*2.2. Bioactivity of the Phlorethols from C. costata*

As a first step, the effect of phlorethols (**CcPh)** from brown alga *C. costata* (0–1000 μg/mL) on the viability of human colorectal carcinoma HT-29 and HCT 116 cell lines, breast cancer MCF-7 cells, and melanoma SK-MEL-28 cells was tested by MTS assay (Figure 1). The treatment of cells by **CcPh** was found to induce a concentration-dependent inhibition of cell's viability. The inhibiting concentration of **CcPh** that caused a 50% reduction in cell viability (IC50) of HT-29 was 92 ± 2.8 μg/mL; HCT, 116–94 ± 3.08 μg/mL; MCF-7, 96 ± 3.3 μg/mL; and melanoma cells SK-MEL-28, 102 ± 4.8 μg/mL.

**Figure 1.** The cytotoxic activity of phlorethols from *C. costata* (**CcPh**) against colorectal carcinoma HT-29 and HCT 116 cells, breast cancer MCF-7 cells, and melanoma SK-MEL-28 cells. The cells were treated with **CcPh** at concentration of 0.001–1 mg/mL for 24 h. Cell viability was estimated using the MTS assay. Data are represented as the mean ± standard deviation (SD), as determined from triplicate experiments.

Recently, the cytotoxic activity of polyphenols' fractions from different species of brown algae was investigated against pancreatic cancer cells Mia-PaCa-2, Panc-1, Panc-3.27, and BxPC-3 [12], human colorectal adenocarcinoma HT-29, Caco-2, breast carcinoma T-47D, MDA-MB-468 cell lines [13], human leukemia HL60 and THP-1, and prostate cancer PC3 cell lines [14]. The IC50 values for investigated polyphenols fractions were in a range from 80 to 200 μg/mL. It was suggested that the number of phenolic ring substituents and phenyl ether linkages as well as the reduction in pH values caused by an oxidation of hydroxyl groups in phenol polymer derivatives mainly influence their cytotoxic activity [15]. Since colorectal carcinoma cells HCT 116 and HT-29 gain resistance under radiation exposure [16,17], we chose them as model to check the hypothesis whether of phlorethols from brown alga *C. costata* is able to effectively sensitize these cells to radiation.

Next, the effect of **CcPh** or X-ray radiation on colony formation in human colorectal cancer cells was determined using the soft agar assay. It was found that the **CcPh** alone decreased the number of colonies in HT-29 by 3%, 7%, and 24% at 10, 20, and 40 μg/mL, respectively and in HCT 116, by 4%, 7% and 18% at the same doses. (Figure 2A,D). It should be noted that **CcPh** did not influence the viability of normal mouse epidermal cells JB6 Cl41 at an effective concentration of 40 μg/mL even after 72-h of treatment (Figure S4).

**Figure 2.** Anticancer and radiosensitizing effects of phlorethols from *C. costata* (**CcPh**) on colony formation in human colorectal carcinoma cells. HT-29 and HCT 116 cells (2.4 <sup>×</sup> <sup>10</sup>4) were treated with (**A,D**) **CcPh** (10, 20, and 40 μg/mL) or (**B,E**) X-ray (2, 4, 8, and 10 Gy) or (**C,F**) a combination of X-ray radiation (2 Gy) and **CcPh** (5, 10, and 20 μg/mL) and subcultured onto Basal Medium Eagle BME soft agar and incubated for 2 weeks. The number of colonies was calculated using the ImageJ software bundled with 64-bit Java 1.8.0\_112 (NIH, Bethesda, Maryland, USA). All experiments were repeated at least three times in each group. Results are expressed as the mean ± standard deviation (SD). The asterisk (\*) indicates a significant decrease in the number of colonies of cancer cells treated with **CcPh** or X-ray compared to PBS-treated cells or **CcPh** in combination with X-ray compared to irradiated cells (\*\* *p* < 0.01, \*\*\* *p* < 0.001).

The exposure of HT-29 by X-ray irradiation at doses of 2, 4, 8, and 10 Gy caused the inhibition of the colonies number by 26%, 55%, 95%, and 97%, respectively and HCT 116 by 18%, 30%, 47%, and 61% at the same doses (Figure 2B,E).

To investigate the radiosensitizing effect of phlorethols **CcPh**, HT-29 and HCT116 cells were treated with **CcPh** at non-toxic concentrations of 5, 10, and 20 μg/mL and exposed to a low dose of X-ray of 2 Gy, which alone slightly inhibited the viability or colony formation in cancer cells.

Under X-ray exposure, the number of colonies of HT-29 and HCT 116 cells were decreased by 24% and 21%, respectively, compared to the non-irradiated cells (control) (Figure 2C,F). The combinatory treatment with X-ray (2 Gy) and **CcPh** (5, 10, and 20 μg/mL) caused significant inhibition of colony formation of HT-29 by 28%, 39%, and 41%, respectively and HCT 116 by 15%, 24%, and 40%, respectively, compared to irradiated cells (Figure 2C,F).

To determine the type of combination effect of X-ray radiation with phlorethols **CcPh**, the combination index (CI) was calculated (Table 1). This revealed that the interactions of radiation and **CcPh** were mostly synergistic (CI < 0.7) [18]. **CcPh** strongly sensitized the HCT 116 cells to radiation as compared with HT-29 cells.


**Table 1.** Combination index (CI) for phlorethols—X-ray irradiation interactions.

\* The Combination Index (CI) is the quantitative measure of the degree of interaction between different treatments. CI values in the range of 0.9–1.1 indicate additive effect; and CI values greater than 1.1, antagonism. CI values in the range from 0.9 to 0.7 indicate slight synergism; and CI values less than 0.7, synergism. CI values were calculated according to the Chou and Talalay mathematical model for drug interactions using the CompuSyn software version 1.0 on the basis of the results of soft agar assay. Data represent mean CI calculated from three independent experiments ± standard deviations.

To the best of our knowledge, this is the first study on the radiosensitizing activity of phlorotannins from brown algae. Since polyphenols from brown algae possessed potent antioxidant activity, their radioprotective effect is a topic of interest of numerous investigations [19]. However there is evidence that several natural compounds are able to exert a dual mode of action after irradiation—radioprotective as well as radiosensitizing—depending on its dose and scheme of treatment [20]. For example, polyphenols of plant origin such as curcumin or resveratrol have been found to protect various systems against ionizing radiation and to sensitize cancer cells to radiation treatment [21].
