*2.4. The E*ff*ect of rHct-S3 on Migration of Colorectal Carcinoma HT-29 Cells*

We investigated the effect of rHct-S3 on the migration of colorectal carcinoma HT-29 cells with high metastatic potential, using a scratch assay. It was demostrated that rHct-S3 supressed migration of HT-29 cells by 33% ± 10.2, 50% ± 7.5, and 99% ± 6.4, respectively, at concentrations of 1, 2, and 4 μM, compared to the control group (Figure 4a,b). In order to reveal the impact of inhibition of proliferation by rHct-S3 on migration, the antiproliferative activity of rHct-S3 against HT-29 cells was checked in 24, 48, 72, and 96 h of treatment (Supplementary Figure S1). It was found that rHct-S3 at concentrations 1, 2, and 4 μM slightly (not more than 10%) decreased the proliferation rate of HT-29 cells after 24 h and 48 h of treatment, while it inhibited cells proliferation by 11% ± 3.0, 26% ± 1.2, and 31% ± 5.0, respectively, after 96 h of treatment. These results indicate that rHct-S3 possess a moderate antiproliferative activity.

To elucidate the potential mechanism of this anti-migratory activity, we evaluated the effect of rHct-S3 on the expression level of the matrix metalloproteinases (MMP)-2 and MMP-9, playing a pivotal role in cancer cell invasion and metastasis. Indeed, actinoporin effectively inhibited the expression of MMP-2 and MMP-9 (Figure 4c) at a concentration of 2 μM. In addition, we estimated whether rHct-S3 affect the activation of caspase-3, a known executor of apoptosis. The upregulation of cleaved caspase-3 was detected in HT-29 cells treated with rHct-S3. Additionally and in line with this, we have detected a degradation of poly (ADP-ribose) polymerase (PARP) as well as Bcl-2 down-regulation and Bax up-regulation (Figure 4d). Thus, rHct-S3 decreases the migratory activity of colorectal carcinoma HT-29 cells by the inhibition of MMP-2 and MMP-9 and induces the apoptosis via the activation of caspase-3.

**Figure 3.** Effect of rHct-S3 on EGF-induced neoplastic cells transformation of JB6 Cl41 cells and colony formation of human colorectal carcinoma HT-29, breast cancer MDA-MB-231, and melanoma SK-MEL-28 cell lines. (**a**,**b**) JB6 Cl41 cells (2.4 <sup>×</sup> 10<sup>4</sup> /mL) treated with/without EGF (1 ng/mL) or investigated compound (1, 2, and 4 μM) in 1 mL of 0.3% Basal medium Eagle (BME) agar containing 10% FBS and overlaid with 3.5 mL of 0.5% BME's agar containing 10% FBS. The culture was maintained at 37 ◦C in a 5% CO2 atmosphere for 2 weeks. (**c**) HT-29, (**d**) MDA-MB-231, (**e**) SK-MEL-28 cells (2.4 <sup>×</sup> <sup>10</sup><sup>4</sup> /mL) treated with/without investigated compound (1, 2, and 4 <sup>μ</sup>M) or cisplatin at 3 <sup>μ</sup>M (positive control) and subjected into a soft agar. The culture was maintained at 37 ◦C in a 5% CO2 atmosphere for 2 weeks. The colonies were counted under a microscope with the aid of the ImageJ software program (*n* = 6 for control and each compound, *n*—quantity of photos). The magnification of representative photos of colonies is ×10. The asterisks (\*\*\* *p* < 0.001) indicate a significant decrease in colony formation in cells treated with compound compared with the non-treated cells (control).

**Figure 4.** Effects of rHct-S3 on migration of HT-29 cells, MMPs and apoptotic proteins. (**a**,**b**) The HT-29 cells migration distance was measured the width of the wound and expressed as a percentage of each control for the mean of wound closure area. All experiments were repeated at least three times in each group (n = 18 for control and each compound, n—quantity of photos). The magnification of representative photos is ×10. The asterisks (\*\*\* *p* < 0.001) indicate a significant decrease in migration of cells treated with rHct-S3 compared with the non-treated cells (control) (**c**,**d**) rHct-S3 inhibited MMP-9, MMP-2 expression and regulated caspase-3, cleaved caspase-3, PARP, Bcl-2 and Bax in HT-29 cells, as determined by Western Blotting with specific antibodies.

#### **3. Discussion**

Actinoporins are the major components of sea anemone venom, which disrupt cell membranes by pore formation [30]. *H. crispa* venom contains numerous actinoporin isoforms, encoded by the multigene family [28]. Hct-S3 is one of the isoform of *H. crispa* actinoporins belonging to Hct-S group with Ser at N-terminus (Figure 5). Earlier, the recombinant analog of Hct-S3 was obtained [26,31] and its hemolytic activity was comparable with well-characterized actinoporins such as RTX-A from *H. crispa* [32], EqII from *A. equina* [33] and StnII from *S. helianthus* [34]. Comparative analysis of amino acid sequences of known actinoporins and Hct-S3 revealed that Hct-S3 shared 87–89% identity with Gigantoxin-4 from *S. gigantea*, and RTX-A and StnI from *S. helianthus*, which possess anticancer activity (Figure 5). However, their anticancer mechanism has not been studied in detail. We attempted to elucidate the mechanism of action of actinoporins, in particular, Hct-S3, in different human cancer cells.

The recombinant analog of Hct-S3 was obtained using a previously developed scheme [26] with a changing of cell disruption approach. The lack of carbohydrates and disulfide bridges simplifies the production of recombinant actinoporins by heterologous expression in *E. coli*. However, there are some difficulties with the isolation of soluble actinoporins due to the protein aggregation as inclusion bodies during cells' ultrasonication. It is known that ultrasonic homogenization is a high-energy process of cell disruption. This fact may lead to the samples heating and result in the denaturation of proteins. Therefore, to minimize the protein denaturation we used a high-pressure homogenization of *E. coli* cells that allow us to increase the yield of soluble form of rHct-S3 by five times.

**Figure 5.** Multiple sequence alignment of actinoporins. EqII (P61914) from *Actinia equina*, FraC (B9W5G6) from *Actinia fragacea*, Gigantoxin-4 (H9CNF5) from *Stichodactyla gigantea*, StnI (P81662) from *Stichodactyla helianthus*, and RTX-A (P58691) from *Heteractis crispa*. The identical and conservative amino acid residues are shown on green and brown background.

Cytotoxic effects of rHct-S3 were studied in normal mouse epidermal, human embryonic kidney cells and human colon carcinoma, breast cancer, and melanoma cells. rHct-S3 exhibited cytotoxic activity against all tested cell lines with comparable IC50 values (Figure 2), which were 100–1000-fold higher than those found for other actinoporins [17,18,35]. Previously, StnI and hemolytic fraction of *S. helianthus* were shown to possess cytotoxic activities against colorectal cancer or breast cancer cells, respectively, while RTX-A demonstrated potent cytotoxic activity against both tested cancer cell lines [16].

Carcinogenesis is known to be a multistage process, which includes the initiation (transformation of normal cells into cancer cells), development (formation of colonies of cancer cells) and progression (growth of colonies of cancer cells) of cancer. Cancer prevention is gaining increasing attention because it may be a promising alternative to cancer treatment sparing complications caused by advanced diseases. The involvement of multiple factors and developmental stages and our increased understanding of cancer at the epigenetic, genetic, molecular, and cellular levels is opening up enormous opportunities to interrupt and reverse the initiation and progression of the disease and provide scientists with numerous targets to arrest by physiological and pharmacologic mechanisms, with the goal of preventing end-stage, invasive disease and impeding or delaying the development of cancer [36]. One of the promising strategies for combating carcinogenesis is to search for substances that can prevent the transformation of normal cells into cancer cells induced by various stimulating factors, e.g., epidermal growth factor (EGF), triphorbol ether (TPA), ultraviolet radiation (UV), etc. The promotion-sensitive mouse epidermal JB6 Cl41 cells are known to respond irreversibly to tumor promoters such as epidermal growth factor (EGF) with induction of anchorage-independent growth in soft agar [37]. Therefore, this well-established culture system was used to study the cancer-preventive activity of rHct-S3. Indeed, the actinoporin delayed the EGF-induced neoplastic transformation of JB6 Cl41 cells (Figure 3a,b) and suppressed colony formation of all cancer cell lines (Figure 3c–e), with the inhibition level of HT-29 and SK-MEL-28 cells comparable to cisplatin. Similar activity was previously demonstrated for RTX-A [16]. This polypeptide prevented malignant transformation of JB6 P<sup>+</sup> Cl41 cells and suppressed the growth of HeLa cell colonies at nanomolar concentrations [16].

The most significant cancer-preventive activity of rHct-S3 was found in colon cancer cells. Therefore, we examined the effects of rHct-S3 on the migration of colon cancer cells, as well as their proliferation, in order to incorporate the influence of cell proliferation in the interpretation of the results of migration assays. In fact, tumor cell migration essentially contributes to invasion and metastatic spread, ultimately resulting in progression of disease. More than 30% of patients with colorectal carcinoma have clinically detectable metastases at the time of primary diagnosis [38]. Since the most serious complication and the main cause of death of patients with colorectal carcinoma are distant metastases, the evolution of antimetastatic activity of potential therapeutic agents continues to be an important and urgent task. The mechanism of metastases formation is complex and not fully understood. The migration, intravasation, extravasation of cancer cells and formation of a new vessels

(neoangiogenesis) to consolidate a secondary tumor at a distant site are the most important steps of the metastasis process [39]. Remarkably, rHct-S3 almost completely suppressed the migration of HT-29 cells at a concentration of 4 μM (Figure 4a,b). Moreover, the actinoporin possessed a moderate antiproliferative activity (Supplementary Figure S1), but its impact on the anti-migratory activity of rHct-S3 was not significant.

During metastasis, the degradation of extracellular matrix (ECM) and components of the basement membrane by proteases facilitates the detachment of cancer cells, their crossing of tissue boundaries, and invasion into adjacent tissue compartments [40]. In recent years, the importance of cancer-associated proteases such as matrix metalloproteinases MMP-2 and MMP-9 in invasion and metastasis has been reported for a variety of solid malignant tumors [41]. Indeed, the actinoporin was found to effectively inhibit an expression of MMP-2 and MMP-9 (Figure 4c) that apparently resulted in the decrease in HT-29 cell migration. Recently, it was shown that caspase-3 is also able to influence the migration and invasion of colorectal cells [42]. In addition, caspase-3 is a key executioner of programmed cell death. In fact, rHct-S3 treatment cleavage of total caspase-3, followed by PARP cleavage, mediate both anti-migratory activity and induction of apoptosis in HT-29 cells (Figure 4d). In line with the pro-apoptotic activity of rHct-S3, an up-regulation of pro-apoptotic Bax and suppression of anti-apoptotic Bcl-2 were observed.

In conclusion, *H. crispa* actinoporin shows promising anticancer activity with a strong inhibiting effect on the migratory potency of cancer cells. We revealed for the first time that the actinoporin was able to inhibit cancer colony formation and cell migration via suppression of MMP-2 and MMP-9 expression and induce cell apoptosis via activation of caspase-3, cleavage of PARP, activation of Bax and suppression of Blc-2 expressions. The results indicate a high potential of the actinoporin to prevent cancer disease progression. Deep investigations of the underlying mechanism of the effect on apoptotic PI3K/AKT/mTOR, and of cell adhesion signaling pathways, are still to be performed.
