Combination of Second-Generation Proteasome Inhibitor Carfilzomib with Bortezomib in Four Different Breast Cancer Cell Lines.

BACKGROUND: Proteasome inhibitors target different pathways in cells and therefore are promising drugs in cancer therapy. The use of these inhibitors is approved mainly in hematological cancers, and recently many clinical trials and preclinical studies have been conducted on efficacy in solid tumors. Carfilzomib is a second-generation inhibitor and was developed to decrease the side effects of bortezomib. Although there are many valid therapies for breast cancer, resistance and recurrence are inevitable in many cases and the proteasomal system plays an important role in related pathways. OBJECTIVE: This study is a preliminary work to evaluate the combined effects of bortezomib and carfilzomib in four different breast cancer cells. METHODS: MDA-MB-231, MCF-7, UACC-2087, and SKBR-3 cell lines were used. Cell viability was determined using bortezomib and carfilzomib alone and in combination. Combination effect values were determined using the Chou- Talalay method. Apoptosis, proteasome activity, cleaved PARP, and HSP70 expressions were analyzed in the determined doses. RESULTS: The response to the combination of the two inhibitors was different in four cell lines. Apoptosis was significantly higher in combination groups compared to carfilzomib in three cell lines except for SKBR-3, and higher in the combination group compared to bortezomib only in UACC-2087. Combination decreased cleaved PARP levels in MDA-MB-231 and MCF-7 and increased SKBR-3 compared to bortezomib. HSP70 levels decreased in combination with UACC-2087 and SKBR-3 compared to carfilzomib. CONCLUSION: Taken together, the combination of the two inhibitors was more apoptotic compared to carfilzomib and apoptosis was higher only in UACC-2087 compared to bortezomib. This apoptosis data can not be directly correlated to the degree of proteasome inhibition, PARP cleavage, and HSP70 response.

Synergistic Induction of Apoptosis by Quercetin and Curcumin in Chronic Myeloid Leukemia (K562) Cells: II. Signal Transduction Pathways Involved.

Flavonoids are phenolic substances with chemo-preventive and chemotherapeutic properties. They are widely found in fruits and vegetables. The polyphenols quercetin and curcumin have antioxidant, anti-inflammatory, anti-carcinogenic, and pro-apoptotic properties. They were successfully used against different human cancers, especially chronic myeloid leukemia cancer cells. We have previously investigated anti-proliferative and apoptotic effects of quercetin and curcumin combination in K562 cells. Our data showed that they had beneficial synergistic effects. Based on these findings, we aimed to clarify signaling pathways involved in synergistic combination treatment with quercetin and curcumin in these cells. Proteins were investigated by Western blotting and by confocal microscopy. Changes in several genes in 10 different pathways related to cell proliferation, apoptosis, cell cycle, inflammation, hypoxia and oxidative stress were observed. Combination of quercetin and curcumin was effective on genes that were particularly related to p53, NF-κB and TGF-α pathways. Down-regulatory (CDKN1B, AKT1, IFN-γ) and up-regulatory (BTG2, CDKN1A, FAS) effects on genes and related protein expressions may provide a multi-targeted therapy potential for chronic myeloid leukemia cancer cells without affecting healthy cells.

Higher proteotoxic stress rather than mitochondrial damage is involved in higher neurotoxicity of bortezomib compared to carfilzomib.

Proteasome inhibitors have great success for their therapeutic potential against hematologic malignancies. First generation proteasome inhibitor bortezomib induced peripheral neuropathy is considered as a limiting factor in chemotherapy and its second-generation counterpart carfilzomib is associated with lower rates of neurotoxicity. The mitochondrial toxicity (mitotoxicity) hypothesis arises from studies with animal models of bortezomib induced peripheral neuropathy. However, molecular mechanisms are not fully elucidated and the role of mitotoxicity in bortezomib and carfilzomib induced neurotoxicity has not been investigated comparatively. Herein, we characterized the neurotoxic effects of bortezomib and carfilzomib at the molecular level in human neuronal cells using LC-MS/MS analysis, flow cytometry, RT-qPCR, confocal microscopy and western blotting. We showed that bortezomib and carfilzomib affected the human neuronal proteome differently, and bortezomib caused higher proteotoxic stress via protein oxidation, protein K48-ubiquitination, heat shock protein expression upregulation and reduction of mitochondria membrane potential. Bortezomib and carfilzomib did not affect the gene expression levels related to mitochondrial dynamics (optic atrophy 1; OPA1, mitofusin 1; MFN1, mitofusin 2; MFN2, fission 1; FIS1, dynamin-related protein 1; DRP1) and overall mitophagy rate whereas, PINK1/Parkin mediated mitophagy gene expressions were altered with both drugs. Bortezomib and carfilzomib caused downregulation of the contents of mitochondrial oxidative phosphorylation complexes, voltage-dependent anion channel 1 (VDAC1) and uncoupling protein 2 (UCP2) similarly. Our findings suggest that, both drugs induce mitotoxicity besides proteotoxic stress in human neuronal cells and the higher incidence of neurotoxicity with bortezomib than carfilzomib is not directly related to mitochondrial pathways.

Cytotoxicity of Different Nano Composite Resins on Human Gingival and Periodontal Ligament Fibroblast Cell Lines: An In Vitro Study.

The aim of this study is to determine the cytotoxicity of three different nano composite resins (CRs) on human gingival fibroblast (hGF) and periodontal ligament fibroblast (hPDLF) cell lines. These CRs selected were nanohybrid organic monomer-based Admira Fusion (AF), nanohybrid Bis-(acryloyloxymethyl) tricyclo 5.2.1.0.sup.2,6 decane-based Charisma Topaz (CT), and supra nano filled resin-based Estelite Quick Sigma (EQS). MTT assay was performed to assess the cytotoxicity of CRs at 24 h and one week. AF and EQS applied on hGF cells at 24 h and one week demonstrated similar cytotoxic outcomes. Cytotoxicity of CT on hGF cells at one week was higher than 24 h (p = 0.04). Cytotoxicity of CT on hGF cells was higher at 24 h (p = 0.002) and one week (p = 0.009) compared to control. All composites showed higher cytotoxicity on hPDLF cells at one week than the 24 h (AF; p = 0.02, CT; p = 0.02, EQS; p = 0.04). AF and EQS demonstrated lower cytotoxicity on hPDLF cells than the control group at 24 h (AF; p = 0.01, EQS; p = 0.001). CT was found more cytotoxic on hPDLF cells than the control (p = 0.01) and EQS group (p = 0.008) at one week. The cytotoxicity of CRs on hGF and hPDLF cells vary, according to the type of composites, cell types, and exposure time.

Synergistic Induction of Apoptosis by Quercetin and Curcumin in Chronic Myeloid Leukemia (K562) Cells.

Chronic myeloid leukemia is a major hematopoietic malignancy characterized by expansion of myeloid cells. In this study, we have investigated whether quercetin, curcumin and their combination induce apoptosis and inhibit growth of K562 cells. We have observed that quercetin and curcumin combination induced apoptosis accompanied by increased ROS and decreased GSH levels as well as loss of mitochondrial membrane potential. Our mRNA and protein expression results suggested that cytochrome c was released from mitochondria causing PARP and caspase-9 cleavages, the hallmarks of mitochondrial apoptotic pathway. We believe that triggering of apoptosis is mostly via mitochondrial pathway and ROS generation may induce impairment of mitochondrial membrane potential. The use of quercetin and curcumin combination potentiates individual apoptotic effects of the polyphenols and reduces their effective dose thereby preventing potential toxic effects on normal cells. Additional preclinical studies and clinical trials are certainly required to further validate their usefulness as potent anticancer agents.

Antitumor and antimetastatic effects of walnut oil in esophageal adenocarcinoma cells.

BACKGROUND: Walnuts contain many components including specific fatty acids, which could be active against cancer. Even though the anticarcinogenic effect of some of the individual fatty acids in walnut oil has been described, the effect of walnut oil itself on esophageal cancer cells hasn't yet been investigated. OBJECTIVE: We aimed to investigate whether walnut oil affects tumor growth and metastatic potential in esophageal cancer cells. METHODS: The human esophageal adenocarcinoma cell line, OE19, was treated with different doses of walnut oil and cell viability, apoptosis/necrosis and cell cycle analyses were performed using WST-1 assay and flow cytometry respectively. Adhesion, colony formation and wound healing assays were performed to assess the antimetastatic effects of walnut oil. NFkB expression was evaluated with western blot analysis. RESULTS: Walnut oil decreased the cell viability of esophageal cancer cells in a dose-dependent manner. 20 mg/mL walnut oil reduced cell viability by ∼50% when compared with control. The analysis revealed that necrosis and accumulation of cells in G0/G1 phase was induced in the cells treated with high doses of walnut oil. It also down-regulated the protein levels of NFkB. Walnut oil suppressed the adhesion, migration and colony formation of the cells. CONCLUSIONS: High-dose short-term administration of walnut oil reduces the cell viability and metastatic ability of esophageal cancer cells, while exhibiting anticarcinogenic effect by inducing necrosis and cell cycle arrest at the G0/G1 phase, probably through suppression of the NFkB pathway. These data indicate that walnut oil, and by extension walnut consumption, may have beneficial effects in esophageal cancer in humans. This should be tested by clinical trials in the future.

Quercetin-Induced Cell Death in Human Papillary Thyroid Cancer (B-CPAP) Cells.

In this study, we have investigated the antiproliferative effect of quercetin on human papillary thyroid cancer cells and determined the apoptotic mechanisms underlying its actions. We have used different concentrations of quercetin to induce apoptosis and measured cell viability. Apoptosis and cell cycle analysis was determined by flow cytometry using Annexin V and propidium iodide. Finally, we have measured changes in caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) protein expression levels as hallmarks of apoptosis and Hsp90 protein expression level as a marker of proteasome activity in treated and control cells. Quercetin treatment of human papillary thyroid cancer cells resulted in decreased cell proliferation and increased rate of apoptosis by caspase activation. Furthermore, it was demonstrated that quercetin induces cancer cell apoptosis by downregulating the levels of Hsp90. In conclusion, we have shown that quercetin induces downregulation of Hsp90 expression that may be involved in the decrease of chymotrypsin-like proteasome activity which, in order, induces inhibition of growth and causes cell death in thyroid cancer cells. Thus, quercetin appears to be a promising candidate drug for Hsp90 downregulation and apoptosis of thyroid cancer cells.

Comparison of antioxidant capacity, protein profile and carbohydrate content of whey protein fractions.

Whey is used as an additive in food industry and a dietary supplement in nutrition. Here we report a comparative analysis of antioxidant potential of whey and its fractions. Fractions were obtained by size exclusion chromatography, before and after enzymatic digestion with pepsin or trypsin. Superoxide radical scavenging, lipid peroxidation inhibition and cupric ion reducing activities of different fractions were checked. Peptides were detected by SDS-PAGE and GC-MS was used to determine carbohydrate content of the fractions. All samples showed antioxidant activity and the second fraction of the trypsin hydrolysate showed the highest superoxide radical scavenging activity. CUPRAC value of this fraction was two-times higher than that of whey filtrate. The first fraction of the pepsin hydrolysate was the most effective inhibitor of lipid peroxidation. Each sample exhibited a different polypeptide profile. Different percentages of carbohydrates were identified in whey filtrate and in all second fractions, where galactose was the major component.

Whey protein versus whey protein hydrolyzate for the protection of azoxymethane and dextran sodium sulfate induced colonic tumors in rats.

Recent studies have shown that whey protein has many useful effects including its anti-cancer effect. In this study we have compared the protective effect of dietary whey protein with whey protein hydrolyzate against azoxymethane and dextran sodium sulfate induced colon cancer in rats. We used a rat model of the colon cancer induced by administration of azoxymethane followed by repeated dextran sodium sulfate ingestion which causes multiple tumor development. Colon tissues were analyzed histologically in addition to biochemical analyses performed by measuring lipid peroxidation, protein oxidation and glutathione levels in both of colon and liver tissues of rats after sacrification. Macroscopic and microscopic tumors were identified in all groups that received azoxymethane followed by repeated dextran sodium sulfate. Group fed with whey protein hydrolyzate showed significantly less macroscopic and microscopic tumor development compared with group fed with whey protein. The protocol applied to generate an appropriate model of colon cancer was successful. Whey protein hydrolyzate was found to be more effective in preventing colon tumor development compared with whey protein.