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Loss of miR-200c up-regulates CYP1B1 and confers docetaxel resistance in renal cell carcinoma.

Chang I, Mitsui Y, Fukuhara S, Gill A, Wong DK, Yamamura S, Shahryari V, Tabatabai ZL, Dahiya R, Shin DM, Tanaka Y - Oncotarget (2015)

Bottom Line: Additionally, miR-200c, which is significantly down-regulated in RCC regulates CYP1B1 expression and activity.These results suggest that CYP1B1 up-regulation mediated by low miR-200c is one of the mechanisms underlying resistance of RCC cells to docetaxel.Therefore, expression of CYP1B1 and miR-200c in RCC may be useful as a prediction for docetaxel response.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Division of Urology, Veterans Affairs Medical Center, San Francisco, California, United States of America.

ABSTRACT
Despite high protein expression and enzymatic activity of cytochrome P450 1B1 (CYP1B1) in renal cell cancer (RCC), its functional significance has not been elucidated. Here we explored the functional role and regulatory mechanism of CYP1B1 in RCC. Reduction of CYP1B1 levels fail to prevent in vitro tumorigenicity such as proliferation, apoptosis, and cell cycle progression of RCC cells. Moreover, the expression levels are not associated with tumor type, stage, Fuhrman grade and 5-year survival probability after surgery. Instead, alteration of CYP1B1 expression regulates the chemosensitivity of RCC cells to docetaxel suggesting its critical contribution to the chemoresistance. Additionally, miR-200c, which is significantly down-regulated in RCC regulates CYP1B1 expression and activity. An inverse association was also observed between the expression levels of miR-200c and CYP1B1 protein in RCC tissues. Finally, alteration of miR-200c levels affects the chemosensitivity of RCC cells. Restoration of docetaxel resistance by exogenous expression of CYP1B1 in miR-200c-over-expressing cells indicates that CYP1B1 is a functional target of miR-200c. These results suggest that CYP1B1 up-regulation mediated by low miR-200c is one of the mechanisms underlying resistance of RCC cells to docetaxel. Therefore, expression of CYP1B1 and miR-200c in RCC may be useful as a prediction for docetaxel response.

No MeSH data available.


Related in: MedlinePlus

Change in CYP1B1 expression alters docetaxel resistance of RCC cells(A and B) After siRNA transfection, CYP1B1 protein expression was determined by Western blot (A) and enzyme activity was measured with P450-Glo assay (B) in A498 cells. *P < 0.05; **P < 0.01 (C and D) After CYP1B1 siRNA transfection, A498 cells were treated with docetaxel (5 μM) for 72 hrs. Apoptotic cell death was measured with annexin V-FITC/7-AAD staining (C) and cell survival was analyzed by MTS assay (D). *P < 0.05 (E and F) After plasmid transfection, CYP1B1 protein expression was determined by Western blot (E) and enzyme activity was measured with P450-Glo assay (F) in 786-O cells. *P < 0.05 (G and H) After CYP1B1 plasmid transfection, 786-O cells were treated with docetaxel (5 μM) for 72 hrs. Apoptotic cell death was measured with annexin V-FITC/7-AAD staining (G) and cell survival was analyzed by MTS assay (H). *P < 0.05; **P < 0.01.
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Figure 2: Change in CYP1B1 expression alters docetaxel resistance of RCC cells(A and B) After siRNA transfection, CYP1B1 protein expression was determined by Western blot (A) and enzyme activity was measured with P450-Glo assay (B) in A498 cells. *P < 0.05; **P < 0.01 (C and D) After CYP1B1 siRNA transfection, A498 cells were treated with docetaxel (5 μM) for 72 hrs. Apoptotic cell death was measured with annexin V-FITC/7-AAD staining (C) and cell survival was analyzed by MTS assay (D). *P < 0.05 (E and F) After plasmid transfection, CYP1B1 protein expression was determined by Western blot (E) and enzyme activity was measured with P450-Glo assay (F) in 786-O cells. *P < 0.05 (G and H) After CYP1B1 plasmid transfection, 786-O cells were treated with docetaxel (5 μM) for 72 hrs. Apoptotic cell death was measured with annexin V-FITC/7-AAD staining (G) and cell survival was analyzed by MTS assay (H). *P < 0.05; **P < 0.01.

Mentions: To determine whether the regulation of CYP1B1 levels affects chemosensitivity to docetaxel, we performed knockdown experiments using CYP1B1 specific siRNA in A498 cells, which express relatively high levels of endogenous CYP1B1 protein. Transfection with two different CYP1B1 siRNAs results in a dramatic reduction in endogenous levels of CYP1B1 mRNA (Supplementary Fig. S1A), protein (Fig. 2A) and enzyme activity (Fig. 2B). CYP1B1 knockdown significantly increased the cytotoxic effect of docetaxel (Fig. 2C) and decreased the survival rate of A498 cells (Fig. 2D). In reciprocal experiments, we examined whether CYP1B1 over-expression increased the chemoresistance of RCC cells to docetaxel. Transient transfection of a plasmid containing human CYP1B1 into 786-O cells, which express relatively low levels of endogenous CYP1B1 protein resulted in a significant increase in protein level (Fig. 2E) and enzyme activity compared to transfection of control vector (Fig. 2F). Over-expression of CYP1B1 reduced the cytotoxic effect of docetaxel (Fig. 2G) and as a consequence, the survival of 786-O cells was also enhanced (Fig. 2H). Collectively, these results indicate that CYP1B1 plays an important role in the response of RCC cells to docetaxel.


Loss of miR-200c up-regulates CYP1B1 and confers docetaxel resistance in renal cell carcinoma.

Chang I, Mitsui Y, Fukuhara S, Gill A, Wong DK, Yamamura S, Shahryari V, Tabatabai ZL, Dahiya R, Shin DM, Tanaka Y - Oncotarget (2015)

Change in CYP1B1 expression alters docetaxel resistance of RCC cells(A and B) After siRNA transfection, CYP1B1 protein expression was determined by Western blot (A) and enzyme activity was measured with P450-Glo assay (B) in A498 cells. *P < 0.05; **P < 0.01 (C and D) After CYP1B1 siRNA transfection, A498 cells were treated with docetaxel (5 μM) for 72 hrs. Apoptotic cell death was measured with annexin V-FITC/7-AAD staining (C) and cell survival was analyzed by MTS assay (D). *P < 0.05 (E and F) After plasmid transfection, CYP1B1 protein expression was determined by Western blot (E) and enzyme activity was measured with P450-Glo assay (F) in 786-O cells. *P < 0.05 (G and H) After CYP1B1 plasmid transfection, 786-O cells were treated with docetaxel (5 μM) for 72 hrs. Apoptotic cell death was measured with annexin V-FITC/7-AAD staining (G) and cell survival was analyzed by MTS assay (H). *P < 0.05; **P < 0.01.
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Related In: Results  -  Collection

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Figure 2: Change in CYP1B1 expression alters docetaxel resistance of RCC cells(A and B) After siRNA transfection, CYP1B1 protein expression was determined by Western blot (A) and enzyme activity was measured with P450-Glo assay (B) in A498 cells. *P < 0.05; **P < 0.01 (C and D) After CYP1B1 siRNA transfection, A498 cells were treated with docetaxel (5 μM) for 72 hrs. Apoptotic cell death was measured with annexin V-FITC/7-AAD staining (C) and cell survival was analyzed by MTS assay (D). *P < 0.05 (E and F) After plasmid transfection, CYP1B1 protein expression was determined by Western blot (E) and enzyme activity was measured with P450-Glo assay (F) in 786-O cells. *P < 0.05 (G and H) After CYP1B1 plasmid transfection, 786-O cells were treated with docetaxel (5 μM) for 72 hrs. Apoptotic cell death was measured with annexin V-FITC/7-AAD staining (G) and cell survival was analyzed by MTS assay (H). *P < 0.05; **P < 0.01.
Mentions: To determine whether the regulation of CYP1B1 levels affects chemosensitivity to docetaxel, we performed knockdown experiments using CYP1B1 specific siRNA in A498 cells, which express relatively high levels of endogenous CYP1B1 protein. Transfection with two different CYP1B1 siRNAs results in a dramatic reduction in endogenous levels of CYP1B1 mRNA (Supplementary Fig. S1A), protein (Fig. 2A) and enzyme activity (Fig. 2B). CYP1B1 knockdown significantly increased the cytotoxic effect of docetaxel (Fig. 2C) and decreased the survival rate of A498 cells (Fig. 2D). In reciprocal experiments, we examined whether CYP1B1 over-expression increased the chemoresistance of RCC cells to docetaxel. Transient transfection of a plasmid containing human CYP1B1 into 786-O cells, which express relatively low levels of endogenous CYP1B1 protein resulted in a significant increase in protein level (Fig. 2E) and enzyme activity compared to transfection of control vector (Fig. 2F). Over-expression of CYP1B1 reduced the cytotoxic effect of docetaxel (Fig. 2G) and as a consequence, the survival of 786-O cells was also enhanced (Fig. 2H). Collectively, these results indicate that CYP1B1 plays an important role in the response of RCC cells to docetaxel.

Bottom Line: Additionally, miR-200c, which is significantly down-regulated in RCC regulates CYP1B1 expression and activity.These results suggest that CYP1B1 up-regulation mediated by low miR-200c is one of the mechanisms underlying resistance of RCC cells to docetaxel.Therefore, expression of CYP1B1 and miR-200c in RCC may be useful as a prediction for docetaxel response.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Division of Urology, Veterans Affairs Medical Center, San Francisco, California, United States of America.

ABSTRACT
Despite high protein expression and enzymatic activity of cytochrome P450 1B1 (CYP1B1) in renal cell cancer (RCC), its functional significance has not been elucidated. Here we explored the functional role and regulatory mechanism of CYP1B1 in RCC. Reduction of CYP1B1 levels fail to prevent in vitro tumorigenicity such as proliferation, apoptosis, and cell cycle progression of RCC cells. Moreover, the expression levels are not associated with tumor type, stage, Fuhrman grade and 5-year survival probability after surgery. Instead, alteration of CYP1B1 expression regulates the chemosensitivity of RCC cells to docetaxel suggesting its critical contribution to the chemoresistance. Additionally, miR-200c, which is significantly down-regulated in RCC regulates CYP1B1 expression and activity. An inverse association was also observed between the expression levels of miR-200c and CYP1B1 protein in RCC tissues. Finally, alteration of miR-200c levels affects the chemosensitivity of RCC cells. Restoration of docetaxel resistance by exogenous expression of CYP1B1 in miR-200c-over-expressing cells indicates that CYP1B1 is a functional target of miR-200c. These results suggest that CYP1B1 up-regulation mediated by low miR-200c is one of the mechanisms underlying resistance of RCC cells to docetaxel. Therefore, expression of CYP1B1 and miR-200c in RCC may be useful as a prediction for docetaxel response.

No MeSH data available.


Related in: MedlinePlus