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Preparation of Mitochondria from Ovarian Cancer Tissues and Control Ovarian Tissues for Quantitative Proteomics Analysis
Published on: November 18, 2019
Quantitative proteomic analysis of mitochondria from human ovarian cancer cells and their paclitaxel-resistant
Ming Chen1, Hong Huang1,2, Haojie He1,3
1Department of Obstetrics and Gynecology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
Abstract:
Paclitaxel resistance is a major obstacle for the treatment of ovarian cancer. The chemoresistance mechanisms are partly related to the mitochondria. Identification of the relevant proteins in mitochondria will help in clarifying the possible mechanisms and in selecting effective chemotherapy for patients with paclitaxel resistance. In the present study, mitochondria from two paclitaxel-sensitive human ovarian cancer cell lines (SKOV3 and A2780) and their corresponding resistant cell lines (SKOV3-TR and A2780-TR) were isolated. Guanidine-modified acetyl-stable isotope labeling and liquid chromatography-hybrid linear ion trap Fourier-transform ion cyclotron resonance mass spectrometry (LC-FTICR MS) were performed to find the expressed differential proteins. Comparative proteomic analysis revealed eight differentially expressed proteins in the ovarian cancer cells and their paclitaxel-resistant sublines. Among them, mimitin and 14-3-3 ζ/δ were selected for further research. The effects of mimitin and 14-3-3 ζ/δ were explored using specific siRNA interference in ovarian cancer cell lines and immunohistochemistry in human tissue specimens. The downregulation of mimitin and 14-3-3 ζ/δ using specific siRNA in paclitaxel-resistant ovarian cancer cells led to an increase in the resistance index to paclitaxel. Multivariate analyses demonstrated that lower expression levels of the mimitin and 14-3-3 ζ/δ proteins were positively associated with shorter progression-free survival (PFS) and overall survival (OS) in patients with primary ovarian cancer (mimitin: PFS: P = 0.041, OS: P = 0.003; 14-3-3 ζ/δ: PFS: P = 0.031, OS: P = 0.011). Mimitin and 14-3-3 protein ζ/δ are potential markers of paclitaxel resistance and prognostic factors in ovarian cancer.
Insights
Mitochondrial proteins mimitin and 14-3-3 ζ/δ are linked to paclitaxel resistance in ovarian cancer. Lower levels of these proteins correlate with poorer patient survival, suggesting they are potential markers for resistance and prognosis.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Paclitaxel resistance is a significant challenge in ovarian cancer treatment.
- Mitochondria play a role in chemoresistance mechanisms.
- Identifying key mitochondrial proteins can elucidate resistance pathways.
Purpose of the Study:
- To identify differential mitochondrial proteins in paclitaxel-resistant ovarian cancer cells.
- To investigate the role of mimitin and 14-3-3 ζ/δ in paclitaxel resistance.
- To evaluate mimitin and 14-3-3 ζ/δ as prognostic markers in ovarian cancer.
Main Methods:
- Isolation of mitochondria from sensitive and resistant ovarian cancer cell lines.
- Proteomic analysis using stable isotope labeling and LC-FTICR MS.
- siRNA interference and immunohistochemistry to assess protein function and expression.
Main Results:
- Eight differentially expressed proteins were identified between sensitive and resistant cells.
- Downregulation of mimitin and 14-3-3 ζ/δ increased paclitaxel resistance.
- Lower expression of mimitin and 14-3-3 ζ/δ correlated with shorter progression-free and overall survival.
Conclusions:
- Mimitin and 14-3-3 ζ/δ are potential biomarkers for paclitaxel resistance in ovarian cancer.
- These proteins serve as prognostic factors for patient survival.
- Targeting these proteins may offer new therapeutic strategies for resistant ovarian cancer.
