Klimenko A et al. (2022), Chlorin Endogenous to the North Pacific Brittle...

ECB-ART-50553

Biomedicines 2022 Jan 08;101:. doi: 10.3390/biomedicines10010134.

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Chlorin Endogenous to the North Pacific Brittle Star Ophiura sarsii for Photodynamic Therapy Applications in Breast Cancer and Glioblastoma Models.

Klimenko A , Rodina EE , Silachev D , Begun M , Babenko VA , Benditkis AS , Kozlov AS , Krasnovsky AA , Khotimchenko YS , Katanaev VL .

Abstract
Photodynamic therapy (PDT) represents a powerful avenue for anticancer treatment. PDT relies on the use of photosensitizers-compounds accumulating in the tumor and converted from benign to cytotoxic upon targeted photoactivation. We here describe (3S,4S)-14-Ethyl-9-(hydroxymethyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid (ETPA) as a major metabolite of the North Pacific brittle stars Ophiura sarsii. As a chlorin, ETPA efficiently produces singlet oxygen upon red-light photoactivation and exerts powerful sub-micromolar phototoxicity against a panel of cancer cell lines in vitro. In a mouse model of glioblastoma, intravenous ETPA injection combined with targeted red laser irradiation induced strong necrotic ablation of the brain tumor. Along with the straightforward ETPA purification protocol and abundance of O. sarsii, these studies pave the way for the development of ETPA as a novel natural product-based photodynamic therapeutic.

PubMed ID: 35052813
PMC ID: PMC8773836
Article link: Biomedicines

Article Images: [+] show captions

	Figure 1. Analytical (A) and semi-preparative (B) chromatography of the butanol fraction of the EtOH extract of O. sarsii, absorption intensity at the wavelength of 366 nm (A) and 210 nm/366 nm (B). The major peak at the retention time of 52 min (A) and 35 min (B) corresponds to ETPA (chlorin) from our previous study [10]. Chromatograms from non-fasted ophiuras are shown in both panels; samples from the fasted ophiuras show identical chromatograms.
	Figure 2. (A) Absorbance spectra of the chlorin compound isolated from the butanol fraction of the EtOH extract from control (red) and fasted (black) O. sarsii reveal absorbance peaks typical for chlorins, identical between the two preparations; (B) cell survival (MTT test) to evaluate the strong phototoxicity of ETPA after red-light irradiation. Data are given as mean ± SEM, n = 8 (3 for HEK-293 cells); (C) ETPA phototoxicity IC50 and the phototoxicity index (PI, calculated as IC50 in the dark/IC50 in the light [17]). Cell survival data in the dark are taken from [10] (breast cancer cell lines) or measured separately (C6 and HEK-293 cells, see Supplementary Figure S4).
	Figure 3. ETPA efficiently generates singlet oxygen: (A) changes in the absorption spectrum of DPIBF (curves 1–4) in the mixture of DPIBF with ETPA in acetone during irradiation by monochromatic red light (660 nm) absorbed by ETPA. Here, “2” corresponds to 2 min, “3” refers to 5 min, and “4” to 15 min irradiation. Power of exciting light was 83 μW. Inset shows the time course of 414 nm absorption fall of DPIBF. ETPA bleaching was not observed; (B) the relative quantum yields of DPIBF oxidation (Vr/n) upon irradiation of TPP and ETPA. The rate of spontaneous DPIBF bleaching in the dark without sensitizer and irradiation is defined as “Control”. For TPP, irradiation time was 10 min, excitation wavelength was 512 nm, irradiation power was 105 μW, and absorbance of TPP at 512 nm was 0.024. For ETPA, irradiation time was 2 min, excitation wavelength was 660 nm, excitation power was 83 μW, and absorbance at 660 nm was 0.095; (C) kinetic trace of photosensitized phosphorescence of singlet oxygen upon excitation of ETPA by 5 μs pulses of violet LED (399 nm) in cartesian (1) and semilogarithmic (2) coordinates. Pulse repetition rate was 5 kHz, average LED power was 30 mW, and irradiation (averaging) time was 10 min. The PMT signal was accumulated using a time-resolved computer photon counting. The duration of one channel was 640 ns, and the number of channels was 256. Absorbance of the solution at 396 nm was 0.246 in a 1 cm quartz cell. Here, “3” indicates phosphorescence emission spectrum estimated using three interchangeable interference filters. I corresponds to the phosphorescence intensity just after the end (5 μs) of the LED pulse. The decay time (τΔ) of the phosphorescence exactly coincided with the known value of singlet oxygen lifetime in acetone; (D) calculation of the quantum yield of singlet oxygen by ETPA in comparison with phenalenone.
	Figure 4. PDT using ETPA in a mouse model of glioblastoma: (A) scheme of the experiment; (B,C) representative T2-weighted MR images from coronal brain sections (0.5 mm thick) obtained 5 days after PDT. The area outlined with a red line refers to hyperintensities regions (edema, B). The area outlined with a blue line refers to PDT-induced glioma necrosis (C); (D) a representative histological section, stained with eosin–hematoxylin, demonstrates the tumor boundaries (outlined in black) and the presence of necrotic loci in the area of laser illumination (outlined in yellow); (E) enlarged area showing the boundaries of necrotic and intact tumor tissues. Images shown are representative of 3 animals.

References [+] :

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