Background
Methods
Animals
Study design
Induction of colon carcinogenesis
Probiotic strains and probiotic dose
Preparation of celecoxib and dose
Chemicals
Experimental procedure
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Group I (Control): Animals were administered with 1 mM EDTA-saline, once a week i.p and 0.5% CMC daily, orally, via orogastric gavage, for 18 weeks.
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Group II (DMH): Animals received DMH (20 mg/kg) in 1 mM EDTA-saline i.p, once a week for 18 weeks.
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Group III (Celecoxib + DMH): These animals were given celecoxib (6 mg/kg) orally, via gavage, daily for one week. Thereafter, DMH was administered, once a week i.p for 18 weeks along with daily administration of celecoxib.
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Group IV (L.acidophilus + Celecoxib + DMH): Animals received L.acidophilus and celecoxib orally, via gavage, daily for one week. Thereafter, a single dose of DMH was given i.p, once a week for 18 weeks along with daily oral feeding of both probiotic and celecoxib.
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Group V (L.rhamnosus GG+ Celecoxib + DMH): Animals were administered with L.rhamnosus GG and celecoxib orally daily for one week. After that, a single dose of DMH was given, i.p once a week for 18 weeks along with daily oral administration of probiotic and celecoxib.
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Group VI (L.acidophilus + L.rhamnosus GG + Celecoxib + DMH): Animals were fed orally with L.acidophilus, L.rhamnosus GG and celecoxib orally, daily for one week. Thereafter, a single dose of DMH was given i.p once a week for 18 weeks along with daily administration of celecoxib and probiotics.
Follow up of the animals
Monitoring of tumor range, tumor incidence, tumor burden and tumor multiplicity
Apoptotic studies
DNA fragmentation
Colonocytes extraction
Determination of apoptotic cells by ethidium bromide/Acridine Orange staining
Caspase activity
Isolation of RNA
Synthesis of complementary DNA (cDNA)
Reverse transcriptase polymerase chain reaction (RT-PCR)
Assessment of apoptotic markers (Bcl-2 & Bax)
Proto-oncogene K-ras
Tumor suppressor gene p53
Statistical analysis
Results
Tumor range, tumor incidence, tumor burden and tumor multiplicity
Groups of animals | Tumor Range | Tumor Incidence (%) | Tumor Burden | Tumor Multiplicity |
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DMH | 4–7 | 100 | 5.8 | 5.8 |
celecoxib+DMH | 3–5 | 100 | 3.9 | 3.9 |
L.acidophilus + celecoxib +DMH | 3–4 | 100 | 3.5 | 3.5 |
L.rhamnosus GG + celecoxib + DMH | 1–3 | 100 | 2.3 | 2.3 |
L.acidophilus + L.rhamnosus GG+ celecoxib +DMH | 2–4 | 100 | 3.2 | 3.2 |