Cytotoxic activity against breast cancer cells of two species of marine sponges from shallows of Yucatan peninsula

Abstract This study aimed to evaluate the in vitro activity of extracts of two marine sponge species, occurring in the shallows of the Yucatan peninsula coast, on two cancer and one normal mammalian cell lines. Hexane, dichloromethane, ethyl acetate and methanol extracts of Halichondria magniconulosa and Halichondria melanadocia were screened for their cytotoxic activity against hormone-dependent breast cancer (MCF-7) and human cervix cancer (SiHa) cell lines. The ethyl acetate extract of H. magniconulosa exhibited significant cytotoxicity against MCF-7 cells to a CC50 of 0.8 µg/mL, as well as high selectivity (SI = 24.5). On the other hand, SiHa cells were moderately sensitive to the dichloromethane and ethyl acetate extracts of the same species. (CC50 = 34.9 and 31.5 µg/mL, respectively). None of the extracts of H. melanadocia were considered active due their CC50’s were ranged from 59.0 to 94.5 µg/mL. Graphical Abstract


Introduction
According to global cancer statistics for 2018, the cancer worldwide prevalence and mortality were 43.5, and 9.6 million cases, respectively. Disturbingly, during the same year, 18.1 million new cases were recorded for both sexes. In all regions of the world, the first most frequently diagnosed cancer in women was breast cancer, closely followed by lung and cervix cancer (Ferlay et al. 2019).
Nowadays, several anti-breast cancer drugs showing some selectivity have been developed; unfortunately, their efficacy can vary by up to 60% and many patients suffer severe adverse reactions (Nandy et al. 2014). Additionally, a poor response to therapy caused by the occurrence of chemoresistance leaving few treatment options (Wang et al. 2014). Consequently, a continuous searching for new effective drugs against breast cancer is essential.
Considering approximately 53% of anticancer drugs developed from 1981 to 2014 are linked in some degree to a natural origin, natural products remain a significant source of scaffolds or compounds capable to inhibit the cancer cell growing (Newman and Cragg 2016). In this sense, marine sponges have been explored searching novel anticancer compounds, and a Japan species belonging to Halicondriidae family, Halichondria okadai, has been found as a natural source of Halichondrin B (Khalifa et al. 2019), the precursor of Eribulin mesylate, recently approved by the FDA for the treatment of breast cancer (H€ usemann et al. 2020).
In Mexico, despite the high biodiversity of its vast coastal zones, the study of marine sponges to determine their value as a source of anticancer compounds is scarce, and species growing at Yucatan peninsula shallows are unexplored. Therefore, as a part of our program on bioprospection of marine invertebrates of the Yucatan Peninsula, Mexico, two Halichondria species (Halichondria magniconulosa and Halichondria melanadocia) were screened for their in vitro cytotoxic activity against breast and human cervix cancer cells. These species are widely distributed through Western Central Atlantic, Gulf of Mexico and the Caribbean Sea. They inhabit shallows coastal environments and commonly grow on red mangrove roots, coral reefs, under rocks, or artificial substrates (Nagelkerken et al. 2008;R€ utzler et al. 2009). H. magniconulosa specimens are fleshy, massive to lobate or subspherical, and grow at depths ranged from 1 to 35 m. Their surface is yellowish and varies from smooth, wrinkled, to having fistules-like projections (1 cm long). On the other hand, H. melanadocia is black to dark green, irregularly massive to lobate or partly ramose. Its surface varies from smooth, to tuberculate or canulose. Specimens of this sponge commonly are found at depths between 1 and 5 m (Hechtel 1965;Collin et al. 2005). Both sponge species suffer predatory activities of spongivorous fishes; thus, H. melanadocia is predated by angelfishes, parrotfishes, filefishes, trunkfishes and cowfishes (Pawlik 1998); while H. magniconulosa is attacked by the two first fishes species (Wulff 2005).

Results and discussion
The American National Cancer Institute suggests a cut-off CC 50 of 30 mg/mL to consider an extract with significant activity (Suffness and Pezzuto 1991). Besides, if an extract exhibited a selectivity index (SI) 10, it could be considered with a low level of harmfulness (Vonthron-S en echeau et al. 2003). Thus, the cytotoxic activity (CC 50 ¼ 0.8 mg/mL) of the ethyl acetate extract of H. magniconulosa against hormone-dependent breast cancer cells (MCF-7) was determined as highly significant. However, the effect of this extract on cancer cervix cells (SiHa) was lesser, showing a moderate CC 50 (31.5 mg/mL). In the same fashion, the dichloromethane extract of H. magniconulosa was moderately cytotoxic against MCF-7 cells (CC 50 ¼ 34.9 mg/mL). Unfortunately, all extracts of H. melanadocia were considered non-active because they exhibited CC 50 's ranged from 59.0 to 94.5 mg/mL (Supporting Information Table S1).
As regards to the harmfulness of the extracts, notably, the ethyl acetate extract of H. magniconulosa showed significant selectivity (SI of 24.5) against MCF-7 cells. Surprisingly, this extract was barely 5.5-fold less selective than Docetaxel. Moreover, it was 40-fold more selective against MCF-7 cells than SiHa cells (SI 0.6).
Ethyl acetate extracts from several marine sponge species belonging to genera different to Halichondria have been evaluated at a concentration of 50 mg/mL for their cytotoxicity against MCF-7 cells, and the majority of those extracts provoked a 30% on cell growth inhibition or less (Beedessee et al. 2012). On the other hand, the ethyl acetate extract of Hyattella cribriformis, sponge from Southeast coast of India (Thondi coast), exhibited a CC 50 of 4.1 mg/mL and selectivity of 3.8 against breast cancer cells (Annamalai et al. 2015). Remarkably, in contrast to results previously described, the cytotoxicity of ethyl acetate extract of H. magniconulosa against MCF-7 cells was 5.5fold (0.8 mg/mL) higher as well as 6.4 times more selective (SI ¼ 24.7).
It is important to note that while Halichondrin B, a potent anticancer polyether macrolide, was isolated from a polar extract of H. okadai (Hirata and Daisuke 1986), the methanolic extracts evaluated in this study exhibited a lack of activity against MCF-7 cells. Contrary, the medium-polar (ethyl acetate) extract of H. magniconulosa was the most active and selective one against the same cancer cell line.

Experimental
See Supporting Information.

Conclusions
According to our best knowledge, this is the first study of Halichondria species growing at shallows of Yucatan peninsula coast focused in searching for compounds against breast and human cervix cancer cells. The ethyl acetate extract of Halichondria magniconulosa exerted notable cytotoxicity and selectivity against hormone-dependent breast cancer cells (MCF-7). Therefore, this result point to consider this species as a potential source of anticancer compounds. Consequently, the chromatographic fractionation of the ethyl acetate extract needs to be performed to isolate and identify its bioactive compounds.