DNA barcoding and phylogeny based comparative evaluation of anti-cancer properties of Caulerpa (J V Lamouroux) spp. from Indian coasts

Abstract

A total of 15 Caulerpa samples were collected from Indian coasts and identified based on morphological and molecular data inferred from ITS, 18S, tufA and rbcL. Seven different species viz. C. scalpelliformis, C. racemosa, C. sertularioides, C. verticillata, C. taxifolia, and C. corynephora; and their geographical isolates were identified. Barcode data for these species was generated using aforementioned molecular markers and used for phylogenetic assessment. Phylogenetic trees using Bayesian inference (BI) and Maximum Likelihood (ML) function were generated for each molecular marker. tufA was found to be most suitable marker for the genus Caulerpa, resolving the species into 17 different lineages, with 15 corresponding to already known sections and 2 new lineages. Besides, a database named DbIndAlgae of Indian algae was generated and all the morphological as well as molecular data generated in this study is uploaded on the database. In addition, the phycochemical analysis revealed the presence of alkaloids, terpenoids, steroids, tannins, saponins, flavonoids, and phenols in different Caulerpa species. The selective cytotoxicity of methanolic extracts of Caulerpa (CMEs) was evaluated on MDA-MB-231, T47-D and H1299 cells, and the results revealed significant cytotoxicity of all species. C. racemosa KNY-254 and C. taxifolia TEN-158 were found to be most potent on MDA-MB-231 cells with IC50 value of 0.226 ± 0.004 and 0.246 ± 0.009 µg/µL. The mitochondrial membrane perturbation was revealed by JC-1 and apoptotic cell death was confirmed by Annexin V/FITC staining. CMEs also induced ROS in MDA-MB-231 cells as depicted by DHE, and increased activity of SOD, decreased activity of gluthatione reductase. The CMEs also exhibit anti-invasion activity and inhibited up to 71% migration across the artificially scratched wound in MDA-MB-231, w.r.t. untreated control cells. Moreover, chemical probing of C. racemosa KNY-254 by LC-MS analysis revealed six previously reported and six unreported molecules. The molecular docking analysis revealed weak to moderate interactions with all of the protein targets viz. Bcl2, AMPK, mTOR, BID, PERK, IGF-1R, PI3K, PTP1B and Akt2, known to play important role in cancer cell signaling. Additionally, a moderately positive correlation amongst the phylogeny and anti-cancer activity was observed suggesting that phylogeny might provide cues for anti-cancer activity, subject to further validations.