Curcurmin induce apoptosis in human cervix epithelioid carcinoma cells
(HeLa) through activation of caspase- 3 and P53

C-S Yu PhDa, JK-S Chan Bsb, J-G Chung PhDc

a", Center of General Education, cDepartment of Microbiology; China
Medical University, Taichung 404, Taiwan, bDepartment of Biochemistry,
University of British Columbia, Vancouver, BC, Canada "

Curcurmin has been demonstrated in rats to prevent azoxymethane-induced
aberrant crypt foci, putative precursor lesions in the colon and in
mice to prevent tumorigenesis induced by azoxymethane in the colon and
by N-ethyl N-nitrosoguanidine in the duodenum and forestomach. The
mechanism of curcurmin affect on human cervix cancer cells was not
addressed. Therefore, curcurmin was used to study the biological
activities on the human cervix epithelioid carcinoma cells (HeLa). On
MTT assay, curcurmin showed obvious cytotoxic effects and inhibit the
proliferation of HeLA cells. The cytotoxic effects of curcurmin was
accompanied by the dose- and time-dependent appearance of
characteristics of apoptosis including DNA fragmentation (gel
electrophoresis) and sub-G1 ratio (flow cytometric assay). The HeLA
cells cotreated with curcurmin caused a rapid transient induction of
caspase-3 activity, but not caspase-1 activity. We also found out that
cleavage of poly(ADP-ribose) polymerase (PARP) and decrease of
pro-caspase-3 protein were detected in curcurmin-treated HeLa cells.
Increased the pro-apoptotic protein (bax), cytochrome C release and
caspases-9 and 3 activity and decreased anti-apoptotic protein (Mcl-1)
were detected in HeLa cells after cotreated with curcurmin. We also
used PCR and multiplex PCR methods to examine the caspase 3 gene
expression and the results show that curcurmin induced caspase-3 gene
expression.

Peter,

Sorry I didn't provide details. The lymphoma first occurred in 1994, and  
recurred in the fall of 2001. First a (slightly edited) transcript of a  
consultation with Dr. R. Klasa of the BC Cancer Agency in Vancouver on  
Feb. 27, 2002. My comments are in parentheses.

                        ONCOLOGY CONSULTATION
      Mr. Hurd is a 70-year-old man with a history of transformed MALT  
lymphoma treated with subtotal gastrectomy and chemotherapy in 1994 and  
complicated by anthracycline-induced cardiomyopathy. He was seen in  
consultation on February 27, 2002 for an opinion regarding management of  
recurrent lymphoma.
      Mr. Hurd presented with dyspeptic symptoms in 1994. An upper Gl  
endoscopy with biopsy showed MALT lymphoma in the antrum of the stomach,  
associated with Helicobacter pylori infection. He was treated with triple  
antibiotic therapy; however, symptoms progressed over six weeks and the  
mass grew on imaging (doubled in size). He developed B symptoms. He  
underwent subtotal gastrectomy and pathology was remarkable for probable  
diffuse large cell lymphoma with negative margins in addition to areas of  
MALT. This pathology was reviewed and was felt to be in keeping with small  
non-cleaved, non-Burkitt's lymphoma. Staging imaging is not available,  
however, bone marrow biopsy pre-chemotherapy was negative.
       Following chemotherapy (CHOP) Mr. Hurd improved significantly. He  
was felt to be in complete remission. Although he has never returned to  
his previous weight, he did gain weight in the interim.
       Although he was asymptomatic, Mr. Hurd read about possible  
cardiomyopathy and requested an assessment. A MUGA scan showed an ejection  
fraction of 27%. He was seen by a cardiologist who prescribed Altace. He  
apparently had a repeat echocardiogram in the spring of 2001, which showed  
an ejection fraction of 31%. He is not limited by cardiomyopathy. (This  
was asymptotic congestive heart failure brought on by the adriamycin in  
CHOP and discovered in a routine heart examination in 1997)
       In the summer of 2001 Mr. Hurd noticed a hard mass on the left side  
of his abdomen. In addition, he developed drenching night sweats, and  
noticed pruritis. He did not lose weight, his appetite was stable, and his  
energy was unremarkable. An upper Gl endoscopy with biopsy was normal and  
blood work was normal. Fine needle aspiration which took place in the fall  
of 2001 was nondiagnostic, and bone marrow biopsy on October 11, 2001 was  
negative for lymphoma.
       On January 23, 2002, he underwent a biopsy by laparotomy. Pathology  
was remarkable for diffuse small cleaved cell lymphoma, CD10 positive,  
CD20 positive, consistent with
intermediate grade lymphoma. A chest x-ray done on February 4, 2002 was  
normal.
A CT scan of the abdomen done on September 18, 2001 showed large masses.
An ultrasound of the abdomen done on February 11, 2002 showed a mass  
measuring "9 x
10 x 33 mm".(This measurememt was clearly a typing error, hence the  
quotes--see measurements below).
     Mr. Hurd's biopsy was complicated by a postoperative abscess  
requiring several trips to the Emergency Room, treatment with antibiotics  
and surgical drainage. .
     Mr. Hurd currently spends more than half the day in bed. He is  
limited by pain on sitting up. He has lost approximately 10 Ib since  
surgery, and his wife notices that he is vague. They have not noticed any  
focal areas of weakness or sensory change.
     On examination, this was a cachectic man who looked unwell. Pulse  
rate was 70 with the occasional skipped beat. Blood pressure was 110/60 in  
the right arm in the supine position. Chest examination was remarkable for  
bibasilar crackles.
     Abdominal examination revealed decreased bowel sounds in a hard  
abdomen, which was
nontender to palpation. Liver span was 10 cm with the edge at the costal  
border. There was
no splenomegaly. There was a midline laparotomy incision. Abdominal  
examination was
most remarkable for a very large mass extending from the left flank across  
the midline and
extending from the umbilicus to under the xiphisternum. Measurements were  
at least 20 cm
in each direction. There was no peripheral lymphadenopathy at any site.  
Chest x-ray from February 4, 2002 was normal. The most recent abdominal  
imaging is an ultrasound from February 11th which shows a large mass. Bone  
marrow biopsy done in November 2001 was negative for lymphoma.

ASSESSMENT
     This is a 70-year-old man with a history of transformed MALT  
lymphoma, the high-grade component being either diffuse large cell  
lymphoma or small non-cleaved, non-Burkitt's lymphoma. He was treated in  
1994 with subtotal gastrectomy followed by eight cycles of CHOP  
chemotherapy with complete remission. Treatment was complicated by  
anthracycline-included cardiomyopathy. He presents with a recurrence of  
lymphoma with a large abdominal mass and ECOG performance status of 3 or  
greater.
     Mr. Hurd's lymphoma recurrence is not a curable condition. However,  
he would likely benefit symptomatically from a course of palliative  
chemotherapy. We are in agreement with Dr. Wass that a trial of alkylating  
agent-based chemotherapy would be a reasonable first-line approach.  
Because of the seven-year interval between requirements for treatment, his  
lymphoma may well be sensitive to alkylating agents. Should he not respond  
to an alkylating agent, he may benefit from a course of palliative  
Etoposide. The mass is too large for palliative radiotherapy, which would  
likely result in significant morbidity, particularly in a gentleman who  
has had a previous subtotal gastrectomy. Finally, he may if these  
treatment approaches fail possibly benefit from treatment with antibodies;  
however, this would not be our favored first-line approach due to the bulk  
of the mass.
                                    END OF TRANSCRIPT

        Following the consultation I was put on a 6 month chemo regime of  
CVP. The abcess mentioned in the transcript caused the stiches in the 1994  
surgery to pop leaving me with a very large hernia which was not operated  
on at the time since I was on chemo.
        The chemo shrunk the tumor by a little less than 1/2. During the  
chemo (which ended in June, 2002) I developed a persistent cough which  
developed because the lymphoma had blocked the thorasic duct and fluid was  
building up in the right lung. My onc decided to start me on fludarabine  
in the fall after an attempt to solve the lung problem. I went into  
hospital in Aug for 8 days while fluid was continuously drained from the  
lung (a liter a day) and I was on a very restricted diet. The lung  
specialist was sure the fluid problem was insolvable unless the cancer was  
checked but discharged me anyway since the diet had somewhat reduced the  
fluid. My GP communicated to my wife that I probably wouldn't recover. I  
came out of the hospital minus another 15 pounds and barely able to  
shuffle around the house, sleeping most of the time. However my extensive  
research on alternative approaches to cancer settled me on trying the  
curcumin which I began on Sept.2, 2002. Tumor mass was now 11x4.5 cm. The  
fludarabine was begun at half dose on Sept. 17. On Oct. 11 my onc was  
amazed to find she could no longer feel the large abdominal mass, only a  
small mass. Also by this time the fluid buildup in the lung had stopped  
indicating the thorasic duct was no longer blocked by lymphoma. Another  
fact was that my energy was increasing remarkably, which the onc said was  
very unusual on fludarabine which usually caused fatigue. This was after  
41 days of curcumin and 24 days of 1/2 dose fludarabine.
         The regime was continued for 6 rounds until Feb. 7/03. Tumor now  
at 5x3 cm, so down about half. It was decided to try 3 more rounds of  
fludarabine ending on May 14/03, at which time a cat scan showed no change  
in size of abdominal mass. The onc was not sure whether tumor was active  
or we had arrived at scar tissue, but decided to stop the chemo. I stayed  
on the curcumin as time went on.
          In late Sept./03 I had abdominal surgery to repair the hernia  
and the surgeon took a very good look at the mesentery. Two biopsies  
showed no active cancer but considerable scar tissue.
           My read on this sequence of events is that the shrinkage of the  
tumor occurred almost entirely between Sept. 2 and Oct. 11/02, when the  
onc noticed the remarkable shrinkage, leaving approximately a 5x3 cm scar  
tissue mass which was seen in the two later cat scans. If this read is  
correct, then was it mostly the curcumin for 41 days or the fludarabine at  
1/2 dose for 24 days or a happy synthesis. I am reinforced in the curcumin  
hypothesis because a year ago I developed lymph node swelling in my neck  
suspiciously like lymphoma, went back on the heavy dose curcumin and it  
disappeared in about a week. At any rate, after being described as  
incurable, I am still cancer free and my onc says that with any luck I am  
cured. This is probably premature, given the nature of lymphoma, and I  
take each day at a time.
            A final few remarks. I tried many alternative approaches to  
cancer and did a lot of research, being a retired university prof of  
math.  I agree that many testimonials are lacking, but you can find a lot  
more info if you dig. My philosophy was that I would try about anything as  
long as I was convinced it wouldn't harm me, and not wait for the double  
blind tests.  The amazing thing about curcumin was that it was in a  
completely different league from most of the other alternatives as far as  
research was concerned. That being said, I have to remember talking to a  
lymphoma survivor who had after much chemo and radiation was given 3 weeks  
to live.  She went on the grape cure (Johanna Brandt) and was completely  
cured in a year. A few months later I heard two cancer researches say they  
had discovered in the skins of grapes one of the most powerful cancer  
agents they had ever tested on mice. Go figure. I understand the grape  
cure has a great following in Europe.
            If the curcumin is to be effective in cancer I think it should  
be done in the heavy doses I used and, most importantly, accompanied by  
bioperine, which increases the absorption by 10-15 times. One research  
article I read said that curcumin would be an excellent chemo but  
unfortunately was not well absorbed in the gut. The author hadn't heard of  
bioperine.

Albert