Ekaterina O. Serebrovskaya

Targeting cancer cells by using an antireceptor antibody-photosensitizer fusion protein

Antibody-photosensitizer chemical conjugates are used successfully to kill cancer cells in photodynamic therapy. However, chemical conjugation of photosensitizers presents several limitations, such as poor reproducibility, aggregation, and free photosensitizer impurities. Here, we report a fully genetically encoded immunophotosensitizer, consisting of a specific anti-p185HER-2-ECD antibody fragment 4D5scFv fused with the phototoxic fluorescent protein KillerRed. Both parts of the recombinant protein preserved their functional properties: high affinity to antigen and light activation of sensitizer. 4D5scFv-KillerRed showed fine targeting properties and efficiently killed p185HER-2-ECD-expressing cancer cells upon light irradiation. It also showed a remarkable additive effect with the commonly used antitumor agent cisplatin, further demonstrating the potential of the approach.

Phototoxic effects of fluorescent protein KillerRed on tumor cells in mice

KillerRed is known to be a unique red fluorescent protein displaying strong phototoxic properties. Its effectiveness has been shown previously for killing bacterial and cancer cells in vitro. Here, we investigated the photototoxicity of the protein on tumor xenografts in mice. HeLa Kyoto cell line stably expressing KillerRed in mitochondria and in fusion with histone H2B was used. Irradiation of the tumors with 593 nm laser led to photobleaching of KillerRed indicating photosensitization reaction and caused significant destruction of the cells and activation of apoptosis. The portion of the dystrophically changed cells increased from 9.9% to 63.7%, and the cells with apoptosis hallmarks from 6.3% to 14%. The results of this study suggest KillerRed as a potential genetically encoded photosensitizer for photodynamic therapy of cancer.

Fluorescent proteins as light-inducible photochemical partners

Green Fluorescent Protein (GFP) and other related fluorescent proteins are generally used as genetically encoded, chemically inert labels in vivo. This review focuses on the emerging application of fluorescent proteins as light-inducible intracellular photochemical partners. The first example of a chemically active GFP-like protein was the phototoxic red fluorescent protein KillerRed, which can be used for precise light-induced killing of cells, protein inactivation, and studying reactive oxygen species signaling in different cellular compartments. Moreover, recent studies revealed that various GFPs can act as light-induced electron donors in photochemical reactions with biologically relevant electron acceptors. These findings have important implications for practical uses of fluorescent proteins as well as for our understanding of the evolution and biology of this protein family.

Flavoprotein miniSOG as a genetically encoded photosensitizer for cancer cells

BACKGROUND Genetically encoded photosensitizers are a promising optogenetic instrument for light-induced production of reactive oxygen species in desired locations within cells in vitro or whole body in vivo. Only two such photosensitizers are currently known, GFP-like protein KillerRed and FMN-binding protein miniSOG. In this work we studied phototoxic effects of miniSOG in cancer cells. METHODS HeLa Kyoto cell lines stably expressing miniSOG in different localizations, namely, plasma membrane, mitochondria or chromatin (fused with histone H2B) were created. Phototoxicity of miniSOG was tested on the cells in vitro and tumor xenografts in vivo. RESULTS Blue light induced pronounced cell death in all three cell lines in a dose-dependent manner. Caspase 3 activation was characteristic of illuminated cells with mitochondria- and chromatin-localized miniSOG, but not with miniSOG in the plasma membrane. In addition, H2B-miniSOG-expressing cells demonstrated light-induced activation of DNA repair machinery, which indicates massive damage of genomic DNA. In contrast to these in vitro data, no detectable phototoxicity was observed on tumor xenografts with HeLa Kyoto cell lines expressing mitochondria- or chromatin-localized miniSOG. CONCLUSIONS miniSOG is an excellent genetically encoded photosensitizer for mammalian cells in vitro, but it is inferior to KillerRed in the HeLa tumor. GENERAL SIGNIFICANCE This is the first study to assess phototoxicity of miniSOG in cancer cells. The results suggest an effective ontogenetic tool and may be of interest for molecular and cell biology and biomedical applications.

Phototoxic effects of lysosome-associated genetically encoded photosensitizer KillerRed

Abstract. KillerRed is a unique phototoxic red fluorescent protein that can be used to induce local oxidative stress by green-orange light illumination. Here we studied phototoxicity of KillerRed targeted to cytoplasmic surface of lysosomes via fusion with Rab7, a small GTPase that is known to be attached to membranes of late endosomes and lysosomes. It was found that lysosome-associated KillerRed ensures efficient light-induced cell death similar to previously reported mitochondria- and plasma membrane–localized KillerRed. Inhibitory analysis demonstrated that lysosomal cathepsins play an important role in the manifestation of KillerRed-Rab7 phototoxicity. Time-lapse monitoring of cell morphology, membrane integrity, and nuclei shape allowed us to conclude that KillerRed-Rab7-mediated cell death occurs via necrosis at high light intensity or via apoptosis at lower light intensity. Potentially, KillerRed-Rab7 can be used as an optogenetic tool to direct target cell populations to either apoptosis or necrosis.

Towards PDT with Genetically Encoded Photosensitizer KillerRed: A Comparison of Continuous and Pulsed Laser Regimens in an Animal Tumor Model.

The strong phototoxicity of the red fluorescent protein KillerRed allows it to be considered as a potential genetically encoded photosensitizer for the photodynamic therapy (PDT) of cancer. The advantages of KillerRed over chemical photosensitizers are its expression in tumor cells transduced with the appropriate gene and direct killing of cells through precise damage to any desired cell compartment. The ability of KillerRed to affect cell division and to induce cell death has already been demonstrated in cancer cell lines in vitro and HeLa tumor xenografts in vivo. However, the further development of this approach for PDT requires optimization of the method of treatment. In this study we tested the continuous wave (593 nm) and pulsed laser (584 nm, 10 Hz, 18 ns) modes to achieve an antitumor effect. The research was implemented on CT26 subcutaneous mouse tumors expressing KillerRed in fusion with histone H2B. The results showed that the pulsed mode provided a higher rate of photobleaching of KillerRed without any temperature increase on the tumor surface. PDT with the continuous wave laser was ineffective against CT26 tumors in mice, whereas the pulsed laser induced pronounced histopathological changes and inhibition of tumor growth. Therefore, we selected an effective regimen for PDT when using the genetically encoded photosensitizer KillerRed and pulsed laser irradiation.

Comparative in vitro study on cytotoxicity of recombinant β-hairpin peptides

Natural antimicrobial peptides (AMPs) are important components of the innate immune system with a wide spectrum of biological activity. In this study, we investigated the cytotoxic effect of three recombinant β‐hairpin cationic AMPs: arenicin‐1 from the polychaeta Arenicola marina, tachyplesin I from the horseshoe crab Tachypleus tridentatus, and gomesin from the spider Acanthoscurria gomesiana. All the three β‐hairpin AMPs were overexpressed in Escherichia coli. Different cell lines were incubated with various concentrations of the investigated AMPs in order to evaluate their cytotoxic activity. Double staining with subsequent flow cytometric analysis was used to determine the predominant way of cell death mediated by each AMP. Hemolytic activity of the peptides was tested against fresh human red blood cells. Our results indicated that all the three AMPs exhibited significant cytotoxic effect against cancer cells that varied depending on the cell line type and, in most cases, on the presence of serum components. Flow cytometric analysis implicitly indicated that tachyplesin I mostly promoted late apoptosis/necrosis, while arenicin‐1 and gomesin induced early apoptosis under the same conditions. Tachyplesin I proved to be the most promising therapeutic candidate as it displayed the highest specific cytotoxicity against cancer cell lines, independent of the serum presence.

Inducing cellular senescence in vitro by using genetically encoded photosensitizers

Cellular senescence, a form of cell cycle arrest, is one of the cellular responses to different types of exogenous and endogenous damage. The senescence phenotype can be induced in vitro by oncogene overexpression and/or DNA damage. Recently, we have reported a novel mechanism of cellular senescence induction by mild genotoxic stress. Specifically, we have shown that the formation of a small number of DNA lesions in normal and cancer cells during S phase leads to cellular senescence-like arrest within the same cell cycle. Here, based on this mechanism, we suggest an approach to remotely induce premature senescence in human cell cultures using short-term light irradiation. We used the genetically encoded photosensitizers, tandem KillerRed and miniSOG, targeted to chromatin by fusion to core histone H2B to induce moderate levels of DNA damage by light in S phase cells. We showed that the cells that express the H2B-fused photosensitizers acquire a senescence phenotype upon illumination with the appropriate light source. Furthermore, we demonstrated that both chromatin-targeted tandem KillerRed (produces O2−) and miniSOG (produces 1O2) induce single-stranded DNA breaks upon light illumination. Interestingly, miniSOG was also able to induce double-stranded DNA breaks.

KillerRed and miniSOG as genetically encoded photosensitizers for photodynamic therapy of cancer

Despite of the success of photodynamic therapy (PDT) in cancer treatment, the problems of low selective accumulation of a photosensitizer in a tumor and skin phototoxicity have not resolved yet. The idea of encoding of a photosensitizer in genome of cancer cells is attractive, particularly because it can provide highly selective light induced cell killing. This work is aimed at the development of new approach to PDT of cancer, namely to using genetically encoded photosensitizers. A phototoxicity of red fluorescent GFP-like protein KillerRed and FMN-binding protein miniSOG was investigated on HeLa tumor xenografts in nude mice. The tumors were generated by subcutaneous injection of HeLa cells stably expressing the phototoxic proteins. The tumors were irradiated with 594 nm or 473 nm laser at 150 mW/cm2 for 20 or 30 min, repeatedly. Fluorescence intensity of the tumors was measured in vivo before and after each treatment procedure. Detailed pathomorphological analysis was performed 24 h after the therapy. On the epi-fluorescence images in vivo photobleaching of both proteins was observed indicating photodynamic reaction. Substantial pathomorphological abnormalities were found in the treated KillerRed-expressing tumor tissue, such as vacuolization of cytoplasm, cellular and nuclear membrane destruction, activation of apoptosis. In contrast, miniSOG-expressing tumors displayed no reaction to PDT, presumably due to the lack of FMN cofactor needed for fluorescence recovery of the flavoprotein. The results are of interest for photodynamic therapy as a proof of possibility to induce photodamages in cancer cells in vivo using genetically encoded photosensitizers.

Lysosome-associated miniSOG as a photosensitizer for mammalian cells.

Genetically encoded photosensitizers represent a promising optogenetic tool for the induction of light-controlled oxidative stress strictly localized to a selected intracellular compartment. Here we tested the phototoxic effects of the flavin-containing phototoxic protein miniSOG targeted to the cytoplasmic surfaces of late endosomes and lysosomes by fusion with Rab7. In HeLa Kyoto cells stably expressing miniSOG-Rab7, we demonstrated a high level of cell death upon blue-light illumination. Pepstatin A completely abolished phototoxicity of miniSOG-Rab7, showing a key role for cathepsin D in this model. Using a far-red fluorescence sensor for caspase-3, we observed caspase-3 activation during miniSOG-Rab7-mediated cell death. We conclude that upon illumination, miniSOG-Rab7 induces lysosomal membrane permeabilization (LMP) and leakage of cathepsins into the cytosol, resulting in caspase-dependent apoptosis.