The p53-independent induction of apoptosis in breast cancer cells in response to proteasome inhibitor bortezomib.
ABSTRACT An important hallmark of cancer cells is acquired resistance toward apoptosis. The apoptotic pathway is the most well-defined cell death program and is characterized by several morphological and biochemical features. The tumor suppressor protein p53 is a critical regulator of apoptosis in many cell types. p53 stimulates a wide network of signals that act through either extrinsic or intrinsic pathways of apoptosis. However, a number of studies have shown that apoptosis can be induced in a p53-independent manner as well. In this study, we examined the mechanism of apoptosis in p53-null breast cancer cells in response to the proteasome inhibitor bortezomib. Initially, we determined the p53 status of 4T1 breast carcinoma and 4THMpc (a highly mestatic derivative of 4T1) cells and verified that both cells are p53 deficient. It was subsequently shown that apoptosis can be induced in both cells in a dose-dependent manner in response to bortezomib treatment, based on DNA fragmentation evidence. Western blot analyses of ubiquitin-protein conjugates additionally showed that the proteasome is potently inhibited by bortezomib in p53-null 4T1 and 4THMpc cells. The results presented in the current study also show that caspase-3 is significantly activated in response to the treatment with bortezomib, implying that induction of apoptosis in these p53-deficient cells is occuring via caspase-3. The additional results presented here suggest that the pro-apoptotic proteins Bad, Noxa, and Puma are not critical regulators of apoptosis induction in p53-null 4T1 and 4THMpc cells. Similarly, there was no difference in the expression level of Mcl-1 in treated cells, suggesting that this anti-apoptotic protein is also uninvolved in the apoptotic response resulting from bortezomib treatment. In contrast, a very significant upregulation of the anti-apoptotic protein Hsp25/27 was detected in these p53-deficient cells after treatment with bortezomib. If the increased expression of Hsp25/27 protein levels are muting the apoptotic effects of the bortezomib treatment, then the apoptosis-inducing effects of such proteasome inhibitors might be increased with approaches simultaneously inhibiting Hsp25/27 protein in p53-deficient cells.
SourceAvailable from: Jaromir Gumulec[Show abstract] [Hide abstract]
ABSTRACT: Breast cancer patients negative for the nuclear oestrogen receptor α have a particularly poor prognosis. Therefore, the 4T1 cell line (considered as a triple-negative model) was chosen to induce malignancy in mice. The aim of the present study was to assess if zinc ions, provided in excess, may significantly modify the process of mammary oncogenesis. Zn(II) ions were chosen because of their documented antitumour effects. Zn(II) is also known to induce the expression of metallothioneins (MT) and glutathion (GSH). A total dose of zinc sulphate per one gram of mouse weight used in the experiment was 0.15 mg. We studied the expression of MT1, MT2, TP53 and MTF-1 genes and also examined the effect of the tumour on antioxidant capacity. Tumour-free mice had significantly higher expression levels of the studied genes (p<0.003). Significant differences were also revealed in the gene expression between the tissues (p<0.001). The highest expression levels were observed in the liver. As compared to brain, lung and liver, significantly lower concentrations of MT protein were found in the primary tumour; an inverse trend was observed in the concentration of Zinc(II). In non-tumour mice, the amount of hepatic hydrosulphuryl groups significantly increased by the exposure to Zn(II), but the animals with tumour induction showed no similar trend. The primary tumour size of zinc-treated animals was 20% smaller (p=0.002); however, no significant effect on metastasis progression due to the zinc treatment was discovered. In conclusion, Zn(II) itself may mute the growth of primary breast tumours especially at their early stages.International Journal of Oncology 02/2015; 46(4):1810-1818. DOI:10.3892/ijo.2015.2883 · 2.77 Impact Factor
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ABSTRACT: In order to determine the networks and biological functions associated with the identified proteins that were differentially expressed in response to 100 nM bortezomib treatment, the data were further analyzed using the Ingenuity Pathway Analysis (IPA) software. Bortezomib-mediated inhibition of the proteasome affected changes in the expression of proteins involved in post-translational modification, protein folding, DNA replication/repair/recombination, energy production and nucleic acid metabolism using IPA software. The merged network showed that ubiquitin (UBC gene encoding polyubiquitin-C, a precursor of ubiquitin protein) interacts with almost all proteins in the network. Interestingly, the proteasomal subunit Psmd14, a critical subunit for the development of stem cells as well, is found to be in direct contact with heat shock 70 kDa protein 1B, heat shock cognate 71 kDa, Psmc3 (a proteasomal subunit involved in diseases of meningitis and HIV-1) and also ubiquitin.Journal of Proteomics 10/2014; DOI:10.1016/j.jprot.2014.09.010 · 3.93 Impact Factor
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ABSTRACT: Suberoyl bis-hydroxamic acid (SBHA) is a histone deacetylase (HDAC) inhibitor and exerts anti-growth effects in several malignancies including breast cancer. Proteasome inhibitors such as Bortezomib and MG-132 constitute novel anticancer agents. In this study, we investigated the synergistic antitumour activity of SBHA in combination with proteasome inhibitors. MCF-7 and MDA-MB-231 breast cancer cells were treated with SBHA, Bortezomib, and MG-132 alone or in combination for 72 h. Cell proliferation, colony formation, apoptosis and gene expression changes were examined. SBHA, Bortezomib, and MG-132 alone significantly inhibited the proliferation and colony formation and induced apoptosis in MCF-7 and MDA-MB-231 cells. Combined treatment showed a good synergistic antitumour effect against breast cancer cells. The p53 protein level was significantly elevated by combined treatment with SBHA and proteasome inhibitors. Moreover, combined treatment increased the expression of Bax, Bcl-xS, and Bak and decreased the expression of Bcl-2. Combination of SBHA with proteasome inhibitors causes synergistic anticancer effects on breast cancer cells. The potential molecular mechanism may involve induction of p53 and modulation of the Bcl-2 family proteins. These findings warrant further investigation of the therapeutic benefits of combination of SBHA with proteasome inhibitors in breast cancer.Cancer Cell International 12/2014; 14(1):107. DOI:10.1186/s12935-014-0107-7 · 1.99 Impact Factor