Extracellular Vesicles and Cancer Multidrug Resistance: Undesirable Intercellular Messengers?

Cancer multidrug resistance (MDR) is one of the main mechanisms contributing to therapy failure and mortality. Overexpression of drug transporters of the ABC family (ATP-binding cassette) is a major cause of MDR. Extracellular vesicles (EVs) are nanoparticles released by most cells of the organism involved in cell-cell communication. Their cargo mainly comprises, proteins, nucleic acids, and lipids, which are transferred from a donor cell to a target cell and lead to phenotypical changes. In this article, we review the scientific evidence addressing the regulation of ABC transporters by EV-mediated cell-cell communication. MDR transfer from drug-resistant to drug-sensitive cells has been identified in several tumor entities. This was attributed, in some cases, to the direct shuttle of transporter molecules or its coding mRNA between cells. Also, EV-mediated transport of regulatory proteins (e.g., transcription factors) and noncoding RNAs have been indicated to induce MDR. Conversely, the transfer of a drug-sensitive phenotype via EVs has also been reported. Additionally, interactions between non-tumor cells and the tumor cells with an impact on MDR are presented. Finally, we highlight uninvestigated aspects and possible approaches to exploiting this knowledge toward the identification of druggable processes and molecules and, ultimately, the development of novel therapeutic strategies.

Role of sirtuins in hepatocellular carcinoma progression and multidrug resistance: Mechanistical and pharmacological perspectives.

Hepatocellular carcinoma (HCC) is the third most common cause of death from cancer worldwide. Therapeutic strategies are still challenging due to the high relapse rate after surgery and multidrug resistance (MDR). It is essential to better understand the mechanisms for HCC progression and MDR for the development of new therapeutic strategies. Mammalian sirtuins (SIRTs), a family of seven members, are related to tumor progression, MDR and prognosis and were proposed as potential prognostic markers, as well as therapeutic targets for treating cancer. SIRT1 is the most studied member and is overexpressed in HCC, playing an oncogenic role and predicting poor prognosis. Several manuscripts describe the role of SIRTs2-7 in HCC; most of them report an oncogenic role for SIRT2 and -7 and a suppressive role for SIRT3 and -4. The scenario is more confusing for SIRT5 and -6, since information is contradictory and scarce. For SIRT1 many inhibitors are available and they seem to hold therapeutic promise in HCC. For the other members the development of specific modulators has just started. This review is aimed to describe the features of SIRTs1-7 in HCC, and the role they play in the onset and progression of the disease. Also, when possible, we will depict the information related to the SIRTs modulators that have been tested in HCC and their possible implication in MDR. With this, we hope to clarify the role of each member in HCC and to shed some light on the most successful strategies to overcome MDR.

Delta-tocotrienol enhances the anti-tumor effects of interferon alpha through reactive oxygen species and Erk/MAPK signaling pathways in hepatocellular carcinoma cells.

The complexity of hepatocellular carcinoma (HCC) signaling and the failure of pharmacological therapeutics reveal the significance of establishing new anti-cancer strategies. Interferon alpha (IFN-α) has been used as adjuvant therapy for reducing HCC recurrence and improving survival. Delta-tocotrienol (δ-tocotrienol), a natural unsaturated isoform of vitamin E, is a promising candidate for cancer treatment. In this study, we evaluated whether the combination of δ-tocotrienol with IFN-α displays significant advantages in the treatment of HCC cells. Results showed that the combination significantly decreased cell viability, migration and invasion of HCC cells compared with single therapies. Combining δ-tocotrienol and IFN-α enhanced the decrease in proliferating cell nuclear antigen (PCNA) and matrix metalloproteinase (MMP) 7 and MMP-9. The combination also produced an enhancement of apoptosis together with increased Bax/Bcl-xL ratio and reactive oxygen species (ROS) generation. δ-tocotrienol induced Notch1 activation and changes in Erk and p38 MAPK signaling status. Blocking experiments confirmed that ROS and Erk are involved, at least in part, in the anti-cancer effects of the combined treatment. In conclusion, the combination of δ-tocotrienol with IFN-α therapy showed promising results for HCC cell treatment, which makes the combination of cytokine-based immunotherapy with natural products a potential strategy against liver cancer.

Sirtuin 1 and 2 inhibitors enhance the inhibitory effect of sorafenib in hepatocellular carcinoma cells.

Multidrug resistance (MDR) counteracts the efficiency of sorafenib, an important first-line therapy for hepatocellular carcinoma (HCC). Sirtuins (SIRTs) 1 and 2 are associated with tumor progression and MDR. We treated 2D and 3D cultures (which mimic the features of in vivo tumors) from HCC cells with sorafenib alone or in the presence of SIRTs 1 and 2 inhibitors (cambinol or EX-527; combined treatments). Cultures subjected to combined treatments showed a greater fall in cellular viability, proliferation (PCNA, cyclin D1 and Ki-67 expression and cell cycle analysis), migration and invasion when compared with cultures treated only with sorafenib. Similarly, combined treatments produced more apoptosis (annexin V/PI, caspase-3/7 activity) than sorafenib alone. Since cell cycle dysregulation and apoptotic blockage are reported mechanisms of MDR, the modulation found in PCNA, cyclin D1, Ki-67 and caspase-3/7 proteins by cambinol and EX-527 are probably playing a role in enhancing the sensitivity of HCC cell lines to sorafenib. EX-527 reduced MRP3 and BCRP expression in sorafenib-treated HCC cells. Since ABC transporters contribute to MDR, MRP3 and BCRP could be also influencing in the response of HCC cells to sorafenib. Overall, 2D and 3D cultures behave similarly except that 3D cultures were less sensitive to treatments, reinforcing the clinical relevance of the current study. Findings presented in this manuscript support a potential application for SIRTs 1 and 2 inhibitors since we demonstrated that these compounds enhance the inhibitory effect of sorafenib upon treatment of hepatocellular carcinoma cells lines.

Participation of 5-lipoxygenase and LTB4 in liver regeneration after partial hepatectomy.

Regeneration is the unmatched liver ability for recovering its functional mass after tissue lost. Leukotrienes (LT) are a family of eicosanoids with the capacity of signaling to promote proliferation. We analyzed the impact of blocking LT synthesis during liver regeneration after partial hepatectomy (PH). Male Wistar rats were subjected to two-third PH and treated with zileuton, a specific inhibitor of 5-lipoxygenase (5-LOX). Our first find was a significant increment of intrahepatic LTB4 during the first hour after PH together with an increase in 5-LOX expression. Zileuton reduced hepatic LTB4 levels at the moment of hepatectomy and also inhibited the increase in hepatic LTB4. This inhibition produced a delay in liver proliferation as seen by decreased PCNA and cyclin D1 nuclear expression 24 h post-PH. Results also showed that hepatic LTB4 diminution by zileuton was associated with a decrease in NF-ĸB activity. Additionally, decreased hepatic LTB4 levels by zileuton affected the recruitment of neutrophils and macrophages. Non-parenchymal cells (NPCs) from zileuton-treated PH-rats displayed higher apoptosis than NPCs from PH control rats. In conclusion, the present work provides evidences that 5-LOX activation and its product LTB4 are involved in the initial signaling events for liver regeneration after PH and the pharmacological inhibition of this enzyme can delay the initial time course of the phenomenon.

ABC Transporters: Regulation and Association with Multidrug Resistance in Hepatocellular Carcinoma and Colorectal Carcinoma.

For most cancers, the treatment of choice is still chemotherapy despite its severe adverse effects, systemic toxicity and limited efficacy due to the development of multidrug resistance (MDR). MDR leads to chemotherapy failure generally associated with a decrease in drug concentration inside cancer cells, frequently due to the overexpression of ABC transporters such as P-glycoprotein (P-gp/MDR1/ABCB1), multidrug resistance-associated proteins (MRPs/ABCCs), and breast cancer resistance protein (BCRP/ABCG2), which limits the efficacy of chemotherapeutic drugs. The aim of this review is to compile information about transcriptional and post-transcriptional regulation of ABC transporters and discuss their role in mediating MDR in cancer cells. This review also focuses on drug resistance by ABC efflux transporters in cancer cells, particularly hepatocellular carcinoma (HCC) and colorectal carcinoma (CRC) cells. Some aspects of the chemotherapy failure and future directions to overcome this problem are also discussed.

Disruption of tumor necrosis factor alpha receptor 1 signaling accelerates NAFLD progression in mice upon a high-fat diet.

Obesity is accompanied by a low-grade inflammation state, characterized by increased proinflammatory cytokines levels such as tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL-1ß). In this regard, there exists a lack of studies in hepatic tissue about the role of TNFα receptor 1 (TNFR1) in the context of obesity and insulin resistance during the progression of nonalcoholic fatty liver disease (NAFLD). The aim of this work was to evaluate the effects of high-caloric feeding (HFD) (40% fat, for 16 weeks) on liver inflammation-induced apoptosis, insulin resistance, hepatic lipid accumulation and its progression toward nonalcoholic steatohepatitis (NASH) in TNFR1 knock-out and wild-type mice. Mechanisms involved in HFD-derived IL-1ß release and impairment of insulin signaling are still unknown, so we determined whether IL-1ß affects liver insulin sensitivity and apoptosis through TNFα receptor 1 (TNFR1)-dependent pathways. We showed that knocking out TNFR1 induces an enhanced IL-1ß plasmatic release upon HFD feed. This was correlated with higher hepatic and epididymal white adipose tissue mRNA levels. In vivo and in vitro assays confirmed an impairment in hepatic insulin signaling, in part due to IL-1ß-induced decrease of AKT activation and diminution of IRS1 levels, followed by an increase in inflammation, macrophage (resident and recruited) accumulation, hepatocyte apoptotic process and finally hepatic damage. In addition, TNFR1 KO mice displayed higher levels of pro-fibrogenic markers. TNFR1 signaling disruption upon an HFD leads to an accelerated progression from simple steatosis to a more severe phenotype with many NASH features, pointing out a key role of TNFR1 in NAFLD progression.

Inhibition of sirtuins 1 and 2 impairs cell survival and migration and modulates the expression of P-glycoprotein and MRP3 in hepatocellular carcinoma cell lines.

Sirtuins (SIRTs) 1 and 2 deacetylases are overexpressed in hepatocellular carcinoma (HCC) and are associated with tumoral progression and multidrug resistance (MDR). In this study we analyzed whether SIRTs 1 and 2 activities blockage was able to affect cellular survival and migration and to modulate p53 and FoxO1 acetylation in HepG2 and Huh7 cells. Moreover, we analyzed ABC transporters P-glycoprotein (P-gp) and multidrug resistance-associated protein 3 (MRP3) expression. We used cambinol and EX-527 as SIRTs inhibitors. Both drugs reduced cellular viability, number of colonies and cellular migration and augmented apoptosis. In 3D cultures, SIRTs inhibitors diminished spheroid growth and viability. 3D culture was less sensitive to drugs than 2D culture. The levels of acetylated p53 and FoxO1 increased after treatments. Drugs induced a decrease in ABC transporters mRNA and protein levels in HepG2 cells; however, only EX-527 was able to reduce MRP3 mRNA and protein levels in Huh7 cells. This is the first work demonstrating the regulation of MRP3 by SIRTs. In conclusion, both drugs decreased HCC cells survival and migration, suggesting SIRTs 1 and 2 activities blockage could be beneficial during HCC therapy. Downregulation of the expression of P-gp and MRP3 supports the potential application of SIRTs 1 and 2 inhibitions in combination with conventional chemotherapy.

Regulation of multidrug resistance proteins by genistein in a hepatocarcinoma cell line: impact on sorafenib cytotoxicity.

Hepatocellular carcinoma (HCC) is the fifth most frequent cancer worldwide. Sorafenib is the only drug available that improves the overall survival of HCC patients. P-glycoprotein (P-gp), Multidrug resistance-associated proteins 2 and 3 (MRP2 and 3) and Breast cancer resistance protein (BCRP) are efflux pumps that play a key role in cancer chemoresistance. Their modulation by dietary compounds may affect the intracellular accumulation and therapeutic efficacy of drugs that are substrates of these transporters. Genistein (GNT) is a phytoestrogen abundant in soybean that exerts its genomic effects through Estrogen-Receptors and Pregnane-X-Receptor (PXR), which are involved in the regulation of the above-mentioned transporters. We evaluated the effect of GNT on the expression and activity of P-gp, MRP2, MRP3 and BCRP in HCC-derived HepG2 cells. GNT (at 1.0 and 10 µM) increased P-gp and MRP2 protein expression and activity, correlating well with an increased resistance to sorafenib cytotoxicity as detected by the methylthiazole tetrazolium (MTT) assay. GNT induced P-gp and MRP2 mRNA expression at 10 but not at 1.0 µM concentration suggesting a different pattern of regulation depending on the concentration. Induction of both transporters by 1.0 µM GNT was prevented by cycloheximide, suggesting translational regulation. Downregulation of expression of the miR-379 by GNT could be associated with translational regulation of MRP2. Silencing of PXR abolished P-gp induction by GNT (at 1.0 and 10 µM) and MRP2 induction by GNT (only at 10 µM), suggesting partial mediation of GNT effects by PXR. Taken together, the data suggest the possibility of nutrient-drug interactions leading to enhanced chemoresistance in HCC when GNT is ingested with soy rich diets or dietary supplements.

FoxO3a nuclear localization and its association with ß-catenin and Smads in IFN-α-treated hepatocellular carcinoma cell lines.

Interferon-α2b (IFN-α2b) reduces proliferation and increases apoptosis in hepatocellular carcinoma cells by decreasing ß-catenin/TCF4/Smads interaction. Forkhead box O-class 3a (FoxO3a) participates in proliferation and apoptosis and interacts with ß-catenin and Smads. FoxO3a is inhibited by Akt, IκB kinase ß (IKKß), and extracellular-signal-regulated kinase (Erk), which promote FoxO3a sequestration in the cytosol, and accumulates in the nucleus upon phosphorylation by c-Jun N-terminal kinase (JNK) and p38 mitogen-activated kinase (p38 MAPK). We analyzed FoxO3a subcellular localization, the participating kinases, FoxO3a/ß-catenin/Smads association, and FoxO3a target gene expression in IFN-α2b-stimulated HepG2/C3A and Huh7 cells. Total FoxO3a and Akt-phosphorylated FoxO3a levels decreased in the cytosol, whereas total FoxO3a levels increased in the nucleus upon IFN-α2b stimulus. IFN-α2b reduced Akt, IKKß, and Erk activation, and increased JNK and p38 MAPK activation. p38 MAPK inhibition blocked IFN-α2b-induced FoxO3a nuclear localization. IFN-α2b enhanced FoxO3a association with ß-catenin and Smad2/3/7. Two-step coimmunoprecipitation experiments suggest that these proteins coexist in the same complex. The expression of several FoxO3a target genes increased with IFN-α2b. FoxO3a knockdown prevented the induction of these genes, suggesting that FoxO3a acts as mediator of IFN-α2b action. Results suggest a ß-catenin/Smads switch from TCF4 to FoxO3a. Such events would contribute to the IFN-α2b-mediated effects on cellular proliferation and apoptosis. These results demonstrate new mechanisms for IFN-α action, showing the importance of its application in antitumorigenic therapies.

Relationship between genotoxic effects of breast cancer treatments and patient basal DNA integrity.

Radiotherapy and chemotherapy cause genotoxic side effects that are highly variable among patients. In this study, we evaluated DNA integrity using the comet assay in peripheral blood lymphocytes from breast cancer patients before ("pre-treatment patients"; n=47) and after ("post-treatment patients"; n=24) radiotherapy and/or chemotherapy treatment and from healthy donors (n=15). Comet evaluation was made by visual (types 0-4) and digital (percentage of DNA remaining in the comet head=% head DNA) analysis. The association between the level of DNA damage and cancer prognostic factors was assessed. The treatments caused a significant increase in DNA damage registered by both visual (p<0.001) and digital (p<0.001) analyses. No significant associations between the level of DNA damage in pre-treatment patients and cancer prognostic factors were found. A significant correlation between the comet results from each patient before and after treatment (r=0.64, p=0.001) was observed. The % head DNA in post-treatment samples from patients with a high level of DNA damage before treatment (30.3±3.1%, p<0.01) was lower than in post-treatment samples from patients with a low-to-medium level of DNA damage before therapy (49.2±4.4%). These results support the usefulness of the comet assay as a sensitive technique to evaluate basal DNA status and DNA damage caused by cancer treatments. The comet assay could contribute to treatment decisions, especially by taking into account the patient's basal DNA damage before therapy.

Mammaglobin A: review and clinical utility.

Mammaglobin A is a protein that belongs to the secretoglobin superfamily. It has highly specific expression in cells from most breast cancers and may be used to detect circulating or disseminated tumor cells. In addition, mammaglobin A is currently under inves tigation as a potential therapeutic target for immune therapies that target breast cancer. The present review will highlight our current understanding of mammaglobin A at the genetic and protein level and its potential clinical applications. Characteristics of breast cancer and methods used to isolate and detect circulating tumor cells will also be presented.

Quercetin prevents liver carcinogenesis by inducing cell cycle arrest, decreasing cell proliferation and enhancing apoptosis.

SCOPE: Quercetin is the most abundant flavonoid in human diet. It has special interest as it holds anticancerous properties. This study aims to clarify the mechanisms involved in quercetin effects during the occurrence of preneoplastic lesions in rat liver. METHODS AND RESULTS: Adult male Wistar rats were subjected to a two-phase model of hepatocarcinogenesis (initiated-promoted group). Initiated-promoted animals also received quercetin 10 and 20 mg/kg body weight (IPQ10 and IPQ20 groups, respectively). Antioxidant defenses were modified by quercetin administration at both doses. However, only IPQ20 group showed a reduction in number and volume of preneoplastic lesions. This group showed increased apoptosis and a reduction in the proliferative index. In addition, IPQ20 group displayed a reduction of cell percentages in G1 and S phases, accumulation in G2, and decrease in M phase, with reduced expression of cyclin D1, cyclin A, cyclin B, and cyclin-dependent kinase 1. Interestingly, peroxisome proliferator activated receptor-α levels were reduced in IPQ20 group. CONCLUSION: The outcomes of this study represent a significant contribution to the current understanding on the preventive mechanisms of quercetin during the early stages of liver cancer development, demonstrating that in addition to its known proapoptotic characteristics, the flavonoid modulates the expression of critical cell cycle regulators and peroxisome proliferator activated receptor-α activity.

Interferon-α2b and transforming growth factor-ß1 treatments on HCC cell lines: Are Wnt/ß-catenin pathway and Smads signaling connected in hepatocellular carcinoma?

Wnt/ß-catenin pathway is often dysregulated in hepatocellular carcinoma (HCC). Activated ß-catenin accumulates in the cytosol and nucleus and forms a nuclear complex with TCF/LEF factors like TCF4. Interferon-α (IFN-α) has recently been recognized to harbor therapeutic potential in prevention and treatment of HCC. Transforming Growth Factor-ß1 (TGF-ß1) is a mediator of apoptosis, exerting its effects via Smads proteins. One mode of interaction between Wnt/ß-catenin and TGF-ß1/Smads pathways is the association of Smads with ß-catenin/TCF4. In this study we analyzed the effects of IFN-α2b and TGF-ß1 treatments on Wnt/ß-catenin pathway, Smads proteins levels, ß-catenin/TCF4/Smads interaction and proliferation and apoptotic death in HepG2/C3A and Huh7 cell lines. IFN-α2b and TGF-ß1 attenuated Wnt/ß-catenin signal by decreasing ß-catenin and Frizzled7 receptor proteins contents and the interaction of ß-catenin with TCF4. Truncated ß-catenin form present in C3A cell line also diminished after treatments. Both cytokines declined Smads proteins and their interaction with TCF4. The overall cellular response to cytokines was the decrease in proliferation and increase in apoptotic death. Treatment with Wnt3a, which elevates ß-catenin protein levels, also generated the increment of Smads proteins contents when comparing with untreated cells. In conclusion, IFN-α2b and TGF-ß1 proved to be effective as modulators of Wnt/ß-catenin pathway in HCC cell lines holding both wild-type and truncated ß-catenin. Since the inhibition of ß-catenin/TCF4/Smads complexes formation may have a critical role in slowing down oncogenesis, IFN-α2b and TGF-ß1 could be useful as potential treatments in patients with HCC.