Abstract
BackgroundPorphyrin TMPyP4 (P4) and its C14H28-alkyl derivative (C14) are G-quadruplex binders and singlet oxygen (1O2) generators. In contrast, TMPyP2 (P2) produces 1O2 but it is not a G-quadruplex binder. As their photosensitizing activity is currently undefined, we report in this study their efficacy against a melanoma skin tumour and describe an in vitro mechanistic study which gives insights into their anticancer activity.MethodsUptake and antiproliferative activity of photoactivated P2, P4 and C14 have been investigated in murine melanoma B78-H1 cells by FACS, clonogenic and migration assays. Apoptosis was investigated by PARP-1 cleavage and annexin-propidium iodide assays. Biodistribution and in vivo anticancer activity were tested in melanoma tumour-bearing mice. Porphyrin binding and photocleavage of G-rich mRNA regions were investigated by electrophoresis and RT-PCR. Porphyrin effect on ERK pathway was explored by Western blots.ResultsThanks to its higher lipophylicity C14 was taken up by murine melanoma B78-H1 cells up to 30-fold more efficiently than P4. When photoactivated (7.2 J/cm2) in B78-H1 melanoma cells, P4 and C14, but not control P2, caused a strong inhibition of metabolic activity, clonogenic growth and cell migration. Biodistribution studies on melanoma tumour-bearing mice showed that P4 and C14 localize in the tumour. Upon irradiation (660 nm, 193 J/cm2), P4 and C14 retarded tumour growth and increased the median survival time of the treated mice by ~50% (P <0.01 by ANOVA), whereas porphyrin P2 did not. The light-dependent mechanism mediated by P4 and C14 is likely due to the binding to and photocleavage of G-rich quadruplex-forming sequences within the 5′-untranslated regions of the mitogenic ras genes. This causes a decrease of RAS protein and inhibition of downstream ERK pathway, which stimulates proliferation. Annexin V/propidium iodide and PARP-1 cleavage assays showed that the porphyrins arrested tumour growth by apoptosis and necrosis. C14 also showed an intrinsic light-independent anticancer activity, as recently reported for G4-RNA binders.ConclusionsPorphyrins P4 and C14 impair the clonogenic growth and migration of B78-H1 melanoma cells and inhibit melanoma tumour growth in vivo. Evidence is provided that C14 acts through light-dependent (mRNA photocleavage) and light-independent (translation inhibition) mechanisms.
Highlights
Porphyrin TMPyP4 (P4) and its C14H28-alkyl derivative (C14) are G-quadruplex binders and singlet oxygen (1O2) generators
In the following we demonstrate that the cationic porphyrins C14 and P4 strongly inhibit the growth of melanoma cells both in vitro and in vivo
Considering that the quantum yield of singlet oxygen generation of P4 was reported to be 0.51 [34], we found that the φΔ of C14 and P2 were respectively 0.51 and 0.18 (Figure 1C)
Summary
Porphyrin TMPyP4 (P4) and its C14H28-alkyl derivative (C14) are G-quadruplex binders and singlet oxygen (1O2) generators. In previous studies from our laboratory we synthesized expanded porphyrins, composed of a macrocycle of five pyrroles, that exhibit a photodynamic activity in cancer cells at micromolar concentrations, either as free molecules or complexed to Zn or Lu [9,10]. A bioinformatic study by Huppert et al [23] revealed that about 3000 5′-UTR of the human transcriptome contains one or more G-quadruplex motifs This brought the authors to the conclusion that these tertiary structures should regulate translation. They demonstrated that an RNA quadruplex-forming sequence within the 5′-UTR of the NRAS transcript inhibited translation in vitro [24]. As G4-RNA can inhibit translation, the use of small molecules to inhibit the function of mRNA looks quite attractive [28]
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