Proteomics is a highly informative approach to analyze cancer-associated transformation in tissues. The main challenge to use a tissue for proteomics studies is the small sample size and difficulties to extract and preserve proteins. The choice of a buffer compatible with proteomics applications is also a challenge. Here we describe a protocol optimized for the most efficient extraction of proteins from the human breast tissue in a buffer compatible with two-dimensional gel electrophoresis (2D-GE). This protocol is based on mechanically assisted disintegration of tissues directly in the 2D-GE buffer. Our method is simple, robust and easy to apply in clinical practice. We demonstrate high quality of separation of proteins prepared according to the reported here protocol.
Transforming growth factor-β (TGFβ) is a potent regulator of tumorigenesis, although mechanisms defining its tumor suppressing and tumor promoting activities are not understood. Here we describe phosphoproteome profiling of TGFβ signaling in mammary epithelial cells, and show that 60 identified TGFβ-regulated phosphoproteins form a network with scale-free characteristics. The network highlighted interactions, which may distribute signaling inputs to regulation of cell proliferation, metabolism, differentiation and cell organization. In this report, we identified two novel and TGFβ-dependent phosphorylation sites of 14-3-3σ, i.e. Ser69 and Ser74. We observed that 14-3-3σ phosphorylation is a feed-forward mechanism in TGFβ/Smad3-dependent transcription. TGFβ-dependent 14-3-3σ phosphorylation may provide a scaffold for the formation of the protein complexes which include Smad3 and p53 at the Smad3-specific CAGA element. Furthermore, breast tumor xenograft studies in mice and radiobiological assays showed that phosphorylation of 14-3-3σ at Ser69 and Ser74 is involved in regulation of cancer progenitor population and radioresistance in breast cancer MCF7 cells. Our data suggest that TGFβ-dependent phosphorylation of 14-3-3σ orchestrates a functional interaction of TGFβ/Smad3 with p53, plays a role in the maintenance of cancer stem cells and could provide a new potential target for intervention in breast cancer.
Background: Bladder cancer is the 4th most common cancer associated death in men in the western world. Cisplatin-based polychemotherapy (1st line) and vinflunine (2nd line) are the only chemotherapy regimens which show survival benefit in patients with metastatic disease. Hence, there is a major need to identify new effective treatment alternatives for patients with this disease. We have developed a prodrug of melphalan, Melphalan-flufenamide (Mel-flufen) which show enhanced cytotoxic efficacy compared to melphalan in several tumour types. Treatment with Mel-flufen gives 10–20 fold higher intracellular drug levels compared to the same dose of melphalan. Mel-flufen is currently undergoing Phase I/II clinical trials. Here we studied if this drug might be a useful in bladder cancer by analyzing its effects in a panel of bladder cancer cell lines. Material and Methods: The bladder cancer cell lines J82, RT4, 5637 and TCC-SUP were profiled for Mel-flufen and melphalan sensitivity using MTT and FMCA cell viability assay. Mel-flufen signaling was measured as caspase-3 and Bak/Bax activation in flow cytometry and PARP cleavage, JNK phosphorylation, p53 and p21WAF1/Cip1 induction on western blot respectively. Results: Mel-flufen caused a dose and time dependent inhibition of cell growth in all the four bladder cancer cell lines studied. Comparison with the parental drug revealed an enhanced cytotoxic effect of Mel-flufen at equimolar doses. Molecular profiling of the apoptotic response showed that Mel-flufen induced activation of the Bcl-2 proteins Bak and Bax followed by caspase-3 activation and induction of apoptotic morphology. Albeit Mel-flufen induced activation of p53, the cytotoxic effect was not dependent on p53 as it was also observed in p53-mutant bladder cancer cell lines. A prominent activation of JNK1/2 was observed in response to Melflufen. Importantly, this JNK activation preceded induction of Melflufen-induced apoptotic signaling and was critical for the induced cytotoxicity in these bladder cancer cell lines. Conclusion: Our data demonstrate that the melphalan prodrug Mel-flufen significantly inhibits bladder cancer cell growth in vitro. Importantly, we show that Mel-flufen is more efficient in inhibiting bladder cancer growth than the parental drug melphalan and causes a more pronounced apoptotic signaling. Taken together these data suggests that Mel-flufen may be a good candidate for bladder cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B40.
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