Synergistic effect of 5-fluorouracil and the flavanoid oroxylin A on HepG2 human hepatocellular carcinoma and on H22 transplanted mice.
OBJECTIVE
To investigate the synergistic inhibitory effects of the combination of 5-fluorouracil (5-FU) with the natural flavanoid oroxylin A on human hepatocellular carcinoma cells HepG2 in vitro and on transplanted murine hepatoma 22 (H22) tumors in vivo and the preliminary mechanisms.


METHODS
The inhibitory effects of 5-FU combined with the natural flavanoid oroxylin A in vitro were detected by MTT assay and the effects in vivo were investigated by transplanted H22 mice model. DAPI staining and Annexin V/propidium iodide (PI) double staining were used to detect the cell morphological changes and apoptosis. The mRNA levels of thymidine synthetase (TS) and dihydropyrimidine dehydrogenase (DPD) in HepG2 cells after oroxylin A and 5-FU combination treatment were observed by quantitative real-time PCR. Western blotting assay was used to reveal the expressions of apoptotic-inducing proteins P53, cleaved PARP, COX-2, Bcl-2, and pro-caspase3.


RESULTS
Oroxylin A in combination with 5-FU presented synergistic effect (CI<1) on HepG2 cells in vitro when the inhibitory rate was higher than 7.5%. The inhibitory rate on H22 murine solid tumor in vivo in the combination group was higher than monotherapy. 5-FU combined with oroxylin A exerted stronger apoptotic induction in HepG2 cells than either single drug treatment. Quantitative real-time PCR discovered the downregulation of TS mRNA and DPD mRNA in HepG2 cells after combination treatment. Western blotting assay revealed oroxylin A enhanced 5-FU-induced apoptosis in HepG2 cells by elevating the expressions of apoptotic-inducing proteins P53 and cleaved PARP and decreasing the expression of apoptotic-inhibitory proteins COX-2, Bcl-2, and pro-caspase3.


CONCLUSIONS
The anti-hepatocellular carcinoma effects in vitro and in vivo of 5-FU and oroxylin A combinations were synergistic and oroxylin A increased the sensitivity of HepG2 cells to 5-FU by modulating the metabolic enzymes of 5-FU and apoptotic-related proteins.
Zhao
Li
L
Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, 24 Tongjiaxiang, 210009, Nanjing, China.
Chen
Zhen
Z
Wang
Jun
J
Yang
Li
L
Zhao
Qing
Q
Wang
Jia
J
Qi
Qi
Q
Mu
Rong
R
You
Qi-Dong
QD
Guo
Qing-Long
QL
D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D051546:Cyclooxygenase 2 / Q000378:metabolism; D042943:Dihydrouracil Dehydrogenase (NADP) / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D005419:Flavonoids / Q000008:administration & dosage / Q000737:chemistry / Q000494:pharmacology*; D005472:Fluorouracil / Q000008:administration & dosage / Q000494:pharmacology*; D006801:Humans; D008114:Liver Neoplasms, Experimental / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D008297:Male; D051379:Mice; D015394:Molecular Structure; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013940:Thymidylate Synthase / Q000235:genetics; D047368:Tumor Burden / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D005419:Flavonoids; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; C080669:5,7-dihydroxy-6-methoxy-2-phenylchromen-4-one; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D042943:Dihydrouracil Dehydrogenase (NADP); D013940:Thymidylate Synthase; D011065:Poly(ADP-ribose) Polymerases; D053148:Caspase 3; D005472:Fluorouracil