273 ISSN 0006-3509, Biophysics, 2021, Vol. 66, No. 2, pp. 273–277. © Pleiades Publishing, Inc., 2021. Russian Text © The Author(s), 2021, published in Biofizika, 2021, Vol. 66, No. 2, pp. 323–328. The Phototoxic Effect of Water-Soluble Porphyrins on Human Clear Cell Renal Cell Carcinoma Line Сaki-1 A. F. Arutyunyan a, *, L. L. Tevonyan a , A. D. Beniaminov a , Y. E. Yegorov a , and D. N. Kaluzhny a a Engelhard Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991 Russia *e-mail: uzhny@mail.ru Received December 4, 2020; revised December 4, 2020; accepted December 11, 2020 Abstract—Two tetrapyridine porphyrins, cationic porphyrin P4 (TMPyP4) and its amphiphilic derivative porphyrin P1 with carboxyl groups, and their zinc-containing analogs ZnP4 and ZnP1 were examined in order to obtain the photosensitizers with a narrower specificity for target tissue. The two classes of molecules were compared with respect to physicochemical properties of and the effect on Caki-1 clear cell renal cell car- cinoma cells in vitro. Micromolar concentrations of the compounds did not cause morphological changes in cells in the absence of light irradiation. Cell treatment with porphyrin porphyrin P4 under blue light led to a rapid cytotoxic effect. The cells began to detach from the substrate, and swellings (blebs) became detectable in their membranes. The rate of the changes was consistent with direct membrane damage by porphyrin P4. Compound ZnP1 did not cause any visible changes in Caki-1 cell morphology in similar conditions. The results were explained by different lipophilicities and, therefore, different intracellular localization of the compounds. Membrane localization was assumed to prevail in the case of porphyrin P4, which is more lipo- philic then ZnP1, and to mediate its fast cytopathic effect. Keywords: porphyrin, reactive oxygen species, cells DOI: 10.1134/S0006350921020020 INTRODUCTION Photodynamic therapy of tumors is intensely developing. Prophyrins are promising molecules to use in photodynamic therapy of cancer because they specifically interact with cell structures and are capa- ble of generating reactive oxygen species [1, 2]. The photochemical effect directly damages the cell targets and thus causes death of cancer cells. Studies are now performed to develop and to identify the photosensi- tizers that affect particular targets. Prophyrins are a class of compounds that are convenient to modify chemically in order to confer desirable properties. Var- ious carriers, including those transferring oxygen in addition to a compound of interest, have been designed to deliver photosensitizers into cells and to overcome hypoxia in the tumor [3]. Tetrapyridine porphyrins are a class of molecules that are capable of interacting with various cell targets, including DNA. Cationic forms are more advanta- geous in terms of higher solubility and more efficient delivery into cells. Gene expression regulation by a low concentration (0.5 μM) of cationic porphyrin TMPyP4 have been studied using whole-genome RNA sequencing. A high proportion of regulated genes were found to be functionally associated with cell adhesion. When used in a higher concentration (2 μM), TMPyP4 caused cell death [4]. TMPyP4 has high affinity for DNA and, in particular, noncanoni- cal G-quadruplex structures, which form in telomeric DNA sequences [5]. TMPyP4 is therefore a potential agent that may reduce telomerase activity by compet- ing for the interaction with the substrate. To choose the photosensitizers that affect DNA in a targeted manner, it is essential to study their possible side effects on alternative cell targets. The cell mem- brane is a primary potential target of amphiphilic pho- tosensitizers. In this work, we studied the light- induced generation of reactive oxygen species and the light-induced effect on Caki-1 human clear cell renal cell carcinoma cells for several cationic porphyrins. MATERIALS AND METHODS Tetrapyridine porphyrin derivatives used in this work are shown in Fig. 1. Compound ZnP4 was obtained by boiling porphyrin P4 (TMPyP4, Sigma Aldrich, United States) in the presence of Zn(OH) 2 . Compound P1 was obtained in the reaction of 2-bro- moacetic acid with tetrapyridine porphyrin (Sigma Aldrich, United States) according to a published pro- tocol [6]. Porphyrin ZnP1 was similarly obtained by boiling P1 in the presence of Zn(OH) 2 [7]. The struc- Abbreviations: DPBF, 1,3-diphenylisobenzofuran. CELL BIOPHYSICS