Sakunthala Muthugounder

Differential Growth Suppression of Human Melanoma Cells by Tea (Camellia sinensis) Epicatechins (ECG, EGC and EGCG)

We previously reported that catechins of green tea have different antiproliferative effects on cell lines derived from gender-dependent cancers; epicatechin 3-gallate (ECG) had the strongest inhibitory effect. In the present study, we examined the effects of epigallocatechin (EGC), epicatechin-gallate (ECG) and EGC 3-gallate (EGCG) on the viability, density, doubling time and cycle number of cell lines derived from melanoma metastasized to lymph nodes (MB-1133 and SE-0154) or distant organs (CH-0356, JK-0346, SA-1171, GE-0208, NS-1176 and LF-0023). These catechins have been documented to have no growth suppressive or apoptotic effects on normal melanocytes (Nihal et al., Int J Cancer 2005;114:513–21). EGCG (50 μM) showed greater inhibitory potency than EGC (50 μM) in SE-0154, NS-1176, GE-0208 and LF-0023 cell lines but the two catechins produced similar inhibitory effects in CH-0356, JK-0346 and SA-1171 cell lines. The IC50 (50% inhibitory concentration) was lower for EGC than EGCG in MB-1133 and CH-0356 cells, higher for EGC than EGCG in GE-0208 cells and comparable (11–12 μM) for both the catechins in LF-0023 cells. When compared with EGC, the cytotoxic effect (% dead cell counts) and the suppression of the growth (change in cell number) of all melanoma cell lines tested were pronounced with EGCG. This investigation validates the hypothesis that anticancer action of the various catechins may vary with the type of malignancy and provides a model for tumor cell heterogeneity based on susceptibility and resistance of tumor cells to different green tea catechins. Therefore, this information is critical for undertaking chemopreventive or chemotherapeutic trials against melanoma and gender-based cancers.

Cryosurgical ablation of liver tumors in colon cancer patients increases the serum total ganglioside level and then selectively augments antiganglioside IgM

Cryosurgical ablation (CSA) of tumors induces disruptive necrosis. Necrosis may release tumor gangliosides into circulation and they may augment serum antiganglioside antibodies depending on the nature of gangliosides released. The hypothesis is tested by determining the level of serum total gangliosides (STG) and their antibody titers in the sera of colon cancer patients with cryoablated liver tumors. As controls, we examined the sera of patients who underwent radiofrequency ablation (RFA) and regular surgery (RS), none of which cause disruptive necrosis. The STG level (expressed as lipid-bound sialic acids, LBSA) is higher (p(2)<0.001) in 35 patients (stage IV) than in 38 healthy case-controls (median 23.48 mg/dL, Q-range 7.1 vs 16.04 mg/dL, Q-range 4.5). The mean STG level increased significantly to 31.2+/-6.0mg/dL (p(2)<0.03) after CSA. Concomitantly, the IgM titer against colon cancer-associated gangliosides (GM(2), GD(1a), GT(1b)), increased significantly, but no increase was observed against normal tissue gangliosides (GM(3) or GM(1)). Also after RFA and RS, no such increase was observed either in the level of STG or in IgM titer against tumor gangliosides. The results suggest that CSA-induced necrosis might have acted as an adjuvant, because purified gangliosides without exogenous adjuvants even after repeated immunization failed to elicit antibody response. The post-CSA decline in the STG level correlated with the increase in the antibodies, suggesting a homeostatic role of the antibodies.

Endogenous immune response to gangliosides in patients with confined prostate cancer

Our study investigated whether endogenous IgM antibodies to gangliosides occur in patients with early stages of prostate cancer (CaP) patients, after defining ganglioside profiles of CaP cell lines. Immune and resorcinol staining detected the presence of gangliosides GM3, GM2, GD3, GD2 and GD1a but not GM1a, GD1b or GT1b in the extracts of normal prostatic epithelial cells (PrEC) and neoplastic androgen‐insensitive (PC‐3, DU145) and ‐sensitive (LNCaP‐FGC and LNCaP‐FGC‐10) CaP cells. Using a sensitive ELISA, developed and validated in our laboratory, the titers of IgM against 8 gangliosides from sera of patients with benign prostatic hyperplasia (BPH) (n = 11), organ‐confined (T1/T2, n = 36) and unconfined (T3/T4, n = 27) CaP and age‐matched healthy men (n = 11) were determined double‐blinded. Using ANOVA and Fishers least significant difference (LSD) methods, the log‐titers among different groups were compared. CaP patients differed from healthy and BPH patients in increased titers against GD1a and decreased titers against GD3. Titers of antibodies to other gangliosides exhibited no difference between CaP patients and others. The specific augmentation of anti‐GD1a IgM in patients with organ‐confined CaP (stage T1/T2) but not in patients with unconfined CaP (stage T3/T4) or BPH or in healthy controls is striking. This finding together with identification of GD1a as a major ganglioside in CaP cell lines and with the accruing studies on the immunosuppressive nature of GD1a indicates that augmentation of anti‐GD1a IgM in confined CaP may signify an early endogenous immune response to eliminate a “danger signal” from tumor microenvironment and circulation.

Human antiganglioside autoantibodies : Validation of ELISA

Abstract: Gangliosides have a hydrophilic sugar chain that contains antigenic determinants and a hydrophobic ceramide. In humans, gangliosides elicit a T‐cell independent IgM response; antiganglioside IgM autoantibodies may be pentameric or polymeric. A correlation between specific neuropathies and antiganglioside autoantibodies has been confirmed. Although many neurologists attempt to lower titers of antiganglioside autoantibodies, oncologists are developing strategies to augment production of IgM antibodies that will remove immunosuppressive gangliosides from the circulation of patients and target gangliosides and kill tumor cells. Antiganglioside IgM antibodies can cause leakage of the blood‐nerve barrier in a concentration‐dependent and complement‐independent manner, bind to neuronal gangliosides to create a neuromuscular block and serve as a marker of axonal damage in neuropathies such as multiple sclerosis. They are also a promising biomarker of early prostate cancer. There is a need to validate the protocol for enzyme‐linked immunosorbent assay (ELISA) of antiganglioside IgM autoantibodies. This validation must consider the purity of gangliosides from different commercial sources, the coating of gangliosides onto a solid matrix in a manner that maximizes exposure of oligosaccharide epitopes to IgM paratopes, techniques to minimize background noise and eliminate nonspecific antibody binding, and carefully defined positive and negative controls. The validated protocol also must include a simple formula to estimate titers for several replicas. Finally, antibody titers must be converted to natural logs for statistical appraisal.

Increased levels of gangliosides in the plasma and ascitic fluid of patients with advanced ovarian cancer

Objectives  To assess the expression of total gangliosides in primary ovarian cancer cell lines, ascitic fluid and plasma of advanced ovarian cancer patients.

Ganglioside signatures of primary and nodal metastatic melanoma cell lines from the same patient.

The primary cutaneous melanoma initially migrates to the regional lymph nodes (LNs). Human melanoma overexpresses gangliosides, the sialylglycosphingolipids. The ganglioside signatures may differ between primary and LN melanomas owing to the differences in the tumor microenvironments. The melanoma cells obtained from the primary and LN of the same patient might be useful to evaluate the above hypothesis. For this purpose, the cryopreserved cell lines from a primary cutaneous melanoma (IGR-39) and its nodal metastasis (IGR-37) from the same patient were used. We have also compared the ganglioside signatures of freshly obtained melanoma cells from primary, LN and organ metastases from different patients. Gangliosides were extracted, purified and identified by resorcinol and specific murine monoclonal antibodies. Comparison of the primary cell line with the nodal metastatic line obtained from the same patient distinctly showed the following features: (i) an increased production of gangliosides, (ii) O-acetylation of GM2 and GD3, (iii) an increased and altered O-acetylation of GD2 and (iv) possibly de-N-acetylation of GD3. These findings suggest that the nodal microenvironment might favor activation of O-acetyl-transferases capable of O-acetylating both &agr;2, 3 and &agr;2, 8 sialic acids of gangliosides. Supporting this, the primary melanoma cells obtained from different patients, showed no O-acetylation of GD3 or GD2. The cell line from groin LN showed the presence of O-acetyl (O-Ac)GD3. The cell lines from thyroid, spleen and jejunum expressed O-AcGD2. In all metastatic melanoma cell lines GD1a is more prevalent than GD3, suggesting that GD1a may be a major melanoma-ganglioside.

Significance of Endogenous Augmentation of Antiganglioside IgM in Cancer Patients

Abstract:  Gangliosides expressed by solid malignancies are shed into the circulation at a rate that varies with tumor stage, burden, and progression. Gangliosides have an immunosuppressive effect; thus an increase in the total ganglioside (TG) serum level may coincide with tumor progression. However, circulating gangliosides also may induce an endogenous IgM response. Unlike conventional pentameric IgM antibodies against peptide antigens, antiganglioside IgM antibodies can be polymeric and may not have a J‐chain. Because these antibodies can remove shed gangliosides from the tumor microenvironment and the circulation, therapy that actively or passively augments serum levels of IgM against tumor‐derived immunosuppressive gangliosides might restore immunocompetence and thereby slow tumor progression. The success of this approach, in passive and active specific therapy of cancer patients, requires analysis of biopsy tissue or sera of therapy recipients to confirm the presence of target gangliosides, such as GM2 or GD3. A patients response to active or passive immunotherapy against a specific ganglioside target(s) can be monitored by serial assessment of serum specimens for TG level and antiganglioside IgM titer(s). This tailored approach to immunotherapy could be incorporated in postoperative adjuvant protocols.