[show abstract][hide abstract] ABSTRACT: N-acetyl-L-cysteine (NAC) is commonly used to identify and test ROS inducers, and to inhibit ROS. In this study, we identified inhibition of proteasome inhibitors as a novel activity of NAC. Both NAC and catalase, another known scavenger of ROS, similarly inhibited ROS levels and apoptosis associated with H2O2. However, only NAC, but not catalase or another ROS scavenger Trolox was able to prevent effects linked to proteasome inhibition, such as protein stabilization, apoptosis and accumulation of ubiquitin conjugates. These observations suggest that NAC has a dual activity as an inhibitor of ROS and proteasome inhibitors. Recently, NAC was used as a ROS inhibitor to functionally characterize a novel anticancer compound, piperlongumine leading to its description as a ROS inducer. In contrast, our experiments showed that this compound depicts features of proteasome inhibitors including suppression of FOXM1, stabilization of cellular proteins, induction of ROS-independent apoptosis and enhanced accumulation of ubiquitin conjugates. In addition, NAC, but not catalase or Trolox interfered with the activity of piperlongumine, further supporting that piperlongumine is a proteasome inhibitor. Most importantly, we showed that NAC, but not other ROS scavengers, directly binds to proteasome inhibitors. To our knowledge, NAC is the first known compound that directly interacts with and antagonizes the activity of proteasome inhibitors. Altogether, our data suggest that as a result of the dual nature of NAC data interpretation might not be straightforward when NAC is utilized as an antioxidant to demonstrate ROS involvement in drug-induced apoptosis.
[show abstract][hide abstract] ABSTRACT: Tumor cells accumulate high level of reactive oxygen species (ROS) because they are metabolically more active than healthy cells. Elevated ROS levels increase tumorigenecity but also render cancer cells more vulnerable to oxidative stress than healthy cells. The oncogenic transcription factor Forkhead Box M1 (FOXM1), which is overexpressed in a wide range of human cancers, was reported to protect cancer cells from the adverse effects of oxidative stress by up-regulating the expression of scavenger enzymes. We therefore hypothesized that the combination of FOXM1 ablation and ROS inducers could selectively eradicate cancer cells. We show that RNA interface-mediated knockdown of FOXM1 further elevates intracellular ROS levels and increases sensitivity of cancer cells to ROS-mediated cell death after treatment with ROS inducers. We also report that the combination of ROS inducers with FOXM1/proteasome inhibitors induces robust apoptosis in different human cancer cells. In addition, we report evidence that the FOXM1/proteasome inhibitor bortezomib in combination with the ROS inducer β-phenylethyl isothiocyanate efficiently inhibits the growth of breast tumor xenografts in nude mice. We conclude that the combination of ROS inducers and FOXM1 inhibitors could be used as a therapeutic strategy to selectively eliminate cancer cells.
American Journal Of Pathology 05/2013; · 4.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Oncogenic transcription factor FOXM1 is overexpressed in the majority of human cancers. In addition, FOXM1 has been implicated in cell migration, invasion, angiogenesis and metastasis. The important role of FOXM1 in cancer affirms its significance for therapeutic intervention. Current data suggest that targeting FOXM1 in mono- or combination therapy may have promising therapeutic benefits for the treatment of cancer. However, challenges with the delivery of anti-FOXM1 siRNA to tumors and the absence of small molecules, which specifically inhibit FOXM1, are delaying the development of FOXM1 inhibitors as feasible anticancer drugs. In this review, we describe and summarize the efforts that have been made to target FOXM1 in cancer and the consequences of FOXM1 suppression in human cancer cells.
[show abstract][hide abstract] ABSTRACT: Nanoparticle-encapsulated thiazole antibiotic, thiostrepton, has been shown to be an effective agent for inhibiting tumor growth in solid tumor models through the inhibition of proteasomal activity by the induction of apoptosis in cancer cells. Here, we show the efficacy of thiostrepton-micelles in inhibiting tumor growth in a DEN/PB-induced liver cancer model. We also demonstrate an enhanced anticancer effect of the combination treatment of thiostrepton with bortezomib, another proteasome inhibitor in this liver cancer model.
[show abstract][hide abstract] ABSTRACT: Bortezomib is well-known for inducing cell death in cancer cells, specifically through the mechanism of proteasome inhibition. Thiostrepton, a thiazole antibiotic, has also been described for its proteasome inhibitory action, although differing slightly to bortezomib in the proteasomal site to which it is active. Previously we had shown the synergic effect of bortezomib and thiostrepton in breast cancer cells in vitro, where sub-apoptotic concentrations of both proteasome inhibitors resulted in synergic increase in cell death when combined as a treatment. Here, we administered such a combination to MDA-MB-231 xenograft tumors in vivo, and found that the effect of complementary proteasome inhibitors reduced tumor growth rates more efficiently than compared with when administered alone. Increased induction of apoptotic activity in tumors was found be associated with the growth inhibitory activity of combination treatment. Further examination additionally revealed that combination-treated tumors exhibited reduced proteasome activity, compared with non-treated and single drug-treated tumors. These data suggest that this drug combination may be useful as a therapy for solid tumors.
Cancer biology & therapy 02/2012; 13(3):184-9. · 3.29 Impact Factor
[show abstract][hide abstract] ABSTRACT: Irradiation and DNA-damaging chemotherapeutic agents are commonly used in anticancer treatments. Following DNA damage FOXM1 protein levels are often elevated. In this study, we sought to investigate the potential role of FOXM1 in programmed cell death induced by DNA-damage. Human cancer cells after FOXM1 suppression were subjected to doxorubicin or γ-irradiation treatment. Our findings indicate that FOXM1 downregulation by stable or transient knockdown using RNAi or by treatment with proteasome inhibitors that target FOXM1 strongly sensitized human cancer cells of different origin to DNA-damage-induced apoptosis. We showed that FOXM1 suppresses the activation of pro-apoptotic JNK and positively regulates anti-apoptotic Bcl-2, suggesting that JNK activation and Bcl-2 down-regulation could mediate sensitivity to DNA-damaging agent-induced apoptosis after targeting FOXM1. Since FOXM1 is widely expressed in human cancers, our data further support the fact that it is a valid target for combinatorial anticancer therapy.
PLoS ONE 01/2012; 7(2):e31761. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: The sensitivity of cancer cells to apoptosis induced by anticancer drugs in vitro may be a predictor of their sensitivity to these drugs in vivo. In this review I summarize recent data describing anticancer drug-induced apoptosis in human melanoma cells. Proteasome inhibitors alone, or in combination with other drugs, efficiently induce apoptosis in melanoma cells. It has been shown that apoptosis induced by proteasome inhibitors is linked to suppression of transcription factor FoxM1 and upregulation of the proapoptotic Noxa protein. In addition, proteasome inhibitors stabilize the antiapoptotic Mcl-1 protein, and its suppression leads to more robust apoptosis in melanoma cells. Drugs targeting B-Raf (BAY 54-9085) or IKKb (BMS-345541) have been tested in melanoma cell lines, and it has been shown that the proapoptotic activity of both drugs depends on the inhibition of NF-kB in melanoma cells. A synthetic analog of dsRNA in complex with a polycation stimulated autophagy via induction of dsRNA helicase MDA-5 followed by apoptosis that was partially modulated by Noxa. These data may provide important information needed for designing more efficient combinations of anticancer drugs against melanoma.
Current topics in medicinal chemistry 01/2012; 12(1):50-2. · 4.47 Impact Factor
[show abstract][hide abstract] ABSTRACT: As an oncogenic transcription factor, the Forkhead box protein M1 (FOXM1) is overexpressed in human tumors. FOXM1 promotes tumorigenesis by regulating genes associated with cell cycle progression and cell proliferation, and its inhibition in cell lines has been shown to sensitize cells to apoptosis. In this report, we examined the possibility of suppressing FOXM1 in tumors in vivo, through the administration of FoxM1-specific siRNA. Firstly, we determined the functionality of siRNA treatment in subcutaneous MDA-MB-231-luc breast cancer tumors. We found that upon encapsulation into a PEI-based delivery agent, fluorescently-labeled siRNA was retained within tumors when administered intratumorally. Injection of anti-luciferase siRNA was also able to suppress tumor-associated luciferase for at least 48 hours. More importantly, repeat administrations of PEI-encapsulated anti-FoxM1 siRNA resulted in the reduced expression of FOXM1 protein levels in tumors. In addition, both the protein levels and mRNA levels of cdc25B and Aurora B Kinase, transcriptional targets of FOXM1 were also reduced in tumors treated with anti-FoxM1 siRNA. p27, an indirect target of FOXM1 associated with growth inhibition was further found be increased in tumors treated with FoxM1-siRNA. Our data suggests that anti-FoxM1 siRNA can be functional when administered into tumors in an in vivo system, and that anti-FoxM1 siRNA holds potential as part of a therapy for cancer treatment.
[show abstract][hide abstract] ABSTRACT: Using mass spectrometric analysis we found that oncogenic transcription factor FOXM1 that is overexpressed in a majority of human cancers interacts with multifunctional protein NPM, which is also overexpressed in a variety of human tumors. Coimmunoprecipitation and glutathione S-transferase pull-down experiments demonstrated that NPM forms a complex with FOXM1 and also identified the regions responsible for their interaction. Immunofluorescence microscopy confirmed the interaction between FOXM1 and NPM in cancer and immortal cells. Furthermore, knockdown of NPM in immortal and cancer cells led to significant down-regulation of FOXM1 similar to its levels in normal cells, suggesting that NPM might modulate FOXM1 level. In addition, in OCI/AML3 leukemia cells where mutant NPM is localized in the cytoplasm we found that typically nuclear FOXM1 was predominantly co-localized with NPM in the cytoplasm, while NPM knockdown led to the disappearance of FOXM1 from the cytoplasm, suggesting that NPM may also determine intracellular localization of FOXM1. Knockdown of FOXM1 or NPM in MIA PaCa-2 pancreatic cancer cells inhibited anchorage-dependent and independent growth in cell culture, and tumor growth in nude mice. In addition, over-expression of FOXM1 reversed the effect of NPM knockdown in vitro. Our data suggest that in cancer cells NPM interacts with FOXM1 and their interaction is required for sustaining the level and localization of FOXM1. Targeting the interaction between FOXM1 and NPM by peptides or small molecules may represent a novel therapeutic strategy against cancer.
Journal of Biological Chemistry 12/2011; 286(48):41425-33. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Proteasome inhibitors stabilize numerous proteins by inhibiting their degradation. Previously we have demonstrated that proteasome inhibitors thiostrepton, MG132 and bortezomib paradoxically inhibit transcriptional activity and mRNA/protein expression of FOXM1. Here we demonstrate that, in addition to FOXM1, the same proteasome inhibitors also decrease mRNA and protein expression of NPM and ARF genes. These data suggest that proteasome inhibitors may suppress gene expression by stabilizing their transcriptional inhibitors.
[show abstract][hide abstract] ABSTRACT: Apoptosis has been widely accepted as the primary mechanism of drug-induced cell death. Recently, a second type of cell death pathway has been demonstrated: autophagy, also called programmed type II cell death. Autophagy is a highly regulated process, by which selected components of a cell are degraded. It primarily functions as a cell survival mechanism under stress. However, persistent stress can also promote extensive autophagy leading to cell death. Forkhead box M1 (FoxM1), an oncogenic transcription factor that is abundantly expressed in a wide range of human cancers. Here we evaluated the role of FoxM1 in sensitivity of human cancer cells to proteasome inhibitor-induced apoptosis and autophagy. We found that FoxM1 knockdown sensitized the human cancer cells to apoptotic cell death induced by proteasome inhibitors, such as, MG132, bortezomib and thiostrepton, while it did not affect the levels of autophagy following treatment with these drugs.
[show abstract][hide abstract] ABSTRACT: The thiazole antiobiotic, thiostrepton, has been found to induce cell death in cancer cells through proteasome inhibition. As a proteasome inhibitor, thiostrepton has also been shown to suppress the expression of FOXM1, the oncogenic forkhead transcription factor overexpressed in cancer cells. In this study, we explored the potential in vivo anticancer properties of thiostrepton, delivered through nanoparticle encapsulation to xenograft models of breast and liver cancer. We encapsulated thiostrepton into micelles assembled from amphiphilic lipid-PEG (polyethylene glycol) molecules, where thiostrepton is solubilized within the inner lipid compartment of the micelle. Upon assembly, hydrophobic thiostrepton molecules are solubilized into the lipid component of the micelle shell, formed through the self-assembly of amphipilic lipid-PEG molecules. Maximum accumulation of micelle-thiostrepton nanoparticles (100 nm in diameter, -16 mV in zeta potential) into tumors was found at 4 hours postadministration and was retained for at least 24 hours. Upon continuous treatment, we found that nanoparticle-encapsulated thiostrepton reduced tumor growth rates of MDA-MB-231 and HepG2 cancer xenografts. Furthermore, we show for the first time the in vivo suppression of the oncogenic FOXM1 after treatment with proteasome inhibitors. Immunoblotting and immunohistochemical staining also showed increased apoptosis in the treated tumors, as indicated by cleaved caspase-3 expression. Our data suggest that the thiazole antibiotic/proteasome inhibitor thiostrepton, when formulated into nanoparticles, may be highly suited as a nanomedicine for treating human cancer.
Molecular Cancer Therapeutics 09/2011; 10(12):2287-97. · 5.60 Impact Factor
[show abstract][hide abstract] ABSTRACT: Thiazole antibiotic, thiostrepton was recently identified as proteasome inhibitor. We investigated the therapeutic potential of the combination of thiostrepton and proteasome inhibitor bortezomib (Velcade) on various human tumor cell lines. Combination of sub-lethal concentrations of thiostrepton and bortezomib induced potent apoptosis and inhibition of long-term colony formation in a wide variety of human cancer cell lines. The synergistic relationship between thiostrepton and bortezomib combination was also quantitatively demonstrated by calculating their combination index values that were much lower than 1 in all studied cell lines. The synergy between these drugs was based on their proteasome inhibitory activities, because thiostrepton modification, thiostrepton methyl ester, which did not have intact quinaldic acid ring and did not inhibit proteasome activity failed to demonstrate any synergy in combination with bortezomib.
PLoS ONE 01/2011; 6(2):e17110. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Proteasome inhibitors are used against human cancer, but their mechanisms of action are not entirely understood. For example, the role of the tumor suppressor p53 is controversial. We reevaluated the role of p53 in proteasome inhibitor-induced apoptosis by using isogenic human cancer cell lines with different p53 status. We found that well-known proteasome inhibitors such as MG132 and bortezomib, as well as the recently discovered proteasome inhibitor thiostrepton, induced p53-independent apoptosis in human cancer cell lines that correlated with p53-independent induction of proapoptotic Noxa but not Puma protein. In addition, these drugs inhibited growth of several cancer cell lines independently of p53 status. Notably, thiostrepton induced more potent apoptosis in HepG2 cells with p53 knockdown than in parental cells with wild-type p53. Our data confirm that proteasome inhibitors generally induce p53-independent apoptosis in human cancer cells.
American Journal Of Pathology 01/2011; 178(1):355-60. · 4.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Thiopeptides are sulfur containing highly modified macrocyclic antibiotics with a central pyridine/tetrapyridine/dehydropiperidine ring with up to three thiazole substituents on positions 2, 3 and 6. Thiazole antibiotics with central pyridine nucleus have a macrocyclic loop connecting thiazole rings at position 2 and 3 described as ring A. In addition antibiotics with central tetrahydropyridine nucleus have a quinaldic acid macrocycle also connected to thiazole on position 2 described as ring B. We have demonstrated before that thiazole antibiotics thiostrepton and Siomycin A act as proteasome inhibitors in mammalian tumor cells. Here we decided to test whether other known thiazole antibiotics such as berninamycin, micrococcin P1 and P2, thiocillin and YM-266183 (lacking the quinaldic acid ring B) demonstrate this activity. We found that none of them act as proteasome inhibitors. Moreover, structural modification of thiostrepton to thiostrepton methyl ester (with open B ring) also did not demonstrate this activity. These data suggest that B ring of thiostrepton and Siomycin A that is absent in other thiazole antibiotics determines the proteasome inhibitory activity of these drugs.
Cancer biology & therapy 01/2011; 11(1):43-7. · 3.29 Impact Factor