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Anti-proliferative effect of methanolic extract of Gracilaria tenuistipitata on oral cancer cells involves apoptosis, DNA damage, and oxidative stress
© Yeh et al.; licensee BioMed Central Ltd. 2012
Received: 21 February 2012
Accepted: 23 August 2012
Published: 31 August 2012
Methanolic extracts of Gracilaria tenuistipitata (MEGT) were obtained from the edible red algae. Previously, we found that water extract of G. tenuistipitata was able to modulate oxidative stress-induced DNA damage and its related cellular responses.
In this study, the methanol extraction product MEGT was used to evaluate the cell growth inhibition in oral cancer cells and its possible mechanism was investigated.
The cell viability of MEGT treated Ca9-22 oral cancer cell line was significantly decreased in a dose–response manner (p < 0.05). The sub-G1 population and annexin V intensity of MEGT-treated Ca9-22 cancer cells were significantly increased in a dose–response manner (p < 0.0005 and p < 0.001, respectively). The γH2AX intensities of MEGT-treated Ca9-22 cancer cells were significantly increased in a dose–response manner (p < 0.05). The reactive oxygen species (ROS) and glutathione (GSH)-positive intensities of MEGT-treated Ca9-22 oral cancer cells were significantly increased and decreased, respectively, in a dose–response manner (p < 0.05). The DiOC2(3) intensity for mitochondrial membrane potential (MMP) of MEGT-treated Ca9-22 cancer cells was significantly decreased in a dose–response manner (p < 0.05).
These results indicated that MEGT had apoptosis-based cytotoxicity against oral cancer cells through the DNA damage, ROS induction, and mitochondrial depolarization. Therefore, MEGT derived from the edible algae may have potential therapeutic effects against oral squamous cell carcinoma (OSCC).
KeywordsRed algae Oral cancer Apoptosis γ-H2AX ROS Mitochondrial membrane potential Glutathione
Oral squamous cell carcinoma (OSCC), the sixth most common cancer worldwide , is characterized by high morbidity and mortality rates. The poor prognosis for OSCC  is mainly due to its poor response to chemotherapy . Therefore, drug discovery for anti-OSCC therapy remains challenging.
Natural products [4–8], especially from marine sources [9–12], offer an abundance for drug development to treat many types of cancer. Algae contain many bioactive primary and secondary metabolites [13, 14] and represent about 9% of marine biomedical compounds . Among them, the bioactive compounds of the genus Gracilaria had been summarized [16, 17] and mainly classified by water [18–20] and ethanol/methanol [21–23] extractions. Most of these studies focus on health promoting effects, such as anti-inflammatory, anti-hypercholesterolemic, antioxidative, and antimicrobial properties rather than on cancer therapy.
The Gracilaria algae is inexpensive in Taiwan because it has been cultivated since 1961 , and therefore, is an abundant resource for research purposes. In our previous work , water extracts of G. tenuistipitata demonstrated a potential protective effect from hydrogen peroxide-induced DNA damage. However, extraction methods may influence the biological effects for some natural products including soy products . Recently, many ethanolic or methanolic extracts of natural products were found to possess antiproliferative effects in cancer; such as Njavara ethanolic extracts for glioma cells , Cassia tora methanolic leaf extracts for HeLa cells , Olea europaea ethanolic extracts for leukemic cells , Plocamium telfairiae methanolic extracts for colon cancer cells , and Indigofera linnaei Ali methanolic extracts for tumor cells . Accordingly, the biological effects for methanolic extracts of G. tenuistipitata (MEGT) were evaluated in this study.
In this study, we propose that MEGT has the potential to modulate the cell proliferation of OSCC. To test this hypothesis, the anticancer potential against the human OSCC cancer cells (Ca9-22) was explored in terms of the cell viability and the alterations of cell cycle, apoptosis, ROS, GSH content, and mitochondrial membrane potential to determine the possible mechanism of action.
The seaweed Gracilaria tenuistipitata was collected in spring of 2009 from a culture farm at Kouhu beach, Yunlin County, Taiwan, and was delivered to the laboratory at 0°C. In the laboratory, the algae were washed with running tap water to remove epiphytes and encrusting material, soaked in distilled water twice, and then lyophilized. The dried sample was pulverized and passed through 60-mesh sieve. The lyophilized sample was then ground to a fine powder and stored at −40°C.
The dried samples (50 g) were immersed in 250 ml methanol three times and were immediately extracted with 1000 ml of 99.9% methanol in a mechanical shaker at room temperature for 24 h. Subsequently, the extract of methanol solution was filtered with Whatman No. 1 filter paper three times. The filtered solution was then collected and evaporated to dryness at 40 ± 2°C in a rotary evaporator (Buchi Laboratoriums-Technik, Switzerland). The dry extract was stored in a sealed container at −40°C until use.
The human OSCC cancer cell line, Ca9-22 [32, 33], was cultured in DMEM medium (Gibco, Grand Island, NY, USA) and supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, 0.03% glutamine and 1 mM sodium pyruvate. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2.
Cell viability assay
MEGT was dissolved in DMSO and added to medium to make the final concentration of DMSO less than 1%. Cell proliferation was determined by the WST-1 kit (Roche) . Cells were plated at a density of 1 × 105 cells/well in a 96-well cell culture plate and treated with methanolic extract at doses of 0.1, 0.25, 0.5, 1 mg/ml for 24 h. After incubation, the WST-1 proliferation reagent (Roche) was added to cells (10 μl per well) and continued to incubate for 1–2 h at 37°C. Plates were checked visually by comparing the colors of the wells with or without MEGT-treated cells.
Cell cycle distribution
Cells were treated with a solvent vehicle of 0.1, 0.25, 0.5 and 1 mg/ml of MEGT for 24 h. After trypsinization, the cells were harvested, washed twice with PBS, and fixed overnight with 70% ethanol. After centrifugation, the cell pellets were stained with 10 μg/ml propidium iodide (PI) (Sigma, St Louis, MO, USA) and 10 μg/ml RNase A in PBS buffer for 15 min at room temperature in the dark. PI intensities were measured using a FACSCalibur flow cytometer (Becton-Dickinson, Mansfield, MA, USA) and analyzed Win-MDI software, version 2.9 (http://facs.scripps.edu/wm29w98.exe).
Flow cytometry-based detection of Annexin V staining
Annexin V staining (Pharmingen, San Diego, CA) was performed to examine the apoptosis status as previously described . A total of 1 × 106 cells per 100-mm petri-dish were treated with vehicle or increasing concentrations of MEGT for 24 h. Finally, cells were stained with 10 μg/ml of annexin V-fluorescein isothiocyanate (FITC) and analyzed using the FACSCalibur flow cytometer.
Flow cytometry-based detection of γ-H2AX staining
The MEGT-treated cells were fixed in 70% ethanol, washed twice in BSA-PBST solution (1% bovine serum albumin and 0.2% Triton X-100 in PBS; Sigma), and incubated with 100 μl of BSA-PBST solution containing 0.2 μg p-Histone H2A.X (Ser 139) monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. After washing twice with BSA-PBST, cells were suspended in Alexa Fluor 488-tagged secondary antibody (Jackson Laboratory, Bar Harbor, ME, USA) at a 1:100 dilution for 1 h. After washing twice with BSA-PBST, cells were resuspended in PBS for analysis using the FACSCalibur flow cytometer.
Flow cytometry-based detection of intracellular reactive oxygen species (ROS)
Intracellular ROS was measured using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) as previously described  with slight modification. MEGT-treated cells were washed with PBS twice and then incubated with 10 μM H2DCF-DA in PBS for 30 min at 37°C. Cells were harvested and washed twice with PBS. After centrifugation, cells were resuspended in PBS and immediately forwarded to analysis by the FACSCalibur flow cytometer with excitation and emission settings of 480 and 525 nm, respectively.
Flow cytometry-based detection of cellular reduced glutathione (GSH) content
Cells (8 × 105) were cultured in 60-mm tissue-culture dishes for 24 h. The culture medium was replaced with new medium when the cells were 80% confluent. After MEGT treatments, the cells were incubated with 5 μM CMF-DA for 20 min at 37°C in the CO2 incubator. The CMF fluorescence is directly related to the intracellular GSH level. After CMF-DA staining, the cells were washed with PBS, collected by centrifugation, and then measured with the FACS-Calibur flow cytometer.
Flow cytometry-based detection of mitochondrial membrane potential
Mitochondrial membrane potential (MMP) was measured using a MitoProbe™TM DiOC2(3) assay kit (Invitrogen, San Diego, CA, USA). MEGT-treated Ca9-22 cells were suspended in 1 ml of warm PBS at approximately 1 × 106 cells/ml, loaded with 5 μl of 10 μM DiOC2(3), and incubated at 37°C, 5% CO2, for 20 to 30 min. Cells were then harvested, washed and resuspended in PBS and analyzed immediately using flow cytometry assay with excitation and emission settings of 488 and 525 nm, respectively.
All data are presented as means ± S.E. Comparison between experimental groups was assessed by one-way ANOVA with Tukey's HSD Post Hoc Test using the software JMP® 9. Differences between treatments of different concentrations containing the same letter are not significant.
Cytotoxicity effects of MEGT-treated Ca9-22 oral cancer cells
Changes of cell cycle distribution in MEGT-treated Ca9-22 oral cancer cells
Apoptosis induction of MEGT-treated Ca9-22 oral cells
Induction of DNA double strand breaks in MEGT-treated Ca9-22 oral cancer cells
ROS induction in MEGT-treated Ca9-22 oral cancer cells
Depletion of intracellular GSH induced by MEGT-treated Ca9-22 oral cancer cells
To evaluate whether GSH was involved in the MEGT-induced ROS change of Ca9-22 oral cancer cells, the flow cytometry based-CMF-DA assay was performed. The percentages and profiles of GSH-positive staining of 0, 0.1, 0.25, 0.5 and 1 mg/ml MEGT for 24 h were calculated (Figure 5C). This revealed GSH-positive intensity of MEGT-treated Ca9-22 oral cancer cells was significantly decreased in a dose–response manner (p < 0.05) (Figure 5D).
Reduction of mitochondrial membrane potential (MMP) in MEGT-treated Ca9-22 oral cancer cells
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.
In this study, the effects of MEGT on an oral cancer cell model were assayed to explore the genotoxic effect, by cell cycle distribution and annexin V detection to explore its apoptotic effect, by γ-H2AX detection to explore its DNA damage effect, by determination of ROS and GSH levels to explore its oxidative stress modifying effect, and by DiOC2(3) staining to explore the MMP depolarization. The results of this study have demonstrated that MEGT induced potent anti‒oral cancer effects thought the induction of apoptosis, DNA damage, and oxidative stress pathways. Based on this finding, MEGT may be a potential natural product for anti-oral cancer treatment.
We thank the National Science Council of Taiwan for grants (NSC98-2314-B-037-035), grants for DOH101-TD-C-111-002, Kaohsiung Medical University Research Foundation (KMUER014 and KMU-M110001), and NSYSU-KMU JOINT RESEARCH PROJECT (#NSYSUKMU 101–006). We also thank Mr. Harrison Yang for English editing.
- Parkin DM, Laara E, Muir CS: Estimates of the worldwide frequency of sixteen major cancers in 1980. Int J Cancer. 1988, 41 (2): 184-197. 10.1002/ijc.2910410205.View ArticlePubMedGoogle Scholar
- Myoung H, Hong SP, Yun PY, Lee JH, Kim MJ: Anti-cancer effect of genistein in oral squamous cell carcinoma with respect to angiogenesis and in vitro invasion. Cancer Sci. 2003, 94 (2): 215-220. 10.1111/j.1349-7006.2003.tb01422.x.View ArticlePubMedGoogle Scholar
- Liou KT, Lin SM, Huang SS, Chih CL, Tsai SK: Honokiol ameliorates cerebral infarction from ischemia-reperfusion injury in rats. Planta Med. 2003, 69 (2): 130-134. 10.1055/s-2003-37707.View ArticlePubMedGoogle Scholar
- Jimenez-Medina E, Garcia-Lora A, Paco L, Algarra I, Collado A, Garrido F: A new extract of the plant Calendula officinalis produces a dual in vitro effect: cytotoxic anti-tumor activity and lymphocyte activation. BMC Cancer. 2006, 6: 119-10.1186/1471-2407-6-119.View ArticlePubMedPubMed CentralGoogle Scholar
- Koppikar SJ, Choudhari AS, Suryavanshi SA, Kumari S, Chattopadhyay S, Kaul-Ghanekar R: Aqueous cinnamon extract (ACE-c) from the bark of Cinnamomum cassia causes apoptosis in human cervical cancer cell line (SiHa) through loss of mitochondrial membrane potential. BMC Cancer. 2010, 10: 210-10.1186/1471-2407-10-210.View ArticlePubMedPubMed CentralGoogle Scholar
- Lee do K, Jang S, Kim MJ, Kim JH, Chung MJ, Kim KJ, Ha NJ: Anti-proliferative effects of Bifidobacterium adolescentis SPM0212 extract on human colon cancer cell lines. BMC Cancer. 2008, 8: 310-10.1186/1471-2407-8-310.View ArticlePubMedGoogle Scholar
- Stingl JC, Ettrich T, Muche R, Wiedom M, Brockmoller J, Seeringer A, Seufferlein T: Protocol for minimizing the risk of metachronous adenomas of the colorectum with green tea extract (MIRACLE): a randomised controlled trial of green tea extract versus placebo for nutriprevention of metachronous colon adenomas in the elderly population. BMC Cancer. 2011, 11: 360-10.1186/1471-2407-11-360.View ArticlePubMedPubMed CentralGoogle Scholar
- Yen CY, Chiu CC, Chang FR, Chen JY, Hwang CC, Hseu YC, Yang HL, Lee AY, Tsai MT, Guo ZL: 4beta-Hydroxywithanolide E from Physalis peruviana (golden berry) inhibits growth of human lung cancer cells through DNA damage, apoptosis and G2/M arrest. BMC Cancer. 2010, 10: 46-10.1186/1471-2407-10-46.View ArticlePubMedPubMed CentralGoogle Scholar
- Molinski T: Marine natural products. Clinical advances in hematology & oncology: H&O. 2009, 7 (6): 383-385.Google Scholar
- Molinski TF, Dalisay DS, Lievens SL, Saludes JP: Drug development from marine natural products. Nat Rev Drug Discov. 2009, 8 (1): 69-85. 10.1038/nrd2487.View ArticlePubMedGoogle Scholar
- Glaser KB, Mayer AM: A renaissance in marine pharmacology: from preclinical curiosity to clinical reality. Biochem Pharmacol. 2009, 78 (5): 440-448. 10.1016/j.bcp.2009.04.015.View ArticlePubMedGoogle Scholar
- Mayer AM, Glaser KB, Cuevas C, Jacobs RS, Kem W, Little RD, McIntosh JM, Newman DJ, Potts BC, Shuster DE: The odyssey of marine pharmaceuticals: a current pipeline perspective. Trends Pharmacol Sci. 2010, 31 (6): 255-265. 10.1016/j.tips.2010.02.005.View ArticlePubMedGoogle Scholar
- Faulkner DJ: Marine natural products. Nat Prod Rep. 2002, 19 (1): 1-48.View ArticlePubMedGoogle Scholar
- Liu M, Hansen PE, Lin X: Bromophenols in marine algae and their bioactivities. Mar Drugs. 2011, 9 (7): 1273-1292. 10.3390/md9071273.View ArticlePubMedPubMed CentralGoogle Scholar
- Jha RK, Zi-rong X: Biomedical compounds from marine organisms. Mar Drugs. 2004, 2: 123-146. 10.3390/md203123.View ArticlePubMed CentralGoogle Scholar
- Armisen R: World-wide use and importance of Gracilaria. J Appl Phycol. 1995, 7: 231-243. 10.1007/BF00003998.View ArticleGoogle Scholar
- de Almeida CL, Falcao Hde S, Lima GR, Montenegro Cde A, Lira NS, de Athayde-Filho PF, Rodrigues LC, de Souza Mde F, Barbosa-Filho JM, Batista LM: Bioactivities from marine algae of the genus gracilaria. Int J Mol Sci. 2011, 12 (7): 4550-4573. 10.3390/ijms12074550.View ArticlePubMedPubMed CentralGoogle Scholar
- Lin YH, Tsai JS, Hung LB, Pan BS: Hypocholesterolemic effect of compounded freshwater clam protein hydrolysate and Gracilaria. Food Chemistry. 2010, 123: 395-399. 10.1016/j.foodchem.2010.04.053.View ArticleGoogle Scholar
- Yangthong M, Hutadilok-Towatana N, Phromkunthong W: Antioxidant activities of four edible seaweeds from the southern coast of Thailand. Plant Foods Hum Nutr. 2009, 64 (3): 218-223. 10.1007/s11130-009-0127-y.View ArticlePubMedGoogle Scholar
- Yeh ST, Lin YC, Huang CL, Chen JC: White shrimp Litopenaeus vannamei that received the hot-water extract of Gracilaria tenuistipitata showed protective innate immunity and up-regulation of gene expressions after low-salinity stress. Fish Shellfish Immunol. 2010, 28 (5–6): 887-894.View ArticlePubMedGoogle Scholar
- Ganesan P, Kumar CS, Bhaskar N: Antioxidant properties of methanol extract and its solvent fractions obtained from selected Indian red seaweeds. Bioresour Technol. 2008, 99 (8): 2717-2723. 10.1016/j.biortech.2007.07.005.View ArticlePubMedGoogle Scholar
- Vijayavel K, Martinez JA: In vitro antioxidant and antimicrobial activities of two Hawaiian marine Limu: Ulva fasciata (Chlorophyta) and Gracilaria salicornia (Rhodophyta). J Med Food. 2010, 13 (6): 1494-1499. 10.1089/jmf.2009.0287.View ArticlePubMedGoogle Scholar
- Souza BW, Cerqueira MA, Martins JT, Quintas MA, Ferreira AC, Teixeira JA, Vicente AA: Antioxidant potential of two red seaweeds from the Brazilian coasts. J Agric Food Chem. 2011, 59 (10): 5589-5594. 10.1021/jf200999n.View ArticlePubMedGoogle Scholar
- Ajisaka T, Chiang YM: Recent status of Gracilaria cultivation in Taiwan. Hydrobiologia. 1993, 260/261: 335-338. 10.1007/BF00049037.View ArticleGoogle Scholar
- Yang JI, Yeh CC, Lee JC, Yi SC, Huang HW, Tseng CN, Chang HW: Aqueous extracts of the edible Gracilaria tenuistipitata are protective against H2O2-induced DNA damage, growth inhibition, and cell cycle arrest. Molecules. 2012, 17 (6): 7241-7254. 10.3390/molecules17067241.View ArticlePubMedGoogle Scholar
- Bolling BW, Blumberg JB, Chen CY: Extraction methods determine the antioxidant capacity and induction of quinone reductase by soy products in vitro. Food Chem. 2009, 116 (1): 351-355. 10.1016/j.foodchem.2009.01.087.View ArticlePubMedPubMed CentralGoogle Scholar
- Rao AS, Reddy SG, Babu PP, Reddy AR: The antioxidant and antiproliferative activities of methanolic extracts from Njavara rice bran. BMC Complement Altern Med. 2010, 10: 4-10.1186/1472-6882-10-4.View ArticlePubMedPubMed CentralGoogle Scholar
- Rejiya CS, Cibin TR, Abraham A: Leaves of Cassia tora as a novel cancer therapeutic–an in vitro study. Toxicol In Vitro. 2009, 23 (6): 1034-1038. 10.1016/j.tiv.2009.06.010.View ArticlePubMedGoogle Scholar
- Fares R, Bazzi S, Baydoun SE, Abdel-Massih RM: The antioxidant and anti-proliferative activity of the Lebanese Olea europaea extract. Plant Foods Hum Nutr. 2011, 66 (1): 58-63. 10.1007/s11130-011-0213-9.View ArticlePubMedGoogle Scholar
- Kim JY, Yoon MY, Cha MR, Hwang JH, Park E, Choi SU, Park HR, Hwang YI: Methanolic extracts of Plocamium telfairiae induce cytotoxicity and caspase-dependent apoptosis in HT-29 human colon carcinoma cells. J Med Food. 2007, 10 (4): 587-593. 10.1089/jmf.2007.002.View ArticlePubMedGoogle Scholar
- Kumar RS, Rajkapoor B, Perumal P: Antitumor and cytotoxic activities of methanol extract of Indigofera linnaei Ali. Asian Pac J Cancer Prev. 2011, 12 (3): 613-618.PubMedGoogle Scholar
- Matsumoto K, Nakamura T, Kramer RH: Hepatocyte growth factor/scatter factor induces tyrosine phosphorylation of focal adhesion kinase (p125FAK) and promotes migration and invasion by oral squamous cell carcinoma cells. J Biol Chem. 1994, 269 (50): 31807-31813.PubMedGoogle Scholar
- Sato F, Wu Y, Bhawal UK, Liu Y, Imaizumi T, Morohashi S, Kato Y, Kijima H: PERIOD1 (PER1) has anti-apoptotic effects, and PER3 has pro-apoptotic effects during cisplatin (CDDP) treatment in human gingival cancer CA9-22 cells. Eur J Cancer. 2011, 47 (11): 1747-1758. 10.1016/j.ejca.2011.02.025.View ArticlePubMedGoogle Scholar
- Liu C, Tsai AL, Chen YC, Fan SC, Huang CH, Wu CC, Chang CH: Facilitation of human osteoblast apoptosis by sulindac and indomethacin under hypoxic injury. J Cell Biochem. 2012, 113 (1): 148-155. 10.1002/jcb.23338.View ArticlePubMedGoogle Scholar
- Chiu CC, Liu PL, Huang KJ, Wang HM, Chang KF, Chou CK, Chang FR, Chong IW, Fang K, Chen JS: Goniothalamin inhibits growth of human lung cancer cells through DNA damage, apoptosis, and reduced migration ability. J Agric Food Chem. 2011, 59 (8): 4288-4293. 10.1021/jf200566a.View ArticlePubMedGoogle Scholar
- Wang H, Joseph JA: Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radic Biol Med. 1999, 27 (5–6): 612-616.View ArticlePubMedGoogle Scholar
- Vermund H, Gollin FF: Mechanisms of action of radiotherapy and chemotherapeutic adjuvants. A review. Cancer. 1968, 21 (1): 58-76. 10.1002/1097-0142(196801)21:1<58::AID-CNCR2820210110>3.0.CO;2-5.View ArticlePubMedGoogle Scholar
- Sun SY, Hail N, Lotan R: Apoptosis as a novel target for cancer chemoprevention. J Natl Cancer Inst. 2004, 96 (9): 662-672. 10.1093/jnci/djh123.View ArticlePubMedGoogle Scholar
- Azuma M, Tamatani T, Ashida Y, Takashima R, Harada K, Sato M: Cisplatin induces apoptosis in oral squamous carcinoma cells by the mitochondria-mediated but not the NF-kappaB-suppressed pathway. Oral Oncol. 2003, 39 (3): 282-289. 10.1016/S1368-8375(02)00116-1.View ArticlePubMedGoogle Scholar
- Yoneda K, Yamamoto T, Osaki T: p53- and p21-independent apoptosis of squamous cell carcinoma cells induced by 5-fluorouracil and radiation. Oral Oncol. 1998, 34 (6): 529-537. 10.1016/S1368-8375(98)00036-0.View ArticlePubMedGoogle Scholar
- Abal M, Andreu JM, Barasoain I: Taxanes: microtubule and centrosome targets, and cell cycle dependent mechanisms of action. Curr Cancer Drug Targets. 2003, 3 (3): 193-203. 10.2174/1568009033481967.View ArticlePubMedGoogle Scholar
- Ferreira CG, Epping M, Kruyt FA, Giaccone G: Apoptosis: target of cancer therapy. Clin Cancer Res. 2002, 8 (7): 2024-2034.PubMedGoogle Scholar
- Malaguarnera L: Implications of apoptosis regulators in tumorigenesis. Cancer Metastasis Rev. 2004, 23 (3–4): 367-387.View ArticlePubMedGoogle Scholar
- Luqmani YA: Mechanisms of drug resistance in cancer chemotherapy. Med Princ Pract. 2005, 14 (Suppl 1): 35-48.PubMedGoogle Scholar
- Shoemaker RH: The NCI60 human tumour cell line anticancer drug screen. Nat Rev Cancer. 2006, 6 (10): 813-823. 10.1038/nrc1951.View ArticlePubMedGoogle Scholar
- Sreelatha S, Jeyachitra A, Padma PR: Antiproliferation and induction of apoptosis by Moringa oleifera leaf extract on human cancer cells. Food Chem Toxicol. 2011, 49 (6): 1270-1275. 10.1016/j.fct.2011.03.006.View ArticlePubMedGoogle Scholar
- Wurstle ML, Laussmann MA, Rehm M: The central role of initiator caspase-9 in apoptosis signal transduction and the regulation of its activation and activity on the apoptosome. Exp Cell Res. 2012, 318 (11): 1213-1220. 10.1016/j.yexcr.2012.02.013.View ArticlePubMedGoogle Scholar
- Reubold TF, Eschenburg S: A molecular view on signal transduction by the apoptosome. Cell Signal. 2012, 24 (7): 1420-1425. 10.1016/j.cellsig.2012.03.007.View ArticlePubMedGoogle Scholar
- Dickey JS, Redon CE, Nakamura AJ, Baird BJ, Sedelnikova OA, Bonner WM: H2AX: functional roles and potential applications. Chromosoma. 2009, 118 (6): 683-692. 10.1007/s00412-009-0234-4.View ArticlePubMedPubMed CentralGoogle Scholar
- Bekker-Jensen S, Mailand N: Assembly and function of DNA double-strand break repair foci in mammalian cells. DNA Repair (Amst). 2010, 9 (12): 1219-1228. 10.1016/j.dnarep.2010.09.010.View ArticleGoogle Scholar
- Chiu CC, Chang HW, Chuang DW, Chang FR, Chang YC, Cheng YS, Tsai MT, Chen WY, Lee SS, Wang CK: Fern plant-derived protoapigenone leads to DNA damage, apoptosis, and G(2)/m arrest in lung cancer cell line H1299. DNA Cell Biol. 2009, 28 (10): 501-506. 10.1089/dna.2009.0852.View ArticlePubMedGoogle Scholar
- Kang MA, So EY, Simons AL, Spitz DR, Ouchi T: DNA damage induces reactive oxygen species generation through the H2AX-Nox1/Rac1 pathway. Cell Death Dis. 2012, 3: e249-10.1038/cddis.2011.134.View ArticlePubMedPubMed CentralGoogle Scholar
- Hamanaka RB, Chandel NS: Mitochondrial reactive oxygen species regulate cellular signaling and dictate biological outcomes. Trends in biochemical sciences. 2010, 35 (9): 505-513. 10.1016/j.tibs.2010.04.002.View ArticlePubMedPubMed CentralGoogle Scholar
- Simon HU, Haj-Yehia A, Levi-Schaffer F: Role of reactive oxygen species (ROS) in apoptosis induction. Apoptosis. 2000, 5 (5): 415-418. 10.1023/A:1009616228304.View ArticlePubMedGoogle Scholar
- Shackelford RE, Kaufmann WK, Paules RS: Oxidative stress and cell cycle checkpoint function. Free Radic Biol Med. 2000, 28 (9): 1387-1404. 10.1016/S0891-5849(00)00224-0.View ArticlePubMedGoogle Scholar
- Ding H, Han C, Guo D, Chin YW, Ding Y, Kinghorn AD, D'Ambrosio SM: Selective induction of apoptosis of human oral cancer cell lines by avocado extracts via a ROS-mediated mechanism. Nutr Cancer. 2009, 61 (3): 348-356. 10.1080/01635580802567158.View ArticlePubMedGoogle Scholar
- Jeong JC, Jang SW, Kim TH, Kwon CH, Kim YK: Mulberry fruit (Moris fructus) extracts induce human glioma cell death in vitro through ROS-dependent mitochondrial pathway and inhibits glioma tumor growth in vivo. Nutr Cancer. 2010, 62 (3): 402-412. 10.1080/01635580903441287.View ArticlePubMedGoogle Scholar
- Bechelli J, Coppage M, Rosell K, Liesveld J: Cytotoxicity of algae extracts on normal and malignant cells. Leukemia Research and Treatment. 2011, Article ID 373519: 1-7.View ArticleGoogle Scholar
- Matthews GM, Howarth GS, Butler RN: Nutrient and antioxidant modulation of apoptosis in gastric and colon cancer cells. Cancer Biol Ther. 2006, 5 (6): 569-572. 10.4161/cbt.5.6.2868.View ArticlePubMedGoogle Scholar
- Higuchi Y: Glutathione depletion-induced chromosomal DNA fragmentation associated with apoptosis and necrosis. J Cell Mol Med. 2004, 8 (4): 455-464. 10.1111/j.1582-4934.2004.tb00470.x.View ArticlePubMedGoogle Scholar
- Kowaltowski AJ, de Souza-Pinto NC, Castilho RF, Vercesi AE: Mitochondria and reactive oxygen species. Free Radic Biol Med. 2009, 47 (4): 333-343. 10.1016/j.freeradbiomed.2009.05.004.View ArticlePubMedGoogle Scholar
- Pramanik KC, Boreddy SR, Srivastava SK: Role of mitochondrial electron transport chain complexes in capsaicin mediated oxidative stress leading to apoptosis in pancreatic cancer cells. PLoS One. 2011, 6 (5): e20151-10.1371/journal.pone.0020151.View ArticlePubMedPubMed CentralGoogle Scholar
- Lee CS, Park SY, Ko HH, Han ES: Effect of change in cellular GSH levels on mitochondrial damage and cell viability loss due to mitomycin c in small cell lung cancer cells. Biochem Pharmacol. 2004, 68 (9): 1857-1867. 10.1016/j.bcp.2004.06.010.View ArticlePubMedGoogle Scholar
- Marinho-Soriano E, Bourret E: Polysaccharides from the red seaweed Gracilaria dura (Gracilariales, Rhodophyta). Bioresour Technol. 2005, 96 (3): 379-382. 10.1016/j.biortech.2004.04.012.View ArticlePubMedGoogle Scholar
- Marinho-Soriano E, Fonseca PC, Carneiro MA, Moreira WS: Seasonal variation in the chemical composition of two tropical seaweeds. Bioresour Technol. 2006, 97 (18): 2402-2406. 10.1016/j.biortech.2005.10.014.View ArticlePubMedGoogle Scholar
- Imbs AB, Vologodskaya AV, Nevshupova NV, Khotimchenko SV, Titlyanov EA: Response of prostaglandin content in the red alga Gracilaria verrucosa to season and solar irradiance. Phytochemistry. 2001, 58 (7): 1067-1072. 10.1016/S0031-9422(01)00321-1.View ArticlePubMedGoogle Scholar
- Dai J, Mumper RJ: Plant phenolics: extraction, analysis and their antioxidant and anticancer properties. Molecules. 2010, 15 (10): 7313-7352. 10.3390/molecules15107313.View ArticlePubMedGoogle Scholar
- Lamoral-Theys D, Pottier L, Dufrasne F, Neve J, Dubois J, Kornienko A, Kiss R, Ingrassia L: Natural polyphenols that display anticancer properties through inhibition of kinase activity. Curr Med Chem. 2010, 17 (9): 812-825. 10.2174/092986710790712183.View ArticlePubMedGoogle Scholar
- Hu ML: Dietary polyphenols as antioxidants and anticancer agents: more questions than answers. Chang Gung medical journal. 2011, 34 (5): 449-460.PubMedGoogle Scholar
- Guo W, Kong E, Meydani M: Dietary polyphenols, inflammation, and cancer. Nutr Cancer. 2009, 61 (6): 807-810. 10.1080/01635580903285098.View ArticlePubMedGoogle Scholar
- Hadi SM, Bhat SH, Azmi AS, Hanif S, Shamim U, Ullah MF: Oxidative breakage of cellular DNA by plant polyphenols: a putative mechanism for anticancer properties. Semin Cancer Biol. 2007, 17 (5): 370-376. 10.1016/j.semcancer.2007.04.002.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/12/142/prepub
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