Previous studies on E. asper revealed that its ether extract is potent angiogenesis inhibitor; but the study was limited to its qualitative assessment only . In the present study, it is the first time that quantification of anti-angiogenic activity of E. asper methanolic extract as well as its cytotoxicity was reported.
Molluscs are known for their bioactive compounds and have become a focus of many studies aimed at discovering and isolating novel natural products . In a recent study, Bojnourdi et al.  investigated the anti-angiogenic properties of shell extract of the mollusc chiton on CAM model and found that it produced maximum 58% anti-angiogenic activity. In another interesting study by Gupta et al. , 22 different marine invertebrate extracts were screened for their anti-angiogenic activity, out of which, Meretrix meretrix (47.01% inhibition), Meretrix casta (64.63% inhibition), Telescopium telescopium (62.02% inhibition) and Bursa crumena (60.48% inhibition) showed significant anti-angiogenic activity in CAM. In the present study, EAME showed 42% inhibition of branching points and 51% inhibition of first order blood vessels in CAM (Fig. 1a, b), which is comparable to the work done by Bojnourdi et al. and Gupta et al. However, our analysis was extended further to find the underlying mechanism behind this anti-angiogenic activity by carrying out histological investigations.
The angiogenesis process in CAM is characterized by migration of mesodermal blood vessels, followed by their invasion through basement membrane and extracellular matrix (ECM) and finally their compartmentalization into new capillary vessels in the ectoderm . In normal growth, the number of mesodermal blood vessels decreases as the capillary plexus increases in the early stages of development. It is also known that the formation of capillary plexus in CAM is accompanied by decrease in CAM thickness as well as reduction of mesenchymal cells . This signifies the importance of our present study in which EAME has suppressed CAM angiogenesis by inhibiting migration and compartmentalization of mesodermal blood vessels leading to lesser capillaries subtending the ectoderm as shown in Fig. 4. Since, the embryo weight in treated remained more or less similar to control, it can be said that EAME did not have any adverse effect on the developing embryo and whatever effect found on the blood vessels was solely due to alteration in angiogenesis process. The reduction in area of CAM (Table 1) after treatment was believed to be because of decrease in vasculature.
A similar study carried out by Pandit et al.  on the feather star Lamprometra palmata palmata revealed the effect of its extract on the histology of CAM. Quantification of CAM histology explained that the capillary plexus in ectoderm decreased along with increase in mesodermal blood vessels and reduction in compartmentalization and after treatment with chloroform fraction of L. palmata palmata. However, it was also found that the mesenchymal cell density showed an irregular pattern and the CAM thickness increased with increasing concentration, which is in contrast to our results where both these aspects showed slight decrease after treatment with EAME.
An essential step of angiogenesis is the secretion of matrix metalloproteinases, which degrades the Extracellular membrane (ECM) proteins and allows migration of endothelial cells through the ECM . Around 23 different MMPs are known to be involved in ECM remodelling, out of which MMP-2 and -9 are crucial regulators of angiogenesis and metastasis . Our results from the CAM model showed that EAME efficiently inhibited CAM angiogenesis by increasing the number of mesodermal blood vessels and decreasing the percentage of capillaries in ectoderm. This suggests that the inhibition of CAM angiogenesis by EAME could be a result, in part, from the failure of ECM protein degradation and therefore impede mesodermal blood vessels migration and its subsequent proliferation into capillaries.
In recent years, quite a few studies were carried out to prove anti-angiogenic effect through suppression of MMPs. One such study by Huang et al.  demonstrated that Phyllanthus urinaria efficiently inhibited embryonic angiogenesis by reducing the MMP-2 activity in the CAM model. Another interesting research conducted by Manjunathan et al.  explained the positive angiogenic activity of human recombinant leptin (HRL) in chick CAM model. Their findings verify that the angiogenic response of HRL was due to increase in MMP-2 levels in CAM which caused breakdown of ECM in favour of migration and proliferation of ECs thus supporting the occurrence of sprouting angiogenesis. Since our results are in agreement with these data, it is highly possible that the inhibition of MMP-2 and -9 is one of the reasons for the reduction in capillaries and increase in mesodermal blood vessels in CAM after EAME treatment.
It is already known that angiogenesis plays an important role in development of tumours. Consequently, our results of CAM assay indicated that EAME might be a promising candidate as a new anti-cancer agent. Hence, further studies were conducted to investigate the possible effect of EAME on cancer cell lines directly. We have explored the cytotoxic potential of E. asper methanolic extract against three solid tumour cell lines and it was observed that EAME possesses moderate cytotoxicity against A549 cell line.
There are quite a few reports related to combined study of anti-angiogenicity and cytotoxicity in natural products. Baharara et al.  reported one such study using extract of the brittle star Ophiocoma erinaceus. He demonstrated that brittle star methanolic extract considerably inhibited blood vessels in CAM as well as significantly inhibited A2780 cell growth at 50–100 µg/mL. In another recent study, Mirian et al.  determined the in vitro anti-angiogenicity and cytotoxicity of oleo gum resin extract of three plants Rhus coriaria, Pistacia vera and Pistacia khinjuk. However, there are hardly any articles exploring the anti-angiogenic and anti-proliferative effects of mollusc extracts. Our study is probably one of the few that demonstrates these dual activities using a mollusc extract.
Although molluscs have been a good source of cytotoxic compounds, most of these are opisthobranch molluscs (nudibranch, sea hares) while the gastropods remain relatively less explored in this context. A recent study carried out by Antonova et al.  reported the significant cytotoxic activity of haemocyanin isolated from two gastropods Helix aspera (HaH) and Helix lucorum (HlH) against cell lines CAL-29 and T-24 (human bladder carcinoma).Another recent report by Bae et al.  focuses on the cytotoxicity of the marine sponge Haliclona sp. extracts on A549 cell line. Their results reveal that the Haliclona extract, at 100 μg/mL inhibited A549 cell viability by 51.6%. It was also confirmed that this cytotoxic effect was due to its influence on the cell proliferation and cell cycle. Our results indicate that EAME showed 40% inhibition of A549 cell line which is comparable to these studies, therefore it is confirmed that EAME shows moderate but significant cytotoxicity against A549.
The cytotoxic effect of EAME on A549 cells was confirmed through study of cell cycle by flow cytometry. One of the crucial aspects of cell cycle regulation is the DNA structure checkpoints, which arrest the cell cycle at the different phases (G1, S, G2 and M) in response to DNA damage or incomplete replication . Our results demonstrate that EAME moderately increased the amount of Sub-G1 DNA content (Table 3) suggesting that EAME induced apoptosis in the A549 cells. Cancer associated apoptosis-defects play a very important role in drug resistance, so regulation of defective apoptosis has become a fundamental aspect in cancer therapy. Hence, almost all cytotoxic drugs currently used, target on inducing apoptosis in malignant cells [33, 34].
There is increasing evidence of chemo-preventive potential of natural products therefore; continuous efforts are being taken to find anticancer chemotherapeutic drugs from edible and natural sources . Since EAME expressed moderate cytotoxicity in A549, it was attempted to further investigate whether this cytotoxicity is related to its anti-proliferative or anti-migratory effect. Accordingly, the effect of different concentrations of EAME on the wound healing process of A549 cell line was evaluated. Our results have clearly illustrated that EAME significantly reduces the wound healing capacity of A549 cell line, thus establishing its anti-proliferative and anti-migratory property.
Recently many reports illustrate the anti-metastatic effect of various natural or synthetic compounds against A549 non-small cell lung carcinoma. Hsieh, et al.  have reported that the anti-proliferative activity of the plant Kalanchoe tubiflora extract on A549 cell line was mediated by its induction of cell cycle arrest at G2 phase via its micro-tubule stabilizing property. An individual study on dihydroaustrasulfone alcohol, a compound isolated from marine coral, has illustrated the anti-proliferative activity of this compound on A549 cell line . It was found that the anti-proliferative activity of this compound is correlated with cell cycle arrest at G1 phase, down-regulation of Akt signalling pathway and suppression of proteases MMP-2 and MMP-9.
As explained earlier, MMP-2 and -9 play essential and varied roles in the progression of several types of cancers. A recent report by Webb et al.  has revealed the contribution of MMP-2 and MMP-9 in retinoblastoma cells. After comprehensive study, they found that MMP-2 and -9 are involved in stimulating cell migration and contributing to cell viability whereas MMP-9 played an additional role in Angiopoietin-2 production thus enhancing angiogenesis in retinoblastoma cells. Subsequently, a growing number of MMP-2/9 inhibitors are being developed as anti-metastatic agent; Fucoidan, an anti-metastatic agent, significantly reduces lung cancer cell migration and invasion by down-regulating the levels of MMP-2 via the suppression of PI3K-Akt-mTOR and NF-kB signalling pathway . Mere15, novel polypeptide, suppressed the expression of MMP-2 and MMP-9 and eventually inhibits cancer cell metastasis . Our results revealed that EAME significantly reduced the expression of MMP-2/9 in A549 cells (Fig. 8). This result suggested that EAME, similar to other anti-cancer agents, induces anti-metastatic activity by down regulating the expression of MMP-2 and MMP-9.
Owing to its dual targeted anti-cancer property, EAME might be a good source of a novel anti-cancer drug in future. Further work would include fractionating the crude methanolic extract and then testing the individual fractions for the corresponding activities. The molecular mechanism associated with the anti-angiogenic and anti-proliferative activities could be studied then using the active fraction(s).