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New thymidylate synthase inhibitors induce apoptosis in melanoma cell lines
S Giudice1, L Benassi, G Bertazzoni
1Department of Dermatology, University of Modena and Reggio Emilia, Italy.
Abstract:
Malignant melanoma is particularly resistant to conventional chemotherapy and radiotherapy. For this reason in the past years a huge variety of new compounds has been developed with potential chemotherapeutic activity which needs to be tested in vitro and in vivo. We investigated the in vitro action of three new experimental antifolate substances (MR7, MR21 and MR36) with a critical target for thymidylate synthase (TS), an essential enzyme for DNA synthesis. The response of two melanoma cell lines (SK-MEL-2 derived from malignant melanoma metastasis and SK-MEL-28 derived from primary malignant melanoma) was examined after treatment with these substances. The antifolate agents induced apoptosis in SK-MEL-2 and SK-MEL-28 cells as confirmed by the TUNEL technique and Comet Assay. Western-blot analysis showed a down-regulation of Bcl-2 protein level and PARP cleavage, otherwise p53 and Bax expressions were not modulated. Moreover, these antifolate-induced apoptosis was accompanied by both pro-caspase-9 and -8 activations. These results were supported by the use of the pan-caspases inhibitor Z-VAD-FMK that almost completely decreased the amount of apoptosis in both the melanoma cell lines treated with antifolate. In conclusion our results show that TS inhibitors are able to induce apoptosis through a caspase-mediated pathway, but without the involvement of the p53/Bax signalling.
Insights
New antifolate drugs targeting thymidylate synthase (TS) effectively induce apoptosis in melanoma cells. This cell death pathway is mediated by caspases but does not involve p53 or Bax signaling.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Malignant melanoma exhibits resistance to conventional therapies.
- Development of novel chemotherapeutic agents is crucial for melanoma treatment.
- Thymidylate synthase (TS) is a key enzyme in DNA synthesis and a target for antifolates.
Purpose of the Study:
- To evaluate the in vitro efficacy of three novel antifolate compounds (MR7, MR21, MR36) against melanoma cell lines.
- To investigate the mechanism of action of these antifolates, focusing on apoptosis induction and signaling pathways.
Main Methods:
- Treatment of two melanoma cell lines (SK-MEL-2 and SK-MEL-28) with experimental antifolates.
- Assessment of apoptosis using TUNEL assay and Comet Assay.
- Western-blot analysis to examine protein expression (Bcl-2, PARP, p53, Bax).
- Evaluation of caspase activation (caspase-9, -8) and inhibition using Z-VAD-FMK.
Main Results:
- Antifolate treatment induced apoptosis in both melanoma cell lines.
- Down-regulation of Bcl-2 and cleavage of PARP were observed.
- p53 and Bax protein expressions remained unchanged.
- Antifolate-induced apoptosis involved the activation of caspase-9 and caspase-8.
- Inhibition of caspases significantly reduced antifolate-induced apoptosis.
Conclusions:
- Novel TS inhibitors effectively induce apoptosis in melanoma cells.
- The observed apoptosis is mediated by a caspase-dependent pathway.
- The p53/Bax signaling pathway is not involved in the antifolate-induced cell death mechanism.
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