Reprogramming energy metabolism and inducing angiogenesis: co-expression of monocarboxylate transporters with VEGF family members in cervical adenocarcinomas.

BACKGROUND: Deregulation of cellular energetic metabolism was recently pointed out as a hallmark of cancer cells. This deregulation involves a metabolic reprogramming that leads to a high production of lactate. Lactate efflux, besides contributing for the glycolytic flux, also acts in the extracellular matrix, contributing for cancer malignancy, by, among other effects, induction of angiogenesis. However, studies on the interplay between cancer metabolism and angiogenesis are scarce. Therefore, the aim of the present study was to evaluate the metabolic and vascular molecular profiles of cervical adenocarcinomas, their co-expression, and their relation to the clinical and pathological behavior. METHODS: The immunohistochemical expression of metabolism-related proteins (MCT1, MCT4, CD147, GLUT1 and CAIX) as well as VEGF family members (VEGF-A, VEGF-C, VEGF-D, VEGFR-1, VEGFR-2 and VEGFR-3) was assessed in a series of 232 cervical adenocarcinomas. The co-expression among proteins was assessed and the expression profiles were associated with patients' clinicopathological parameters. RESULTS: Among the metabolism-related proteins, MCT4 and CAIX were the most frequently expressed in cervical adenocarcinomas while CD147 was the less frequently expressed protein. Overall, VEGF family members showed a strong and extended expression with VEGF-C and VEGFR-2 as the most frequently expressed and VEGFR-1 as the less expressed member. Co-expression of MCT isoforms with VEGF family members was demonstrated. Finally, MCT4 was associated with parametrial invasion and HPV18 infection, CD147 and GLUT1 with distant metastasis, CAIX with tumor size and HPV18 infection, and VEGFR-1 with local and lymphnode metastasis. CONCLUSIONS: The results herein presented provide additional evidence for a crosstalk between deregulating cellular energetics and inducing angiogenesis. Also, the metabolic remodeling and angiogenic switch are relevant to cancer progression and aggressiveness in adenocarcinomas.

Characterization of monocarboxylate transporters (MCTs) expression in soft tissue sarcomas: distinct prognostic impact of MCT1 sub-cellular localization.

BACKGROUND: Soft tissue sarcomas (STSs) are a group of neoplasms, which, despite current therapeutic advances, still confer a poor outcome to half of the patients. As other solid tumors, STSs exhibit high glucose consumption rates, associated with worse prognosis and therapeutic response. As highly glycolytic tumors, we hypothesized that sarcomas should present an increased expression of lactate transporters (MCTs). METHODS: Immunohistochemical expression of MCT1, MCT2, MCT4 and CD147 was assessed in a series of 86 STSs and the expression profiles were associated with patients' clinical-pathological parameters. RESULTS: MCT1, MCT4 and CD147 were mainly observed in the plasma membrane of cancer cells (around 60% for MCTs and 40% for CD147), while MCT2 was conspicuously found in the cytoplasm (94.2%). Importantly, we observed MCT1 nuclear expression (32.6%). MCT1 and MCT4, alone or co-expressed with CD147 in the plasma membrane, were associated with poor prognostic variables including high tumor grade, disease progression and shorter overall survival. Conversely, we found MCT1 nuclear expression to be associated with low grade tumors and longer overall survival. CONCLUSIONS: The present work represents the first report of MCTs characterization in STSs. We showed the original finding of MCT1 expression in the nucleus. Importantly, opposite biological roles should be behind the dual sub-cellular localization of MCT1, as plasma membrane expression of MCT1 is associated with worse patients' prognosis, while nuclear expression is associated with better prognosis.

Lactate transporters and vascular factors in HPV-induced squamous cell carcinoma of the uterine cervix.

BACKGROUND: Tumour microenvironment is a fundamental aspect of tumour behaviour, modulating important events as cancer cell migration and invasion, as well as angiogenesis and metastisation. Among other microenvironment features, hypoxia and acidity play important roles in this modulation. As the metabolic reprogramming of cancer cells induces extracellular acidity, which in turn induces angiogenesis, and hypoxia induces both the metabolic reprogramming and angiogenesis, the present study aims to evaluate the immunohistochemical expression of a variety of metabolic and vascular markers as common targets of the hypoxic microenvironment in a series of cervical squamous cells carcinoma, as well as using an in vitro 3D culture model. METHODS: Immunohistochemical expression of MCT1, MCT4, CD147, GLUT1 and CAIX was assessed in a series of 28 chronic cervicitis, 34 LSIL, 29 HSIL, 38 cases of squamous cells carcinoma (SCC), as well as in in vitro 3D culture of keratinocytes expressing HPV genes. Furthermore, VEGF family members' expression was assessed in the SCC cases. The expression profiles were associated with patients' clinicopathological parameters. RESULTS: We found an increase of MCT4 expression along progression to malignancy in cervical samples. Also, MCT4 was associated with CD147 and CAIX expression. VEGF-A expression was more frequently found in cases without MCT1 expression. Both MCT4 and CD147 were more frequently expressed in younger patients at diagnosis while no associations were found between VEGF family and clinicopathological parameters. Finally, we show evidence for the upregulation of MCT4, as well as CD147 and CAIX, after HPV transfection. CONCLUSIONS: The results herein presented point at MCT4 as a promising therapeutic target in squamous cells carcinoma of the uterine cervix. Importantly, we show a possible association between lactate transport and angiogenesis, which should be further explored.

The monocarboxylate transporter inhibitor α-cyano-4-hydroxycinnamic acid disrupts rat lung branching.

BACKGROUND/AIMS: The human embryo develops in a hypoxic environment. In this way, cells have to rely on the glycolytic pathway for energy supply, leading to an intracellular accumulation of monocarboxylates such as lactate and pyruvate. These acids have an important role in cell metabolism and their rapid transport across the plasma membrane is crucial for the maintenance of intracellular pH homeostasis. This transport is mediated by a family of transporters, designated by monocarboxylate transporters (MCTs), namely isoforms 1 and 4. MCT1/4 expression is regulated by the ancillary protein CD147.The general aim of this study was to characterize the expression pattern of MCT1/4, CD147 and the glucose transporter GLUT1 during human fetal lung development and elucidate the role of MCTs in lung development. METHODS: The expression pattern of MCT1/4 and GLUT1 was characterized by immunohistochemistry and fetal lung viability and branching were evaluated by exposing rat fetal lung explants to CHC, an inhibitor of MCT activity. RESULTS: Our findings show that all the biomarkers are differently expressed during fetal lung development and that CHC appears to have an inhibitory effect on lung branching and viability, in a dose dependent way. CONCLUSION: We provide evidence for the role of MCTs in embryo lung development, however to prove the dependence of MCT activity further studies are waranted.

Portuguese propolis disturbs glycolytic metabolism of human colorectal cancer in vitro.

BACKGROUND: Propolis is a resin collected by bees from plant buds and exudates, which is further processed through the activity of bee enzymes. Propolis has been shown to possess many biological and pharmacological properties, such as antimicrobial, antioxidant, immunostimulant and antitumor activities. Due to this bioactivity profile, this resin can become an alternative, economic and safe source of natural bioactive compounds.Antitumor action has been reported in vitro and in vivo for propolis extracts or its isolated compounds; however, Portuguese propolis has been little explored. The aim of this work was to evaluate the in vitro antitumor activity of Portuguese propolis on the human colon carcinoma cell line HCT-15, assessing the effect of different fractions (hexane, chloroform and ethanol residual) of a propolis ethanol extract on cell viability, proliferation, metabolism and death. METHODS: Propolis from Angra do Heroísmo (Azores) was extracted with ethanol and sequentially fractionated in solvents with increasing polarity, n-hexane and chloroform. To assess cell viability, cell proliferation and cell death, Sulforhodamine B, BrDU incorporation assay and Anexin V/Propidium iodide were used, respectively. Glycolytic metabolism was estimated using specific kits. RESULTS: All propolis samples exhibited a cytotoxic effect against tumor cells, in a dose- and time-dependent way. Chloroform fraction, the most enriched in phenolic compounds, appears to be the most active, both in terms of inhibition of viability and cell death. Data also show that this cytotoxicity involves disturbance in tumor cell glycolytic metabolism, seen by a decrease in glucose consumption and lactate production. CONCLUSION: Our results show that Portuguese propolis from Angra do Heroísmo (Azores) can be a potential therapeutic agent against human colorectal cancer.

The clinicopathological significance of monocarboxylate transporters in testicular germ cell tumors.

BACKGROUND: Metabolic reprogramming is one of the hallmarks of cancer. The hyperglycolytic phenotype is often associated with the overexpression of metabolism-associated proteins, such as monocarboxylate transporters (MCTs). MCTs are little explored in germ cell tumors (GCTs), thus, the opportunity to understand the relevance of these metabolic markers and their chaperone CD147 in this type of tumor arises. The main aim of this study was to evaluate the expression of MCT1, MCT2, MCT4 and CD147 in testicular GCT samples and the clinicopathological significance of these metabolism related proteins. RESULTS: MCT1, MCT4 and CD147 were associated with higher stages, higher M and N stages and histological type, while MCT4 was also associated with higher risk stratification, presence of vascular invasion, and lower overall and event free survival. MCT4 silencing in JEG-3 had no significant effect in cell viability, proliferation and death, as well as extracellular levels of glucose and lactate. However, MCT4-silenced cells showed an increase in migration and invasion. CONCLUSION: The proteins herein studied, with the exception of MCT2, were associated with characteristics of worse prognosis, lower global and event free survival of patients with GCTs. Also, in vitro MCT4 silencing stimulated cell migration and invasion. MATERIALS AND METHODS: Immunohistochemical expression was evaluated on samples from 149 adult patients with testicular GCT, arranged in Tissue Microarrays (TMAs), and associated with the clinicopathological data. Also, MCT4 silencing studies using siRNA were performed in JEG-3 cells.

Internalization studies on zeolite nanoparticles using human cells.

Zeolites are crystalline porous materials with a regular framework which have non-toxic effects on a variety of human cell lines and have been explored for cell imaging and drug delivery. Understanding the interaction between zeolite nanoparticles and cells is imperative for improving their potentialities, since the process of internalization of these particles is still poorly understood. In this study, the intracellular trafficking and internalization kinetics of zeolite L into breast cancer cells and normal epithelial mammary cells were analysed using scanning electron microscopy (SEM), confocal microscopy and transmission electron microscopy (TEM). We also studied the involvement of endocytic pathways using two pharmacological inhibitors, chlorpromazine and dynasore. Zeolite nanoparticles were taken up by both cell types and the cellular uptake was fast, and started immediately after 5 min of incubation. Interestingly, the uptake was dependent on the cell type since in breast cancer cells it was faster and more efficient, with a higher number of nanoparticles being internalized by cancer cells over time, compared to that in the epithelial mammary cells. TEM results showed that the internalized nanoparticles were mainly localized in the cell vacuoles. The data obtained upon using endocytic pharmacological inhibitors suggest that the zeolite L uptake is mediated by caveolin.

Targeting lactate transport suppresses in vivo breast tumour growth.

BACKGROUND: Most cancers, including breast cancer, have high rates of glucose consumption, associated with lactate production, a process referred as "Warburg effect". Acidification of the tumour microenvironment by lactate extrusion, performed by lactate transporters (MCTs), is associated with higher cell proliferation, migration, invasion, angiogenesis and increased cell survival. Previously, we have described MCT1 up-regulation in breast carcinoma samples and demonstrated the importance of in vitro MCT inhibition. In this study, we performed siRNA knockdown of MCT1 and MCT4 in basal-like breast cancer cells in both normoxia and hypoxia conditions to validate the potential of lactate transport inhibition in breast cancer treatment. RESULTS: The effect of MCT knockdown was evaluated on lactate efflux, proliferation, cell biomass, migration and invasion and induction of tumour xenografts in nude mice. MCT knockdown led to a decrease in in vitro tumour cell aggressiveness, with decreased lactate transport, cell proliferation, migration and invasion and, importantly, to an inhibition of in vivo tumour formation and growth. CONCLUSIONS: This work supports MCTs as promising targets in cancer therapy, demonstrates the contribution of MCTs to cancer cell aggressiveness and, more importantly, shows, for the first time, the disruption of in vivo breast tumour growth by targeting lactate transport.

TCTEX1D4 interactome in human testis: unraveling the function of dynein light chain in spermatozoa.

Studies were designed to identify the TCTEX1D4 interactome in human testis, with the purpose of unraveling putative protein complexes essential to male reproduction and thus novel TCTEX1D4 functions. TCTEX1D4 is a dynein light chain that belongs to the DYNT1/TCTEX1 family. In spermatozoa, it appears to be important to sperm motility, intraflagellar transport, and acrosome reaction. To contribute to the knowledge on TCTEX1D4 function in testis and spermatozoa, a yeast two-hybrid assay was performed in testis, which allowed the identification of 40 novel TCTEX1D4 interactors. Curiously, another dynein light chain, TCTEX1D2, was identified and its existence demonstrated for the first time in human spermatozoa. Immunofluorescence studies proved that TCTEX1D2 is an intra-acrosomal protein also present in the midpiece, suggesting a role in cargo movement in human spermatozoa. Further, an in silico profile of TCTEX1D4 revealed that most TCTEX1D4 interacting proteins were not previously characterized and the ones described present a very broad nature. This reinforces TCTEX1D4 as a dynein light chain that is capable of interacting with a variety of functionally different proteins. These observations collectively contribute to a deeper molecular understanding of the human spermatozoa function.

Characterization of monocarboxylate transporter activity in hepatocellular carcinoma.

AIM: To assess the immunoexpression of hypoxia-related markers in samples from cirrhosis and primary and metastatic hepatocellular carcinoma (HCC). METHODS: From a total of 5836 autopsies performed at the Pathology Department - University of Sao Paulo School of Medicine Hospital - from 2003 to 2009, 188 presented primary liver tumors. Immunohistochemical reactivity for monocarboxylate transporters (MCTs)-1, 2 and 4, CD147 and glucose transporter-1 (GLUT1) was assessed in necropsies from 80 cases of HCC. Data were stored and analyzed using the IBM SPSS statistical software (version 19, IBM Company, Armonk, NY). All comparisons were examined for statistical significance using Pearson's χ (2) test and Fisher's exact test (when n < 5). The threshold for significant P values was established as P < 0.05. RESULTS: Plasma membrane expression of MCT4 and overall expression of GLUT1 showed progressively higher expression from non-neoplastic to primary HCC and to metastases. In contrast, overall expression of MCT2 was progressively decreased from non-neoplastic to primary HCC and to metastases. MCT1 (overall and plasma membrane expression), MCT2 and CD147 plasma membrane expression were associated with absence of cirrhosis, while plasma membrane expression of CD147 was also associated with absence of HBV infection. MCT2 overall expression was associated with lower liver weight, absence of metastasis and absence of abdominal dissemination. Additionally, MCT4 plasma membrane positivity was strongly associated with Ki-67 expression. CONCLUSION: MCT4 and GLUT1 appear to play a role in HCC progression, while MCT2 is lost during progression and associated with better prognosis.

Differential sensitivities to lactate transport inhibitors of breast cancer cell lines.

The tumour microenvironment is known to be acidic due to high glycolytic rates of tumour cells. Monocarboxylate transporters (MCTs) play a role in extracellular acidification, which is widely known to be involved in tumour progression. Recently, we have described the upregulation of MCT1 in breast carcinomas and its association with poor prognostic variables. Thus, we aimed to evaluate the effect of lactate transport inhibition in human breast cancer cell lines. The effects of α-cyano-4-hydroxycinnamate, quercetin and lonidamine on cell viability, metabolism, proliferation, apoptosis, migration and invasion were assessed in a panel of different breast cancer cell lines. MCT1, MCT4 and CD147 were differently expressed among the breast cancer cell lines and, as expected, different sensitivities were observed for the three inhibitors. Interestingly, in the most sensitive cell lines, lactate transport inhibition induced a decrease in cell proliferation, migration and invasion, as well as an increase in cell death. Results were validated by silencing MCT1 expression using siRNA. The results obtained here support targeting of lactate transport as a strategy to treat breast cancer, with a special emphasis on the basal-like subtype, which so far does not have a specific molecular therapy.