br Evaluation of the cytotoxicity by Lactate Dehydrogenase L
3.4. Evaluation of the cytotoxicity by Lactate Dehydrogenase (LDH) leakage assay
Cellular damage induced by rutin, fucoidan, Ru + Fu mixture and Ru-Fu complex was measured by assessing the extent of LDH release into the culture media. LDH, a stable cytosolic enzyme that is released upon cell lysis. LDH catalyses the conversion of lactate to pyruvate via NAD+ reduction to NADH. Diaphorase, an enzyme uses NADH to re-duce tetrazolium salt (INT) to a red formazan product which is mea-sured at 490 nm. The formation of formazan is proportional to the ex-tent of cytotoxicity. As compared to the control, rutin, fucoidan and Ru + Fu mixtures significantly induced cytotoxicity as observed from the LDH release levels from the HeLa LOXO-101 (Fig. 4e). In addition, the cytotoxic eﬀect of the Ru-Fu complex was much more pronounced against HeLa cells than individual compounds and mixture of com-pounds (Fig. 4e). These results are validating the findings of MTT assay of the study. Reports implied that the molecular conformation of fu-coidan might influence the binding features of sulphate groups, and this could be an important factor for modulating the inhibition potency against cancer cell growth . Similarly, it was reported that sig-nificant release of LDH was noted in HeLa cells when treated with the combination of three compounds such as myricetin and methyl eugenol with cisplatin . MTT and LDH assays of citrus phenol treated HeLa cells showed that the cell proliferation was inhibited significantly at all time-points. MTT and LDH cytotoxic studies of rutin on a variety of cancer cell lines, including colon cancer (SW480), neuroblastoma (LAN-5), colorectal (CRC) and hepatic carcinoma (HTC) have shown that the chemo-preventive eﬀect of rutin is mainly through the induction of cytotoxicity .
Fig. 5. (a). Induction of Cellular apoptosis is observed by AO/EtBr staining of HeLa cells. Images show the morphological features and stages of viable (v) (green), apoptosis/apoptotic bodies (A/Ab) (orange) and necrotic (N) (orange to red) in cells treated with the complex of Ru-Fu. Scale bar represents 100 μm. (b). Bar diagram shows percentage of apoptotic cells after treatment with rutin (Ru 15 μg/ml), fucoidan (Fu 50 μg/ml), rutin-fucoidan mixtures (Ru 15 + Fu 50) and rutin-fucoidan complex (Ru-Fu 20 μg/ml) for 24 h. The error bars denote mean values ± SD of three independent experiments, each performed in triplicates. (**) p ≤0.01 compared with control cells.
Fig. 6. (a). DCHF-DA staining of cells for ROS accumulation inducing apoptosis. Scale bar represents 100 μm. (b). Release of ROS (%) from HeLa cells treated with compounds and complex of the study. The error bars denote mean values ± SD of three independent experiments, each performed in triplicates. (**) p ≤0.01 compared with untreated cells (control). r> 3.5. Analysis of Apoptotic induction potential by AO/EtBr staining
Fluorescence microscopy was used to diﬀerentiate the apoptotic and necrotic cells on the basis of cell morphology and membrane integrity. After treating Hela cells with rutin, fucoidan, Ru + Fu mixture and Ru-Fu complex for 24 h, the cells were stained with AO/EtBr for observing morphological changes through induction of apoptosis (Fig. 5a and b).
In Fig. 5a, the cells in early apoptotic stage exhibited an orange co-loured nucleus with fragmented or condensed chromatin structures, whereas the cells in late apoptosis showed fragmented and condensed red colored nuclei. These apoptotic characteristics were more evident in Ru-Fu treated HeLa cells than the cells treated with rutin, fucoidan and Ru + Fu mixture indicating the eﬃcacy of the developed complex in apoptotic induction. However, the untreated (control) HeLa cells did