Anka Thies

This study investigates the effects of mistletoe lectin-I (ML-I) on melanoma growth and spread in vivo. The human melanoma cell line MV3 was xenografted into severe combined immunodeficient mice and vehicle solution or purified ML-I was administered at 30, 150 and 500 ng per kg body weight (20 mice per group) daily. After 19 days, mice were killed, primary tumours (PTs) and lungs were dissected out, and tumour weights, number of lung metastases (LMs), number of tumour-infiltrating dendritic cells (DCs), and apoptosis rates in the melanoma cells and in the DCs were assessed. A 35% reduction of PT weight (P=0.03) and a 55% decrease in number of LMs (P=0.016) were evident for low-dose ML-I (30 ng kg⁻¹) treatment but not for higher doses. Mistletoe lectin-I increased apoptosis rates in the melanoma cells of PTs at all doses, while no induction of apoptosis was noted in the LMs. Low-dose ML-I significantly increased the number of DCs infiltrating the PTs (PPin vivo, primarily due to immunomodulatory effects.

The NFkappaB signalling pathway plays an important role in chemoresistance and decreased apoptosis. One indirect way to inhibit the NFkappaB pathway is to slow down the proteasomal degradation of its inhibitor IkappaB, thus preventing NFkappaB from translocation into the nucleus. Hence, the effect of the proteasome inhibitor bortezomib (Velcade) on the cell proliferation of the MV3, FemX-1 and G361 human melanoma cell lines and its action in combination with the PPAR-gamma agonist rosiglitazone or the mistletoe lectin ML-I, both having anti-proliferative effects on melanoma cells in single agent use, was investigated. Proliferation of melanoma cells under the different treatment regimes over a broad concentration range (0.0001-100 microg/ml) was assessed by means of the XTT cell proliferation assay. At a concentration of 0.1 microg/ml bortezomib significantly reduced the proliferation rate of all melanoma cells to 1-13% of the control, which was mediated through increased apoptosis and inhibition of NFkappaB expression. Furthermore, the combination of bortezomib and rosiglitazone was the most potent and increased the effectiveness against melanoma cell growth by 63-71% (compared to single use of rosiglitazone) and by 27-39% (compared to single use of bortezomib), respectively. This combination strategy might be a promising approach for future melanoma therapy.

Metastasis formation is a complex process and as such can only be modelled in vivo. As markers indicating metastatic spread in syngenic mouse models differ significantly from those in man, this study aimed to develop a human melanoma xenograft mouse model that reflects the clinical situation. Six human melanoma cell lines (LOX, MV3, FEMX-1, G361, MeWo and UISO-Mel6) were xenografted into severe combined immunodeficient mice and tumour growth, metastatic behaviour and number of lung metastases were assessed. Tumours and metastases were analysed for HPA binding and expression of CEACAM-1 and L1, all markers indicative of metastasis in clinical studies. Development of primary tumour nodules ranged from 3 weeks (MV3) to 3 months (MeWo). Whereas G361 and FEMX-1 rarely formed lung metastases, MeWo, MV3 and LOX were moderately and UISO-Mel6 was highly metastatic. Similar to clinical studies, HPA, CEACAM1 and L1 indicated metastatic spread in the xenograft melanoma model, but were not all simultaneously expressed in all cell lines. Considering the strongest expression of one marker combined with an absent or low expression of the other two markers, we conclude that LOX is the cell line of choice for analyses of the functional role of HPA-binding glycoconjugates, UISO-Mel6 is ideally suited to study CEACAM1 function in melanoma spread and L1 function can best be modelled using MeWo.

Aberrant glycosylation is a common feature of metastatic sub‐clones of malignant tumours and in uveal melanoma in particular, the HNK‐1 glycotope has been positively correlated with poor prognosis. So far, no such correlation has been investigated in cutaneous melanoma. In order to do so, HNK‐1 expression was evaluated immunohistochemically in 100 primary cutaneous melanomas and correlated with metastasis after up to 10‐years' follow‐up. Furthermore, HNK‐1 expression was analysed in metastatic deposits (19 distant cutaneous metastases and six sentinel lymph node metastases), as well as in benign nevi. Kaplan–Meier analysis revealed a positive association between HNK‐1 expression and metastasis (p < 0.005) and multivariate Cox regression analysis adjusted for the standard prognostic markers ulceration and vertical tumour thickness confirmed HNK‐1 expression as an independent prognostic marker. HNK‐1 expression was preserved in 42% of the distant cutaneous metastases, but metastatic cells in lymph nodes were devoid of HNK‐1 immunoreactivity. None of the benign pigmented lesions exhibited HNK‐1 immunoreactivity. Expression of the HNK‐1 glycotope in cutaneous malignant melanoma is an independent prognostic marker of metastasis. Differential HNK‐1 expression at the metastatic sites implies that its expression is modulated by the surrounding environment. As HNK‐1 is also transiently expressed during migration of melanocyte precursor cells derived from the neural crest, recapitulation of this transient expression might occur during metastatic spread of cutaneous malignant melanoma. Copyright © 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

PURPOSE: The cell adhesion molecule CEACAM1 is involved in intercellular adhesion and subsequent signal transduction events in a number of epithelia. CEACAM1 downregulation has been demonstrated in colorectal and prostate carcinomas. This study sought to analyze whether its expression in malignant melanoma is associated with metastasis.PATIENTS AND METHODS: CEACAM1 expression was immunohistochemically evaluated in 100 primary cutaneous malignant melanomas and correlated with metastasis in a 10-year follow-up. Furthermore, CEACAM1 expression was analyzed in metastatic lesions (11 distant metastases and six sentinel lymph node metastases). Univariate Kaplan-Meier analysis and multivariate Cox proportional hazard regression analysis adjusted for standard prognostic indicators were performed to assess the prognostic relevance of CEACAM1 expression.RESULTS: A total of 28 of 40 patients with CEACAM1-positive primary melanomas developed metastatic disease, compared with only six of 60 patients with CEACAM1-negative melanomas. Often, the strongest CEACAM1 expression was observed at the invading front. In addition, CEACAM1 expression was preserved in the metastatic lesions. Kaplan-Meier analysis revealed a highly significant association between CEACAM1 expression and metastasis (P < .0001); multivariate Cox regression analysis, including CEACAM1 expression status adjusted for tumor thickness, presence of ulceration, and mitotic rate, confirmed that CEACAM1 is an independent factor for the risk of metastasis and demonstrated that the predictive value of CEACAM1 expression is superior to that of tumor thickness.CONCLUSION: Expression of the cell adhesion molecule CEACAM1 in the primary tumors in melanoma patients is associated with the subsequent development of metastatic disease. This raises the possibility of a functional role for this cell adhesion molecule in the metastatic spread it indicates.

Glycoconjugates, as detected by lectin histochemistry, have been implicated in metastasis formation in many neoplasias. However, no data concerning the three mistletoe lectins (MLs) and the spread of malignant melanoma have been published. The binding status of ML-I, -II and -III was histochemically assessed in 100 malignant melanomas and correlated with metastasis in a 10 year follow-up period. Furthermore, the staining intensity of the three MLs, scored from negative (-) to very intense (+ + +), was evaluated. Kaplan-Meier analsis revealed that very intense binding (+ + +) of ML-I was positively-correlated with metastasis (p=0.044). Since ML-I is specific for galactose, high density galactose expression in malignant melanoma is a predictor of poor prognosis.