Direct effect of croton oil on intestinal epithelial cells and colonic smooth muscle cells.
OBJECTIVE
To investigate the direct effect of croton oil (CO) on human intestinal epithelial cell (HIEC) and guinea pig colonic smooth muscle cells in vitro.


METHODS
Growth curves of HIEC were drawn by MTT colorimetry. The dynamics of cell proliferation was analyzed with flow cytometry, and morphological changes were observed under light and electron microscopy after long-term (6 weeks) treatment with CO. Expression of cyclooxygenase-2 (COX-2) mRNA was detected by dot blot in HIEC treated with CO. Genes related to CO were screened by DD-PCR, and the direct effect of CO on the contractility of isolated guinea pig colonic smooth muscle cells was observed.


RESULTS
High concentration (20-40 mg x L(-1)) CO inhibited cell growth significantly (1, 3, 5, 7d OD sequence: (20 mg x L(-1)) 0.040+/-0.003, 0.081+/-0.012, 0.147+/-0.022,0.024+/-0.016; (40 mg x L(-1)) 0.033+/-0.044, 0.056+/-0.012, 0.104+/-0.010, 0.189+/-0.006; OD control 0.031+/-0.008, 0.096+/-0.012, 0.173+/-0.009, 0.300+/-0.016, P<0.01), which appeared to be related directly to the dosage. Compared with the control, the fraction number of cells in G1 phase decreased from 0.60 to 0.58, while that in S phase increased from 0.30 to 0.34 and DNA index also increased after 6 weeks of treatment with CO (the dosage was increased gradually from 4 to 40 mg x L(-1)). Light microscopic observation revealed that cells had karyomegaly, less plasma and karyoplasm lopsidedness. Electron microscopy also showed an increase in cell proliferation and in the quantity of abnormal nuclei with pathologic mitosis. Expression of COX-2 mRNA decreased significantly in HIEC treated with CO. Thirteen differential cDNA fragments were cloned from HIEC treated with CO, one of which was 100 percent homologous with human mitochondrial cytochrome C oxidase subunit II. The length of isolated guinea pig colonic smooth muscle cells was significantly shortened after treatment with CO (P<0.05).


CONCLUSIONS
At a high CO concentration (>20 mg x L(-1)), cell growth and proliferation are inhibited in a dosage-dependent manner. Increase in cell proliferation and in malignant conversion of the cellular phenotype is observed in cells cultured chronically with CO. COX-2 mRNA expression decreases significantly, while human mitochondrial cytochrome C oxidase subunit IImRNA expression increases significantly in HIEC treated with CO. CO also has a direct effect on the contractility of Guinea pig colonic smooth muscle cells.
Wang
Xin
X
Institute of Digestive Disease, Xijing Hospital, Fourth Military Medical University,Xi'an 710033, Shaanxi Province,China.
Lan
Mei
M
Wu
Han-Ping
HP
Shi
Yong-Quan
YQ
Lu
Ju
J
Ding
Jie
J
Wu
Kai-Cun
KC
Jin
Jian-Ping
JP
Fan
Dai-Ming
DM
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003106:Colon / Q000166:cytology / Q000187:drug effects; D003436:Croton Oil / Q000494:pharmacology*; D051546:Cyclooxygenase 2; D003879:Dermatologic Agents / Q000494:pharmacology*; D005769:Gastrointestinal Motility / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006168:Guinea Pigs; D006801:Humans; D066298:In Vitro Techniques; D007413:Intestinal Mucosa / Q000166:cytology / Q000187:drug effects*; D007527:Isoenzymes / Q000235:genetics; D008565:Membrane Proteins; D009119:Muscle Contraction / Q000187:drug effects; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects*; D016133:Polymerase Chain Reaction; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis
D003879:Dermatologic Agents; D007527:Isoenzymes; D008565:Membrane Proteins; D012333:RNA, Messenger; D003436:Croton Oil; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases