Assessment of Photo-Induced Cytotoxic Activity of Cachrys sicula and Cachrys libanotis Enriched-Coumarin Extracts against Human Melanoma Cells

Abstract

Photochemotherapy is one of the most interesting current therapeutic approaches for the treatment of melanoma. Different classes of naturally occurring phytochemicals demonstrated interesting photoactive properties. The aim of this study was to evaluate the photocytotoxic potential of two Cachrys species from Southern Italy: C. sicula and C. libanotis (Apiaceae). The enriched-coumarin extracts were obtained from aerial parts through both traditional maceration and pressurized cyclic solid-liquid (PCSL) extraction using Naviglio extractor^®. Qualitative and quantitative analyses of furanocoumarins were performed with GC-MS. The photocytotoxic effects were verified on C32 melanoma cells irradiated at a dose of 1.08 J/cm². The apoptotic responses were also assessed. Moreover, phenolic content and the in vitro antioxidant potential were estimated. Xanthotoxin, bergapten, and isopimpinellin were identified. All the samples induced concentration-dependent photocytotoxic effects (IC₅₀ ranging from 3.16 to 18.18 μg/mL). The C. libanotis sample obtained with Naviglio extractor^® was the most effective one (IC₅₀ = 3.16 ± 0.21 μg/mL), followed by C. sicula sample obtained with the same technique (IC₅₀ = 8.83 ± 0.20 μg/mL). Both Cachrys samples obtained through PCSL induced up-regulation of apoptotic signals such as BAX (Bcl2-associated X protein) and PARP (poly ADP-ribose polymerase) cleavage. Moreover, these samples proved to be more photoactive, giving a greater upregulation of p21 protein in the presence of UVA radiation. Obtained results suggest that investigated species could be promising candidates for further investigations aimed to find new potential drugs for the photochemotherapy of skin cancer.

Keywords: Apiaceae; Cachrys spp.; furanocoumarins; green extraction technology; photochemotherapy; skin cancer.

Figure 1

Structure of identified furanocoumarins.

Figure 2

(a) Concentration-dependent photocytotoxic effects induced by Cachrys samples. Cells were irradiated for 1 h at a dose of 1.08 J/cm²; (b) non-linear regression analyses: CSM, C. sicula L. maceration; CSN, C. sicula L. Naviglio extractor^®; CLM, C. libanotis L. maceration, CLN, C. libanotis L. Naviglio extractor^®. Data were expressed as means ± SEM (n = 4). *** p < 0.001, ** p < 0.01 (Dunnett’s test) compared to control (untreated irradiated cells).

Figure 3

C32 cells morphology 48 h after UVA irradiation at a dose of 1.08 J/cm². (a) Control, irradiated cells in RPMI 0.5% MeOH, without sample; (b) irradiated cells, C. sicula L. obtained with Naviglio extractor^®, 25 μg/mL; (c) irradiated cells, C. libanotis L. extracted through Naviglio extractor^®, 25 μg/mL. Cells were visualized with an inverted microscope AE20 Motic and images were captured with a VisiCam digital camera. Arrows show cells became rounded and shrunken. Magnification, 10×.

Figure 4

Western Blot analysis of p21, BAX and PARP (poly ADP-ribose polymerase) protein levels in C32 cells treated or not with CSN and CLN extracts for 24 h, both in the presence and absence of UV. The histograms refer to the densitometric analysis (OD) of the Western blot shown in the figure.

Figure 5

Western Blot analysis of p21, BAX, and PARP (poly ADP-ribose polymerase) protein levels in C32 cells treated or not with CSM and CLM extracts for 24 h, both in the presence and absence of UV. The histograms refer to the densitometric analysis (OD) of the Western blot shown in the figure.