Administration sequence-dependent antitumor effects of paclitaxel and 5-fluorouracil in the human gastric cancer cell line MKN45.
BACKGROUND
The clinical outcome of gastric cancer patients has been improved by combination of 5-fluorouracil (5-FU) and paclitaxel (PXL). However, the optimal schedule of this combination has not been determined.


METHODS
The efficacies of sequential administrations of 5-FU and PXL on the gastric cancer cell line MKN45 were investigated using a WST-8 colorimetric assay. The cell cycle distribution of each drug was evaluated by flow-cytometry. Furthermore, the mechanism of antitumor activity enhancement by the administration sequence was investigated by western blotting.


RESULTS
MKN45 cell growth was significantly inhibited by each drug in a dose- and time-dependent manner. The cytotoxicities of PXL followed by 5-FU were significantly greater than those of 5-FU followed by PXL. The flow-cytometric analysis revealed that PXL exposure caused viable cell accumulation in G2/M phase in a dose-dependent manner. Western blotting showed that PXL exposure followed by 5-FU up-regulated Chk1 and Wee1 protein expressions until PXL removal and 5-FU exposure, when these expressions gradually decreased to their basal levels. 14-3-3sigma protein expression was significantly up-regulated upon PXL treatment followed by 5-FU. Interestingly, Mad2 protein expression with PXL treatment followed by 5-FU gradually increased after the PXL removal and 5-FU exposure.


CONCLUSIONS
PXL followed by 5-FU administration may be the optimal sequence for treatment of gastric cancer. The enhanced viable cell accumulation after PXL pretreatment may be related to G2 arrest. After PXL removal and 5-FU exposure, the cells progressing to M phase may undergo cell death by mitotic catastrophe due to DNA damage caused by 5-FU exposure.
Toiyama
Yuji
Y
The Second Department of Surgery, Mie University School of Medicine, Tsu, Mie, Japan.
Tanaka
Koji
K
Konishi
Naomi
N
Mohri
Yasuhiko
Y
Tonouchi
Hitoshi
H
Miki
Chikao
C
Kusunoki
Masato
M
D048948:14-3-3 Proteins / Q000378:metabolism; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D000071877:Checkpoint Kinase 1; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry / Q000379:methods; D005472:Fluorouracil / Q000008:administration & dosage / Q000494:pharmacology*; D016195:G2 Phase / Q000187:drug effects; D006801:Humans; D064246:Mad2 Proteins; D009687:Nuclear Proteins / Q000378:metabolism; D017239:Paclitaxel / Q000008:administration & dosage / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D013997:Time Factors
D048948:14-3-3 Proteins; D000964:Antimetabolites, Antineoplastic; D000972:Antineoplastic Agents, Phytogenic; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; C499349:WEE1 protein, human; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017239:Paclitaxel; D005472:Fluorouracil