Many genotoxic drugs express their anticancer effects through direct or indirect DNA damage and thus resulting in cell death . Therefore, many chemopreventive agents focus on targeting apoptosis-inducing pathways . For example, several chemotherapeutic drugs, such as cisplatin , 5-fluorouracil , and taxol , have been used for head and neck cancers and are found to induce specific apoptotic pathways. Moreover, drug resistance is partly explained by the ability of cancer cells to evade apoptosis [42–44]. Accordingly, drugs with apoptosis-inducing ability in OSCC may reduce potential drug resistance.
This study shows a novel finding that MEGT has a cytotoxic effect on Ca9-22 oral cancer cells. The cell proliferation inhibiting effect of MEGT in Ca9-22 oral cancer cells is dose-dependent. The IC50 value of MEGT-treated Ca9-22 oral cancer cell line is 326 μg/ml (24 h). For comparison, the reported IC50 values (treatment times) of water/ethanol/methanol/ethyl acetate extracts for several natural product studies based on cancer cells were found in similar ranges: Moringa oleifera leaf water extract for HeLa derivative KB cells  was 150 μg/ml (48 h) ; Cassia tora methanolic leaf extract for HeLa cells was 191 μg/ml (48 h) ; Olea europaea ethanolic extract for leukemic (Jurkat) cells was 0.9 mg dw (48 h) ; Indigofera linnaei Ali methanolic extract for cervical, liver, breast, and colon cancer (HeLa, HepG2, MCF-7, and HT-29) cells were less than 100 μg/ml (72 h) ; and Colpomenia sinuosa, Halimeda discoidae, and Galaxaura oblongata ethyl acetate extract for liver cancer (HuH-7) cells were 112.38, 230.53, and 123.54 μg/ml (72 h) and for leukemia (HL-60) cells were 53.35, 226.35, and 132.73 μg/ml, respectively.
In experiments with cancer cells other than oral cancer, the incubation times differ from the 24 h incubation period used in this study. Accordingly, the results of lower IC50 doses obtained in these studies may be due to the longer incubation times used. In general, the treated dosage of the current developed MEGT is similar to the range of the dosage of natural product-derived extracts used in these cancer studies.
Moreover, the IC50 value of clinical chemotherapeutic drug cisplatin was 174 μg/ml (48 h) for HeLa cells . Accordingly, these results suggest that the MEGT is a potential natural product to be used as a chemotherapeutic agent as determined by its effect on Ca9-22 oral cancer cells. Moreover, more natural products with anti-oral cancer activity may be examined for their possible synergistic effects to enhance the efficiency of OSCC chemotherapy.
The possible mechanism for MEGT-induced cell growth inhibition may be partly due to apoptosis in OSCC cells demonstrated by the increases seen using sub-G1 and annexin V-staining. To further validate the role of apoptosis, the caspase signaling pathway [47, 48] may need to be examined. The increase of γ-H2AX-based DNA double strand breaks  under MEGT administration suggested that DNA damage is involved in MEGT-induced apoptosis. To monitor the γ-H2AX foci  and the tailing degrees of the images in single cell electrophoresis (comet assay)  by fluorescent microscopy, the evidence of DNA damage may be further observed. Furthermore, the DNA damage response cascade and ROS signaling pathway are two of the main pathways leading to cell death [52, 53]. DNA damage has also been reported to induce ROS generation through the H2AX-Nox1/Rac1 pathway . ROS is an important mediator of apoptosis  and cell cycle checkpoint functions . Accordingly, the MEGT-induced intracellular ROS changes were examined in OSCC cells.
Many anti-cancer drugs target cells, at least in part, by generating high levels of intracellular ROS [56, 57]. In the example of brown algae, ethyl acetate extracts from Colpomenia sinuosa were reported to induce apoptosis and intracellular ROS in leukemia U937 cells . Consistently, the current study demonstrated that MEGT induced the ROS in oral cancer cells in a dose–response manner.
In the study of ethyl acetate extracts from brown algae Colpomenia sinuosa, the research focused on the sub-G1 phase as an indication of apoptosis without further exploring the overall alterations in the cell cycle. In contrast, ethanol extracts of blue-green algae Aphanizomenon flos-aquae displayed the G0/G1 arrest in HL-60 leukemia cancer cell lines in 24 h incubation . Similarly, we found that the MEGT-treated Ca9-22 cells at the concentration of 0.25 mg/ml significantly increased the percentage of G1 phase cells and concurrently decreased the percentages of S phase cells and cells following G2/M, supporting the hypothesis that MEGT may lead to a G1 phase arrest before apoptosis. Therefore, algal extract may elicit cell cycle arrest responses.
GSH is protective against intracellular ROS, which induces apoptosis in response to cell injury. Depletion of GSH increases susceptibility to ROS, and results in damage  such as DNA fragmentation . Accordingly, many chemotherapeutic agents are developed by the apoptosis-inducible strategy through ROS-mediated cell damage. Consistently, the ROS level of Ca9-22 oral cancer cells after MEGT treatment for 24 h was increased, suggesting that MEGT may induce apoptosis through increasing intracellular ROS and GSH depletion.
In addition, mitochondria also play an important role in ROS and apoptotic events . For example, capsaicin, the main capsaicinoid found in chili peppers, was found to disrupt mitochondrial membrane potential (MMP) and mediate oxidative stress leading to apoptosis in pancreatic cancer cells . Mitomycin c can kill the small cell lung cancer cells by increasing MMP and decreasing intracellular GSH contents due to oxidative stress . The current study demonstrated that MEGT significantly decreased the MMP in oral cancer cells in a dose–response manner, suggesting that MEGT-induced MMP disruption ability may be directly or indirectly influencing ROS generation in oral cancer cells.
Extracts derived from natural products isolated by different solvents may have different cytotoxic efficacies. For example, the IC50 value of ethanol extract of G. tenuistipitata in Ca9-22 cells was 8-fold higher than that of the methanol extract (data not shown), whereas the water extract of G. tenuistipitata did not display any cytotoxicity to H1299 cells . Therefore, methanol extracts of G. tenuistipitata were chosen in this study. Although our results have demonstrated the anti-proliferative effect of MEGT on oral cancer cells, there are some limitations in this study. For example, it has been reported that the polysaccharides from the G. dura, the chemical composition of G. cervicornis, and the prostaglandin content of G. verrucosa are subject to seasonal variation. Accordingly, the possibility of a seasonal variation in the biological effects of MEGT cannot be excluded.
Phenolics are the most abundant secondary metabolites of plants. Methanol is generally efficient for the extraction of lower molecular weight polyphenols . Preliminary analysis indicated that MEGT were rich in polysaccharides and polyphenols (data not shown). The anticancer activities of polyphenols, such as pro-apoptotic and DNA damaging effects have been reported in many literatures [68–71]. Therefore, we expect that the polyphenols are the candidates for the active principles in MEGT which warrants further investigation.