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.
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- "are significantly induced in response to proteasome inhibitor bortezomib. The overexpression of heat shock proteins in response to proteasome inhibitor MG-132 or bortezomib was reported in a number of studies   . Interestingly, a number of proteins involved in the ubiquitin-proteasome system and/or protein catabolic processes were also significantly up-regulated after treatment with bortezomib. "
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: Bortezomib is a highly selective and reversible inhibitor of the 26S proteasome. It has been approved for the treatment of patients with relapsed and refractory multiple myeloma. A number of studies have been conducted to evaluate the activity and safety of bortezomib either alone or in combination with several cytotoxic agents and radiation. In the current study, the efficacy of bortezomib alone or in combination with cisplatin and 5‑fluorouracil was evaluated in 4T1 breast cancer cells, a highly metastatic murine cancer cell line. Using MTT assay, IC50 values of cisplatin and 5‑fluorouracil were determined to be 14.2 and 8.9 µM for cisplatin and 5‑fluorouracil, respectively. The effects of different concentrations of cisplatin and 5‑fluorouracil in combination with two different concentrations of bortezomib were examined in the 4T1 cells. Statistically significant differences were found when 1 or 5 µM cisplatin was combined with 10 or 50 nM bortezomib. Similarly, 1 µM 5‑fluorouracil or 5 µM 5‑fluorouracil in combination with 10 nM bortezomib caused significant cell death as compared to treatment with single agents. However, 1 or 5 µM 5‑fluorouracil did not potentiate the effects of higher concentrations of bortezomib (50 nM). The effect of the combination of cisplatin, 5‑fluorouracil and bortezomib was determined by soft agar assay. It was confirmed that a combination of cisplatin and bortezomib was more effective than each drug as a monotherapy. Therefore, the combination of cisplatin and bortezomib should be tested further in clinical settings.Molecular Medicine Reports 05/2013; DOI:10.3892/mmr.2013.1466 · 1.48 Impact Factor
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ABSTRACT: A greater reduction in cancer risk associated with mushroom diet rich in fungus polysaccharides is generally accepted. Meanwhile, edible Pleurotus abalonus as a member of Abalone mushroom family is a popular nutritional supplement that purportedly prevents cancer occurrence. However, these anecdotal claims are supported by limited studies describing tumor-inhibitory responses to the promising polysaccharides, and the molecular mechanisms underlying these properties have not yet been elucidated. We here fractionated the crude polysaccharide preparation from the fruiting bodies of P. abalonus into three fractions, namely PAP-1, PAP-2 and PAP-3, and tested these fractions for antiproliferative activity in human breast cancer MCF-7 cells. The largest PAP-3, an acidic polysaccharide fraction with a molecular mass of 3.68×10(5) Da, was the most active in inhibiting MCF-7 cancer cells with an IC50 of 193 µg/mL. The changes in cell normal morphology were observed by DAPI staining and the PAP-3-induced apoptosis was confirmed by annexin V/propidium iodide staining. The apoptosis was involved in mitochondria-mediated pathway including the loss of mitochondrial membrane potential (Δψm), the increase of Bax/Bcl-2 ratio, caspase-9/3 activation, and poly(ADP-ribose) polymerase (PARP) degradation, as well as intracellular ROS production. PAP-3 also induced up-regulation of p53, and cell cycle arrest at the S phase. The incubation of MCF-7 cells with antioxidant superoxide dismutase (SOD) and N-acetylcysteine (NAC) significantly attenuated the ROS generation and apoptosis caused by PAP-3, indicating that intracellular ROS plays a pivotal role in cell death. These findings suggest that the polysaccharides, especially acidic PAP-3, are very important nutritional ingredients responsible for, at least in part, the anticancer health benefits of P. abalonus via ROS-mediated mitochondrial apoptotic pathway. It is a major breakthrough bringing new insight of the potential use of the polysaccharides as health-care food or medicine to provide significant natural defense against human cancer.PLoS ONE 05/2013; 8(5):e64266. DOI:10.1371/journal.pone.0064266 · 3.53 Impact Factor