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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Ethanolic extract of Ferula gummosa is cytotoxic against cancer cells by inducing apoptosis and cell cycle arrest a
b
c
d
Hoda Gudarzi , Mona Salimi , Saeed Irian , Amir Amanzadeh , b
e
Hirsa Mostafapour Kandelous , Keyhan Azadmanesh & Misha f
Salimi a
Department of Biology, Faculty of Science, Kharazmi University, Tehran, Iran b
Department of Physiology and Pharmacology, Pasteur Institute of Iran, Tehran, Iran c
Department of Cellular and Molecular Biology, Faculty of Science, Kharazmi University, Tehran, Iran d
National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran
e
Department of Virology, Pasteur Institute of Iran, Tehran, Iran
f
Department of Biotechnology, Islamic Azad University of Pharmaceutical Sciences Branch, Tehran, Iran Published online: 21 Aug 2014.
To cite this article: Hoda Gudarzi, Mona Salimi, Saeed Irian, Amir Amanzadeh, Hirsa Mostafapour Kandelous, Keyhan Azadmanesh & Misha Salimi (2014): Ethanolic extract of Ferula gummosa is cytotoxic against cancer cells by inducing apoptosis and cell cycle arrest, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.951854 To link to this article: http://dx.doi.org/10.1080/14786419.2014.951854
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This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.951854
SHORT COMMUNICATION Ethanolic extract of Ferula gummosa is cytotoxic against cancer cells by inducing apoptosis and cell cycle arrest Hoda Gudarzia, Mona Salimib*, Saeed Irianc, Amir Amanzadehd, Hirsa Mostafapour Kandelousb, Keyhan Azadmaneshe and Misha Salimif*
Downloaded by [University of Southern Queensland] at 11:57 16 October 2014
a
Department of Biology, Faculty of Science, Kharazmi University, Tehran, Iran; bDepartment of Physiology and Pharmacology, Pasteur Institute of Iran, Tehran, Iran; cDepartment of Cellular and Molecular Biology, Faculty of Science, Kharazmi University, Tehran, Iran; dNational Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran; eDepartment of Virology, Pasteur Institute of Iran, Tehran, Iran; f Department of Biotechnology, Islamic Azad University of Pharmaceutical Sciences Branch, Tehran, Iran (Received 27 May 2014; final version received 2 August 2014) Ferula gummosa Boiss. has medicinal applications in treating a wide range of diseases including cancer. The objective of this study was to evaluate the antiproliferative activities of the seed and gum extracts of F. gummosa as well as to study the effect of the potent extract on the induction of apoptosis and cell cycle arrest. Our results demonstrated that the ethanolic extract had the lowest IC50 value at 72 h (0.001 ^ 1.2 mg/mL) in BHY cells. Moreover, flowcytometry and annexin-V analysis revealed that the ethanolic extract induced apoptosis and cell-cycle arrest in BHY cells at G1/S phase. In addition, colorimetric methods exhibited the highest amount of total phenolics and flavonoids in the aqueous and gum extracts (0.12 ^ 0.037, 0.01 ^ 2.51 mg/g of dry powder). Generally, the results obtained indicate that F. gummosa ethanol extract may contain effective compounds which can be used as a chemotherapeutic agent. Keywords: Ferula gummosa; cancer; apoptosis; MTT assay; flowcytometry
1. Introduction The global burden of cancer continues to increase largely because of the ageing and growth of the world population alongside an increasing adoption of cancer-causing behaviours (Jemal et al. 2011). Standard cancer therapy generally combines surgery, multi-therapeutic agents and ionising radiation. The anticancer agents induce cell cycle arrest and/or cell death by apoptotic or non-apoptotic mechanisms including necrosis (Okada & Mak 2004; Brown & Attardi 2005). Blockage of tumour cell proliferation and apoptosis are referred to the potential anticancer agents by plant natural products (Lee et al. 2003). Due to the less toxicity and cost effectiveness, traditional herbal medicine increasingly attracts more attentions as an alternative cancer therapy (Gao et al. 2011). Ferula gummosa Boiss is a member of the Apiaceae family and has been used for the treatment of stomach pain, epilepsy and as a wound-healing remedy in Iranian traditional medicine (Mandegary et al. 2004). Previous studies reported the antibacterial effects of the essential oil from F. gummosa seeds (Eftekhar et al. 2004). Moreover, the antioxidant and antihaemolytic activities of stem, fruit and leaf extracts of F. gummosa have been already reported (Nabavi et al. 2010). Gharaei et al. have recently evaluated the anti-proliferative and
*Corresponding authors. [email protected] q 2014 Taylor & Francis
Email:
[email protected],
[email protected];
2
H. Gudarzi et al.
apoptosis-inducing activities of ethanolic extract of the leaves and flowers of F. gummosa against the gastric cancer cells (Gharaei et al. 2011). Considering this supposition and that there is no report of the antiproliferative effects of F. gummosa seed extracts, we decided to investigate the cytotoxic effects of the ethanolic, acetonic and aqueous extracts obtained from the seeds and gum of F. gummosa Boiss against three cancer cell lines: MCF-7 (human breast adenocarcinoma), BHY (human oral squamous cancer) and SKMEL-3 (human malignant melanoma) or HGF (human gingival fibroblast) cell line as a control.
Downloaded by [University of Southern Queensland] at 11:57 16 October 2014
2. Results and discussion Growing evidence suggests that irregularities in apoptotic signalling pathway are major contributors in many cancer cells and malignant tissues (Lopez-Beltran et al. 2007). As compounds exhibiting apoptosis-inducing activity are considered potential anticancer agents (Frankfurt & Krishan 2003), many attempts have been made to explore new drugs through isolation of apoptosis-inducing agents from natural products. Previous studies demonstrated that Ferula species are a rich source of phytochemicals including coumarin derivatives, sesquiterpenes and sulphur compounds (Iranshahy & Iranshahi 2011). In this regard, terpenoid compounds are by far the most prevalent components of this genus (Sahebkar & Iranshahi 2010). In addition, preliminary phytochemical analysis of the seed and acetone extract of the root of F. gummosa indicated the presence of large amounts of terpenoids and alkaloids as well as small amounts of cardenolids (Mandegary et al. 2004). Besides that, according to the standards of the National Cancer Institute, crude extracts with IC50 less than 20 mg/mL are considered to be active against tested cancer cells/lines (Geran et al. 1972). These data prompted us to prepare various extracts with different polarities in order to test their cytotoxic effects. 2.1. F. gummosa extracts exhibit cytotoxic properties In order to evaluate the cytotoxic properties of the acetonic, ethanolic and aqueous extracts of F. gummosa seeds as well as the ethanolic extract of F. gummosa gum, the MTT assay was performed on three cancer cell lines including BHY, SKMEL-3 and MCF-7. The effects of different extracts on human cancer cells were examined by increasing the concentrations of the extracts from 0.0001 to 0.5 mg/mL for 24 –72 h. Our results indicated that treatment with the ethanolic and acetonic extracts significantly inhibited the growth of BHY and SKMEL-3 cells in a time- and dose-dependent manner. This trend was the same for MCF-7 cells (data not shown). However, the aqueous and gum extracts exhibited weak antiproliferative activity even at a high concentration of 0.5 mg/mL against three cell lines. The IC50 values are listed in Table 1. As the results indicated, some differences were observed in terms of cell line and type of extract. In other words, BHY was well inhibited by the ethanolic and acetonic extracts and the ethanolic extract demonstrated a considerable potency in inhibition of cell growth at 24, 48 and 72 h (Table 1). Moreover, there was a difference in the IC50 values obtained at 24, 48 and 72 h. The lowest IC50 values were observed at 72 h. Furthermore, the IC50 value obtained for the ethanolic extract against normal cell line (HGF) was higher in comparison to cancer cell lines (IC50 ¼ 0.1 mg/mL, Table 2). These results suggest a specific cytotoxic effect mainly against oral cancer cell line by the ethanolic extract. 2.2. Induction of apoptosis in BHY cells by ethanolic extract Through the annexin-V assay, we observed the externalisation of phosphatidylserine to the outer cell membrane in the ethanolic extract-treated BHY cells at 12 IC50 after 72 h incubation, which is commonly traced as a biochemical marker for apoptosis. As shown in Supplementary Figure S1
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Table 1. IC50 values (mg of extract/mL) for antiproliferative activity of F. gummosa different extracts towards BHY, MCF-7 and SKML-3 cells. Extracts Cells
Time (h)
Ethanol
Acetone
Aqueous
Gum
BHY
24 48 72 24 48 72 24 48 72
0.01 ^ 1.14 0.005 ^ 1.3 0.001 ^ 1.2 0.01 ^ 1.2 0.01 ^ 1.14 0.01 ^ 1.17 0.03 ^ 1.1 0.006 ^ 1.5 0.005 ^ 1.2
0.04 ^ 1.11 0.035 ^ 1.2 0.004 ^ 1.2 0.06 ^ 1.17 0.02 ^ 1.02 0.02 ^ 1.07 0.05 ^ 1.1 0.02 ^ 1.1 0.01 ^ 1.1
. 0.5 . 0.5 . 0.5 . 0.5 . 0.5 . 0.5 . 0.5 . 0.5 . 0.5
.0.5 .0.5 .0.5 .0.5 .0.5 .0.5 .0.5 .0.5 .0.5
Downloaded by [University of Southern Queensland] at 11:57 16 October 2014
MCF-7 SKML-3
Note: Values are presented as mean ^ SE of three independent experiments, performed in triplicate.
Table 2. IC50 values (mg of extract/mL) for cytotoxic activity of ethanolic and acetonic of F. gummosa seed extracts towards normal HGF cells. Extract Cell
Time (h)
Ethanol
Acetone
HGF
24 48 72
. 0.5 . 0.5 0.1 ^ 1.2
. 0.5 . 0.5 . 0.5
Note: Values are presented as mean ^ SE of three independent experiments, performed in triplicate.
and Table 3, 0.56% of cells were annexin V þ /PI 2 and 1.79% of cells were annexin V þ / PI þ in solvent-control cells. After treatment with 0.0005 mg/mL of the ethanolic extract for 72 h, the number of cells which were annexin V þ /PI 2 and annexin V þ /PI þ was considerably increased. Meanwhile, the necrotic population (annexin V 2 /PI þ ) was increased by 2.0%. These results collectively indicated that treatment with the ethanolic extract at 12 IC50 for 72 h resulted in a clear shift from live cell population to early and late apoptotic population.
2.3. Ethanolic extract of F. gummosa induces cell cycle arrest Induction of apoptosis via blockage of cell cycle progression by anticancer agents prevents the proliferation of cancer cells, which may be exploited for cancer therapy (Tan et al. 2009). Hence, we studied the effect of F. gummosa ethanolic seed extract on the cell cycle of BHY cells. Our results showed that the ethanolic extract at 0.002 mg/mL (2 times the IC50) could not trigger BHY cell death after 48 h of incubation. Following 48 h of treatment, the sub-G1 sub-population Table 3. Percentage of oral squamous cancer cells in each state after treatment with ethanolic extract at 0.0005 mg/mL for 72 h of incubation. Treatment
Vital cells (%) An– /PI –
Early apoptosis (%) An þ /PI –
Late apoptosis (%) An þ /PI þ
Necrosis (%) An– /PI þ
Control Solvent Extract
96.15 ^ 0.82 90.73 ^ 4.53 85.63 ^ 1.83
0.41 ^ 0.22 0.56 ^ 0.03 1.72 ^ 0.78
0.87 ^ 0.15 1.79 ^ 0.98 3.9 ^ 1.52
2.6 ^ 0.54 3.35 ^ 0.01 5.39 ^ 1.01
4
H. Gudarzi et al.
Table 4. Effect of ethanol extract on cell cycle progression with respect to control. BHY Treatment Control 0.002 mg/mL
Sub-G1 (mean ^ SE)
Go/G1 (mean ^ SE)
S (mean ^ SE)
G2/M (mean ^ SE)
29.95 ^ 1.2 32.0 ^ 1.07
29.98 ^ 0.7 33.17 ^ 0.5
30.7 ^ 1.4 27.55 ^ 0.4
14.4 ^ 0.25 13.28 ^ 0.9
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Notes: At the indicated cell, distribution of the cells in sub-G1, Go/G1, S and G2/M phase was analysed by flowcytometry. Results are expressed as total cells. Data represent means of triplicate experiment.
insignificantly increased from 29.95% to 32%. Concomitantly, there was an increase in G0/G1 and a decrease in S-phase sub-populations from 29.95% to 33.17% and 30.7% to 27.5%, respectively, which were not considerable (Supplementary Figure S2 and Table 4), suggesting an apoptotic pathway which is independent of the cell cycle blockage. 2.4. Total phenolic and flavonoid contents Polyphenols are well documented to act as chemopreventive agents exerting a direct, proapoptotic effect on tumour cells, which block the growth of several types of human cancer cell lines at different phases of the cell cycle (Ramkumar et al. 2013). Our results displayed no direct correlation between cytotoxic properties of F. gummosa seed and gum extracts and polyphenol contents (Supplementary Table S1). The results obtained suggest a strong anti-proliferative activity of the ethanolic seed extract, which may be due to the presence of bioactive components, such as coumarins, terpenoids and/or alkaloids, which are known to induce anti-proliferative outcomes. 3. Conclusion In summary, our results present the first evidence on the antiproliferative effect of the ethanolic seed extract of F. gummosa as demonstrated by in vitro MCF-7, BHY and SKMEL-3 growth inhibition. The ethanolic extract could also induce cell apoptosis at early and late stages. However, more detailed molecular mechanisms underlying the ethanolic-induced BHY apoptotic cell death remain to be elucidated. More importantly, isolation of the effective ingredients is required to be determined. Supplementary material Experimental details relating to this article are available online. References Brown JM, Attardi LD. 2005. The role of apoptosis in cancer development and treatment response. Nat Rev Cancer. 5:231–237. Eftekhar F, Yousefzadi M, Borhani K. 2004. Antibacterial activity of the essential oil from Ferula gummosa seed. Fitoterapia. 75:758–759. Frankfurt OS, Krishan A. 2003. Apoptosis-based drug screening and detection of selective toxicity to cancer cells. Anticancer Drug. 14:555– 561. Gao Y, Su Y, Qu L, Xu S, Meng L, Cai SQ, Shoua C. 2011. Mitochondrial apoptosis contributes to the anti-cancer effect of Smilax glabra Roxb. Toxicol Lett. 207:112–120. Geran RI, Greenberg NH, Macdonald MM, Schumacher AM, Abbott BJ. 1972. Protocols for screening chemical agents and natural products against animal tumors and other biological systems. Cancer Chemother Rep. 33:1–17. Gharaei R, Ghobadi S, Akrami H. 2011. Study of apoptosis inducing effects of ethanol extract of Ferula gummosa leaf. Clin Biochem. 44:S332.
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Iranshahy M, Iranshahi M. 2011. Traditional uses, phytochemistry and pharmacology of asafoetida (Ferula assa-foetida oleo-gum-resin): a review. J Ethnopharmacol. 134:1–10. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. 2011. Global cancer statistics. Cancer J Clin. 61:69–90. Lee JC, Kim J, Jang YS. 2003. Ethanol-eluted extract of Rhus verniciflua stokes inhibits cell growth and induces apoptosis in human lymphoma cells. J Biochem Mol Biol. 36:337–343. Lopez-Beltran A, Maclennan GT, de la Haba-Rodriguez J, Montironi R, Cheng L. 2007. Research advances in apoptosismediated cancer therapy: a review. Anal Quant Cytol Histol. 29:71–78. Mandegary A, Sayyah M, Heidari M. 2004. Antinociceptive anti-inflammatory activity of the seed and root extracts of Ferula gummosa Boiss in mice and rats. Daru. 12:58–62. Nabavi SF, Ebrahimzadeh MA, Nabavi SM, Eslami B. 2010. Antioxidant activity of flower, stem and leaf extracts of Ferula gummosa Boiss. Grasas Y Aceites. 61:244–250. Okada H, Mak TW. 2004. Pathways of apoptotic and non-apoptotic death in tumour cells. Nat Rev Cancer. 4:592–603. Ramkumar KM, Manjula C, Elango B, Krishnamurthi K, Saravana Devi S, Rajaguru P. 2013. In vitro cytotoxicity of Gymnema montanum in human leukaemia HL-60 cells; induction of apoptosis by mitochondrial membrane potential collapse. Cell Prolif. 46:263–271. Sahebkar A, Iranshahi M. 2010. Biological activities of essential oils from the genus Ferula (Apiaceae). Asian Biomed. 4:835–847. Tan ML, Ooi JP, Ismail N, Moad AI, Muhammad TS. 2009. Programmed cell death pathways and current antitumor targets. Pharmaceut Res. 26:1547–1560.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Ethanolic extract of Ferula gummosa is cytotoxic against cancer cells by inducing apoptosis and cell cycle arrest a
b
c
d
Hoda Gudarzi , Mona Salimi , Saeed Irian , Amir Amanzadeh , b
e
Hirsa Mostafapour Kandelous , Keyhan Azadmanesh & Misha f
Salimi a
Department of Biology, Faculty of Science, Kharazmi University, Tehran, Iran b
Department of Physiology and Pharmacology, Pasteur Institute of Iran, Tehran, Iran c
Department of Cellular and Molecular Biology, Faculty of Science, Kharazmi University, Tehran, Iran d
National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran
e
Department of Virology, Pasteur Institute of Iran, Tehran, Iran
f
Department of Biotechnology, Islamic Azad University of Pharmaceutical Sciences Branch, Tehran, Iran Published online: 21 Aug 2014.
To cite this article: Hoda Gudarzi, Mona Salimi, Saeed Irian, Amir Amanzadeh, Hirsa Mostafapour Kandelous, Keyhan Azadmanesh & Misha Salimi (2014): Ethanolic extract of Ferula gummosa is cytotoxic against cancer cells by inducing apoptosis and cell cycle arrest, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.951854 To link to this article: http://dx.doi.org/10.1080/14786419.2014.951854
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [University of Southern Queensland] at 11:57 16 October 2014
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.951854
SHORT COMMUNICATION Ethanolic extract of Ferula gummosa is cytotoxic against cancer cells by inducing apoptosis and cell cycle arrest Hoda Gudarzia, Mona Salimib*, Saeed Irianc, Amir Amanzadehd, Hirsa Mostafapour Kandelousb, Keyhan Azadmaneshe and Misha Salimif*
Downloaded by [University of Southern Queensland] at 11:57 16 October 2014
a
Department of Biology, Faculty of Science, Kharazmi University, Tehran, Iran; bDepartment of Physiology and Pharmacology, Pasteur Institute of Iran, Tehran, Iran; cDepartment of Cellular and Molecular Biology, Faculty of Science, Kharazmi University, Tehran, Iran; dNational Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran; eDepartment of Virology, Pasteur Institute of Iran, Tehran, Iran; f Department of Biotechnology, Islamic Azad University of Pharmaceutical Sciences Branch, Tehran, Iran (Received 27 May 2014; final version received 2 August 2014) Ferula gummosa Boiss. has medicinal applications in treating a wide range of diseases including cancer. The objective of this study was to evaluate the antiproliferative activities of the seed and gum extracts of F. gummosa as well as to study the effect of the potent extract on the induction of apoptosis and cell cycle arrest. Our results demonstrated that the ethanolic extract had the lowest IC50 value at 72 h (0.001 ^ 1.2 mg/mL) in BHY cells. Moreover, flowcytometry and annexin-V analysis revealed that the ethanolic extract induced apoptosis and cell-cycle arrest in BHY cells at G1/S phase. In addition, colorimetric methods exhibited the highest amount of total phenolics and flavonoids in the aqueous and gum extracts (0.12 ^ 0.037, 0.01 ^ 2.51 mg/g of dry powder). Generally, the results obtained indicate that F. gummosa ethanol extract may contain effective compounds which can be used as a chemotherapeutic agent. Keywords: Ferula gummosa; cancer; apoptosis; MTT assay; flowcytometry
1. Introduction The global burden of cancer continues to increase largely because of the ageing and growth of the world population alongside an increasing adoption of cancer-causing behaviours (Jemal et al. 2011). Standard cancer therapy generally combines surgery, multi-therapeutic agents and ionising radiation. The anticancer agents induce cell cycle arrest and/or cell death by apoptotic or non-apoptotic mechanisms including necrosis (Okada & Mak 2004; Brown & Attardi 2005). Blockage of tumour cell proliferation and apoptosis are referred to the potential anticancer agents by plant natural products (Lee et al. 2003). Due to the less toxicity and cost effectiveness, traditional herbal medicine increasingly attracts more attentions as an alternative cancer therapy (Gao et al. 2011). Ferula gummosa Boiss is a member of the Apiaceae family and has been used for the treatment of stomach pain, epilepsy and as a wound-healing remedy in Iranian traditional medicine (Mandegary et al. 2004). Previous studies reported the antibacterial effects of the essential oil from F. gummosa seeds (Eftekhar et al. 2004). Moreover, the antioxidant and antihaemolytic activities of stem, fruit and leaf extracts of F. gummosa have been already reported (Nabavi et al. 2010). Gharaei et al. have recently evaluated the anti-proliferative and
*Corresponding authors. [email protected] q 2014 Taylor & Francis
Email:
[email protected],
[email protected];
2
H. Gudarzi et al.
apoptosis-inducing activities of ethanolic extract of the leaves and flowers of F. gummosa against the gastric cancer cells (Gharaei et al. 2011). Considering this supposition and that there is no report of the antiproliferative effects of F. gummosa seed extracts, we decided to investigate the cytotoxic effects of the ethanolic, acetonic and aqueous extracts obtained from the seeds and gum of F. gummosa Boiss against three cancer cell lines: MCF-7 (human breast adenocarcinoma), BHY (human oral squamous cancer) and SKMEL-3 (human malignant melanoma) or HGF (human gingival fibroblast) cell line as a control.
Downloaded by [University of Southern Queensland] at 11:57 16 October 2014
2. Results and discussion Growing evidence suggests that irregularities in apoptotic signalling pathway are major contributors in many cancer cells and malignant tissues (Lopez-Beltran et al. 2007). As compounds exhibiting apoptosis-inducing activity are considered potential anticancer agents (Frankfurt & Krishan 2003), many attempts have been made to explore new drugs through isolation of apoptosis-inducing agents from natural products. Previous studies demonstrated that Ferula species are a rich source of phytochemicals including coumarin derivatives, sesquiterpenes and sulphur compounds (Iranshahy & Iranshahi 2011). In this regard, terpenoid compounds are by far the most prevalent components of this genus (Sahebkar & Iranshahi 2010). In addition, preliminary phytochemical analysis of the seed and acetone extract of the root of F. gummosa indicated the presence of large amounts of terpenoids and alkaloids as well as small amounts of cardenolids (Mandegary et al. 2004). Besides that, according to the standards of the National Cancer Institute, crude extracts with IC50 less than 20 mg/mL are considered to be active against tested cancer cells/lines (Geran et al. 1972). These data prompted us to prepare various extracts with different polarities in order to test their cytotoxic effects. 2.1. F. gummosa extracts exhibit cytotoxic properties In order to evaluate the cytotoxic properties of the acetonic, ethanolic and aqueous extracts of F. gummosa seeds as well as the ethanolic extract of F. gummosa gum, the MTT assay was performed on three cancer cell lines including BHY, SKMEL-3 and MCF-7. The effects of different extracts on human cancer cells were examined by increasing the concentrations of the extracts from 0.0001 to 0.5 mg/mL for 24 –72 h. Our results indicated that treatment with the ethanolic and acetonic extracts significantly inhibited the growth of BHY and SKMEL-3 cells in a time- and dose-dependent manner. This trend was the same for MCF-7 cells (data not shown). However, the aqueous and gum extracts exhibited weak antiproliferative activity even at a high concentration of 0.5 mg/mL against three cell lines. The IC50 values are listed in Table 1. As the results indicated, some differences were observed in terms of cell line and type of extract. In other words, BHY was well inhibited by the ethanolic and acetonic extracts and the ethanolic extract demonstrated a considerable potency in inhibition of cell growth at 24, 48 and 72 h (Table 1). Moreover, there was a difference in the IC50 values obtained at 24, 48 and 72 h. The lowest IC50 values were observed at 72 h. Furthermore, the IC50 value obtained for the ethanolic extract against normal cell line (HGF) was higher in comparison to cancer cell lines (IC50 ¼ 0.1 mg/mL, Table 2). These results suggest a specific cytotoxic effect mainly against oral cancer cell line by the ethanolic extract. 2.2. Induction of apoptosis in BHY cells by ethanolic extract Through the annexin-V assay, we observed the externalisation of phosphatidylserine to the outer cell membrane in the ethanolic extract-treated BHY cells at 12 IC50 after 72 h incubation, which is commonly traced as a biochemical marker for apoptosis. As shown in Supplementary Figure S1
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Table 1. IC50 values (mg of extract/mL) for antiproliferative activity of F. gummosa different extracts towards BHY, MCF-7 and SKML-3 cells. Extracts Cells
Time (h)
Ethanol
Acetone
Aqueous
Gum
BHY
24 48 72 24 48 72 24 48 72
0.01 ^ 1.14 0.005 ^ 1.3 0.001 ^ 1.2 0.01 ^ 1.2 0.01 ^ 1.14 0.01 ^ 1.17 0.03 ^ 1.1 0.006 ^ 1.5 0.005 ^ 1.2
0.04 ^ 1.11 0.035 ^ 1.2 0.004 ^ 1.2 0.06 ^ 1.17 0.02 ^ 1.02 0.02 ^ 1.07 0.05 ^ 1.1 0.02 ^ 1.1 0.01 ^ 1.1
. 0.5 . 0.5 . 0.5 . 0.5 . 0.5 . 0.5 . 0.5 . 0.5 . 0.5
.0.5 .0.5 .0.5 .0.5 .0.5 .0.5 .0.5 .0.5 .0.5
Downloaded by [University of Southern Queensland] at 11:57 16 October 2014
MCF-7 SKML-3
Note: Values are presented as mean ^ SE of three independent experiments, performed in triplicate.
Table 2. IC50 values (mg of extract/mL) for cytotoxic activity of ethanolic and acetonic of F. gummosa seed extracts towards normal HGF cells. Extract Cell
Time (h)
Ethanol
Acetone
HGF
24 48 72
. 0.5 . 0.5 0.1 ^ 1.2
. 0.5 . 0.5 . 0.5
Note: Values are presented as mean ^ SE of three independent experiments, performed in triplicate.
and Table 3, 0.56% of cells were annexin V þ /PI 2 and 1.79% of cells were annexin V þ / PI þ in solvent-control cells. After treatment with 0.0005 mg/mL of the ethanolic extract for 72 h, the number of cells which were annexin V þ /PI 2 and annexin V þ /PI þ was considerably increased. Meanwhile, the necrotic population (annexin V 2 /PI þ ) was increased by 2.0%. These results collectively indicated that treatment with the ethanolic extract at 12 IC50 for 72 h resulted in a clear shift from live cell population to early and late apoptotic population.
2.3. Ethanolic extract of F. gummosa induces cell cycle arrest Induction of apoptosis via blockage of cell cycle progression by anticancer agents prevents the proliferation of cancer cells, which may be exploited for cancer therapy (Tan et al. 2009). Hence, we studied the effect of F. gummosa ethanolic seed extract on the cell cycle of BHY cells. Our results showed that the ethanolic extract at 0.002 mg/mL (2 times the IC50) could not trigger BHY cell death after 48 h of incubation. Following 48 h of treatment, the sub-G1 sub-population Table 3. Percentage of oral squamous cancer cells in each state after treatment with ethanolic extract at 0.0005 mg/mL for 72 h of incubation. Treatment
Vital cells (%) An– /PI –
Early apoptosis (%) An þ /PI –
Late apoptosis (%) An þ /PI þ
Necrosis (%) An– /PI þ
Control Solvent Extract
96.15 ^ 0.82 90.73 ^ 4.53 85.63 ^ 1.83
0.41 ^ 0.22 0.56 ^ 0.03 1.72 ^ 0.78
0.87 ^ 0.15 1.79 ^ 0.98 3.9 ^ 1.52
2.6 ^ 0.54 3.35 ^ 0.01 5.39 ^ 1.01
4
H. Gudarzi et al.
Table 4. Effect of ethanol extract on cell cycle progression with respect to control. BHY Treatment Control 0.002 mg/mL
Sub-G1 (mean ^ SE)
Go/G1 (mean ^ SE)
S (mean ^ SE)
G2/M (mean ^ SE)
29.95 ^ 1.2 32.0 ^ 1.07
29.98 ^ 0.7 33.17 ^ 0.5
30.7 ^ 1.4 27.55 ^ 0.4
14.4 ^ 0.25 13.28 ^ 0.9
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Notes: At the indicated cell, distribution of the cells in sub-G1, Go/G1, S and G2/M phase was analysed by flowcytometry. Results are expressed as total cells. Data represent means of triplicate experiment.
insignificantly increased from 29.95% to 32%. Concomitantly, there was an increase in G0/G1 and a decrease in S-phase sub-populations from 29.95% to 33.17% and 30.7% to 27.5%, respectively, which were not considerable (Supplementary Figure S2 and Table 4), suggesting an apoptotic pathway which is independent of the cell cycle blockage. 2.4. Total phenolic and flavonoid contents Polyphenols are well documented to act as chemopreventive agents exerting a direct, proapoptotic effect on tumour cells, which block the growth of several types of human cancer cell lines at different phases of the cell cycle (Ramkumar et al. 2013). Our results displayed no direct correlation between cytotoxic properties of F. gummosa seed and gum extracts and polyphenol contents (Supplementary Table S1). The results obtained suggest a strong anti-proliferative activity of the ethanolic seed extract, which may be due to the presence of bioactive components, such as coumarins, terpenoids and/or alkaloids, which are known to induce anti-proliferative outcomes. 3. Conclusion In summary, our results present the first evidence on the antiproliferative effect of the ethanolic seed extract of F. gummosa as demonstrated by in vitro MCF-7, BHY and SKMEL-3 growth inhibition. The ethanolic extract could also induce cell apoptosis at early and late stages. However, more detailed molecular mechanisms underlying the ethanolic-induced BHY apoptotic cell death remain to be elucidated. More importantly, isolation of the effective ingredients is required to be determined. Supplementary material Experimental details relating to this article are available online. References Brown JM, Attardi LD. 2005. The role of apoptosis in cancer development and treatment response. Nat Rev Cancer. 5:231–237. Eftekhar F, Yousefzadi M, Borhani K. 2004. Antibacterial activity of the essential oil from Ferula gummosa seed. Fitoterapia. 75:758–759. Frankfurt OS, Krishan A. 2003. Apoptosis-based drug screening and detection of selective toxicity to cancer cells. Anticancer Drug. 14:555– 561. Gao Y, Su Y, Qu L, Xu S, Meng L, Cai SQ, Shoua C. 2011. Mitochondrial apoptosis contributes to the anti-cancer effect of Smilax glabra Roxb. Toxicol Lett. 207:112–120. Geran RI, Greenberg NH, Macdonald MM, Schumacher AM, Abbott BJ. 1972. Protocols for screening chemical agents and natural products against animal tumors and other biological systems. Cancer Chemother Rep. 33:1–17. Gharaei R, Ghobadi S, Akrami H. 2011. Study of apoptosis inducing effects of ethanol extract of Ferula gummosa leaf. Clin Biochem. 44:S332.
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