Differential Radiomodulating Action of Olea europaea L. cv. Caiazzana Leaf Extract on Human Normal and Cancer Cells: A Joint Chemical and Radiobiological Approach

Abstract

The identification of a natural compound with selectively differential radiomodulating activity would arguably represent a valuable asset in the striving quest for widening the therapeutic window in cancer radiotherapy (RT). To this end, we fully characterized the chemical profile of olive tree leaf polyphenols from the Caiazzana cultivar (OLC), autochthonous to the Campania region (Italy), by ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HR-MS). Oleacein was the most abundant molecule in the OLC. Two normal and two cancer cells lines were X-ray-irradiated following 24-h treatment with the same concentration of the obtained crude extract and were assessed for their radioresponse in terms of micronucleus (MN) induction and, for one of the normal cell lines, of premature senescence (PS). Irradiation of pre-treated normal cells in the presence of the OLC reduced the frequency of radiation-induced MN and the onset of PS. Conversely, the genotoxic action of ionising radiation was exacerbated in cancer cells under the same experimental conditions. To our knowledge, this is the first report on the dual action of a polyphenol-rich olive leaf extract on radiation-induced damage. If further confirmed, these findings may be pre-clinically relevant and point to a substance that may potentially counteract cancer radioresistance while reducing RT-associated normal tissue toxicity.

Keywords: Olea europaea L. cv. Caiazzana; cancer; ionising radiation; normal tissue; oleacein; radiomodulation; radioprotection; radiosensitization; radiotherapy.

Figure 1

(A) Leaves of O. europaea cv. Caiazzana (i) and OLC extract therefrom (ii); (B) HPLC-UV chromatograms with the UV-DAD spectrum of a secoiridoid compound; (C) calculated amounts (%) of quantified compounds in OLC.

Figure 2

Tandem mass spectra and relative proposed comprehensive fragmentation pattern of (A) oleuropein (22); (B) oleuropein hexoside (18); (C) elenoyl oleuropein (31). Theoretical m/z values are reported under each structure.

Figure 3

TOF-MS/MS spectra of (A) oleacein and its acetal derivatives 26 (B), 29 (C), and 30 (D). The proposed fragmentation pattern of all these compounds is in panel (E). Theoretical m/z values are reported under each structure.

Figure 4

OLC antioxidant activity (AA%) vs. 2,2′-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radical cation (♦), and vs. 2,2-diphenyl-1-picrylhydrazy (DPPH) radical (●). Fe (III) reducing power (RP) by PFRAP assay is also reported (■). Values reported are the mean ± SD of three independent measurements.

Figure 5

Time–course occurrence of X-ray-induced premature senescence (PS) in HUVECs in the presence (+ with OLC) and the absence of (− without OLC) of the extract. OLC mediates reduction of PS for all radiation doses at the three times assayed post-irradiation. Reported values refer to two separate experiments. Error bars refer to standard errors (SE) of the mean.

Figure 6

10× magnification micrograph showing the typology of observed DNA damage per cell line and treatment conditions (no OLC vs. OLC): (a,d) unirradiated binucleated (BN) cells from normal MCF-10A and HUVEC cell lines, respectively; (b,e) BN MCF-10A and HUVECs irradiated with 4 Gy of X-rays in the absence of extract presenting multiple micronuclei (MN); (c,f) BN MCF-10A and HUVEC irradiated with the same dose but after OLC treatment and showing just one MN in each BN cell; (g,j) BN cells from cancer DU-145 and PANC-1 cell lines, respectively; (h,k) BN cells from DU-145 and PANC-1 respectively, irradiated with 4 Gy of X-rays without extract and showing only a couple of MN; (i,l) BN cells from DU-145 and PANC-1, 4Gy-irradiated in the presence of OLC showing a considerable amount of DNA damage in the form of several MN per BN cell.

Figure 7

MN frequency in normal cell lines: (A) Dose-dependent MN induction in HUVECs; (B) Dose-dependent MN induction in MCF-10A cells. Irradiation in the presence of the OLC (green bars) yielded consistently less DNA damage compared to non OLC-treated samples (brown bars). MN frequency values are depicted as mean ± SE for at least two independent experiments per cell lines.

Figure 8

MN frequency in cancer cell lines: (A) Dose-dependent MN induction in prostate DU145 cancer cells; (B) dose-dependent MN induction in pancreatic PANC-1 cancer cells. Irradiation in the presence of the extract (green bars) causes more DNA damage compared to that measured in non OLC-treated samples (brown bars). Error bars represent SE from at least two independent experiments per cell line.