Polyphenol from millet bran increases the sensitivity of colorectal cancer cells to oxaliplatin by blocking the ganglioside GM3 catabolism.

Colorectal cancer (CRC) is an aggressive malignancy with very limited therapeutic approaches. Drug resistance develops as a frequent characteristic in many patients with CRC, which leads to a decrease in the therapeutic efficacy of anticancer agents. Our previous evidences showed that bound polyphenol from millet bran (BPIS) possesses the potential of inhibiting cancer cell proliferation, and its main anticancer components are ferulic acid (FA) and p-coumaric acid (p-CA). In the present study, we found that BPIS significantly increases the sensitivity of human drug-resistant CRC cell line to oxaliplatin (OXA), a commonly used chemotherapy drug against CRC. Mechanistically, we indicated that BPIS significantly impairs the expression of a gene encoding multidrug resistance protein 1 (MDR1), a well-known permeability glycoprotein (P-gp), by preventing ganglioside GM3 catabolism. Neuraminidase 3 (NEU3) is a key enzyme catalyzing the conversion of ganglioside GM3 to ceramide trihexosides (Gb3), whose expression is increased in drug-resistant HCT-116/L cells. BPIS treatment increased GM3 level, but reduced Gb3 and P-gp levels by inhibiting NEU3 expression, which subsequently boosted the chemotherapy sensitivity of drug-resistant HCT-116/L cells to OXA. These findings reveal that BPIS increases the chemo-sensitivity by remodeling NEU3-mediated ganglioside GM3 catabolism, and it may be applied as a novel drug for facilitating the effectiveness of chemotherapeutic agents in CRC.

GM3(Neu5Gc) ganglioside: an evolution fixed neoantigen for cancer immunotherapy.

Numerous molecules have been considered as targets for cancer immunotherapy because of their levels of expression on tumor cells, their putative importance for tumor biology, and relative immunogenicity. In this review we focus on the ganglioside GM3(Neu5Gc), a glycosphingolipid present on the outer side of the plasma membrane of vertebrate cells. The reasons for selecting GM3(Neu5Gc) as a tumor-specific antigen and its use as a target for cancer immunotherapy are discussed, together with the development of antitumor therapies focused on this target by the Center of Molecular Immunology (CIM, Cuba).

Antitumor and cytotoxic properties of a humanized antibody specific for the GM3(Neu5Gc) ganglioside.

Gangliosides are sialic acid-bearing glycosphingolipids expressed on all mammalian cell membranes, and participate in several cellular processes. During malignant transformation their expression changes, both at the quantitative and qualitative levels. Of particular interest is the overexpression by tumor cells of Neu5Gc-gangliosides, which are absent, or detected in trace amounts, in human normal cells. The GM3(Neu5Gc) ganglioside in particular has been detected in many human tumors, and it is considered one of the few tumor specific antigen. We previously demonstrated that a humanized antibody specific for this molecule, named 14F7hT, retained the binding and cytotoxic properties of the mouse antibody. In this work, we confirm that 14F7hT exerts a non-apoptotic cell death mechanism in vitro and shows its potent in vivo antitumor activity on a solid mouse myeloma model. Also, we demonstrate, in contrast to the murine counterpart, the capacity of this antibody to induce antibody-dependent cell-mediated cytotoxicity using human effector cells, which increases its potential for the treatment of GM3(Neu5Gc)-expressing human tumors.

Racotumomab-alum vaccine for the treatment of non-small-cell lung cancer.

Racotumomab-alum vaccine is an anti-idiotypic vaccine able to mimic the tumor-associated antigen NeuGcGM3. Different Phase I clinical trials and compassionate use studies demonstrated its low toxicity and capacity to induce a strong anti-NeuGcGM3 response, able to bind and directly kill tumor cells expressing the antigen. A Phase II/III randomized double-blind clinical trial in advanced non-small cell lung cancer patients showed a significant improvement in overall survival and progression-free survival for racotumomab-alum versus placebo. Patients who developed anti-NeuGcGM3 antibodies capable of binding and killing NeuGcGM3 expressing tumor cells showed significantly longer median survival times. The impact of using racotumomab-alum as switch maintenance followed by second-line therapy is currently being explored in a new randomized, multinational Phase III study.

Membrane microdomains and insulin resistance.

A new concept, that "metabolic disorders, such as type 2 diabetes, are membrane microdomain disorders caused by aberrant expression of gangliosides", has arisen. By examining this working hypothesis, we demonstrate the molecular pathogenesis of type 2 diabetes and insulin resistance focusing on the interaction between insulin receptor and gangliosides in microdomains and propose the new therapeutic strategy "membrane microdomain ortho-signaling therapy".

Ganglioside GM3 is involved in neuronal cell death.

Gangliosides abundant in the nervous system have been implicated in a broad range of biological functions, including the regulation of cell proliferation and death. Glutamate-induced cell death, which is accompanied by an accumulation of reactive oxygen species (ROS), is a major contributor to pathological cell death within the nervous system. However, the mechanism underlying this neuronal cell death has not been fully elucidated. In this study, we report that ganglioside GM3 is involved in neuronal cell death. GM3 was up-regulated in the mouse hippocampal cell line HT22 death caused by glutamate. Increment in GM3 levels by both the exogenous addition of GM3 and the overexpression of the GM3 synthase gene induced neuronal cell death. Overexpression of GM3 synthase by microinjecting mRNA into zebrafish embryos resulted in neuronal cell death in the central nervous system (CNS). Conversely, RNA interference-mediated silencing of GM3 synthase rescued glutamate-induced neuronal death, as evidenced by the inhibition of massive ROS production and intracellular calcium ion influx. 12-lipoxygenase (12-lipoxygenase) (12-LOX) was recruited to glycosphingolipid-enriched microdomains (GEM) in a GM3-dependent manner during oxidative glutamate toxicity. Our findings suggest that GM3 acts as not only a mediator of oxidative HT22 death by glutamate but also a modulator of in vivo neuronal cell death.

Ganglioside GM3 inhibits the high glucose- and TGF-beta1-induced proliferation of rat glomerular mesangial cells.

Abrupt proliferation of glomerular mesangial cells (GMCs) is a common feature in the early stage of diabetic glomerulopathy, and ganglioside GM3 (NeuAcalpha3Galbeta4Glcbeta1Cer) is thought to regulate the proliferation of many cell types. Recently, we have reported ganglioside GM3 as a modulator of glomerular hypertrophy in streptozotocin-induced diabetic rats []. This study examined whether modulation of cellular ganglioside GM3 could regulate the high glucose- and transforming growth factor-beta1 (TGF-beta1)-induced proliferation of GMCs. To pharmacologically modulate the cellular ganglioside GM3, GMCs originated from rat kidneys were cultured with exogenous ganglioside GM3 or d-threo-PDMP, an inhibitor of ganglioside synthesis, in the RPMI 1640 media containing normal (5.6 mM, NG) or high (25 mM, HG) glucose. HG, TGF-beta1 (10 ng/ml) and d-threo-PDMP (20 microM) significantly stimulated the mesangial cell proliferation, whereas these increments were remarkable attenuated by exogenous ganglioside mixture (0.1-0.2 mg/ml) or GM3 (20-100 microM) in a dose-dependent manner. The mesangial cell proliferation caused by HG, TGF-beta1 and d-threo-PDMP was closely correlated with decreases in both cellular sialic acid contents and ganglioside GM3 synthase activity. Based upon the mobility on high-performance thin-layer chromatography (HPTLC), GMCs showed a complex pattern of ganglioside expression that consisted, at least, of five different components of gangliosides, mainly ganglioside GM3. HG, TGF-beta1 and d-threo-PDMP induced a significant reduction of ganglioside expression with apparent changes in the composition of ganglioside GM3, and semi-quantitative analysis by HPTLC showed that ganglioside GM3 expression reduced to about 35-54% of control. These results provide a pathophysiological link between mesangial cell proliferation and ganglioside GM3 expression, indicating that exogenously added ganglioside GM3 inhibits the high-ambient glucose- and TGF-beta1-induced proliferation of cultured GMCs.

Mechanism for the negative regulation of cell proliferation by ganglioside GM3 in high glucose-treated glomerular mesangial cells.

We recently identified ganglioside GM3 as a modulator of glomerular hypertrophy in streptozotocin-induced diabetic rats (Life Sci., 72: 1997-2006, 2003). This study examined whether alteration of ganglioside GM3 expression could modulate the high glucose-induced proliferation of glomerular mesangial cells (GMCs). GMCs isolated from rat kidneys were cultured under normal (5.6 mM) or high (25 mM) glucose condition for 24-72 hrs. Cell proliferation was predominantly stimulated when GMCs were cultured with high glucose as well as 20 microM of d-threo-PDMP, an inhibitor of ganglioside biosynthesis, for 24 hrs, whereas raising ambient glucose significantly reduced the mesangial sialic acid contents. Based upon mobility on high-performance thin-layer chromatography (HPTLC), GMCs showed a complex pattern of ganglioside expression that consisted of three major components of gangliosides, mainly GM3. High glucose induced a significant reduction of ganglioside expression with apparent changes in the composition of major ganglioside GM3, and semi-quantitative analysis by HPTLC showed that ganglioside GM3 was reduced to 62% of GMCs cultured under normal glucose condition. A prominent immunofluorescence microscopy using anti-GM3 monoclonal antibody also showed a dramatic disappearance of immunoreactivity in high glucose-treated GMCs. Moreover, high glucose significantly lowered the Km values of GM3 synthase (16 microM vs. 49 microM), but did not change the Vmax. These results provide the pathophysiological relationship between the high glucose-induced proliferation of GMCs and the decreased expression of ganglioside GM3, indicating a mechanism for the negative regulation of mesangial proliferation by ganglioside GM3. This mechanism may play an important role in the development of diabetic glomerulopathy.

GM3 content modulates the EGF-activated p185c-neu levels, but not those of the constitutively activated oncoprotein p185neu.

The functional relationship between ganglioside GM(3) and two tyrosine-kinase receptors, the normal protein p185(c-neu) and the mutant oncogenic protein p185(neu), was examined in HC11 cells and in MG1361 cells, respectively. In the former, p185(c-neu) expression and activation are controlled by EGF addition to the culture medium and by epidermal growth factor receptor (EGFR) activity, whereas the latter express unchangingly high levels of constitutively activated p185(neu). Studies were carried out using (+/-)-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride ([D]-PDMP), which inhibits ganglioside biosynthesis resulting in ganglioside depletion, and addition of exogenous GM(3) to the culture medium. In HC11 cells treated with only [D]-PDMP, p185(c-neu) levels remain similar to control cells, whereas levels of tyrosine-phosphorylated p185(c-neu) increase after treatment with [D]-PDMP in combination with EGF. When exogenous GM(3) is added in combination with [D]-PDMP and EGF, the enhanced phosphorylated-p185(c-neu) returns to control levels. Interestingly, EGFR levels also vary and, analogously to phosphorylated-p185(c-neu), the increase of EGFR content consequent to the [D]-PDMP and EGF addition is reversed by exogenous GM(3). In contrast, the addition of neither [D]-PDMP nor exogenous GM(3) modifies expression and tyrosine-phosphorylation levels of p185(neu) in MG1361 cells. These findings indicate that changes in GM(3) content modulate the tyrosine-phosphorylated p185(c-neu) levels in a reversible manner, but this is not specific for p185(c-neu) because EGFR levels are also modified. Furthermore, these data suggest that GM(3) may play a functional role by affecting the internalisation pathway of p185(c-neu)/EGFR heterodimers, but not of p185(neu) homodimers.

GM3 ganglioside inhibits endothelin-1-mediated signal transduction in C6 glioma cells.

We found that sparse and confluent C6 glioma cells differ both in GM3 content, which increases with cell density, and in endothelin-1 (ET-1)-induced phosphoinositide hydrolysis, which was markedly higher in the sparse cells than in the confluent. Also after manipulation of the cellular GM3 content through treatment with exogenous GM3 or with drugs known to affect GM3 metabolism, the ET-1 effect was inversely related to GM3 cellular levels. Cell treatment with an anti-GM3 mAb resulted in the enhancement of ET-1-induced phospholipase C activation and restored the capacity of GM3-treated cells to respond to ET-1. These findings suggest that the GM3 ganglioside represents a physiological modulator of ET-1 signaling in glial cells.

3'-Azidothymidine significantly alters glycosphingolipid synthesis in melanoma cells and decreases the shedding of gangliosides.

In this report, we establish that 3'-azido-3'-deoxythymidine (AZT) treatment of melanoma cells greatly alters the pattern of glycosphingolipid biosynthesis. In SK-MEL-30 cells, synthesis of the gangliosides GM3 and GD3 was significantly inhibited (60% and 50% of control, respectively) and the production of their precursor, lactosylceramide, was stimulated by 2.5-fold. Control experiments established that phospholipid synthesis was not affected by AZT treatment, consistent with AZT treatment only affecting lipid biosynthetic reactions that involve glycosylation. Likely as a consequence of decreased rates of ganglioside synthesis, AZT treatment of SK-MEL-30 cells also significantly suppressed the amount of gangliosides shed from the membranes of these cells. Since shedding of gangliosides has been proposed to allow melanoma cells to avoid destruction by the immune system and alterations of glycosphingolipid levels are likely important for the malignant cell phenotype, these results may have important implications regarding the potential use of AZT or related glycosylation inhibitors as cancer chemotherapeutics.

Gangliosides GD1a and GM3 induce interleukin-10 production by human T cells.

Gangliosides are sialic acid-containing glycosphingolipids and exhibit various physiologic functions. Gangliosides GD1a and GM3 strongly induced interleukin-10 (IL-10) protein secretion and mRNA expression in T cells from normal human subjects while the other gangliosides were ineffective. IL-10 induction by both gangliosides was completely blocked by protein tyrosine kinase (PTK) inhibitors, herbimycin A, genistein, and tyrphostin AG 1288, but not by other signal transduction inhibitors. These results suggest that GD1a and GM3 may induce IL-10 production in T cells by regulating the PTK-dependent signaling pathway. These gangliosides may thus act as important immunoregulators via IL-10.

Angiogenic and angiostatic microenvironment in tumors--role of gangliosides.

Gangliosides are important components of the cell membrane that are usually shed in the surrounding microenvironment by neoplastic cells. Gangliosides can also modulate the angiogenic response of microvessels stimulated by angiogenic factors. The experiments reported here make a contribution to the assessment of the nature of this angiogenic modulation, by demonstrating that a) GM3 gangliosides can block the proliferation of endothelium induced by neoplastic cells from human tumors of five different origins; b) this block also occurs when the endothelial cells are preincubated with GM3 and disappears when the cells are returned to a medium poor in GM3; c) in the presence of GM3 the capacity of the endothelial cells to bind to fibronectin and to collagen types I and IV was sharply reduced; d) concentrations of GM3 able to block endothelial cell growth are counteracted by addition to the medium of GT1b ganglioside. The data suggest that the prevalence of a microenvironment rich in GM3 prevents proliferation of vascular endothelium, but the appropriate presence of another ganglioside, such as GT1b, nullifies the effect. Modulation of the angiogenic response of vascular endothelium to angiogenic factors released by tumors is probably dependent on the distribution and activity of growth factor receptors on the endothelial cell surface. The nature and concentration of the gangliosides in the endothelial microenvironment have a decisive influence on this event and possibly on the progression of tumor-induced angiogenesis.

Growth inhibition of human lung adenocarcinoma cells by antibodies against epidermal growth factor receptor and by ganglioside GM3: involvement of receptor-directed protein tyrosine phosphatase(s).

Growth of the EGF receptor-expressing non-small-cell lung carcinoma cell line H125 seems to be at least partially driven by autocrine activation of the resident EGF receptors. Thus, the possibility of an EGF receptor-directed antiproliferative treatment was investigated in vitro using a monoclonal antibody (alpha EGFR ior egf/r3) against the human EGF receptor and gangliosides which are known to possess antiproliferative and anti-tyrosine kinase activity. The moderate growth-inhibitory effect of alpha EGFR ior egf/r3 was strongly potentiated by the addition of monosialoganglioside GM3. Likewise, the combination of alpha EGFR ior egf/r3 and GM3 inhibited EGF receptor autophosphorylation activity in H125 cells more strongly than either agent alone. A synergistic inhibition of EGF receptor autophosphorylation by alpha EGFR ior egf/r3 and GM3 was also observed in the human epidermoid carcinoma cell line A431. In both cell lines, the inhibition of EGF receptor autophosphorylation by GM3 was prevented by pretreatment of the cells with pervanadate, a potent inhibitor of protein tyrosine phosphatases (PTPases). Also, GM3 accelerated EGF receptor dephosphorylation in isolated A431 cell membranes. These findings indicate that GM3 has the capacity to activate EGF receptor-directed PTPase activity and suggest a novel possible mechanism for the regulation of cellular PTPases.