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1.  Biodistribution and Efficacy Studies of the Proteasome Inhibitor BSc2118 in a Mouse Melanoma Model 
Translational Oncology  2014;7(5):570-579.
Inhibition of the proteasome offers many therapeutic possibilities in inflammation as well as in neoplastic diseases. However, clinical use of proteasome inhibitors is limited by the development of resistance or severe side effects. In our study we characterized the anti-tumor properties of the novel proteasome inhibitor BSc2118. The sensitivity of tumor lines to BSc2118 was analyzed in comparison to bortezomib using crystal violet staining in order to assess cell viability. The In Vivo distribution of BSc2118 in mouse tissues was tracked by a fluorescent-modified form of BSc2118 (BSc2118-FL) and visualized by confocal microscopy. Inhibition of the 20S proteasome was monitored both in cultured cell lines and in mice, respectively. Finally, safety and efficacy of BSc2118 was evaluated in a mouse melanoma model. BSc2118 inhibits proliferation of different tumor cell lines with a similar potency as compared with bortezomib. Systemic administration of BSc2118 in mice is well tolerated, even when given in a dose of 60 mg/kg body weight. After systemic injection of BSc2118 or bortezomib similar proteasome inhibition patterns are observed within the murine organs. Detection of BSc2118-FL revealed correlation of distribution pattern of BSc2118 with inhibition of proteasomal activity in cells or mouse tissues. Finally, administration of BSc2118 in a mouse melanoma model shows significant local anti-tumor effects. Concluding, BSc2118 represents a novel low-toxic agent that might be alternatively used for known proteasome inhibitors in anti-cancer treatment.
doi:10.1016/j.tranon.2014.07.002
PMCID: PMC4225687  PMID: 25389452
2.  Peroxiredoxin-1 protects estrogen receptor α from oxidative stress-induced suppression and is a protein biomarker of favorable prognosis in breast cancer 
Introduction
Peroxiredoxin-1 (PRDX1) is a multifunctional protein, acting as a hydrogen peroxide (H2O2) scavenger, molecular chaperone and immune modulator. Although differential PRDX1 expression has been described in many tumors, the potential role of PRDX1 in breast cancer remains highly ambiguous. Using a comprehensive antibody-based proteomics approach, we interrogated PRDX1 protein as a putative biomarker in estrogen receptor (ER)-positive breast cancer.
Methods
An anti-PRDX1 antibody was validated in breast cancer cell lines using immunoblotting, immunohistochemistry and reverse phase protein array (RPPA) technology. PRDX1 protein expression was evaluated in two independent breast cancer cohorts, represented on a screening RPPA (n = 712) and a validation tissue microarray (n = 498). In vitro assays were performed exploring the functional contribution of PRDX1, with oxidative stress conditions mimicked via treatment with H2O2, peroxynitrite, or adenanthin, a PRDX1/2 inhibitor.
Results
In ER-positive cases, high PRDX1 protein expression is a biomarker of improved prognosis across both cohorts. In the validation cohort, high PRDX1 expression was an independent predictor of improved relapse-free survival (hazard ratio (HR) = 0.62, 95% confidence interval (CI) = 0.40 to 0.96, P = 0.032), breast cancer-specific survival (HR = 0.44, 95% CI = 0.24 to 0.79, P = 0.006) and overall survival (HR = 0.61, 95% CI = 0.44 to 0.85, P = 0.004). RPPA screening of cancer signaling proteins showed that ERα protein was upregulated in PRDX1 high tumors. Exogenous H2O2 treatment decreased ERα protein levels in ER-positive cells. PRDX1 knockdown further sensitized cells to H2O2- and peroxynitrite-mediated effects, whilst PRDX1 overexpression protected against this response. Inhibition of PRDX1/2 antioxidant activity with adenanthin dramatically reduced ERα levels in breast cancer cells.
Conclusions
PRDX1 is shown to be an independent predictor of improved outcomes in ER-positive breast cancer. Through its antioxidant function, PRDX1 may prevent oxidative stress-mediated ERα loss, thereby potentially contributing to maintenance of an ER-positive phenotype in mammary tumors. These results for the first time imply a close connection between biological activity of PRDX1 and regulation of estrogen-mediated signaling in breast cancer.
doi:10.1186/bcr3691
PMCID: PMC4226972  PMID: 25011585
3.  Statins impair glucose uptake in human cells 
Objective
Considering the increasing number of clinical observations indicating hyperglycemic effects of statins, this study was designed to measure the influence of statins on the uptake of glucose analogs by human cells derived from liver, adipose tissue, and skeletal muscle.
Design
Flow cytometry and scintillation counting were used to measure the uptake of fluorescently labeled or tritiated glucose analogs by differentiated visceral preadipocytes, skeletal muscle cells, skeletal muscle myoblasts, and contact-inhibited human hepatocellular carcinoma cells. A bioinformatics approach was used to predict the structure of human glucose transporter 1 (GLUT1) and to identify the presence of putative cholesterol-binding (cholesterol recognition/interaction amino acid consensus (CRAC)) motifs within this transporter. Mutagenesis of CRAC motifs in SLC2A1 gene and limited proteolysis of membrane GLUT1 were used to determine the molecular effects of statins.
Results
Statins significantly inhibit the uptake of glucose analogs in all cell types. Similar effects are induced by methyl-β-cyclodextrin, which removes membrane cholesterol. Statin effects can be rescued by addition of mevalonic acid, or supplementation with exogenous cholesterol. Limited proteolysis of GLUT1 and mutagenesis of CRAC motifs revealed that statins induce conformational changes in GLUTs.
Conclusions
Statins impair glucose uptake by cells involved in regulation of glucose homeostasis by inducing cholesterol-dependent conformational changes in GLUTs. This molecular mechanism might explain hyperglycemic effects of statins observed in clinical trials.
doi:10.1136/bmjdrc-2014-000017
PMCID: PMC4212557  PMID: 25452863
Glucose Uptake; GLUT1; Pharmacological Therapy
4.  PHOTODYNAMIC THERAPY OF CANCER: AN UPDATE 
Photodynamic therapy (PDT) is a clinically approved, minimally invasive therapeutic procedure that can exert a selective cytotoxic activity toward malignant cells. The procedure involves administration of a photosensitizing agent followed by irradiation at a wavelength corresponding to an absorbance band of the sensitizer. In the presence of oxygen, a series of events lead to direct tumor cell death, damage to the microvasculature and induction of a local inflammatory reaction. Clinical studies revealed that PDT can be curative particularly in early-stage tumors. It can prolong survival in inoperable cancers and significantly improve quality of life. Minimal normal tissue toxicity, negligible systemic effects, greatly reduced long-term morbidity, lack of intrinsic or acquired resistance mechanisms, and excellent cosmetic as well as organ function-sparing effects of this treatment make it a valuable therapeutic option for combination treatments. With a number of recent technological improvements, PDT has the potential to become integrated into the mainstream of cancer treatment.
doi:10.3322/caac.20114
PMCID: PMC3209659  PMID: 21617154
photodynamic therapy; laser; photosensitizer; cancer; singlet oxygen
5.  Statins Impair Glucose Uptake in Tumor Cells1 
Neoplasia (New York, N.Y.)  2012;14(4):311-323.
Statins, HMG-CoA reductase inhibitors, are used in the prevention and treatment of cardiovascular diseases owing to their lipid-lowering effects. Previous studies revealed that, by modulating membrane cholesterol content, statins could induce conformational changes in cluster of differentiation 20 (CD20) tetraspanin. The aim of the presented study was to investigate the influence of statins on glucose transporter 1 (GLUT1)-mediated glucose uptake in tumor cells. We observed a significant concentration- and time-dependent decrease in glucose analogs' uptake in several tumor cell lines incubated with statins. This effect was reversible with restitution of cholesterol synthesis pathway with mevalonic acid as well as with supplementation of plasma membrane with exogenous cholesterol. Statins did not change overall GLUT1 expression at either transcriptional or protein levels. An exploratory clinical trial revealed that statin treatment decreased glucose uptake in peripheral blood leukocytes and lowered 18F-fluorodeoxyglucose (18F-FDG) uptake by tumor masses in a mantle cell lymphoma patient. A bioinformatics analysis was used to predict the structure of human GLUT1 and to identify putative cholesterol-binding motifs in its juxtamembrane fragment. Altogether, the influence of statins on glucose uptake seems to be of clinical significance. By inhibiting 18F-FDG uptake, statins can negatively affect the sensitivity of positron emission tomography, a diagnostic procedure frequently used in oncology.
PMCID: PMC3349257  PMID: 22577346
6.  Proteasome inhibition potentiates antitumor effects of photodynamic therapy in mice through induction of ER stress and unfolded protein response 
Cancer research  2009;69(10):4235-4243.
Photodynamic therapy (PDT) is an approved therapeutic procedure that exerts cytotoxic activity towards tumor cells by inducing production of reactive oxygen species such as singlet oxygen. PDT leads to oxidative damage of cellular macromolecules, including numerous proteins that undergo multiple modifications such as fragmentation, cross-linking and carbonylation that result in protein unfolding and aggregation. Since the major mechanism for elimination of carbonylated proteins is their degradation by proteasomes, we hypothesized that a combination of PDT with proteasome inhibitors might lead to accumulation of carbonylated proteins in endoplasmatic reticulum (ER), aggravated ER stress and potentiated cytotoxicity towards tumor cells. Indeed, we observed that Photofrin-mediated PDT leads to robust carbonylation of cellular proteins and induction of unfolded protein response (UPR). Pre-treatment of tumor cells with three different proteasome inhibitors, including bortezomib, MG132 and PSI gave increased accumulation of carbonylated and ubiquitinated proteins in PDT-treated cells. Proteasome inhibitors effectively sensitized tumor cells of murine (EMT6 and C-26) as well as human (HeLa) origin to PDT-mediated cytotoxicity. Significant retardation of tumor growth with 60-100% complete responses was observed in vivo in two different murine tumor models (EMT6 and C-26) when PDT was combined with either bortezomib or PSI. Altogether these observations indicate that combination of PDT with proteasome inhibitors leads to potentiated antitumor effects. The results of these studies are of immediate clinical application as bortezomib is a clinically approved drug that undergoes extensive clinical evaluations for the treatment of solid tumors.
doi:10.1158/0008-5472.CAN-08-3439
PMCID: PMC2785802  PMID: 19435917
photodynamic therapy; Photofrin; proteasome; bortezomib; cancer
7.  Zinc protoporphyrin IX, a heme oxygenase-1 inhibitor, demonstrates potent antitumor effects but is unable to potentiate antitumor effects of chemotherapeutics in mice 
BMC Cancer  2008;8:197.
Background
HO-1 participates in the degradation of heme. Its products can exert unique cytoprotective effects. Numerous tumors express high levels of HO-1 indicating that this enzyme might be a potential therapeutic target. In this study we decided to evaluate potential cytostatic/cytotoxic effects of zinc protoporphyrin IX (Zn(II)PPIX), a selective HO-1 inhibitor and to evaluate its antitumor activity in combination with chemotherapeutics.
Methods
Cytostatic/cytotoxic effects of Zn(II)PPIX were evaluated with crystal violet staining and clonogenic assay. Western blotting was used for the evaluation of protein expression. Flow cytometry was used to evaluate the influence of Zn(II)PPIX on the induction of apoptosis and generation of reactive oxygen species. Knock-down of HO-1 expression was achieved with siRNA. Antitumor effects of Zn(II)PPIX alone or in combination with chemotherapeutics were measured in transplantation tumor models.
Results
Zn(II)PPIX induced significant accumulation of reactive oxygen species in tumor cells. This effect was partly reversed by administration of exogenous bilirubin. Moreover, Zn(II)PPIX exerted potent cytostatic/cytotoxic effects against human and murine tumor cell lines. Despite a significant time and dose-dependent decrease in cyclin D expression in Zn(II)PPIX-treated cells no accumulation of tumor cells in G1 phase of the cell cycle was observed. However, incubation of C-26 cells with Zn(II)PPIX increased the percentage of cells in sub-G1 phase of the cells cycle. Flow cytometry studies with propidium iodide and annexin V staining as well as detection of cleaved caspase 3 by Western blotting revealed that Zn(II)PPIX can induce apoptosis of tumor cells. B16F10 melanoma cells overexpressing HO-1 and transplanted into syngeneic mice were resistant to either Zn(II)PPIX or antitumor effects of cisplatin. Zn(II)PPIX was unable to potentiate antitumor effects of 5-fluorouracil, cisplatin or doxorubicin in three different tumor models, but significantly potentiated toxicity of 5-FU and cisplatin.
Conclusion
Inhibition of HO-1 exerts antitumor effects but should not be used to potentiate antitumor effects of cancer chemotherapeutics unless procedures of selective tumor targeting of HO-1 inhibitors are developed.
doi:10.1186/1471-2407-8-197
PMCID: PMC2478682  PMID: 18620555
8.  A NOVEL FUNCTION OF VCP (VALOSIN CONTAINING PROTEIN; P97) IN THE CONTROL OF N-GLYCOSYLATION OF PROTEINS IN THE ENDOPLASMIC RETICULUM 
Summary
α chain of T-cell receptor (TCR) is a typical ERAD (ER-associated degradation) substrate degraded in the absence of other TCR subunits. Depletion of derlin1 fails to induce accumulation of αTCR despite inducing accumulation of α1-antitrypsin, another ERAD substrate. Furthermore, while depletion of VCP does not affect levels of α1-antitrypsin, it induces an increase in levels of αTCR. RNAi of VCP induces preferential accumulation of αTCR with less mannose residues, suggesting its retention within the ER. Mass spectrometric analysis of cellular N-linked glycans revealed that depletion of VCP decreases the level of high mannose glycoproteins, increases the levels of truncated low-mannose glycoproteins and induces changes in the abundance of complex glycans assembled in post-ER compartments. Since proteasome inhibition was unable to mimic those changes, they can not be regarded as a simple consequence of inhibited ERAD but represent a complex effect of VCP on the function of the ER.
doi:10.1016/j.abb.2007.04.010
PMCID: PMC2040342  PMID: 17493577
T-cell receptor; ER-associated degradation (ERAD); valosin-containing protein (VCP); derlin; retrotranslocation; ubiquitin; proteasome; protein degradation
9.  Statins Impair Antitumor Effects of Rituximab by Inducing Conformational Changes of CD20 
PLoS Medicine  2008;5(3):e64.
Background
Rituximab is used in the treatment of CD20+ B cell lymphomas and other B cell lymphoproliferative disorders. Its clinical efficacy might be further improved by combinations with other drugs such as statins that inhibit cholesterol synthesis and show promising antilymphoma effects. The objective of this study was to evaluate the influence of statins on rituximab-induced killing of B cell lymphomas.
Methods and Findings
Complement-dependent cytotoxicity (CDC) was assessed by MTT and Alamar blue assays as well as trypan blue staining, and antibody-dependent cellular cytotoxicity (ADCC) was assessed by a 51Cr release assay. Statins were found to significantly decrease rituximab-mediated CDC and ADCC of B cell lymphoma cells. Incubation of B cell lymphoma cells with statins decreased CD20 immunostaining in flow cytometry studies but did not affect total cellular levels of CD20 as measured with RT-PCR and Western blotting. Similar effects are exerted by other cholesterol-depleting agents (methyl-β-cyclodextrin and berberine), but not filipin III, indicating that the presence of plasma membrane cholesterol and not lipid rafts is required for rituximab-mediated CDC. Immunofluorescence microscopy using double staining with monoclonal antibodies (mAbs) directed against a conformational epitope and a linear cytoplasmic epitope revealed that CD20 is present in the plasma membrane in comparable amounts in control and statin-treated cells. Atomic force microscopy and limited proteolysis indicated that statins, through cholesterol depletion, induce conformational changes in CD20 that result in impaired binding of anti-CD20 mAb. An in vivo reduction of cholesterol induced by short-term treatment of five patients with hypercholesterolemia with atorvastatin resulted in reduced anti-CD20 binding to freshly isolated B cells.
Conclusions
Statins were shown to interfere with both detection of CD20 and antilymphoma activity of rituximab. These studies have significant clinical implications, as impaired binding of mAbs to conformational epitopes of CD20 elicited by statins could delay diagnosis, postpone effective treatment, or impair anti-lymphoma activity of rituximab.
Jakub Golab and colleagues found that statins significantly decrease rituximab-mediated complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity against B cell lymphoma cells.
Editors' Summary
Background.
Lymphomas are common cancers of the lymphatic system, the tissues and organs that produce and store the white blood cells (lymphocytes) that fight infections. In healthy people, the cells in the lymph nodes (collections of lymphocytes in the armpit, groin, and neck) and other lymphatic organs divide to form new cells only when the body needs them. Lymphomas form when a T or B lymphocyte starts to divide uncontrollably. The first sign of lymphoma is often a painless swelling in the armpit, groin, or neck caused by lymphocyte overgrowth in a lymph node. Eventually, the abnormal (malignant) lymphocytes, which provide no protection against infectious diseases, spread throughout the body. Treatments for lymphoma include chemotherapy (drugs that kill rapidly dividing cells) and radiotherapy. In addition, a drug called rituximab was recently developed for the treatment of some types of B cell lymphoma. Rituximab is a monoclonal antibody, a laboratory-produced protein. It binds to a protein called CD20 that is present on the surface of both normal and malignant B lymphocytes and induces cell killing through processes called “complement-dependent cytotoxity” (CDC) and “antibody-dependent cellular cytotoxity” (ADCC).
Why Was This Study Done?
Although rituximab lengthens the lives of patients with some types of B cell lymphoma, it is not a cure—the lymphoma usually recurs. Researchers are trying to increase the effectiveness of rituximab by combining it with other anticancer agents. One group of drugs that might be combined with rituximab is the “statins,” drugs that reduce the risk of heart disease by lowering the level of cholesterol (a type of fat) in the blood. In laboratory experiments, statins kill some cancer cells, in part by altering the fat composition of their outer (plasma) membrane. In addition, some population-based studies suggest that statin treatment might slightly decrease the risk of developing some kinds of cancer, including lymphoma. Statins are already undergoing clinical evaluation in combination with chemotherapy for the treatment of lymphoma, but in this study, the researchers investigate the influence of statins on rituximab-induced killing of B cell lymphomas.
What Did the Researchers Do and Find?
When the researchers tested the ability of rituximab and statin combinations to kill B cell lymphoma cells growing in dishes, they found that statins decreased rituximab-dependent CDC and ADCC of these cells. Statin treatment, they report, did not alter the total amount of CD20 made by the lymphoma cells or the amount of CD20 in their plasma membranes, but it did reduce the binding of another anti-CDC20 monoclonal antibody to the cells. Because both this antibody and rituximab bind to a specific three-dimensional structure in CD20 (a “conformational epitope”), the researchers hypothesized that statins might alter rituximab-induced killing by affecting the shape of the CD20 molecule on the lymphoma cell surface. To test this idea, they used two techniques—atomic force microscopy and limited proteolysis. The data obtained using both approaches confirmed that statins induce shape changes in CD20. Finally, the researchers took B cells from five patients who had taken statins for a short time and showed that this treatment had reduced the amount of anti-CD20 monoclonal antibody able to bind to these cells.
What Do These Findings Mean?
These findings indicate that statins change the shape of the CD20 molecules on the surface of normal and malignant B lymphocytes, probably by changing the amount of cholesterol in the cell membrane. This effect of statins has several clinical implications, which means that cancer specialists should check whether patients with known or suspected B cell lymphoma are taking statins to treat high cholesterol. First, the impaired binding of monoclonal antibodies to conformational epitopes of CD20 in patients being treated with statins might delay the diagnosis of B cell lymphomas (CD20 binding to lymphocytes is used during the diagnosis of lymphomas). Second, some patients with B cell lymphoma may receive an incorrect diagnosis and may not be offered rituximab. Finally, because statins impair the anti-lymphoma activity of rituximab, a possibility that needs to be investigated in clinical studies, cancer specialists should check that patients with B cell lymphoma are not taking statins before prescribing rituximab.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050064.
The MedlinePlus has an encyclopedia page on lymphoma and a list of links to other sources of information on lymphoma (in English and Spanish)
The US National Cancer Institute provides information about lymphoma and about statins and cancer prevention (in English and Spanish)
The UK charity Cancerbackup provides information for patients and caregivers on different types of B-cell lymphoma and on rituximab
The US Leukemia and Lymphoma Society also provides information for patients and caregivers about lymphoma
doi:10.1371/journal.pmed.0050064
PMCID: PMC2270297  PMID: 18366248
10.  Valosin-containing Protein (p97) Is a Regulator of Endoplasmic Reticulum Stress and of the Degradation of N-End Rule and Ubiquitin-Fusion Degradation Pathway Substrates in Mammalian Cells 
Molecular Biology of the Cell  2006;17(11):4606-4618.
Valosin-containing protein (VCP; p97; cdc48 in yeast) is a hexameric ATPase of the AAA family (ATPases with multiple cellular activities) involved in multiple cellular functions, including degradation of proteins by the ubiquitin (Ub)–proteasome system (UPS). We examined the consequences of the reduction of VCP levels after RNA interference (RNAi) of VCP. A new stringent method of microarray analysis demonstrated that only four transcripts were nonspecifically affected by RNAi, whereas ∼30 transcripts were affected in response to reduced VCP levels in a sequence-independent manner. These transcripts encoded proteins involved in endoplasmic reticulum (ER) stress, apoptosis, and amino acid starvation. RNAi of VCP promoted the unfolded protein response, without eliciting a cytosolic stress response. RNAi of VCP inhibited the degradation of R-GFP (green fluorescent protein) and Ub-G76V-GFP, two cytoplasmic reporter proteins degraded by the UPS, and of α chain of the T-cell receptor, an established substrate of the ER-associated degradation (ERAD) pathway. Surprisingly, RNAi of VCP had no detectable effect on the degradation of two other ERAD substrates, α1-antitrypsin and δCD3. These results indicate that VCP is required for maintenance of normal ER structure and function and mediates the degradation of some proteins via the UPS, but is dispensable for the UPS-dependent degradation of some ERAD substrates.
doi:10.1091/mbc.E06-05-0432
PMCID: PMC1635394  PMID: 16914519

Results 1-10 (10)