The effect of tricyclic antidepressants on cutaneous melanoma cell lines and primary cell cultures.

The tricyclic antidepressants have previously been shown to exert activity against glioma cells in vitro. Initial studies in cell lines suggested that this might extend to melanoma cells. We have therefore conducted a study in primary cell cultures from metastatic cutaneous melanoma deposits using a well established ATP-based tumour chemosensitivity assay to confirm and extend these findings. Two cell lines and eight primary cell cultures from metastatic melanoma deposits were exposed to three tricyclic drugs, amitriptyline, nortriptyline and clomipramine, at concentrations ranging from 200 to 6.25 µmol/l in the ATP-based tumour chemosensitivity assay. All three drugs showed activity, although nortriptyline was more active than clomipramine or amitriptyline in both cell lines and primary cell cultures, with an IC50 of 9, 27 and 33 µmol/l, respectively. Tricyclic agents show activity against melanoma in vitro. This could be related to the lysosomal effects based on their cationic amphiphilic properties, or effects at the mitochondrial membrane.

Approaches to mitochondrially mediated cancer therapy.

For some malignant cancers even combined surgical, radiotherapeutic and chemotherapeutic approaches are not curative, indeed, in certain tumour types even a modest survival benefit is difficult to achieve. There are various biological reasons which underlie this profound resistance but the propensity of cancer cells to repair breaks caused by DNA-damaging radiation and cytotoxic drugs is of major significance in this context. Such highly resistant tumours include the malignant gliomas which are intrinsic to and directly affect the brain and spinal cord. In evaluating approaches which do not elicit tumour cell death directly by DNA damage, it is intriguing to consider mitochondrially mediated apoptosis as a potentially effective alternative. Since the mitochondrial membrane potentials in cancer cells are frequently reduced in comparison with those of non-neoplastic cells this allows a window of opportunity for small molecule agents to enter the tumour cell mitochondria and reduce oxygen consumption with subsequent release of cytochrome c and activation of a caspase pathway to apoptosis which is cancer cell specific. In the quest for agents which can selectively destroy neoplastic cells in this manner, whilst leaving normal adjacent cells intact, various tricyclic drugs have come under scrutiny. In a range of laboratory assays we, and others, have established that certain cancers and, in particular, malignant glioma, are intrinsically sensitive to this approach. We have also established the cellular, molecular and biochemical mechanisms underlying this process. While such archival tricyclics as the antidepressants, clomipramine and amitriptyline, have been used in these experiments their commercial development in cancer therapy has not been forthcoming and their clinical use in glioma has been confined to anecdotal cases. In addition, the dose-dependant role of agents such as anticonvulsants and steroids commonly used in glioma patients in modulating efficacy of the tricyclics is a matter for continued investigation. Other ways of targeting the mitochondrion for cancer therapy include exploitation of the 18kDa translocator protein (peripheral-type benzodiazepine receptor) within the mitochondrial permeability transition pore and enzymatic or molecular modification of a species of ganglioside (GD3/GD3(A)) expressed on the surface of neoplastic cells which are determinants of mitochondrially mediated apoptosis. It is hoped that such approaches may lead to clinical programmes which will improve the prognosis for patients suffering from highly resistant neoplasms.

Storage of cell lines.

The successful storage of cell lines depends upon many factors, including the condition of the cells to be frozen and the experience of the operator. Attempting to freeze down unhealthy, contaminated or poorly labelled cells can have huge implications for a research laboratory. This chapter outlines the importance of good record keeping, vigilant monitoring, aseptic technique, and high-quality reagents in the successful storage and downstream propagation of cell lines.

The molecular basis of the chemosensitivity of metastatic cutaneous melanoma to chemotherapy.

BACKGROUND: Chemotherapy benefits relatively few patients with cutaneous melanoma. The assessment of tumour chemosensitivity by the ATP-based tumour chemosensitivity assay (ATP-TCA) has shown strong correlation with outcome in cutaneous melanoma, but requires fresh tissue and dedicated laboratory facilities. AIM: To examine whether the results of the ATP-TCA correlate with the expression of genes known to be involved in resistance to chemotherapy, based on the hypothesis that the molecular basis of chemosensitivity lies within known drug resistance mechanisms. METHOD: The chemosensitivity of 47 cutaneous melanomas was assessed using the ATP-TCA and correlated with quantitative expression of 93 resistance genes measured by quantitative reverse transcriptase PCR (qRT-PCR) in a Taqman Array after extraction of total RNA from formalin-fixed paraffin-embedded tissue. RESULTS: Drugs susceptible to particular resistance mechanisms showed good correlation with genes linked to these mechanisms using signatures of up to 17 genes. Comparison of these signatures for DTIC, treosulfan and cisplatin showed several genes in common. HSP70, at least one human epidermal growth factor receptor, genes involved in apoptosis (IAP2, PTEN) and DNA repair (ERCC1, XPA, XRCC1, XRCC6) were present for these agents, as well as genes involved in the regulation of proliferation (Ki67, p21, p27). The combinations tested included genes represented in the single agent signatures. CONCLUSIONS: These data suggest that melanoma chemosensitivity is influenced by known resistance mechanisms, including susceptibility to apoptosis. Use of a candidate gene approach may increase understanding of the mechanisms underlying chemosensitivity to drugs active against melanoma and provide signatures with predictive value.

Patterns of expression of DNA repair genes and relapse from melanoma.

PURPOSE: To use gene expression profiling of formalin-fixed primary melanoma samples to detect expression patterns that are predictive of relapse and response to chemotherapy. EXPERIMENTAL DESIGN: Gene expression profiles were identified in samples from two studies (472 tumors). Gene expression data for 502 cancer-related genes from these studies were combined for analysis. RESULTS: Increased expression of DNA repair genes most strongly predicted relapse and was associated with thicker tumors. Increased expression of RAD51 was the most predictive of relapse-free survival in unadjusted analysis (hazard ratio, 2.98; P = 8.80 × 10(-6)). RAD52 (hazard ratio, 4.73; P = 0.0004) and TOP2A (hazard ratio, 3.06; P = 0.009) were independent predictors of relapse-free survival in multivariable analysis. These associations persisted when the analysis was further adjusted for demographic and histologic features of prognostic importance (RAD52 P = 0.01; TOP2A P = 0.02). Using principal component analysis, expression of DNA repair genes was summarized into one variable. Genes whose expression correlated with this variable were predominantly associated with the cell cycle and DNA repair. In 42 patients treated with chemotherapy, DNA repair gene expression was greater in tumors from patients who progressed on treatment. Further data supportive of a role for increased expression of DNA repair genes as predictive biomarkers are reported, which were generated using multiplex PCR. CONCLUSIONS: Overexpression of DNA repair genes (predominantly those involved in double-strand break repair) was associated with relapse. These data support the hypothesis that melanoma progression requires maintenance of genetic stability and give insight into mechanisms of melanoma drug resistance and potential therapies.