title: Vitamin D3 enhances the tumouricidal effects of 5-Fluorouracil through multipathway mechanisms in azoxymethane rat model of colon cancer.
abstract: BACKGROUND
  Vitamin D3 and its analogues have recently been shown to enhance the anti-tumour effects of 5- Fluorouracil (5-FU) both in vitro and in xenograft mouse model of colon cancer. This study measured the potential mechanism(s) by which vitamin D3 could synergise the tumouricidal activities of 5-FU in azoxymethane (AOM) rat model of colon cancer.
  
  
  METHODS
  Seventy-five male Wistar rats were divided equally into 5 groups: Control, AOM, AOM-treated by 5-FU (5-FU), AOM-treated by vitamin D3 (VitD3), and AOM-treated by 5-FU + vitamin D3 (5-FU/D). The study duration was 15 weeks. AOM was injected subcutaneously for 2 weeks (15 mg/kg/week). 5-FU was injected intraperitoneally in the 9th and 10th weeks post AOM (8 total injections were given: 12 mg/kg/day for 4 successive days, then 6 mg/kg every other day for another 4 doses) and oral vitamin D3 (500 IU/rat/day; 3 days/week) was given from week 7 post AOM till the last week of the study. The colons were collected following euthanasia for gross and histopathological examination. The expression of β-catenin, transforming growth factor-β1 (TGF-β1), TGF-β type 2 receptor (TGF-βR2), smad4, inducible nitric oxide synthase (iNOS), and heat shock protein-90 (HSP-90) proteins was measured by immunohistochemistry. In colonic tissue homogenates, quantitative RT-PCR was used to measure the mRNA expression of Wnt, β-catenin, Dickkopf-1 (DKK-1) and cyclooxygenase-2 (COX-2) genes, while ELISA was used to measure the concentrations of TGF-β1, HSP-90 and COX-2 proteins.
  
  
  RESULTS
  Monotherapy with 5-FU or vitamin D3 significantly decreased the number of grown tumours induced by AOM (P < 0.05); however, their combination resulted in more significant tumouricidal effects (P < 0.05) compared with monotherapy groups. Mechanistically, vitamin D3/5-FU co-therapy significantly decreased the expression of Wnt, β-catenin, iNOS, COX-2 and HSP-90 and significantly increased the expression of DKK-1, TGF-β1, TGF-βR2, smad4 (P < 0.05), in comparison with their corresponding monotherapy groups.
  
  
  CONCLUSIONS
  Vitamin D3 and 5-FU synergise together and exhibit better anticancer effects by modulating Wnt/β-catenin pathway, TGF-β1 signals, iNOS, COX-2 and HSP-90. Further studies are required to illustrate the clinical value of vitamin D supplementation during the treatment of colon cancer with 5-FU in human patients.
authors.affiliation: Laboratory Medicine Department, Faculty of Applied Medical Sciences, Umm Al-Qura University, Al-Abdeyah, PO Box 7607, Makkah, Kingdom of Saudi Arabia. bassem.refaat@yahoo.co.uk.
authors.forename: Bassem
authors.initials: B
authors.lastname: Refaat
authors.affiliation: Laboratory Medicine Department, Faculty of Applied Medical Sciences, Umm Al-Qura University, Al-Abdeyah, PO Box 7607, Makkah, Kingdom of Saudi Arabia. dr_adel_elshemy2006@yahoo.com.|Department of Pharmacology, Faculty of Medicine, Assiut University, Assiut, Egypt. dr_adel_elshemy2006@yahoo.com.
authors.forename: Adel Galal
authors.initials: AG
authors.lastname: El-Shemi
authors.affiliation: Clinical Nutrition Department, Faculty of Applied Medical Sciences, Umm Al-Qura University, Al-Abdeyah, PO Box 7607, Makkah, Kingdom of Saudi Arabia. o.kensara@gmail.com.
authors.forename: Osama Adnan
authors.initials: OA
authors.lastname: Kensara
authors.affiliation: Laboratory Medicine Department, Faculty of Applied Medical Sciences, Umm Al-Qura University, Al-Abdeyah, PO Box 7607, Makkah, Kingdom of Saudi Arabia. amrmohamed2004@yahoo.com.|Clinical Laboratory Diagnosis, Department of Animal Medicine, Faculty of Veterinary Medicine, Assiut University, Assiut, 71526, Egypt. amrmohamed2004@yahoo.com.
authors.forename: Amr Mohamed
authors.initials: AM
authors.lastname: Mohamed
authors.affiliation: Laboratory Medicine Department, Faculty of Applied Medical Sciences, Umm Al-Qura University, Al-Abdeyah, PO Box 7607, Makkah, Kingdom of Saudi Arabia. shakir_sust@hotmail.com.
authors.forename: Shakir
authors.initials: S
authors.lastname: Idris
authors.affiliation: Laboratory Medicine Department, Faculty of Applied Medical Sciences, Umm Al-Qura University, Al-Abdeyah, PO Box 7607, Makkah, Kingdom of Saudi Arabia. jawad7776@gmail.com.
authors.forename: Jawwad
authors.initials: J
authors.lastname: Ahmad
authors.affiliation: Laboratory Medicine Department, Faculty of Applied Medical Sciences, Umm Al-Qura University, Al-Abdeyah, PO Box 7607, Makkah, Kingdom of Saudi Arabia. athar.h.k@hotmail.com.
authors.forename: Athar
authors.initials: A
authors.lastname: Khojah
mesh_terms: D000818:Animals; D002762:Cholecalciferol / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000627:therapeutic use*; D003110:Colonic Neoplasms / Q000188:drug therapy*; D004195:Disease Models, Animal; D005472:Fluorouracil / Q000008:administration & dosage / Q000627:therapeutic use*; D006801:Humans; D008297:Male; D051379:Mice; D051381:Rats; D017208:Rats, Wistar; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D023041:Xenograft Model Antitumor Assays; D051176:beta Catenin / Q000378:metabolism*
chemical_list: D016212:Transforming Growth Factor beta; D051176:beta Catenin; D002762:Cholecalciferol; D005472:Fluorouracil