br Further we evaluated the e ect of rutin fucoidan
Further, we evaluated the eﬀect of rutin, fucoidan, Ru + Fu mixture and Ru-Fu complex on the loss of ψm in HeLa LY3009120 using Rh123 fluorescent dye (Fig. 7a). The treatment of cells with compounds caused loss of ψm and apoptosis induction as the DAPI staining (blue color) of cells showed the more condensed and fragmented nuclear bodies whereas these features were absent in untreated (control) cells (Fig. 7 a and b). Rh123 staining (green) denotes the loss of ψm in cells that uptake Rh123. The loss of ψm is due to mitochondrial membrane depolarization, an event characteristic of early and irreparable stage of apoptosis. DAPI staining also showed that the treatment of HeLa cells by the study molecules displayed the apoptotic features such as con-densed as well as fragmented nuclei, whereas untreated HeLa cells showed normal nuclei (Fig. 7a). Apoptosis induction capability of the study molecules and Ru-Fu complex could be attributed to changes in the ψm and the enhanced eﬀect was observed with the treatment of cells by Ru-Fu complex. It is shown that mitochondria play a crucial role in an intrinsic apoptotic pathway through releasing Cytochrome c for the activation of caspase cascade, an important event in initiating apoptosis [69,70]. The results of this study revealed that Ru-Fu could enhance the development of mitochondrial dysfunctions to initiate apoptosis. It was demonstrated that treatment of mice Ehrlich ascites carcinoma (EAC) cells with rutin and Rutin–zinc (II) complex with paclitaxel significantly decreased the ψm . Several reports en-umerated fucoidan-induced apoptosis in cancer cells through an
increase in the relative ratio of Bax/Bcl-2 expression and consequent mitochondrial dysfunction . In addition, fucoidan-mediated apop-tosis was reported to be induced due to an increase in ROS generation and its associated events such as the ψm decrease, and phosphoryla-tion of p38, JNK and ERK1/2 kinase proteins . Interestingly, in contrast, another study showed that fucoidan restored the normal cell functions such as mitochondrial integrity and metabolic activity in wistar rats bearing tumor induced by carcinogen diethyl-nitrosamine (DEN) . Hence, we suggest that Ru-Fu complex potentially induces not only apoptosis by distorting the mitochondrial integrity in cancer cells but also rejuvenates the metabolic activity of healthy cells com-plimented with its non-toxic nature. Fucoidan-induced nuclear frag-mentation has been well reported in various cancer cells [55,71,74]. Flavonoids are capable of translocation into the nucleus of cancer cells and induce chromatin condensation and DNA fragmentation . The flavonoid quercetin along with EGCG synergistically induces apoptosis via chromatin condensation and fragmentation in various cancer cells [75,76].
3.8. Eﬀect on cell apoptosis
Double fluorescence staining of control and treated HeLa cells using Annexin-V-FITC/PI and subsequent flow cytometry analyses dis-tinguished the apoptotic cells from viable cells (Fig. 8). Cells in early phases of apoptosis were stained with annexin-V-FITC, whereas dead cells and cells in late apoptotic phases were stained with both annexin-V-FITC and PI. These reagents gain entry into the cell only after the damage of the plasma membrane. The characteristic dot-plots illus-trating the population of apoptotic cells are shown in Fig. 8. The
Fig. 9. Eﬀects of molecules on cell cycle regulation in HeLa cell proliferation. Images indicate the cells in each phase of the cell cycle at sub-IC50 dosage of molecules. Data on cell cycle distribution represent the percentage of cell death in the total population. Data show percentage of live cells in each phase of the cell cycle (G0/G1, S, and G2/M). The cell cycle arrest in the G0/G1 and S phases is associated with an increased abundance of cell distributions (*) p ≤0.05 and (**) p ≤ 0.01, compared with untreated cells (control groups). Error bars denote mean ± SD of three independent experiments performed in triplicates.