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ORIGINAL ARTICLE
Year : 2019  |  Volume : 30  |  Issue : 3  |  Page : 107-113

Label-free quantitative phosphoproteomics reveals the role of beta-estradiol in sunitinib-resistant renal cell carcinoma growth via perturbing transforming growing factor-beta pathway


1 School of Medicine, College of Medicine, Fu Jen Catholic University, New Taipei City, Taiwan
2 School of Medicine, College of Medicine, Fu Jen Catholic University; Department of Surgery, Sijhih Cathay General Hospital, New Taipei City, Taiwan
3 Division of Urology, Department of Surgery, Cathay General Hospital, Taipei, Taiwan
4 School of Medicine, College of Medicine, Fu Jen Catholic University; Division of Urology, Department of Surgery, Cathay General Hospital, Taipei, Taiwan

Correspondence Address:
Yen-Chieh Wang
Division of Urology, Department of Surgery, Cathay General Hospital, Taipei 10630
Taiwan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/UROS.UROS_129_18

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Context: Sunitinib is the first-line targeted therapy for metastatic renal cell carcinoma (RCC). However, resistance to sunitinib often occurred in patients receiving sunitinib treatment. On the other hand, 17-beta-estradiol (estrogen or E2) has been demonstrated to repress RCC growth in vitro, whether E2 can also affect the growth of sunitinib-resistant RCC remains unknown. Aims: In this study, the role of E2 in inhibiting sunitinib-resistant RCC growth and the underlining acting mechanisms was explored. Settings and Design: Sunitinib resistance was first induced in vitro in ACHN cells. The effect of E2 on cellular growth was then assayed. Label-free phosphoproteomics was also conducted. Subjects and Methods: ACHN cells were first challenged with 10-μM sunitinib up to 4 months to induce drug resistance. Then, E2 at different concentrations were tested in both parental and sunitinib-resistant ACHN cells. To conduct phosphoproteomics study, the total cell lysates from E2-treated ACHN cells were harvested, trypsin digested, and the phosphopeptides were enriched by Fe-IMAC. Statistical Analysis Used: For comparing the E2-induced cell growth inhibition, Student's t-test was used, and P < 0.05 was considered statistically significant. As for label-free phosphoproteomics, false discovery rate <0.01 and phosphosite possibility >0.75 were considered as positive identifications. Results: E2 at the physiological concentration, that is, 10 nM, can repress the cell growth in both parental and sunitinib-resistant ACHN cells. Further, label-free phosphoproteomics revealed that transforming growth factor beta (TGF-β) pathway, cell cycle, and cytoskeleton bindings were enhanced in sunitinib-resistant cells but can be reduced by E2 treatment. On the other hand, programmed cell death and apoptosis were repressed in sunitinib-resistant cells, and E2 at 10 nM did not reverse the effect. We further validated the expression of SMAD3, an important molecule in TGF-β pathway, and found that SMAD3 decreased in sunitinib-resistant cells but can be upregulated by E2 treatment. Conclusions: Our study demonstrated that E2 can inhibit the cell growth in sunitinib-resistant RCC cells at physiological concentration by upregulating SMAD3 in the TGF-β pathway, which may lead to growth inhibition in RCC.


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