WELCOME

Department of Medical Biochemistry University of Madras

Taramani campus, Chennai - 600 113

By

Dr. D. SAKTHISEKARANProfessor of Biochemistry

CHEMOTHERAPEUTIC EFFICACY OF INDIAN NONI FRUIT JUICE AGAINST N – NITROSODIETHYLAMINE INDUCED AND

PHENOBARBITAL PROMOTED EXPERIMENTAL HEPATOCELLULAR CARCINOMA.

Cancer  Cancer is a disease characterized by uncontrollable,

irreversible, independent, autonomous, uncoordinated and relatively unlimited and abnormal over growth of tissues.  Cancer spreads by invasion to the surrounding tissues and by metastasis to distant sites.

Tumors can be of 2 major types:

Benign tumors are not cancer.

Malignant tumors are cancer.

Benign tumors are generally slow growing expansive masses often with a “Pushing margin” and enclosed within a fibrous capsule.

Malignant tumors are usually rapidly growing,

invading local tissue and spreading to distant sites.

Seven Danger Signals

1. Any Sore that does not heal.

2. A Lump or Thickening in the breast or elsewhere.

3. Unusual bleeding or discharge from any body opening.

4. Any change in a Wart or Mole.

5. Persistent indigestion or difficulty in swallowing.

6. Persistent Hoarseness or Cough.

7. Any change in normal bowel habits.

 Experimental

Cancer

 Inducing agent

 Animal used

 Period of Induction

  Fibrosarcoma

 Cell lines

 

 Wistar Albino

Rats

 90 days

Hepatocellular

Carcinoma

 N-nitrosodiethylamine

(200mg/kg body wt, given i.p) 

 Wistar Albino

Rats

 120 days

 

 Breast Cancer 7,12 Dimethyl benz(a)anthracene

(100mg/kg body wt. given i.p.) 

 Female Sprague

Dawley Rats

 90 days

 Lung Cancer

 Benzo(a)pyrene

(50 mg/kg body wt. orally) 

 Swiss Albino

Mice

 120 days

 

 Oral Cancer

 

 7,12 Dimethyl benz(a)anthracene(0.5% in paraffin oil brushed thrice a

week on the buccal pouch)

 Golden Syrian

Hamsters

 105 days

Chemical carcinogens

Cancer Treatment

Radiotherapy Chemotherapy Surgery

CANCER CHEMOTHERAPY

Type of Cancer : HCC

Anti cancer drug : Noni Fruit juice

ABOUT NONI Noni is a tropical fruit from the tree Morinda vitriolic, popularly known as Indian mulberry. Noni was used in traditional medicines for centuries for a broad range of health ailments (Tamil: Venn Nuna).

Noni’s healing properties are widely recorded in ayurveda and siddha medicinal texts. There are more than 5000 medicinal plants belonging to 180 families, used around the world for healing different diseases. Noni is rich in vitamins A, C, E, B, B2, B6, B12, Calcium, Iron, niacin, folic acid, pantothenic acid, phosphorus, magnesium, zinc, copper, and other minerals like chromium, manganese, carbohydrates and 150 isolated neutraceuticals. It is the best source of anti-oxidant.

Benefits of noniRegular use of noni / rejuvenates the body, revitalizes the cells,

restores energy, relieves pain, reduces inflammation, reduces inflammation, releases stress, purifies blood, stimulates immune system, improves digestion, enhances well being, regulates cell function, maintains healthy skin, hair and scalp, protects you from toxins and pollutants, reduces the risk of developing cancer improves memory and concentration, inhibits tumor growth, reduces the chances of premature onset of age-related diseases such as arthritis, heart disease, diabetes or strokes, protects against viral and bacterial strains that have become antibiotic resistant, aids in digestion, that means we absorb more nutrients at the cellular level.

Noni supports the immune system, the circulatory system, the digestive system, the metabolic system, the tissues and cells of our body.

N-Nitrosodiethylamine (DEN)

N-nitrosodiethylamine is a widely occurring nitrosamine which is present in tobacco and various processed foods. These (nitroso) compounds can also be formed invivo in physiological conditions. N-nitrosodiethylamine (DEN), one of the most important environmental carcinogens in this class, primarily induces tumors of liver.

It is widely accepted that metabolic activation of nitrosamines by cytochrome P450 enzymes to reactive electrophiles is required for their cytotoxic, mutagenic and carcinogenic activity. Because of its relatively simple metabolic pathway and potent carcinogenic activity, DEN has found widespread use as an experimental model in the field of carcinogenesis and in chemoprevention.

Experimental design:The animals were divided into six groups and were treated as follows.

Group I: Normal control animals (received normal rat chow).Group II: Hepatoma induced animals (single intraperitoneal injection of DEN;

200mg/kg body weight in saline at 10th weeks of age. Two weeks after the administration of DEN, Phenobarbital (0.05%) was administered as the

promoter of carcinogenesis. The promoter was incorporated into the rat chow upto 14 successive weeks).Group III: Hepatoma induced animals (as in group 2) treated with Noni; throughout the

study. Noni was supplemented 20 weeks i.e., 4 weeks before the administration of DEN and 16 weeks after administration of DEN.

Group IV: Hepatoma induced animals (as in group 2) pre-treated with Noni for 4 weeks before the administration of DEN (as in group 4).

Group V: Hepatoma induced animals (as in group 2) post-treated with Noni (Noni was supplemented for 14 weeks in the promotion phase).

Group VI: control animals treated with Noni alone (for 14 weeks as in group 7).After the experimental period, the animals will be sadrificed by cervical decapitation. The liver and kidney will be immediately removed and washed in ice-cold saline and a portion of the tissues will be homogenised in 0.1 M, Tris-HCl buffer, pH 7.4 to give a 10% homogenate. It will be used for the estimation of the following biochemical parameters.

1. Biochemical assays

a. Cancer marker enzymes

Biochemical tumor markers viz. 5’nucleotidase, gamma glutamyl

transferase, aryl hydrocarbon hydroxylase, adenosine deaminase, lactate

dehydrogenase, -glucuronidase, shall be assayed using standard protocols.

b. Xenobiotic enzymes

Xenobiotic enzymes like cytochrome b5, cytochrome P450, NADPH

cytochrome C reductase and UDP glucuronyl transferase will be analyzed in

Hepatoma tissues using standard procedures.

c. Lipid peroxidation (LPO) and Antioxidant status

d. Antioxidant status

Antioxidant enzymes and low molecular weight antioxidants including

catalase, SOD, and GPx, GST, GR, GSH vitamin E and C

will be analyzed in Hepatoma tissues using standard

procedures.

e. Membrane bound enzymes

Erythrocyte membrane bound ATPases including Na+/K+ ATPase, Ca2+

ATPase and Mg+ ATPase will be assayed according to internationally

accepted protocols.

f. TCA Cycle enzymes

Mitochondrial enzymes like Isocitrate dehydrogenase, Malate

dehydrogenase, Succinate dehydrogenase, -Keto glutarate

dehydrogenase will be assayed in cancer bearing tissues.

g. Carbohydrate Metabolising enzymes

Glycolytic enzymes such as hexokinase, phosphogluco Isomerase,

Aldolase and Gluconeogenic enzymes will also be assayed using standard

protocols.

h. Protein profile of the blood and tissues will be

i. studied by Poly Acrylamide gel Electrophoresis (PAGE).

2. Histopathological studies

3. Expression of p53, Bcl2 using RT PCR.

4. DNA single-strand breaks will be observed by Comet assay.

5. DNA adduct formation will be estimated by measuring the

intracellular content of 8-hydroxy –2’deoxyguanosine (8OHdG) by

HPLC.

6. Apoptotic index will be assessed using DNA fragmentation assay.

7. Cell proliferation will be analyzed using PCNA by

Immunohistochemistry.

8. The level of caspase-3 will be analysed by ELISA and immunoblotting

techniques.

9. The expression of p53 and Bcl2 will be analysed by

Western Blotting.