pH-dependent cytotoxic effects of extracts of the marine sponge Polymastia janeirensis on cancer cell lines

Abstract The purpose of this work was to study the cytotoxic effects of marine sponge Polymastia janeirensis, which has been observed in the field to release an orange substance that is toxic to fish. The result showed that aqueous extract (pH 7.0) was highly cytotoxic to glioma (U87) and neuroblastoma (SHSY5Y) cancer cell lines (IC50 < 1.0 μg/mL). In addition, this extract showed potent antioxidant and procoagulant (decreased the clotting time by 1.7-fold) activities. Interestingly, the cytotoxic effects were pH-dependent since the viability of the cancer cells was not affected with the extract (pH 5.5). The close similarity between the aqueous extract (pH 7.0) and the orange liquid that is released by the sponge indicates that this potential chemical defence of P. janeirensis deserves further investigation.


Introduction
The marine environment has been shown to be a rich source of unprecedented chemical structures, especially for cancer therapy (Kumar et al. 2013;Pejin et al. 2014; Ebada et al.

ABSTRACT
The purpose of this work was to study the cytotoxic effects of marine sponge Polymastia janeirensis, which has been observed in the field to release an orange substance that is toxic to fish. The result showed that aqueous extract (pH 7.0) was highly cytotoxic to glioma (U87) and neuroblastoma (SHSY5Y) cancer cell lines (IC 50 < 1.0 μg/mL). In addition, this extract showed potent antioxidant and procoagulant (decreased the clotting time by 1.7-fold) activities. Interestingly, the cytotoxic effects were pH-dependent since the viability of the cancer cells was not affected with the extract (pH 5.5). The close similarity between the aqueous extract (pH 7.0) and the orange liquid that is released by the sponge indicates that this potential chemical defence of P. janeirensis deserves further investigation. 2015; Zhang et al. 2015). Of particular interest is the marine sponge Polymastia janeirensis Boury-Esnault, 1973; which is not frequently targeted by predators in its environment. It releases a liquid with a vivid orange colour when severed, according to field observations. To date, biological effects have only been described for its crude extracts, which have shown cytotoxic effects (Monks et al. 2002;Frota Junior et al. 2009).
Among the many types of cancer, that of the brain deserves special attention, mainly because of the lack of effective treatment options for patients (Stupp et al. 2009). The aetiology of cancer has been suggested to be closely linked to an intrinsic increase in reactive oxygen species (ROS). This imbalance causes oxidative damage to lipids, proteins and DNA and also leads to altered blood clotting (Trachootham et al. 2009).
Thus, to contribute to the study of the anticancer potential of P. janeirensis, this research investigates the biological effects of its fractions and extracts on human glioma and neuroblastoma cell lines. Moreover, considering the link between cancer, disturbances in coagulation, and imbalances in ROS, we also evaluated their effects on blood coagulation in addition to their redox properties. To the best of our knowledge, this is the first report focused on evaluation of a pH-dependent activity, which is correlated with the chemical defence of a marine sponge. Table 1 shows the results of cytotoxic effects of the P. janeirensis extracts and fractions on cancer cell lines. The aqueous extract (pH 7.0) had greater cytotoxic effects on the glioma and neuroblastoma cell lines, with an IC 50 of <1.0 μg/mL. The most interesting aspect of this activity is its extreme dependence on water pH. When the extracts were prepared with water at a pH of 5.5, the viabilities of the cancer cells were not affected.

Results and discussions
To better understand the pH-dependent cytotoxic activity, additional evaluations were performed using a range of pH levels (5.5-8.0). The extracts prepared with pH above 6.5 showed similar results for both cancer cell lines (IC 50 < 1.0 μg/mL) (Table S1). However, aqueous extracts (pH 6.0) revealed an IC 50 slightly higher, around 2.0 μg/mL; demonstrating that from this pH the activity begins to decline, reaching no activity at pH 5.5. Based on these results, we can infer that the water pH is extremely important to extract the active compounds. Therefore, two pH values (7.0 and 5.5) of P. janeirensis aqueous extract were chosen for evaluating the antioxidant and anticoagulant effects.
The main finding of our research was the identification of the pH-dependent cytotoxic activity, which is likely correlated with P. janeirensis chemical defence mechanism. The aqueous extract at pH 7.0 had a strong orange colour, which resembled that of the liquid that is released by the sponge when severed, whilst the aqueous extract at pH 5.5 had an intense red colour. Studies have shown that the pH of seawater (5.5-8.3) can influence the survival of marine sponges and a low pH can induce permeability changes in these sponges that can affect normal cell functioning (Belarbi et al. 2003). P. janeirensis profoundly affected coagulation: the EtOAc, hexane and aqueous fractions exerted anticoagulant activities, whilst the aqueous extracts presented procoagulant effects ( Figures S1 and S2). The control plasma took over 6 min to initiate coagulation and aqueous extracts at pH levels of 7.0 and 5.5 showed decreased clotting times of 3.5 and 5 min, respectively ( Figure S2).
The redox properties assessment of P. janeirensis extracts and fractions revealed significant dose-dependent antioxidant effects ( Figure S3). The comparison of the CL profile with that of Trolox ( Figure S4 A-B) indicated that the EtOAc fraction and aqueous extracts (pH 7.0 and 5.5) inhibited free radical production for longer periods, but the aqueous extracts increased their production at the end of the process. The EtOAc fraction exhibited the highest antioxidant capacity, followed by the aqueous extracts at pH levels of 5.5 and 7.0 and the aqueous fraction (Table 1).
The link between procoagulant activity and an increased rate of apoptosis in tumours of the endothelium should also be emphasised. Apoptotic cells externalise phosphatidylserine, a class of anionic lipids, on the outside of their cell membranes. Those phosphatidylserine leads to a buildup of thrombin in the affected region, triggering coagulation (Strieth et al. 2008). In summary, our results of procoagulant effect of aqueous extract (pH 7.0) supports the mechanism of action through a caspase-9 apoptotic pathway that was previously described for the aqueous extract of P. janeirensis (Frota Junior et al. 2009).

Conclusion
The results demonstrate that P. janeirensis possesses pH-dependent cytotoxic activities. Moreover, the strong potential of the aqueous extract to modulate free radicals and exert procoagulant effects supports the presence of a cytotoxic mechanism that acts on cancer cells. We can therefore infer that P. janeirensis is a promising source of chemical prototypes that may be developed into novel forms of cancer therapy.

Supplementary material
Experimental details and figures relating to this paper are available online.