing!
• Never say die and never give up. As long as you are still
breathing, know that there is still hope even when you are
told that you have no more hope. Hang on to your hope
and believe that you still have hope. Some people may accuse
me of trying to give people false hope. It is cruel to raise
false hopes. But on the other hand, some doctors tell you
that: You only have three to six months to live. There is no
medicine, no cure. Just prepare to die. Some patients are told
to enrol themselves with the palliative centre for further
management (and you know the reason why!). Do you not
think such insensitive comments or actions are even more
cruel than raising false hopes?
This book has many stories about false hopelessness. Goh had
colon cancer that had spread to the liver. He could hardly walk
and his stomach was bloated. He was told to go home to die.
He was in the hospital for two months and was put on morphine
only. The doctors on ward rounds even refused to see him. Yet
this no hope patient recovered and became much healthier than
he was before he had cancer. Two-month old baby Carisa had
Stage 4 neuroblastoma and was given three months to live. At
the time of this writing, Baby Carisa is now two years old and is
still alive. Melisa was ill with cervical cancer that had spread to
the liver and lungs. She was asked to write her will as the
doctor concluded that she only had three to six months to live.
It is now more than two years and she is alive and well. The
unbelievable and amazing thing about this case is that her thirty
spots in her liver have all disappeared. Raju had bone cancer of
an unknown origin. He was unable to move and had to sleep
sitting up on a deck chair. The doctor gave him six months to
live. After two months on herbs, he came back alive and was
able to visit India twice. He is now able to drive around normally.
Indeed, if at all there is one message that this booklet is meant
to convey, it is HOPE!
I am fully aware that during distressed moments, cancer patients
become very gullible and will grab at any straw that promises
the slightest chance of hope for living. I have heard of vultures
descending on your home, even in the middle of the night, offering
magic potions with a promise of cure. The mother of a boy who
had cancer told me of one salesman who would ring her four to
five times a day to keep tabs on how the son was doing. And each
time the phone rang, it was with the advice to take more and more
of the capsules he was selling. So the son ended up taking sixty
capsules of the product a day, besides another dozen of other
supplements. Use your discretion and be cautious.
From the point of view of science and medicine, hope and feelings
do not count or exist. These attributes do not belong to the
physical body for they do not show up on the X-ray film or CT
scan. Therefore they are not supposed to exist. Yet, you and I
know that there are things such as hope, love, feeling and
inspiration. These are attributes of the soul and mind. They do
exist and they matter - irrespective of what Rene Descartes
wanted you to believe or what the medical world says. The
feeling of no hope kills. So, I end this discussion by saying that
there is no such thing as false hope. But there is such a thing as
false hopelessness because no mortal on earth can play God.
Only God decides who dies or when you die!
When the terminally ill patients come to me I used to tell them
this: Don’t worry. All of us have to die someday. It is a matter of when
– tomorrow, next month or the next ten years. But, let me tell you
that you need not yet die just because you have terminal cancer. I am
younger than you and do not have any cancer but I may even die
earlier than you. So don’t worry so much. Death is not an issue here.
What matters now and the future is that while you are still alive you
do not have to suffer. Let us pray that we can help you lead a normal,
pain-free life. When the time comes and you have to go home, let us
pray that you die peacefully. Petrea King of Quest for Life
Foundation, Australia said: Life is not a competition about how
long we survive. It’s about the quality with which we live.
This is what Cancer Care Therapy is all about. We see healing
at various levels: physical, mental and spiritual. We may not be
able to cure the physical body but we may be able to touch and
heal the mind and the soul.
• Empower yourself. Knowledge is power goes a saying and I
truly believe this is true. So, I advise you to read and ask
questions. Seek knowledge so that you understand your
illness. Research has shown that those who feel hopeless,
helpless and accept their fate lying down survive poorly. I tell
cancer patients to stand up and live! Do not just be contented
to remain at the bottom of the pile with the following
mentality: My doctor says this and my doctor says that. You will
then end up following everything that he says. I am not
asking you to defy your doctor’s instructions but I also know
that you may be led by the nose without having a clue of
what is going on. It may not always be good for you.
Let us look at the case of Melisa again. She had approximately
thirty spots of varying sizes scattered all over her liver. The
oncologist recommended that she went for liver surgery. Melisa
asked the oncologist: How are you going to dig out all the spots in
my liver? Of course this question irritated the learned doctor,
but saved Melisa’s life! The question may sound stupid but it
packs a lot of sense. In the absence of a satisfactory answer,
Melisa declined the surgery. And she was absolutely right. Guat
had breast cancer. When she was asked to go for chemotherapy
or radiotherapy after her mastectomy, she asked the surgeon:
Can these cure me? Can you guarantee that? The doctor was
dumbfounded; there was no guarantee. That being the case,
Guat declined further medical treatments. To her, she preferred
to die, if that is what it all came to, in peace without the agony of
the side effects of the medical treatments.
• Reevaluate your strategy. In my books, I have written about
gut feelings and common sense. Body-mind healers advocate
the use of intuition or the sixth sense when making important
decisions in life. I have come to realise that not all decisions
made based on scientific data are wise or correct in many of life’s
situations. Statistics and data can be deceptive, cold, dead and
insensitive to human feelings. I advocate the use of simple
common sense. For example, ask yourself if the treatment that
you are undergoing is bringing you anywhere. Is it benefiting
you or killing you? Of course, when you go for any invasive
treatment, your hope is always to achieve a cure. Ask your
doctor if there is such a thing as a cure for your condition.
Some patients are given chemotherapy just for palliative reasons,
not cure. The treatment is supposed to make your life better
or more bearable. Reevaluate that strategy and determine if
this is true.
Dr. Jeffry Tobias (in Cancer) wrote: …one important decision ….
to stop, (know) when to say no more. Indeed it is wise to know
when to withdraw and to say enough is enough, if and when the
treatment is spinning you in a circle. There was one lung cancer
patient who came and told me that his oncologist spent only
half a minute with him every time he went for his chemotherapy.
My advice to him was: Find another oncologist who can be more
caring and who could give you more of his time and expertise. How
much can a half-a-minute doctor help you? Can you expect such
a busy-no-time doctor to save your life? Evaluate my comments
and make your own decisions about all these issues, for
sometimes what it takes is only common sense to save your life.
• Make wise decisions. Gurdjieff said: The wise man is not
educated and the educated man is not wise. These words of
wisdom are true. To enable you to make wise decisions, you
need to be aware of the following:
• Do not make decisions based on or out of fear.
• Seek more information, from different sources and
viewpoints, if possible, before you make any important
decision.
• Weigh out the options, both pros and cons. Do not simply
see the good side of things only. Take into account the
worst possible scenario as well.
• Connect with your inner self. Seek out your intuition,
common sense or gut feelings. Take time to be alone,
to be silent. Relax and let the inner voice within you
speak to you. If you are too busy or preoccupied with
others, you cannot hear your own inner voice.
• Arrive at a decision that you can live with. It is your
body, it is your liver or your breast that is to be taken
off. Ask yourself if you can live with that decision. It is
your life that is at stake. Doctors and other people can
only help but you are the one who suffers the brunt of
whatever is done to you.
• Once a decision is made, stay on course. Then, reevaluate
your strategy if it harms you.
• Trust and flow – easy does it! Dr. Joseph Murphy (in The
Power of Your Subconscious Mind) said: According to your
belief, it is done unto you. The causes of failures in healing are
mental coercion, lack of confidence, doubt and hesitation.
All these reflect negative attitudes. If you come with the
main goal of going all out for a cure, chances are that you will
fail to get one. In your intense desire to cure yourself, you
can become very tense and may not have peace of mind.
Have you ever experienced a time when you are trying very
hard to crack and solve a problem? Your mind comes to a
dead end. Try to take it easy. Relax and go to sleep. When
you wake up the next morning, things will look easier and
you will get to solve your problem in no time. This is because
the subconscious mind does not respond to mental coercion.
You do not have to teach your body how to heal itself. It is
intelligent enough and wise enough to know how to heal
itself. Relax and let the body do its job without your
unnecessary interference. Ask and pray for good health and
you get just that. Again, I say, take it easy.
• Forgive and let go. Negative thoughts such as hurtful
memories, bitterness, hatred, anger, etc., beget negative
reactions and they block the free flow of life. Let your life be
expressed in terms of love, forgiveness, sharing, caring,
harmony, peace, beauty and abundance. By loving and forgiving
yourself and others you let go of your negative emotions
and this is the first necessary ingredient of healing.
Three Options for Cancer Management
The doctor removed a lump from the breast of a lady and found
it to be malignant. Next, the doctor wanted to remove the
whole breast. After mastectomy the usual recipe would be:
chemotherapy, radiotherapy and probably tamoxifen. This lady and
her husband came to seek my advice. I made it clear to them that
they had to make their own decision, I could only advise and give
information. At first, they should relax, take time to reflect and
need not be hasty in wanting to do something very quickly, for
each step taken may mean a lot in terms of a possible recovery,
alleviating emotional problems, sufferings, etc. Of course, the
easiest option would be to lop off the breast and follow exactly
what the doctors say. To some patients, this is the obvious and best
option, but some patients would prefer or want to explore other
options for healing.
Perhaps those who wish to only have a brief one-page advice of
what they should do if they have cancer can simply take note of
the following summary.
• If you have cancer, know that no one on earth can guarantee
you a cure. At best, there will only be remission.
• The remission can last for a few months or for a few years.
The cancer may come back again.
• In general, it appears that neither modern medicine nor
holistic medicine can claim great success at curing cancer.
At most, there is a 30% - 40% success rate. What is meant
by success? When people mention a success rate, the term
is vague. Anyway, the fact is that about 60% of cancer patients
would, unfortunately, be left high and dry not knowing what
else to do after their treatments have failed them.
• Even if you are well after you have undergone the necessary
treatments, it is foolish to assume that you are done with
cancer. Cancer may not be done with you, yet. Take
appropriate and necessary steps to prevent recurrence or
to prolong your remission period.
• What seems to be a realistic aim in managing cancer is to be
able to live a quality life, i.e., to be as normal or healthy as
possible knowing that cancer cells may still be in your body. It
may be wiser for you to learn how to live with your own cancer
cells.
• The doctors may prescribe drugs, e.g., tamoxifen, to prevent
the cancer from coming back. This is not necessarily effective,
although popping pills into the mouth is an easy task. The
holistic healers ask you to change your lifestyle and diet,
take herbs, exercise, etc. For many people, these are hard
chores to put into practice, for herbs may taste repulsive to
some people. But in the long run, a holistic approach to
health may be better for you.
Chemotherapy & Brain Confusion
WASHINGTON (Reuters) - Chemotherapy causes changes in the brain's metabolism and blood flow that can last as long as 10 years, a discovery that may explain the mental fog and confusion that affect many cancer survivors, researchers said on Thursday.
The researchers, from the University of California, Los Angeles, found that women who had undergone chemotherapy 5 to 10 years earlier had lower metabolism in a key region of the frontal cortex.
Experts estimate at least 25 percent of chemotherapy patients are affected by symptoms of confusion, so-called chemo brain, and a recent study by the University of Minnesota reported an 82 percent rate, the statement said.
"People with 'chemo brain' often can't focus, remember things or multitask the way they did before chemotherapy," Silverman said. "Our study demonstrates for the first time that patients suffering from these cognitive symptoms have specific alterations in brain metabolism."
Comment from Cancer Answers: We have many cancer patients who come and tell us they became forgetful and "slow" after their chemotherapy treatment.
Source: American Society of Clinical Ocnology
Typhonium flagelliforme inhibits cancer cell growth in vitro and induces apoptosis: An evaluation by the bioactivity guided approach
A b s t r a c t
Aim of the Study: Typhonium flagelliforme (Lodd.) Blume (Araceae) is a Malaysian plant used locally tocombat cancer. In order to evaluate its antiproliferative activity in vitro and to possibly identify the activechemical constituents, a bioactivity guided study was conducted on the extracts of this plant.Materials and Methods: The active extracts of Typhonium flagelliforme were fractionated by flash columnchromatography and each fractionwas evaluated for antiproliferative activity using MTT assay. The apoptoticeffect of the active fraction was determined microscopically and by using TUNEL colorimetric assay.GC–MS and NMR were used to determine the chemical constituents of this active fraction.Results: Several fractions of the hexane and dichloromethane extracts were found to inhibit the growth ofNCI-H23 non-small cell lung carcinoma cell line significantly, with IC50 <15g/ml. style="color: rgb(0, 51, 0);" size="5">
Introduction
Introduction
Typhonium flagelliforme (Lodd.) Blume (Araceae) is a herbal plantwhich grows up to 30cm in height. It has an oblong whitish tuber,triangular leaves and a spathe which is dilated and round at thebase enclosing the yellowish spadix (Ridley, 1967; Hsuan et al.,1998). This plant grows wild inwasteland and is native to the SouthEast Asian countries and the southern part of India and Sri Lanka(Nicolson and Sivadasan, 1981).Typhonium flagelliforme, previously mistakenly referred asTyphonium divaricatum (Nicolson and Sivadasan, 1981), is commonlyknown as Rodent Tuber in Malaysia. Being describedethnomedically as toxic, warming and phlegm resolving, this plantis used to soothe swelling, coughing and more predominantly for∗ Corresponding author at: Centre for Drug Research, Universiti Sains Malaysia,Gelugor, 11800 Penang, Malaysia. Tel.: +60 4 6552829; fax: +60 4 6577957.E-mail addresses: nava@usm.my, vncnava@gmail.com (V. Navaratnam).the treatment of cancer (Teo and Ch’ng, 1999; Lee andWong, 2004).As a general practice, the juice of the fresh whole Typhonium flagelliformeplant is prepared in honey to be consumed as a drink(Teo and Ch’ng, 1999). There are also other practices where theleaves are wrapped in Longan flesh and taken raw (Lee and Wong,2004).Several chemical constituents had been identified from Typhoniumflagelliforme. The hexane extract was reported to containsaturated hydrocarbons and aliphatic acids (Choo et al., 2001a),while the ethyl acetate extract was found to contain aromatic fattyacids (Chen et al., 1997). No biological activities were indicated forthese compounds. In addition, phenylpropanoid glycosides, sterols,and a cerebroside which has antihepatotoxic activitywere reportedfrom the root of this plant (Huang et al., 2004).The polar extractswere investigated in vivo and found to be ableto ease expectoration, it is also antiasthmatic, anti-inflammatory,analgesic and sedative (Zhong et al., 2001). Pharmacological studiesconducted on rats also indicated that the juice extract was able toprevent hepatocarcinogenesis (Karuppiah et al., 1999).
The anticancer activity of Typhonium flagelliforme however,had only been investigated in vitro on two murine cancer celllines, namely P388 lymphocytic leukemia (Choo et al., 2001b)and a lymphoid cell line for which the cell line designation wasnot mentioned (Neoh, 1992). Both the studies indicated that thenon-polar extracts possessed significant antiproliferative activity.Since no study had been carried out on carcinoma cell linesand the effect of this plant on non-cancerous cell lines was alsounknown, we evaluated the antiproliferative effect of Typhoniumflagelliforme on a human lung carcinoma cell line (NCI-H23) and anon-tumorigenic fibroblast cell line (BALB/c 3T3) using the bioactivityguided approach.We also evaluated the apoptotic activity anddetermined the major chemical constituents of an active and cellspecific fraction.
The anticancer activity of Typhonium flagelliforme however,had only been investigated in vitro on two murine cancer celllines, namely P388 lymphocytic leukemia (Choo et al., 2001b)and a lymphoid cell line for which the cell line designation wasnot mentioned (Neoh, 1992). Both the studies indicated that thenon-polar extracts possessed significant antiproliferative activity.Since no study had been carried out on carcinoma cell linesand the effect of this plant on non-cancerous cell lines was alsounknown, we evaluated the antiproliferative effect of Typhoniumflagelliforme on a human lung carcinoma cell line (NCI-H23) and anon-tumorigenic fibroblast cell line (BALB/c 3T3) using the bioactivityguided approach.We also evaluated the apoptotic activity anddetermined the major chemical constituents of an active and cellspecific fraction.
Materials and methods
Plant material
Fresh Typhonium flagelliforme plants were collected from Kampung Baru Cina, Sungai Buloh, Selangor. The taxonomic identity ofthe plant was authenticated by Mr. Baharuddin Sulaiman, a taxonomistfrom the School of Biological Sciences, Universiti SainsMalaysia, where a voucher specimen of the plant was deposited
Extraction of the plant material
Based on our initial findings, extraction of the fresh plant ofTyphonium flagelliforme by maceration produced an extract withhighest antiproliferative activity compared with other extractiontechniques, such as soxhlet and ultrasonication using fresh or dryplant. As a result, this extraction method was used for the study.Fresh whole plant of Typhonium flagelliforme (3.5 kg) was cut witha mill grinder into fine pieces and macerated with hexane (8 l) for1 week at room temperature (ca. 25–27 ◦C). After removing thehexane extract, the residue was macerated with dichloromethane(DCM) (8 l) followed bymethanol (MeOH) (8 l), each for aweek. Theextractswere filtered before evaporating to dryness under reducedpressure at 35 ◦C with a Buchi R110 Rotavapor (Buchi LabortechnikAG, Flawil, Switzerland). The MeOH extractwas further lyophilizedwith a Freezone 12 l freeze dryer (Labconco Corp., MO, USA). The% yield of the crude extracts was calculated as: (weight of crudeextract/weight of fresh plant)×100%.
Bioactivity guided sample purification
All extracts obtained from three different extraction solvents(hexane,DCMand MeOH)were subjected to cell proliferation assayaccording to the procedure described in Section 2.6. The extractswhich showed significant inhibitory effect at 100g/ml were furtherevaluated at six lower concentrations in order to obtain thegrowth inhibition profile, from which the IC50 values were determined.From the bioassay results, hexane and DCM extractswere foundto be active. As such, purification was done by using normal phaseflash column chromatography on these two extracts. The stationaryphase wasmade up of a glass column (50mmi.d.) packed withsilica gel 60, 0.040–0.063mm (Merck, Darmstadt, Germany) (ca.1 g extract/100 g silica gel). Compressed oxygen-free nitrogen gaswas used to drive the flow of the solvent at a pressure of 1 bar.The mobile phase consisted of combinations of hexane, DCM andMeOH, and the eluting strength of the solvent was increased graduallyby increasing the composition of the more polar solvent.For purification of the hexane extract, the initial solvent compositionwas hexane (100% v; 2100 ml) and then it was changed tohexane–DCM (4:1 v/v; 500 ml), followed by hexane–DCM (3:2 v/v;200ml), hexane–DCM (2:3 v/v; 200 ml), hexane–DCM (1:4 v/v;400ml), DCM (100% v; 900 ml), DCM–MeOH (95:5 v/v; 700 ml),DCM–MeOH (9:1 v/v; 1300 ml) and finally to DCM–MeOH (4:1 v/v;1500 ml). For the purification of the DCM extract, the initial solventcomposition was hexane–DCM (9:1 v/v; 1350ml) and thenit was changed to hexane–DCM (4:1 v/v; 400 ml), followed byhexane–DCM (3:2 v/v; 600 ml), hexane–DCM (2:3 v/v; 600 ml),hexane–DCM (1:4 v/v; 600 ml),DCM(100% v; 800ml),DCM–MeOH(95:5 v/v; 800 ml), DCM–MeOH (9:1 v/v; 800 ml), DCM–MeOH(4:1 v/v; 800 ml) and finally to DCM–MeOH (1:1 v/v; 800 ml).The eluent was collected in fractions of 40 ml. The chemicalcomposition of each fraction was evaluated by using thin-layerchromatography (TLC) and visualized with UV (254 nm and365 nm). Since Typhonium flagelliforme is known also to containfatty acids (Chen et al., 1997), long chain unsaturated hydrocarbons(Choo et al., 2001a,b), glycosides and steroids (Huang et al.,2004)which could hardly be detected by UV, anisaldehyde–sulfuricacid reagent was used to identify these compounds. Based on theTLC profiles, those fractions with similar compositionswere pooledtogether and concentrated under reduced pressure. A total of 17combined fractions were obtained from the hexane extract (designatedas H/F1, H/F2, . . ., H/F17) and a total of 21 combined fractionswere obtained fromthe DCM extract (designated as D/F1, D/F2, . . .,D/F21). D/F21 was obtained by removing silica gel from the top ofthe column to a depth of 4 cm and extracting the residue adsorbedon the silica gel by using two aliquots of 200ml of DCM. The TLC Rfvalues of the chemical constituents of each fraction and the weightof the fractions are given in Table 1 (for fractions of hexane extract)and Table 2 (for fractions of DCM extract).Fractions number H/F1 and H/F13 were further purified withsilica gel 60 preparative-TLC plates of 1mm thickness (Merck,Darmstadt, Germany) to yield purer subfractions. H/F1 was developedwith hexane and resulted in 10 bands which were visualizedwith UV 254 nm (Rf = 0.09, 0.20, 0.25, 0.32, 0.36, 0.42, 0.46,0.56, 0.70, 0.93). These bands were scraped from the prep-TLC plate and the compounds contained in these bands wereextracted with hexane to yield 10 subfractions (designated asH/F1/01, H/F1/02, . . ., H/F1/10). Fraction 13 was developed withhexane–chloroform–ethyl acetate, 4:5:1 v/v/v and resulted in fourbands which could not be visualized under UV. However, afterspraying a small section of the TLC plate with anisaldehyde–sulfuricacid reagent, the sprayed section of the bands became visible(Rf = 0.08, 0.35, 0.68, 0.88). The unsprayed sections of the bandswere then scraped from the plate and the compounds wereextracted with DCM to yield four subfractions (designated asH/F13/01, . . ., H/F13/04).The fractions and subfractions obtained through flash columnchromatography were subjected to antiproliferation assay on NCIH23and following that, 10 most active samples were selected forevaluation of their cell line specificity. Subsequently, the fractionwhich showed specific action towards NCI-H23 was evaluated forits apoptotic effect towards the cell line.
Cell culture
NCI-H23 (human non-small cell lung carcinoma cell line,ATCC No.: CRL-5800) was contributed by Dr. Tengku SifzizulTengkuMuhammad (School of Biological Sciences, Universiti SainsMalaysia). This cell line was cultured in RPMI-1640 (Sigma, MO,USA) supplemented with 10% fetal bovine serum (Invitrogen Corp.,Auckland, N.Z.). The non-tumorigenic cell line, BALB/c 3T3 (mousefibroblast cell line, ATCC No.: CCL-163) was purchased from theAmerican Type Culture Collection (ATCC).
Sample preparation for cell proliferation assay
Stock solutions of the test samples were prepared at concentrationsof 20 mg/ml in either ethanol (EtOH) or dimethyl sulfoxide(DMSO) depending on their solubility and these were kept at−20 ◦C. Immediately before the assay, dilution was made from thestock solutions in complete culture medium to produce test solutionswith a final concentration of 100g/ml for initial screening.When a test solution was found to inhibit the growth of NCI-H23almost completely (>85% inhibition) at this concentration, it wasfurther evaluated for its antiproliferative effect at six lower concentrationsranging from 0.78–100g/ml. Solutions of 0.5% EtOH and0.5% DMSO in complete culture medium were used as the vehicle control
MTT cell proliferation assay
Based on some experiences in our laboratory, NCI-H23 wasfound to respond most strongly towards Typhonium flagelliformeextracts compared to other cell lines. Thus, this cell line was usedthroughout the study to screen for antiproliferative components.In order to evaluate whether the antiproliferative action wasspecific to tumor cells, the active fractions were also tested ona non-tumorigenic cell line, BALB/c 3T3. Due to the difference ingrowth rates for both NCI-H23 and BALB/c 3T3 cell lines, the initialseeding densities for both the cell lines were optimized to ensurethat the final absorbance reading falls between the linearity rangeof the detector. 6×103 cells/well ofNCI-H23 and 1.5×103 cells/wellof BALB/c 3T3 were seeded in 96-well microtiter plates. The plateswere incubated for 24 h for cell recovery and attachment before testsamples and the vehicle control were added to the cells. This wasfollowed by 72 h of incubation. 20l of 5 mg/mlMTT (Acros Organics,Belgium) was then added to the cells followed by further 3 hof incubation. The medium was then aspirated and the formazanproduct generated from the viable cells was dissolved in DMSObefore being measured at 570 nmagainst the referencewavelengthof 650 nm. The growth inhibitory effect of the test samples wasdetermined by comparing the optical density of the treated sampleagainst the optical density of the control. The inhibition concentration,IC50 of the test samples was calculated using Probit Analysis(SPSS Version 12.0.1, Chicago, IL, USA). All the experiments werecarried out in triplicates and three independent experiments wereperformed for each sample.
Assessment of apoptosis in cell culture
Cell morphological examinationNCI-H23 cells treated with the selected fraction were observedunder the phase contrastmicroscope at 320×magnifications. Cytoplasmicshrinkage, membrane blebbing and the ultimate formationof smaller membrane bound vesicles were observed as signs of opoptosis
DNA nick-end labellingFor further confirmation of the occurrence of apoptosis,terminal deoxynucleotidyl tranferase (TdT) mediated biotin-16-deoxyuridine triphosphate (dUTP) Nick-End Labelling (TUNEL)assay was performed using DeadEndTM Colorimetric TUNEL System(Promega, Madison, WI, USA). Cells cultured on a chamberslide were treated with the selected fraction for 24 h at concentrationsclose to the IC50. The assay was then conducted accordingto the manufacturer’s instructions. Briefly, treated cells were fixedwith 4% paraformaldehyde solution in phosphate buffer saline(PBS). After washing with fresh PBS, the cells were permeabilizedwith 0.2% Triton X-100 solution. The fragmented 3-OH DNAends were then labeled by incorporating biotinylated dUTP inrTdT reaction mixture at 37 ◦C for 1 h in the incubator, followedby addition of 0.3% hydrogen peroxide for blocking endogenousperoxidase. Streptavidin HRP (1:500 in PBS) was then added tothe slide. After incubating at room temperature (ca. 25 ◦C) for30 min, cell nuclei with fragmented DNAs were stained withhydrogen peroxide and chromagen diaminobenzidine for visualisation.
Fresh Typhonium flagelliforme plants were collected from Kampung Baru Cina, Sungai Buloh, Selangor. The taxonomic identity ofthe plant was authenticated by Mr. Baharuddin Sulaiman, a taxonomistfrom the School of Biological Sciences, Universiti SainsMalaysia, where a voucher specimen of the plant was deposited
Extraction of the plant material
Based on our initial findings, extraction of the fresh plant ofTyphonium flagelliforme by maceration produced an extract withhighest antiproliferative activity compared with other extractiontechniques, such as soxhlet and ultrasonication using fresh or dryplant. As a result, this extraction method was used for the study.Fresh whole plant of Typhonium flagelliforme (3.5 kg) was cut witha mill grinder into fine pieces and macerated with hexane (8 l) for1 week at room temperature (ca. 25–27 ◦C). After removing thehexane extract, the residue was macerated with dichloromethane(DCM) (8 l) followed bymethanol (MeOH) (8 l), each for aweek. Theextractswere filtered before evaporating to dryness under reducedpressure at 35 ◦C with a Buchi R110 Rotavapor (Buchi LabortechnikAG, Flawil, Switzerland). The MeOH extractwas further lyophilizedwith a Freezone 12 l freeze dryer (Labconco Corp., MO, USA). The% yield of the crude extracts was calculated as: (weight of crudeextract/weight of fresh plant)×100%.
Bioactivity guided sample purification
All extracts obtained from three different extraction solvents(hexane,DCMand MeOH)were subjected to cell proliferation assayaccording to the procedure described in Section 2.6. The extractswhich showed significant inhibitory effect at 100g/ml were furtherevaluated at six lower concentrations in order to obtain thegrowth inhibition profile, from which the IC50 values were determined.From the bioassay results, hexane and DCM extractswere foundto be active. As such, purification was done by using normal phaseflash column chromatography on these two extracts. The stationaryphase wasmade up of a glass column (50mmi.d.) packed withsilica gel 60, 0.040–0.063mm (Merck, Darmstadt, Germany) (ca.1 g extract/100 g silica gel). Compressed oxygen-free nitrogen gaswas used to drive the flow of the solvent at a pressure of 1 bar.The mobile phase consisted of combinations of hexane, DCM andMeOH, and the eluting strength of the solvent was increased graduallyby increasing the composition of the more polar solvent.For purification of the hexane extract, the initial solvent compositionwas hexane (100% v; 2100 ml) and then it was changed tohexane–DCM (4:1 v/v; 500 ml), followed by hexane–DCM (3:2 v/v;200ml), hexane–DCM (2:3 v/v; 200 ml), hexane–DCM (1:4 v/v;400ml), DCM (100% v; 900 ml), DCM–MeOH (95:5 v/v; 700 ml),DCM–MeOH (9:1 v/v; 1300 ml) and finally to DCM–MeOH (4:1 v/v;1500 ml). For the purification of the DCM extract, the initial solventcomposition was hexane–DCM (9:1 v/v; 1350ml) and thenit was changed to hexane–DCM (4:1 v/v; 400 ml), followed byhexane–DCM (3:2 v/v; 600 ml), hexane–DCM (2:3 v/v; 600 ml),hexane–DCM (1:4 v/v; 600 ml),DCM(100% v; 800ml),DCM–MeOH(95:5 v/v; 800 ml), DCM–MeOH (9:1 v/v; 800 ml), DCM–MeOH(4:1 v/v; 800 ml) and finally to DCM–MeOH (1:1 v/v; 800 ml).The eluent was collected in fractions of 40 ml. The chemicalcomposition of each fraction was evaluated by using thin-layerchromatography (TLC) and visualized with UV (254 nm and365 nm). Since Typhonium flagelliforme is known also to containfatty acids (Chen et al., 1997), long chain unsaturated hydrocarbons(Choo et al., 2001a,b), glycosides and steroids (Huang et al.,2004)which could hardly be detected by UV, anisaldehyde–sulfuricacid reagent was used to identify these compounds. Based on theTLC profiles, those fractions with similar compositionswere pooledtogether and concentrated under reduced pressure. A total of 17combined fractions were obtained from the hexane extract (designatedas H/F1, H/F2, . . ., H/F17) and a total of 21 combined fractionswere obtained fromthe DCM extract (designated as D/F1, D/F2, . . .,D/F21). D/F21 was obtained by removing silica gel from the top ofthe column to a depth of 4 cm and extracting the residue adsorbedon the silica gel by using two aliquots of 200ml of DCM. The TLC Rfvalues of the chemical constituents of each fraction and the weightof the fractions are given in Table 1 (for fractions of hexane extract)and Table 2 (for fractions of DCM extract).Fractions number H/F1 and H/F13 were further purified withsilica gel 60 preparative-TLC plates of 1mm thickness (Merck,Darmstadt, Germany) to yield purer subfractions. H/F1 was developedwith hexane and resulted in 10 bands which were visualizedwith UV 254 nm (Rf = 0.09, 0.20, 0.25, 0.32, 0.36, 0.42, 0.46,0.56, 0.70, 0.93). These bands were scraped from the prep-TLC plate and the compounds contained in these bands wereextracted with hexane to yield 10 subfractions (designated asH/F1/01, H/F1/02, . . ., H/F1/10). Fraction 13 was developed withhexane–chloroform–ethyl acetate, 4:5:1 v/v/v and resulted in fourbands which could not be visualized under UV. However, afterspraying a small section of the TLC plate with anisaldehyde–sulfuricacid reagent, the sprayed section of the bands became visible(Rf = 0.08, 0.35, 0.68, 0.88). The unsprayed sections of the bandswere then scraped from the plate and the compounds wereextracted with DCM to yield four subfractions (designated asH/F13/01, . . ., H/F13/04).The fractions and subfractions obtained through flash columnchromatography were subjected to antiproliferation assay on NCIH23and following that, 10 most active samples were selected forevaluation of their cell line specificity. Subsequently, the fractionwhich showed specific action towards NCI-H23 was evaluated forits apoptotic effect towards the cell line.
Cell culture
NCI-H23 (human non-small cell lung carcinoma cell line,ATCC No.: CRL-5800) was contributed by Dr. Tengku SifzizulTengkuMuhammad (School of Biological Sciences, Universiti SainsMalaysia). This cell line was cultured in RPMI-1640 (Sigma, MO,USA) supplemented with 10% fetal bovine serum (Invitrogen Corp.,Auckland, N.Z.). The non-tumorigenic cell line, BALB/c 3T3 (mousefibroblast cell line, ATCC No.: CCL-163) was purchased from theAmerican Type Culture Collection (ATCC).
Sample preparation for cell proliferation assay
Stock solutions of the test samples were prepared at concentrationsof 20 mg/ml in either ethanol (EtOH) or dimethyl sulfoxide(DMSO) depending on their solubility and these were kept at−20 ◦C. Immediately before the assay, dilution was made from thestock solutions in complete culture medium to produce test solutionswith a final concentration of 100g/ml for initial screening.When a test solution was found to inhibit the growth of NCI-H23almost completely (>85% inhibition) at this concentration, it wasfurther evaluated for its antiproliferative effect at six lower concentrationsranging from 0.78–100g/ml. Solutions of 0.5% EtOH and0.5% DMSO in complete culture medium were used as the vehicle control
MTT cell proliferation assay
Based on some experiences in our laboratory, NCI-H23 wasfound to respond most strongly towards Typhonium flagelliformeextracts compared to other cell lines. Thus, this cell line was usedthroughout the study to screen for antiproliferative components.In order to evaluate whether the antiproliferative action wasspecific to tumor cells, the active fractions were also tested ona non-tumorigenic cell line, BALB/c 3T3. Due to the difference ingrowth rates for both NCI-H23 and BALB/c 3T3 cell lines, the initialseeding densities for both the cell lines were optimized to ensurethat the final absorbance reading falls between the linearity rangeof the detector. 6×103 cells/well ofNCI-H23 and 1.5×103 cells/wellof BALB/c 3T3 were seeded in 96-well microtiter plates. The plateswere incubated for 24 h for cell recovery and attachment before testsamples and the vehicle control were added to the cells. This wasfollowed by 72 h of incubation. 20l of 5 mg/mlMTT (Acros Organics,Belgium) was then added to the cells followed by further 3 hof incubation. The medium was then aspirated and the formazanproduct generated from the viable cells was dissolved in DMSObefore being measured at 570 nmagainst the referencewavelengthof 650 nm. The growth inhibitory effect of the test samples wasdetermined by comparing the optical density of the treated sampleagainst the optical density of the control. The inhibition concentration,IC50 of the test samples was calculated using Probit Analysis(SPSS Version 12.0.1, Chicago, IL, USA). All the experiments werecarried out in triplicates and three independent experiments wereperformed for each sample.
Assessment of apoptosis in cell culture
Cell morphological examinationNCI-H23 cells treated with the selected fraction were observedunder the phase contrastmicroscope at 320×magnifications. Cytoplasmicshrinkage, membrane blebbing and the ultimate formationof smaller membrane bound vesicles were observed as signs of opoptosis
DNA nick-end labellingFor further confirmation of the occurrence of apoptosis,terminal deoxynucleotidyl tranferase (TdT) mediated biotin-16-deoxyuridine triphosphate (dUTP) Nick-End Labelling (TUNEL)assay was performed using DeadEndTM Colorimetric TUNEL System(Promega, Madison, WI, USA). Cells cultured on a chamberslide were treated with the selected fraction for 24 h at concentrationsclose to the IC50. The assay was then conducted accordingto the manufacturer’s instructions. Briefly, treated cells were fixedwith 4% paraformaldehyde solution in phosphate buffer saline(PBS). After washing with fresh PBS, the cells were permeabilizedwith 0.2% Triton X-100 solution. The fragmented 3-OH DNAends were then labeled by incorporating biotinylated dUTP inrTdT reaction mixture at 37 ◦C for 1 h in the incubator, followedby addition of 0.3% hydrogen peroxide for blocking endogenousperoxidase. Streptavidin HRP (1:500 in PBS) was then added tothe slide. After incubating at room temperature (ca. 25 ◦C) for30 min, cell nuclei with fragmented DNAs were stained withhydrogen peroxide and chromagen diaminobenzidine for visualisation.
