The p53-independent induction of apoptosis in breast cancer cells in response to proteasome inhibitor bortezomib.

Art and Science Faculty, Department of Biology, Dumlupınar University, Kütahya, Turkey, .
Tumor Biology (Impact Factor: 2.52). 04/2012; 33(5):1385-92. DOI: 10.1007/s13277-012-0386-3
Source: PubMed

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.

1 Bookmark
  • [Show abstract] [Hide abstract]
    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; · 1.17 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The ubiquitin proteasome pathway is the most significant intracellular proteolytic pathway. The target proteins are usually ubiquitinated prior to degradation by the proteasome; however, ubiquitin-independent targeting mechanisms have also been reported (e.g., antizyme-mediated degradation of ornithine decarboxylase). Aberrations in the components of the ubiquitin proteasome pathway are commonly observed in many cancers. Uncontrolled growth of cancer cells can results either from stabilization of oncoproteins (e.g., c-jun) or increased degradation of tumor suppressor proteins (e.g., p53). In addition, due to the pleiotropic functions of ubiquitin proteasome pathway in cells, there is of great interest in developing inhibitors blocking specifically and potently this pathway for cancer treatment. This review summarize the recent literature and several patented inventions on the ubiquitin proteasome pathway with respect to its role in cancer development and treatment.
    Recent Patents on Anti-Cancer Drug Discovery 06/2013; 8(3):298-309. · 2.70 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Toll-like receptor-9 (TLR9) is an intracellular DNA receptor that is widely expressed in breast and other cancers. We previously demonstrated that low tumor TLR9 expression upon diagnosis is associated with significantly shortened disease-specific survival times in patients with triple-negative breast cancer (TNBC). There are no targeted therapies for this subgroup of patients whose prognosis is among the worst in breast cancer. Due to the previously detected in vitro anti-invasive effects of chloroquine in these cell lines, the present study aimed to investigate the in vivo effects of chloroquine against two clinical subtypes of TNBC that differ in TLR9 expression. Chloroquine suppressed matrix metalloproteinase (MMP)-2 and MMP-9 mRNA expression and protein activity, whereas MMP-13 mRNA expression and proteolytic activity were increased. Despite enhancing TLR9 mRNA expression, chloroquine suppressed TLR9 protein expression in vitro. Daily treatment of mice with intraperitoneal (i.p.) chloroquine (80 mg/kg/day) for 22 days, did not inhibit the growth of control siRNA or TLR9 siRNA MDA-MB-231 breast cancer cells. In conclusion, despite the favorable in vitro effects on TNBC invasion and viability, particularly in hypoxic conditions, chloroquine does not prevent the growth of the triple-negative MDA-MB-231 cells with high or low TLR9 expression levels in vivo. This may be explained by the activating effects of chloroquine on MMP-13 expression or by the fact that chloroquine, by suppressing TLR9 expression, permits the activation of currently unknown molecular pathways, which allow the aggressive behavior of TNBC cells with low TLR9 expression in hypoxia.
    Oncology letters 12/2013; 6(6):1665-1672. · 0.24 Impact Factor