LDH inhibition impacts on heat shock response and induces senescence of hepatocellular carcinoma cells

In normal cells, heat shock response (HSR) is rapidly induced in response to a variety of harmful conditions and represents one of the most efficient defense mechanism. In cancer tissues, constitutive activation converts HSR into a life-threatening process, which plays a major role in helping cell survival and proliferation. Overexpression of heat shock proteins (HSPs) has been widely reported in human cancers and was found to correlate with tumor progression. Hepatocellular carcinoma is one of the conditions in which HSR activation was shown to have the highest clinical significance. Transcription of HSPs is induced by HSF-1, which also activates glycolytic metabolism and increases the expression of LDH-A, the master regulator of the Warburg effect. In this paper, we tried to explore the relationship between HSR and LDH-A. In cultured hepatocellular carcinoma cells, by using two enzyme inhibitors (oxamate and galloflavin), we found that the reduction of LDH-A activity led to decreased level and function of the major HSPs involved in tumorigenesis. Galloflavin (a polyphenol) also inhibited the ATPase activity of two of the examined HSPs. Finally, hindering HSR markedly lowered the alpha-fetoprotein cellular levels and induced senescence. Specific inhibitors of single HSPs are currently under evaluation in different neoplastic diseases. However, one of the effects usually observed during treatment is a compensatory elevation of other HSPs, which decreases treatment efficacy. Our results highlight a connection between LDH and HSR and suggest LDH inhibition as a way to globally impact on this tumor promoting process

Increased Mortality Rate and Not Impaired Ribosomal Biogenesis is Responsible for Proliferative Defect in Dyskeratosis Congenita Cell Lines

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Lactate Can Modulate the Antineoplastic Effects of Doxorubicin and Relieve the Drug's Oxidative Damage on Cardiomyocytes

Background: Doxorubicin (DOXO) is currently administered as the first-choice therapy for a variety of malignancies. Cancer cells exhibit enhanced glycolysis and lactate production. This metabolite affects gene expression and can play a role in chemoresistance. Aim of this study: We investigated whether the enhanced lactate levels that characterize neoplastic tissues can modify the response of cancer cells to DOXO. Methods: After exposing cancer cells to increased lactate levels, we examined whether this metabolite could interfere with the principal mechanisms responsible for the DOXO antineoplastic effect. Results: Increased lactate levels did not affect DOXO-induced topoisomerase poisoning but offered protection against the oxidative damage caused by the drug. This protection was related to changes in gene expression caused by the combined action of DOXO and lactate. Oxidative damage significantly contributed to the heavy cardiotoxicity following DOXO treatment. In cultured cardiomyocytes, we confirmed that DOXO-induced DNA damage and oxidative stress can be significantly mitigated by exposing the cells to increased lactate levels. Conclusions: In addition to contributing to elucidating the effects of the combined action of DOXO and lactate, our results suggest a possible method to reduce the heavy drug cardiotoxicity, a major side effect leading to therapy discontinuation

Epigallocatechin-3-gallate and 6-OH-11-O-Hydroxyphenanthrene Limit BE(2)-C Neuroblastoma Cell Growth and Neurosphere Formation In Vitro

We conducted an in vitro study combining a rexinoid, 6-OH-11-O-hydroxyphenanthrene (IIF), and epigallocatechin-3-gallate (EGCG), which is the main catechin of green tea, on BE(2)-C, a neuroblastoma cell line representative of the high-risk group of patients. Neuroblastoma is the most common malignancy of childhood: high-risk patients, having N-MYC over-expression, undergo aggressive therapy and show high mortality or an increased risk of secondary malignancies. Retinoids are used in neuroblastoma therapy with incomplete success: the association of a second molecule might improve the efficacy. BE(2)-C cells were treated by EGCG and IIF, individually or in combination: cell viability, as evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, was reduced, EGCG+IIF being the most effective treatment. Apoptosis occurred and the EGCG+IIF treatment decreased N-MYC protein expression and molecular markers of invasion (MMP-2, MMP-9 and COX-2). Zymography demonstrated nearly 50% inhibition of MMP activity. When BE(2)-C cells were grown in non-adherent conditions to enrich the tumor-initiating cell population, BE(2)-C-spheres were obtained. After 48 h and 72 h treatment, EGCG+IIF limited BE(2)-C-sphere formation and elicited cell death with a reduction of N-MYC expression. We concluded that the association of EGCG to IIF might be applied without toxic effects to overcome the incomplete success of retinoid treatments in neuroblastoma patients

EGFR inhibition by (-)-epigallocatechin-3-gallate and IIF treatments reduces breast cancer cell invasion

Epidermal growth factor receptor (EGFR) expression is an important marker in breast carcinoma pathology and is considered a pivotal molecule for cancer cell proliferation, invasion and metastasis. We investigated the effects of epigallocatechin-3-gallate (EGCG), the most active green tea catechin, in combination with 6-OH-11-O-hydroxyphenanthrene (IIF), a synthetic retinoid X receptor-\u3b3 (RXR\u3b3) agonist, on three breast carcinoma cell lines: MCF-7, MCF-7TAM and MDA-MB-231. EGFR and AKT activation and molecular markers of cell motility and migration (CD44, extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN), MMP-2, MMP-9 and tissue inhibitor of metalloproteinases (TIMPs)) were studied after EGCG and IIF treatments. The EGCG + IIF treatment was the most active in down-regulating EGFR phosphorylation at Tyr(1068) in all the investigated cell lines; p473AKT was also down-regulated in MCF-TAM cells. EGCG + IIF was also the most active treatment in reducing the expression of markers of invasion and migration in all the three cell lines: CD44, EMMPRIN, MMP-2 and -9 expression decreased, whereas TIMPs were up-regulated. Zymography and scratch assay also confirmed the reduced invasion tendency. We considered that EGCG and IIF treatments could alter the molecular network based on EGFR, CD44 and EMMPRIN expression interdependence and reduced the migration tendency in MCF-7, MCF-7TAM and MDA-MB-231 cells. These events only occurred in association with AKT inactivation in MCF-7TAM cells. In conclusion, the combination of EGCG and IIF significantly attenuated the invasive behaviour of breast carcinoma cells

A RXR Ligand 6-OH-11-O-Hydroxyphenanthrene with Antitumour Properties Enhances (−)-Epigallocatechin-3-gallate Activity in Three Human Breast Carcinoma Cell Lines

(−)-Epigallocatechin-3-gallate (EGCG) and chemotherapeutic agents cotreatment can improve cytotoxicity against cancer cells. We showed that EGCG and the rexinoid 6-OH-11-O-hydroxyphenanthrene (IIF), given together, were cytotoxic toward MCF-7, MCF-7TAM, and MDA-MB-231, three breast carcinoma cell lines showing different molecular characteristics. Cell growth arrest and apoptosis were greater after EGCG and IIF cotreatment than after individual administration. Cytotoxicity was related to upregulation of 67-kDa laminin receptor (LR67), one of the principal molecular targets of EGCG, and activation of the nuclear retinoic X receptors (RXRs) pathway. Furthermore, the transcription factor Forkhead box O3 (Foxo3a), a protein able to trigger apoptosis through upregulation of genes necessary for cell death, was activated. EGCG and IIF cotreatment produced a significant nuclear import of Foxo3a from the cytoplasm in MCF-7, MCF-7TAM, and MDA-MB-231 cells. In MCF-7TAM cells only, Foxo3a nuclear localization was associated with p473AKT downregulation. For the first time we showed that when EGCG and IIF, two harmless molecules, were given together, they might increase cytotoxicity in three breast carcinoma cell lines, two of them being representative of poorly responsive breast carcinoma types

The activation of lactate dehydrogenase induced by mTOR drives neoplastic change in breast epithelial cells.

mTOR kinase and the A isoform of lactate dehydrogenase (LDH-A) are key players controlling the metabolic characteristics of cancer cells. By using cultured human breast cells as a "metabolic tumor" model, we attempted to explore the correlation between these two factors. "Metabolic tumors" are defined as neoplastic conditions frequently associated with features of the metabolic syndrome, such as hyper-insulinemia and hyper-glycemia. MCF-7 cells (a well differentiated carcinoma) and MCF-10A cells (a widely used model for studying normal breast cell transformation) were used in this study. These cells were exposed to known factors triggering mTOR activation. In both treated cultures, we evaluated the link between mTOR kinase activity and the level of LDH expression / function. Furthermore, we elaborated the metabolic changes produced in cells by the mTOR-directed LDH-A up-regulation. Interestingly, we observed that in the non-neoplastic MCF-10A culture, mTOR-directed up-regulation of LDH-A was followed by a reprogramming of cell metabolism, which showed an increased dependence on glycolysis rather than on oxidative reactions. As a consequence, lactate production appeared to be enhanced and cells began to display increased self-renewal and clonogenic power: signals suggestive of neoplastic change. Enhanced clonogenicity of cells was abolished by rapamycin treatment, and furthermore heavily reduced by LDH enzymatic inhibition. These results highlighted a mechanistic link between metabolic alterations and tumorigenesis, whereby suggesting LDH inhibition as a possible chemo-preventive measure to target the metabolic alterations driving neoplastic change

Epigallocatechin-3-gallate Increases RXR\u3b3-mediated Pro-apoptotic and Anti-invasive Effects in Gastrointestinal Cancer Cell Lines

Molecules with synergistic effects often enhance the benefits of cancer therapy. We observed that the major catechin of green tea, (-)-Epigallocatechin-3-gallate (EGCG), induced retinoid X receptor-\u3b3 (RXR\u3b3) expression in the SK-Ch-A1 cholangiocarcinoma cell line and in two colon carcinoma cell lines (LoVo and the derivative multi-drug resistant LoVoMDR). On this basis, we analyzed the effects of EGCG in combination with an RXR\u3b3 ligand, 6-OH-11-O-hydroxyphenantrene (IIF), or with a ligand of retinoic acid receptor, all-trans-retinoic acid (RA). IIF alone and in combination with EGCG activated the retinoic X response elements and induced the germ cell nuclear factor. In parallel, EGCG induced 67 kDa laminin receptor expression alone and in combination with IIF. We observed a synergistic growth inhibition with EGCG and IIF in combination at lower doses. These effects were accompanied by apoptosis activation through the mitochondrial pathway. Moreover, in LoVo cell line we observed an induction of Forkhead box O3 expression, another molecule involved in apoptosis activation. Finally, metalloproteinase activity and extracellular matrix metalloproteinase inducer (EMMPRIN) expression were inhibited and tumor cell invasion was strongly reduced in the SK-Ch-A1 cell line after treatment with EGCG and IIF. In conclusion, the use of specific RXR ligands in combination with catechins could open a new perspective in gastrointestinal tumor chemoprevention