Molecular mechanism implicated in Pemetrexed-induced apoptosis in human melanoma cells

<p>Abstract</p> <p>Background</p> <p>Metastatic melanoma is a lethal skin cancer and its incidence is rising every year. It represents a challenge for oncologist, as the current treatment options are non-curative in the majority of cases; therefore, the effort to find and/or develop novel compounds is mandatory. Pemetrexed (Alimta®, MTA) is a multitarget antifolate that inhibits folate-dependent enzymes: thymidylate synthase, dihydrofolate reductase and glycinamide ribonucleotide formyltransferase, required for <it>de novo</it> synthesis of nucleotides for DNA replication. It is currently used in the treatment of mesothelioma and non-small cell lung cancer (NSCLC), and has shown clinical activity in other tumors such as breast, colorectal, bladder, cervical, gastric and pancreatic cancer. However, its effect in human melanoma has not been studied yet.</p> <p>Results</p> <p>In the current work we studied the effect of MTA on four human melanoma cell lines A375, Hs294T, HT144 and MeWo and in two NSCLC cell lines H1299 and Calu-3. We have found that MTA induces DNA damage, S-phase cell cycle arrest, and caspase- dependent and –independent apoptosis. We show that an increment of the intracellular reactive oxygen species (ROS) and p53 is required for MTA-induced cytotoxicity by utilizing N-Acetyl-L-Cysteine (NAC) to blockage of ROS and p53-defective H1299 NSCLC cell line. Pretreatment of melanoma cells with NAC significantly decreased the DNA damage, p53 up-regulation and cytotoxic effect of MTA. MTA was able to induce p53 expression leading to up-regulation of p53-dependent genes Mcl-1 and PIDD, followed by a postranscriptional regulation of Mcl-1 improving apoptosis.</p> <p>Conclusions</p> <p>We found that MTA induced DNA damage and mitochondrial-mediated apoptosis in human melanoma cells in vitro and that the associated apoptosis was both caspase-dependent and –independent and p53-mediated. Our data suggest that MTA may be of therapeutic relevance for the future treatment of human malignant melanoma.</p

Thymidylate synthase expression determines pemetrexed targets and resistance development in tumour cells.

Although treatment options for cancer patients are increasing every year, the drug resistance problem remains very present. It is very difficult to find a drug that acts equally on tumours of the same histology as the individual's genetic characteristics often determine the response to treatment. Furthermore, tumours that initially respond to anti-tumour therapy are able to adapt and develop resistance to the drug, while others do not. In addition, this usually implies resistance development to agents to which the cells have not been exposed, a phenomenon called cross-resistance or multidrug resistance. Given this situation, it has been suggested that the most appropriate treatment would be able to act in parallel on multiple pathways constitutively altered in tumour cells. Pemetrexed is a multitargeted antifolate that exerts its activity against folate-dependent enzymes involved in de novo pyrimidine and purine synthesis. It is currently in use in combination with cisplatin against malignant pleural mesothelioma and non-squamous non-small cell lung cancer with favourable results. By real-time RT-PCR gene expression assays and restoration viability assays we demonstrated that Pemetrexed targets folate-dependent enzymes involved in de novo biosynthesis of purines differently depending on the intrinsic genetic characteristics of the tumour. These differences did not, however, interfere either with the initial response to the drug or with the activation of apoptotic pathways. In addition, these genetic fingerprints can differentiate two groups of tumours: those capable of developing resistance to antifolate, and not capable. These results may be useful to employ targets gene expression as resistance markers, a valuable tool for identifying patients likely to receive combination therapy to prevent the development of resistance

Estrogen receptor 1 gene expression and its combination with estrogen receptor 2 or aromatase expression predicts survival in non-small cell lung cancer.

The biological roles of estrogen receptor 1 (ERS1), estrogen receptor 2 (ERS2), and aromatase (CYP19A1) genes in the development of non-small cell lung cancer (NSCLC) is unclear, as is the use of their expression as a prognostic factor. The aim of this study was to investigate the prognostic value of estrogen receptors and aromatase mRNA expression, along with aromatase protein concentration, in resected NSCLC patients. Tumor and non-tumor lung tissue samples were analyzed for the mRNA expression of ERS1, ERS2 and CYP19A1 by RT-PCR. Aromatase concentration was measured with an ELISA. A total of 96 patients were included. ERS1 expression was significantly higher in non-tumor tissue than in tumor samples. Two gene expression categories were created for each gene (and protein): high and low. ERS1 high category showed increased overall survival (OS) when compared to the low expression category. Aromatase protein concentration was significantly higher in tumor samples. Higher ERS1 expression in tumor tissues was related to longer overall survival. The analysis of gene expression combinations provides evidence for longer OS when both ERS1 and ERS2 are highly expressed. ESR1, alone or in combination with ERS2 or CYP19A1, is the most determining prognostic factor within the analyzed 3 genes. It seems that ERS1 can play a role in NSCLC prognosis, alone or in combination with other genes such as ERS2 or Cyp19a1. ERS2 in combination with aromatase concentration could have a similar function

Table summarizing the genetic characteristics of studied cell lines.

<p>According to COSMIC database from <b>Sanger Institute</b> and “<b>The TP53 web site</b>”.</p

Viability and proliferation XTT after 48 h of exposure to MTA alone or in combination with dTh, Hx, or/and AICA.

<p>The percentage of viable cells is shown relative to viability of MTA-unexposed cells (control conditions). These results are representative of three independent experiments. <b>A)</b> Viability assays before and after MTA exposure with the pyrimidine biosynthesis pathway restored by addition of Hx alone or in combination with dTh. <b>B)</b> Viability assays before and after MTA exposure with purine biosynthesis pathway restored through the addition of AICA alone or in combination with dTh. <b>C)</b> Heatmap of six MTA-related genes where up- and down-regulation fold changes corresponding to each colour are indicated on the scale on the right of the figure.</p

A schematic diagram of the pyrimidine and purine biosynthesis pathways is shown, where the MTA-targets are indicated with a lightning bolt symbol.

<p>The effect of the addition to the culture medium of the preformed purine Hx, the preformed pyrimidine dTh and/or the purine pathway intermediate AICA as a new source for the restoration of the MTA-interrupted biosynthesis of purines and pyrimidines was different depending on the cell line.</p

MTA exerts its activity in different pathways increasing the apoptotic stimulus.

<p>Firstly, there is an accumulation of the AMP analogue ZMP that induces the activation of the AMPK pathway, starting a cascade of signalling that affects mTOR and PI3P/Akt pathways; mTOR is inactivated and the accumulation of its downstream unphosphorylated substrates facilitates the apoptosis process. Akt also remains inactive, unable to block p53 and to activate mTOR. On the other hand, the inhibition of TS, DHFR, GARFT and AICART induces oxidative stress and DNA damage which in turn is detected by p53 and caspase-dependent and independent mitochondrial apoptosis that is activated as has been previously reported. Together all processes lead to an imbalance between cell death and survival stimuli that result in enhanced apoptotic signalling.</p