**1. Introduction**

Cancer is a major public burden with tens of millions people being diagnosed around the world every year. Eventually, more than half of the patients succumb to their disease despite new developments [1]. In 2018, 9.6 million people died from cancer according to the World Health Organization [2], with increasing numbers in developing countries. Lung, breast, colorectal, and prostate cancer belong to the most frequently diagnosed malignancies worldwide [2]. These diseases claim the lives of more than a million people annually.

Although scientific and technological progress allowed the development of new approaches such as gene-therapy [3], stem cell transplantation [4], immunotherapy [5], and therapy by nanoparticles [6] for cancer treatment, the traditional cancer therapy including a combination of surgery, radio- and chemotherapy is still the most commonly used [7–9]. Standard cancer therapy is accompanied by various drawbacks, such as a lack of tumor-specific drug delivery systems, regular application of toxic anticancer drugs leading to adverse side effects, in particular normal cell death, drug resistance, as well as cancer recurrence after surgical removal of solid tumors [5,10]. Therefore, the search for more effective anticancer compounds is ongoing.

In this context, actinoporins, cytolytic toxins derived from sea anemones (marine venomous cnidarians), were identified as promising candidates for cancer therapy. This unique group of small basic α-pore-forming proteins includes a compact β-fold lacking disulfide bounds formed by 12 β-sheets and two α-helices—one of which, functional and more extended, is located at the N-terminus, and the second one, short, is at the C-terminus [11]. Cytotoxicity of actinoporins relies on the formation of pores within sphingomyelin-containing membranes, which disrupts ion gradients that lead to osmotic swelling and ultimately to cell death [12–14]. Cytolytic activity of actinoporins was observed in different cells including platelets, fibroblasts, parasite cells, lactotrophs and some cancer cells [15,16]. Due to pore-forming ability and selective binding to sphingomyelin on the cell membrane surface, high stability to temperature and proteolytic cleavage, they are currently considered as antibacterial and anticancer agents as well as components of immunotoxins [17,18]. These immunotoxins include StnI from *Stichodactyla helianthus* [17], Gigantoxin-4 from *Stichodactyla gigantea* [19], EqII from *Actinia equina* [20] and its mutant EqTx-II(I18C) [21], as well as FraC from *Actinia fragacea* [22].

In our previous studies, we reported the structure and first functional analyses of actinoporins isolated from *Heteractis crispa* (=*Radianthus macrodactylus*) [23–28]. We were able to demonstrate cytotoxic activity of actinoporin RTX-A in monocytic leukemia (THP-1) cells, cervix carcinoma (HeLa), breast (MDA-MB-231) and colon (SNU-C4) cancer cells. In addition, epidermal growth factor (EGF)-induced tumor transformation of mouse epidermal JB6 P<sup>+</sup> Cl41 cells was observed. Activity was mediated by induction of p53-independent apoptosis as well as inhibition of the oncogenic nuclear factors AP-1 and NF-κB [16]. Moreover, we found the combinatorial library of *H. crispa* actinoporins encoded by the multigene family including at least 47 representatives [26–28]. Here, we report the in vitro anticancer activity of the recombinant analog of Hct-S3, the most abundant isoform of *H. crispa* actinoporins.

## **2. Results**
