ABSTRACT

Nepeta binaloudensis Jamzad, Lamiaceae, is a rare medicinal plant endemic to Iran. In spite of many studies about the chemical constituents and antibacterial effects of this species, no report has been provided about its cytotoxic and anticancer activities. In this study we have evaluated the effects of EtOH 70%, hexane and aqueous extracts of N. binaloudensis on the cell proliferation and n-hexane extract on the expression of adenosine deaminase and ornithine decarboxylase 1 genes in breast cancer cell lines (MCF-7, MDA-MB-231) compared to non-cancer line (MCF-10A). The cell lines were subjected to increasing doses of the extracts ranging from 10 to 320 µg/ml. Cell viability was quantified by MTS assay. Expression of adenosine deaminase and ornithine decarboxylase 1 genes was analyzed by real time PCR. N. binaloudensis inhibited the growth of malignant cells in a time and dose-dependent manner. Among extracts of N. binaloudensis, the hexane extract was found to be more toxic compared to other extracts. Results showed a marked decrease in the expression of ornithine decarboxylase 1 and adenosine deaminase genes in cancer cell lines. At 60 µg/ml concentration of N. binaloudensis hexane extract ornithine decarboxylase 1 and adenosine deaminase mRNA expression were reduced 4.9 fold and 3.5 fold in MCF-7 cell line and 3.6 fold and 2.6 fold in MDA-MB-231 cell line compared to control, respectively. The result of our study highlights the potential influences of N. binaloudensis hexane extract on ornithine decarboxylase 1 and adenosine deaminase genes expression in breast cancer cells and its relation to inhibition of cancer cell growth.

Keywords:
Adenosine deaminase; Anticancer; Cytotoxicity; Ornithine decarboxylase

Introduction

Cancer is the most common cause of death in the world. The majority of deaths from cancer occur in women with breast cancer. Statistics indicate that in 2014 about 29% of cancers were breast cancers among American women (Siegel et al., 2014Siegel, R., Ma, J., Zou, Z., Jemal, A., 2014. Cancer statistics. CA Cancer J. Clin. 64, 9-29.). In Iran also breast cancer rates have increased in females (Mousavi et al., 2009Mousavi, S.M., Gouya, M.M., Ramazani, R., Davanlou, M., Hajsadeghi, N., Seddighi, Z., 2009. Cancer incidence and mortality in Iran. Ann. Oncol. 20, 556-563.). Many investigations are performed to detect the genes which are involved in breast cancer. One of the most important genes is ornithine decarboxylase (ODC) which encodes a key enzyme in polyamines synthesis (Zhu et al., 2012Zhu, Q., Jin, L., Casero, R.A., Davidson, N.E., Huang, Y., 2012. Role of ornithine decarboxylase in regulation of estrogen receptor alpha expression and growth in human breast cancer cells. Breast Cancer Res. Treat. 136, 57-66.; Xu et al., 2015Xu, L., Long, J., Wang, P., Liu, K., Mai, L., Guo, Y., 2015. Association between the ornithine decarboxylase G316A polymorphism and breast cancer survival. Oncol. Lett. 10, 485-491.). Several studies indicate that there are close correlation between ODC activity in the cells and regulation of cell proliferation (Deng et al., 2008Deng, W., Jiang, X., Mei, Y., Sun, J., Ma, R., Liu, X., Sun, X., 2008. Role of ornithine decarboxylase in breast cancer. Acta Biochim. Biophys. Sin. 40, 235-243.; Elmets and Athar, 2010Elmets, C.A., Athar, M., 2010. Targeting ornithine decarboxylase for the prevention of nonmelanoma skin cancer in humans. Cancer Prev. Res. 3, 8-11.; Hayes et al., 2011Hayes, C.S., Defeo, K., Dang, H., Trempus, C.S., Morris, R.J., Gilmour, S.K., 2011. A prolonged and exaggerated wound response with elevated ODC activity mimics early tumor development. Carcinogenesis 32, 1340-1348.). Consequently, any changes in ODC activity and polyamines level have effects on cell differentiation and proliferation. In breast cancer cells, estrogens induce ODC activity. So, the level of ODC and estrogen is increased in these cells (Zhu et al., 2012Zhu, Q., Jin, L., Casero, R.A., Davidson, N.E., Huang, Y., 2012. Role of ornithine decarboxylase in regulation of estrogen receptor alpha expression and growth in human breast cancer cells. Breast Cancer Res. Treat. 136, 57-66.). In addition, Adenosine Deaminase gene (ADA) encodes the enzyme that catalyzes hydrolyzation of adenosine to inosin and participates in purines metabolism, which has a crucial role in development of immune system and maturation of mammalian cells (Ri et al., 2010Ri, G., Ohno, S., Furutani, M., Furutani, Y., Tsukahara, T., Hagita, N., Matsuoka, R., 2010. An indication for correlation between the serum ADA level and gastric cancer risk. Anticancer Res. 30, 2347-2349.; Roberts and Roberts, 2012Roberts, E.L., Roberts, O.T., 2012. Plasma adenosine deaminase isoform 2 in cancer patients undergoing chemotherapy. Br. J. Biomed. Sci. 69, 11-13.). Recent studies suggest that the level of ADA enzyme is increased in breast cancer cells in contrast with normal cells (Aghaei et al., 2010Aghaei, M., Karami-Tehrani, F., Salami, S., Atri, M., 2010. Diagnostic value of adenosine deaminase activity in benign and malignant breast tumors. Arch. Med. Res. 41, 14-18.; Mahajan et al., 2013Mahajan, M., Tiwari, N., Sharma, R., Kaur, S., Singh, N., 2013. Oxidative stress and its relationship with adenosine deaminase activity in various stages of breast cancer. Indian J. Clin. Biochem. 28, 51-54.). Therefore, decreasing of ODC and ADA gene expression and respective enzyme activities can be a good strategy for breast cancer therapy.

Using medicinal plant extracts as anticancer drugs is a more effective way for cancer therapy. About 50% of prescribed drugs in the world originate from plants (Van Slambrouck et al., 2007Van Slambrouck, S., Daniels, A.L., Hooten, C.J., Brock, S.L., Jenkins, A.R., Ogasawara, M.A., Steelant, W.F., 2007. Effects of crude aqueous medicinal plant extracts on growth and invasion of breast cancer cells. Oncol. Rep. 17, 1487-1492.). Nepeta binaloudensis Jamzad, Lamiaceae, is an endemic and rare medicinal plant which grows exclusively in the Binalud Mountains in Khorasan Razavi province in Northeast of Iran (Tundis et al., 2013Tundis, R., Nadjafi, F., Menichini, F., 2013. Angiotensin-converting enzyme inhibitory activity and antioxidant properties of Nepeta crassifolia Boiss & Buhse and Nepeta binaloudensis Jamzad. Phytother. Res. 27, 572-580.). This plant has been applied widely in traditional medicine for the treatment of respiratory disease, digestive problems and osteoarthritis (Koocheki and Rezvani Moghaddam, 2012Koocheki, A., Rezvani Moghaddam, P., 2012. First experiments on cultivation of Nepeta binaloudensis Jamzad – an example of domestication of a highly endangered medicinal plant of Iran. Z. Arznei- Gewurzpfla 17, 64-71.). Previous chemical investigation of N. binaloudensis has shown the presence of nepetalactone and 1,8 cineole (Rustaiyan and Nadji, 1999Rustaiyan, A., Nadji, K., 1999. Composition of the essential oils of Nepeta ispahanica Boiss, and Nepeta binaloudensis Jamzad from Iran. Flavour Frag. J. 14, 35-37.). Some studies were done about antibacterial activities of N. binaloudensis but its cytotoxicity and anticancer effects have not been studied yet. Therefore, the purpose of the study was to determine the anticancer effects of N. binaloudensis extract on two breast cancer cell lines (MCF-7, MDA-MB-231) and normal breast cell line (MCF-10A). Also, the present study is focused on evaluating the level of ODC1 and ADA gene expression in the normal and cancerous breast cells.

Material and methods

Plant collection and extraction

Shoots of Nepeta binaloudensis Jamzad, Lamiaceae, were collected from Binalud Mountains in Neyshabur, Khorasan Razavi province, northeast of Iran. The plant was identified by Mr. M.R. Joharchi, from Ferdowsi University of Mashhad Herbarium (FUMH). Voucher specimen (No. 1025) was deposited with herbarium of Islamic Azad University, Neyshabur Branch. The extraction process was performed from dried shoots. The dried shoots (100 g) were percolated with 100 ml of each solvent (EtOH 70%, hexane and H₂O) for 4 h separately. The extracts were filtered and the solvents were evaporated under vacuum at 45 ºC to afford crude extracts. Dry extracts were dissolved in dimethyl sulfoxide (DMSO) and then subjected to cytotoxic and gene expression assays.

Cell lines and culture medium

Two human breast cancer cell lines (MCF-7, MDA-MB-231) and one normal breast cell line (MCF-10A) were obtained from cell culture laboratory of the Faculty of Sciences, Ferdowsi University (Mashhad, Iran) and Pasteur Institute (Tehran, Iran). The cells were cultured in 25 cm² flasks with RPMI-1640 (RPMI Sigma) and supplemented with 10% fetal bovine serum (FBS, Gibco) and 100 U/ml penicillin and 100 µg/ml streptomycin solutions, at 37 ºC with 5% CO₂ and 95% humidity. The medium was exchanged every day and the cells were passaged every 3–4 days.

Cytotoxicity assay

The cytotoxic activity of the extracts was determined using MTT assay (2, 3, 5 diphenil tetrazolium bromide). Cells were plated with density of 3 × 10⁴ cells/dish in 96 well plates and were incubated for 24 h at 37 ºC. Then, the cells were treated with different concentrations of extracts (0, 10, 20, 40, 80, 160 and 320 µg/ml) and were incubated for 24, 48 and 72 h. A 2 mM MTT solution was added to each well and plate was incubated for 4 h. The medium was then discarded and 100 ml DMSO (dimethyl sulfoxide) was added and the plate was shaken for 10 min. Finally, optical density was determined in 540 nm using ELISA microplate reader (Awareness, Palm City, FL, USA). Each experiment was performed in triplicate. Results are expressed as the percentage growth inhibition with respect to the untreated cells.

Colony formation assay

The effect of hexane extract on MCF7 and MDA-MB-231 cells was investigated by colony formation assay (Franken et al., 2006Franken, N.A., Rodermond, H.M., Stap, J., Haveman, J., van Bree, C., 2006. Clonogenic assay of cells in vitro. Nat. Protoc. 1, 2315-2319.). Briefly, the cells (500 cells/ml) were allowed to grow in 60 mm Petri dishes for 12 h. Subsequently, the cells were treated for 48 h with hexane extract (10, 30 and 60 µg/ml), or 0.1% DMSO. The colonies were fixed and stained with 0.2% crystal violet and counted under stereomicroscope.

Real-time PCR

The expression levels of ODC1 and ADA genes, key genes in breast cancer cells, were analyzed using Real time PCR assay. The cells were cultured in three groups and treated with increasing concentrations (0–60 µg/ml) of the hexane extract dissolved in DMSO for 48 h. After 48 h total cellular RNA was extracted from the treated and untreated (control) cells using Easy BLUE total RNA extraction kit (iNtRON). The treatments were repeated for three times. The quality of RNA was determined by absorption at 260 nm using T80 UV-Visible Spectrophotometer (PG Instruments). For each specimen 2 µg of RNA was reverse transcribed into cDNA with reverse transcription reaction mixture of Power cDNA Synthesis Kit (iNtRON). Primers of ODC1, ADA and βACT were designed using Gene Runner software (version 4).

Primer sequences for ODC1 were: ODC1-FW: 5′-GTGGGTGATTGGATGCTCTTTG-3′ ODC1-RV: 5′-ACCAGGCTAACTACTCGCTCAA-3′.

Primer sequences for ADA were: ADA-FW: 5′-ACCAGGCTAACTACTCGCTCAA-3′ ADA-RV: 5′-TCAGTAAAGCCCATGTCCCGTT-3′.

Primer sequences for βACT were: ACT-FW: 5′-TCCATCATGAAGTGTGACGT-3′ ACT-RV: 5′-GAGCAATGATCTTGATCTTCAT-3′.

Real-time PCR was carried out using SYBR Green method on an ABI Step-One instrument (Applied Biosystems, USA). The cDNA was taken as a template for PCR amplification of ADA and ODC1 genes and βACT gene (control gene). Each reaction was repeated for three times. A PCR reaction mixture of 20 µl contained 10 µl SYBR Green master mix, 0.4 µl Reverse primer and 0.4 µl Forward primer, 6.2 µl dH₂O (RNase free) and 3 µl cDNA (4 ng). Three pairs of primers were used separately. The thermal cycling conditions were as follow: 1 denaturation cycle of 95 ºC for 10 min and 40 cycles of 95 ºC for 15 s, 60 ºC for 60 s (annealing and extension temperature). A negative control was used in each run to assess specificity of primers and possible contamination. The realtime PCR data were analyzed using the relative gene expression (ΔΔCt) method, as described in Applied Biosystems User Bulletin No. 2 (Livak and Schmittgen, 2001). Briefly, the data are presented as the fold change in gene expression normalized to the reference gene (β-actin) and were determined using the equation fold change = 2^-ΔΔCt.

Statistical analysis

One-way analysis of variance and Fisher's LSD were used for data analysis. All results were expressed as mean ±SEM, and p values lower than 0.05 were judged as significant.

Results

Cytotoxicity of various extracts of Nepeta binaloudensis

The cytotoxic activity of N. binaloudensis extracts were studied against cultured MCF-10A (Normal), MBA-MD-231 and MCF-7 (cancer) breast cell lines using MTT assay. The cell lines were subjected to increasing doses of EtOH 70%, hexane and H₂O extracts ranging from 10 to 320 µg/ml for 24, 48 and 72 h. The results showed EtOH 70% and hexane extracts decreased viability of malignant cells in a concentration and time-dependent manner. Whereas H₂O extract showed no marked cytotoxicity on the cell lines tested. EtOH and hexane extracts in the same concentrations caused less toxicity to MCF-10A than tumor cells indicating a degree of specificity for malignant cell lines (Fig. 1). Concentrations inducing 50% cell growth inhibition (IC₅₀) against MBA-MD-231 and MCF-7 cells are presented in Table 1. MCF-7 cells were found to be more sensitive to the cytotoxic effects of the extracts compared to MBA-MD-231cells.

Fig. 2 shows the photomicrographic images of MCF-7 and MBA-MD-231cell lines treated by hexane extract of N. binaloudensis. Cytotoxicity effects were coupled with morphological changes including decrease in cell volume and shrinking of the cells.

Paclitaxel (700 nM) was used as a positive control. Paclitaxel at this concentration decreased the viability of MCF-7 and MBA-MD-231 cells to 6.3% ± 0.6 and 12.2 ± 0.3 in comparison with untreated control received an equal volume of the solvent respectively (data not shown).

In vitro clonogenic survival assay

As given in Fig. 3 there is a significant dose dependent inhibition in colony formation in MCF-7 and MBA-MD-231 after hexane extract treatments. The results were showed a decrease in colony formation by 30–50% in MBA-MD-231 while a 45–70% decrease was observed in MCF-7 when treated with 30 µg/ml and 60 µg/ml n-hexane extract respectively.

Changes in the expression of ODC1 and ADA

Our results showed that exposure of cells to increasing concentrations of N. binaloudensis hexane extract for 48 h caused significant decrease in the mRNA levels of ODC1 and ADA in cancer cell lines but not non-cancer cells. The effect of N. binaloudensis on selected genes expression was shown to be concentration-dependent. N. binaloudensis hexane extract at concentrations of 10, 30 and 60 µg/ml significantly decreased the ODC1 expression by 1.4, 2.3 and 4.97 fold, respectively in MCF-7 cell line. Moreover, this extract significantly reduced mRNA levels of ODC1 in MDA-MB-231 cell line by 1.2, 1.7 and 3.6 fold with10, 30 and 60 µg/ml, respectively (Fig. 4).

Similar to ODC1, N. binaloudensis extract significantly decreased the expression of ADA at all the concentrations. In MCF-7 cell line N. binaloudensis extract decreased the expression of ADA in a dose-dependent manner by 1.32, 1.83 and 3.49 fold with 10, 30 and 60 µg/ml, respectively. As shown in Fig. 5, at 10, 30 and 60 µg/ml concentration of N. binaloudensis extract ADA mRNA expression was reduced 1.21, 1.56 and 2.56 fold in MDA-MB-231 cell line, respectively (Fig. 5). In cancer cell lines, mRNA expression of MCF-7 was reduced more than that of MDA-MB-231.

Discussion

Several studies suggest an increased polyamine concentration in breast cancer tissue compared to normal breast tissue and polyamines biosynthesis inhibitors decrease the growth of cancerous tumors (Brown et al., 2009Brown, I., Halliday, S., Greig, H., Heys, S.D., Wallace, H.M., Schofield, A.C., 2009. Genetic polymorphism in ornithine decarboxylase and risk of breast cancer. Fam. Cancer 8, 307-311.; Arisan et al., 2012Arisan, E.D., Obakan, P., Coker, A., Palavan-Unsal, N., 2012. Inhibition of ornithine decarboxylase alters the roscovitine-induced mitochondrial-mediated apoptosis in MCF-7 breast cancer cells. Mol. Med. Rep. 5, 1323-1329.; Zhu et al., 2012Zhu, Q., Jin, L., Casero, R.A., Davidson, N.E., Huang, Y., 2012. Role of ornithine decarboxylase in regulation of estrogen receptor alpha expression and growth in human breast cancer cells. Breast Cancer Res. Treat. 136, 57-66.). Ornithine decarboxylase (ODC1), is a key enzyme in polyamine biosynthesis which decarboxylates Ornithine to putrescine (Wang and Jiang, 2014Wang, X., Jiang, L., 2014. Effects of ornithine decarboxylase antizyme 1 on the proliferation and differentiation of human oral cancer cells. Int. J. Mol. Med. 34, 1606-1612.). Furthermore, adenosine deaminase (ADA) enzyme, which catalyzes the hydrolytic deamination of adenosine to inosine, plays an important role in purine metabolism and Adenosine homeostasis (Garcia-Gil et al., 2015Garcia-Gil, M., Tozzi, M.G., Varani, S., Della Verde, L., Petrotto, E., Balestri, F., Camici, M., 2015. The combination of adenosine deaminase inhibition and deoxyadenosine induces apoptosis in a human astrocytoma cell line. Neurochem. Int. 80, 14-22.). It has been proved to be a direct correlation between breast cancer and increased ADA enzyme activity in cancer tissue of patients (Gocmen et al., 2009Gocmen, E., Tez, M., Ozturk, S., Koc, M., Demirci, S., 2009. Activities of adenosine deaminase and 5′-nucleotidase in cancereous and non-cancereous human gastric tissues. Bratisl. Lek. Listy 110, 416-418.; Ri et al., 2010Ri, G., Ohno, S., Furutani, M., Furutani, Y., Tsukahara, T., Hagita, N., Matsuoka, R., 2010. An indication for correlation between the serum ADA level and gastric cancer risk. Anticancer Res. 30, 2347-2349.; Ogata et al., 2011Ogata, Y., Aoe, K., Hiraki, A., Murakami, K., Kishino, D., Chikamori, K., Tanimoto, M., 2011. Is adenosine deaminase in pleural fluid a useful marker for differentiating tuberculosis from lung cancer or mesothelioma in Japan, a country with intermediate incidence of tuberculosis?. Acta Med. Okayama 65, 259-263.). Therefore, ODC1 and ADA genes are the appropriate target in anticancer research.

According to previous studies and the importance of recognizing anti-cancer mechanisms of plant extracts, in present study the effects of various extracts of N. binaloudensis on the cell proliferation and the expression of ADA and ODC genes in breast cancer cell lines (MCF-7, MDA-MB-231) with respect to non-cancer lines (MCF-10A) were evaluated.

The results showed that after treatment of cells (at 24, 48 and 72 h) with extracts of hexane and EtOH 70%, excluding H₂O extract, a linear relationship between the doses with the percentage of cell viability was observed. According to the results of this study hexane and EtOH extract of this plant has anti-cancer effects.

Different studies have shown the antiproliferative activity of Nepeta species including N. ucrainica (Khakdan and Rassam, 2014Khakdan, F., Rassam, G.A., 2014. Chemical composition, antibacterial activity, and cytotoxicity of essential oil from Nepeta ucrainica L. spp. kopetdaghensis. Ind. Crop. Prod. 58, 315-321.), N. cataria (Formisano et al., 2011Formisano, C., Rigano, D., Senatore, F., 2011. Chemical constituents and biological activities of Nepeta species. Chem. Biodivers. 8, 1783-1818.) and N. glomerata (Rigano et al., 2011Rigano, D., Arnold, N.A., Conforti, F., Menichini, F., Formisano, C., Piozzi, F., Senatore, F., 2011. Characterisation of the essential oil of Nepeta glomerata Montbret et Aucher ex Bentham from Lebanon and its biological activities. Nat. Prod. Res. 25, 614-626.). Chemical composition analyses of N. binaloudensis oil revealed that 1,8-cineol (68.31%), α-terpineol (5.24%), β-pinene (4.74%), δ-terpineol (2.57%), α-pinene (1.54%) were main components (Mohammadpour et al., 2013Mohammadpour, N., Emami, S.A., Asili, J., 2013. Identification of volatile oil components of Nepeta binaloudensis Jamzad by GC-MS and 13C-NMR methods and evaluation of its antimicrobial activity. J. Essent. Oil Bear. Plants 16, 102-107.). The cytotoxic effects of these monoterpenes have been previously shown against different cell lines. 1,8-cineole, the main compound present in the N. binaloudensis oil, has been reported to inhibit the growth of liver-derived (HepG2) and extrahepatic (A549) cell lines (Rodenak Kladniew et al., 2014Rodenak Kladniew, B., Polo, M., Montero Villegas, S., Galle, M., Crespo, R., Garcia de Bravo, M., 2014. Synergistic antiproliferative and anticholesterogenic effects of linalool, 1,8-cineole, and simvastatin on human cell lines. Chem. Biol. Interact. 214, 57-68.). The results of another study indicated 1,8-cineole suppressed human colorectal cancer proliferation by inducing apoptosis (Murata et al., 2013Murata, S., Shiragami, R., Kosugi, C., Tezuka, T., Yamazaki, M., Hirano, A., Koda, K., 2013. Antitumor effect of 1,8-cineole against colon cancer. Oncol. Rep. 30, 2647-2652.). Alpha terpineol another key component of the oil, has also been reported to inhibits the growth of tumor cells through a mechanism that involves inhibition of the NF-κB pathway (Hassan et al., 2010Hassan, S.B., Gali-Muhtasib, H., Goransson, H., Larsson, R., 2010. Alpha terpineol: a potential anticancer agent which acts through suppressing NF-kappaB signalling. Anticancer Res. 30, 1911-1919.). δ-Terpineol inhibits cell growth and induces apoptosis in human liver cancer BEL-7402 cells (Wu et al., 2014Wu, Z.L., Yin, Z.Q., Du, Y.H., Feng, R.Z., Ye, K.C., Wei, Q., Wang, Y., 2014. gamma-Terpineol inhibits cell growth and induces apoptosis in human liver cancer BEL-7402 cells in vitro. Int. J. Clin. Exp. Pathol. 7, 6524-6533.). Anti-tumor effect of α-pinene on human hepatoma cell lines through inducing G2/M cell cycle arrest has been reported (Chen et al., 2015Chen, W., Liu, Y., Li, M., Mao, J., Zhang, L., Huang, R., Ye, L., 2015. Anti-tumor effect of alpha-pinene on human hepatoma cell lines through inducing G2/M cell cycle arrest. J. Pharmacol. Sci. 127, 332-338.). It was demonstrated that α-pinene inhibited BEL-7402 cells by arresting cell growth in the G2/M phase of the cell cycle, down regulating Cdc25C mRNA and protein expression, and reducing cycle dependence on kinase 1(CDK1) activity (Chen et al., 2014Chen, W.Q., Xu, B., Mao, J.W., Wei, F.X., Li, M., Liu, T., Zhang, L.R., 2014. Inhibitory effects of alpha-pinene on hepatoma carcinoma cell proliferation. Asian Pac. J. Cancer. Prev. 15, 3293-3297.).

Among the extracts, hexane extract exhibited the largest cytotoxic effects on cancer cells. This can be described by the low polarity of hexane, which extract low-polar compounds that are either favorably absorbed through the cell or have cytotoxic activity. Therefore, it is assumed that most of the cytotoxic compounds were concentrated in the hexane extract (Tayarani-Najaran et al., 2013Tayarani-Najaran, Z., Asili, J., Aioubi, E., Emami, S.A., 2013. Growth inhibition and apoptosis induction of Salvia chloroleuca on MCF-7 breast cancer cell line. Iran. J. Pharm. Res. 12, 789-799.).

The results of our study revealed that the cytotoxicity of the hexane extract of N. binaloudensis on the estrogen receptor-positive cell line (MCF7) (ER+/PR+/HER2-) was stronger than that of ER/PR/HER2 triple negative breast cancer cell line (MDA-MB231). Such findings are similar to those found in the work of Srisawat et al. (2015)Srisawat, T., Sukpondma, Y., Graidist, P., Chimplee, S., Kanokwiroon, K., 2015. The dose dependent in vitro responses of MCF-7 and MDA-MB-231 cell lines to extracts of Vatica diospyroides Symington type SS fruit include effects on mode of cell death. Pharmacogn. Mag. 11, S148. who found a higher apoptosis and growth inhibition rate in MCF-7 cells than in MDA-MB-231 cells. The higher cytotoxic effect of N. binaloudensis extract on MCF-7 was probably due to lack of estrogen stimulation in this experiment.

The decrease of the cells viability could be because of an increase of apoptosis and/or decreased cell reproduction (Islam et al., 2013Islam, M., Sharma, S., Kumar, B., Teknos, T.N., 2013. Atorvastatin inhibits RhoC function and limits head and neck cancer metastasis. Oral Oncol. 49, 778-786.). In our study, a colony formation assay was performed to evaluate the hexane extract capability of arresting proliferation of cancerous cells by lowering reproductive ability. Our results revealed that cytotoxic effects of hexane extract could be due to a decrease of cell proliferation.

According to the cytotoxicity results, hexane extract exhibited the largest cytotoxic effects on cancer cells. Therefore, in gene expression studies hexane extract was used in safe concentrations (0–60 µg/ml). Significant reduction in expression of ODC1 gene was observed in breast cancer cell lines (MCF-7 and MDA-MB-231) after 48 h treatment with N. binaloudensis hexane extract. Other studies have shown the inhibitory effects of plant materials on ODC activity and expression in cancerous cell lines and tissues. Liao et al. (2008)Liao, Y.F., Hung, H.C., Hour, T.C., Hsu, P.C., Kao, M.C., Tsay, G.J., Liu, G.Y., 2008. Curcumin induces apoptosis through an ornithine decarboxylase-dependent pathway in human promyelocytic leukemia HL-60 cells. Life Sci. 82, 367-375. found that enzyme activity and protein expression of ODC were reduced in curcumin treated leukemia HL-60 cells. They suggested that curcumin-induced apoptosis occurs through a down-regulating mechanism of ODC. In another study, Hakimuddin et al. (2008)Hakimuddin, F., Tiwari, K., Paliyath, G., Meckling, K., 2008. Grape and wine polyphenols down-regulate the expression of signal transduction genes and inhibit the growth of estrogen receptor–negative MDA-MB231 tumors in nu/nu mouse xenografts. Nutr. Res. 28, 702-713. reported more than 3-fold reduction in ODC1 mRNA level in tumors of wine polyphenol-treated mice.

However, the mechanism by which N. binaloudensis extract reduces ODC1 gene expression is unknown. In the ODC1 promoter, there are multiple binding sites for Ap-1, Ap-2 and c-Myc (Liao et al., 2008Liao, Y.F., Hung, H.C., Hour, T.C., Hsu, P.C., Kao, M.C., Tsay, G.J., Liu, G.Y., 2008. Curcumin induces apoptosis through an ornithine decarboxylase-dependent pathway in human promyelocytic leukemia HL-60 cells. Life Sci. 82, 367-375.). The mechanisms of the inhibition of ODC1 expression by N. binaloudensis extract likely involve transcriptional inhibition of these factors.

ODC enzyme activity is related to the production of polyamines such as spermine that function as a free radical scavenger (Smirnova et al., 2012Smirnova, O.A., Isaguliants, M.G., Hyvonen, M.T., Keinanen, T.A., Tunitskaya, V.L., Vepsalainen, J., Ivanov, A.V., 2012. Chemically induced oxidative stress increases polyamine levels by activating the transcription of ornithine decarboxylase and spermidine/spermine-N1-acetyltransferase in human hepatoma HUH7 cells. Biochimie 94, 1876-1883.). Inhibition of ODC gene expression by N. binaloudensis extract decreases the concentration of polyamines. Thus, reduction of polyamines could increase intracellular ROS and cause apoptosis in cancer cells (Wang et al., 2011Wang, M.F., Liao, Y.F., Hung, Y.C., Lin, C.L., Hour, T.C., Lue, K.H., Liu, G.Y., 2011. Hydroxydibenzoylmethane induces apoptosis through repressing ornithine decarboxylase in human promyelocytic leukemia HL-60 cells. Exp. Mol. Med. 43, 189-196.).

ADA gene expression decreased in the cancerous human breast cell lines treated by N. binaloudensis hexane extract. Many researchers reported that plant extracts have inhibitory effect on ADA expression and activity. For example, Durak et al. (2005)Durak, I., Cetin, R., Devrim, E., Erguder, I.B., 2005. Effects of black grape extract on activities of DNA turn-over enzymes in cancerous and non cancerous human colon tissues. Life Sci. 76, 2995-3000. showed that black grape extracts significantly inhibited the ADA enzyme activity in cancerous colon tissues. In another study, it has been found that expression of the adenosine deaminase gene was consistently decreased by all the berries in intestinal adenoma tissue of rats (Mutanen et al., 2008Mutanen, M., Pajari, A.M., Paivarinta, E., Misikangas, M., Rajakangas, J., Marttinen, M., Oikarinen, S., 2008. Berries as chemopreventive dietary constituents a mechanistic approach with the ApcMin/+ mouse. Asia Pac. J. Clin. Nutr. 17, 123-125.). Tomato juice has been reported to causes significant inhibition on ADA activity in the prostate cancer tissues (Durak et al., 2003Durak, İ., Biri, H., Avcı, A., Sözen, S., Devrim, E., 2003. Tomato juice inhibits adenosine deaminase activity in human prostate tissue from patient with prostate cancer. Nutr. Res. 23, 1183-1188.).

Our data demonstrate that decrease of adenosine deaminase expression abolishes advantage of cancer cells in obtaining more nucleotides in DNA synthesis and proliferation (Namuslu et al., 2014Namuslu, M., Kocaoglu, H., Celik, H.T., Avci, A., Devrim, E., Genc, Y., Durak, I., 2014. Effects of aqueous soybean, mistletoe and red clover extracts on activities of adenosine deaminase and xanthine oxidase enzyme. Bratisl. Lek. Listy 115, 367-371.). Also, inhibition of ADA expression causes increased availability of adenosine which is a protective molecule in case of tumorous condition (Mahajan et al., 2013Mahajan, M., Tiwari, N., Sharma, R., Kaur, S., Singh, N., 2013. Oxidative stress and its relationship with adenosine deaminase activity in various stages of breast cancer. Indian J. Clin. Biochem. 28, 51-54.).

In conclusion, the study described here demonstrates that N. binaloudensis hexane extract has inhibitory effects on ODC1 and ADA gene expression in breast cancer cell line. Therefore, a possible mechanism for observed cytotoxicity effect of this plant extract could be expression inhibition of these genes. This is the first report about the cytotoxicity of N. binaloudensis hexane extract by inhibition of ODC1 and ADA in mRNA level, hence further studies will be necessary to supplement our findings.

Acknowledgment

This work was financially supported by Neyshabur Branch, Islamic Azad University Neyshabur, Iran.

References

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