br It is observed that the
It is observed that the combination of compounds increased the anti-proliferative eﬀect of individual compounds by two fold in HeLa cells. A combination index (CI) of 0.75 was achieved by the combination of Ru + Fu suggesting the synergism exhibited by these compounds (Table 2). The Ru + Fu mixture and Ru-Fu complexes did not increase cytotoxicity in normal human keratinocyte CL 316243 when compared to treatment of cells by individual compounds (Fig. 4d). Numerous reports suggest that rutin induces dose-dependent inhibition of the prolifera-tion of various cancer cell lines [8,12,47]. There are several reports
Fig. 4. (a). Cytotoxicity of rutin treatment in HeLa and HaCaT cells. Both the cells were treated with rutin in the increased concentrations of 10–100 μg/ml.
(b). Cytotoxic eﬀects of fucoidan in HeLa and HaCaT cells and these cells were treated with fucoidan in the concentration range of 10–1000 μg/ml. (c). Cytotoxic eﬀects of Ru-Fu (Rutin-Fucoidan complex) in HeLa and HaCaT cells and these cells were treated with Ru-Fu complex solution in the concentration range of 10–100 μg/ml. The asterisks indicate the IC50 concentrations of com-pounds and complex. (d). Eﬀect of 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) on HaCaT and HeLa cells proliferation. (e). Eﬀects of rutin (Ru 15 μg/ml), fucoidan (Fu 50 μg/ml) alone, Ru + Fu mixture (Ru 15 + Fu 50) and rutin-fucoidan complex (Ru-Fu 20 μg/ml) on LDH leakage (%) in HeLa cells. The error bars denotes mean values ± SD of three independent experi-ments, each performed in triplicates (*p ≤0.05 and ** p ≤0.01).
Interaction between rutin (Ru) + fucoidan (Fu) in combination. The combi-nation indexes (CI) were interpreted as follows: < 1 synergy; 1 additive and > 1 antagonism.
(alone, μg/ml) (in combination, μg/ml)
indicating that sulphate groups of fucoidan play a vital role in the suppression of cancer cell growth by binding with cationic proteins on the cell surface, and it is also reported that the structure of this natural polysaccharide allows it to be used in conjugation with anticancer agents [48–50]. Hence, a combination of these compounds may en-hance their anti-cancer eﬃcacy with less toxicity. Several promising combinations of natural and synthetic anticancer agents have been demonstrated in vitro and in animal models [51,52]. The combination Biomedicine & Pharmacotherapy 109 (2019) 1181–1195
of flavonoid quercetin with EGCG has been shown to synergistically enhance the inhibition of prostate cancer cell growth both in vitro and in vivo . Vishchuk et al. substantiated that the combination of fu-coidan with resveratrol, a phytoalexin polyphenol synergistically in-creased the inhibition of HCT 116 human colon cancer cells growth . Another study proposed that fucoidan could enhance the activity of other chemotherapeutic agents such as cisplatin, tamoxifen or pa-clitaxel against MDA-MB-231 and MCF-7 breast cancer cells .
The present study also provides a novel regimen to enhance the anticancer eﬀect of flavonoid through the formation of complex with enhancer agent fucoidan. Reports also suggested that rutin could form complexes with other metal ions and showed higher cytoprotective eﬀects than parent flavonoid. Much of these reports describe the sy-nergistic anti-carcinogenic eﬀect of rutin or fucoidan in combination with other anticancer molecules, but reports are scarce on the forma-tion of anticancer complex through the interaction of these molecules. Lu et al. have well demonstrated the cytotoxic eﬀect of protamine-fu-coidan complex nanoparticles against metastatic breast cancer cells (MDA-MB-231) . Ikeda et al. reported that Rutin-zinc (II) complex exhibited cytotoxicity against leukemia, multiple myeloma and mela-noma cell lines, but did not show any cytotoxicity against normal cells in vitro . However, the combined anticancer eﬀects through the formation of rutin-polysaccharide complex have not been reported. Our unique observations suggest that the Ru-Fu complex formed of rutin and fucoidan through non covalent interactions interestingly exhibits higher anti-proliferative eﬀect against cancer cells than the eﬀects of the mere combination of compounds in a mixture. The enhanced sy-nergistic bioactivity of the Ru-Fu complex is due to the interaction of compounds which provides much stability to the complex without af-fecting the functions of other. In contrast, the mere combination of compounds has lower extent of enhancement of the synergistic eﬀect than the Ru-Fu complex though the compounds are stable and anti-carcinogenic individually and this suggests that the stable interaction among them is possible to display the enhanced therapeutic eﬀect.