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ORIGINAL ARTICLE
Year : 2019  |  Volume : 15  |  Issue : 5  |  Page : 1073-1079

The synergistic cytotoxic effects of doxorubicin and Viola odorata extract on human breast cancer cell line T47-D


1 Department of Animal Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran
2 Department of Cell and Molecular Sciences, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran

Date of Web Publication4-Oct-2019

Correspondence Address:
Mohammad Nabiuni
Kharazmi University, No. 43, South Mofatteh Ave., Postal Code: 15719-14911, Tehran
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_990_17

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 > Abstract 


Background: Breast cancer accounts for one-third of cancer cases in women. Doxorubicin (Dox) is one of the chemotherapeutical compounds widely used to treat breast cancer. Chemical drugs have several side effects and their continuous administration leads to drug resistance in patients. To decrease such side effects in cancer treatment, combination therapy as well as application of natural and herbal compounds has been taken into consideration. The aim of this study was to investigate the cytotoxic effect of Viola odorata (Vo) extract on T47-D human breast cancer cells, alone and in combination with Dox.
Materials and Methods: The cytotoxic effects of V. odorata and Dox were studied by morphological examination and 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Flowcytometric analysis was performed to determine the type of cell death. Moreover, scratch healing assay was conducted to investigate antimigration effect of V. odorata.
Results: The results of MTT assay showed that V. odorata and Dox-induced cell death in T47-D cells in a dose- and time-dependent manner. Morphological analysis revealed that V. odorata and Dox-induced features of apoptotic cell death in T47-D cells. These results were confirmed by flow cytometry analysis. Scratch healing assay revealed that migration rate was reduced in the V. odorata- treated cells.
Conclusions: Our findings suggest that components of V. odorata exert antitumor effects on human breast cancer and could be administered with lower doses of antitumor agent Dox, in combination therapy, to decrease its side effects.

Keywords: Apoptosis, breast cancer, combination therapy, doxorubicin, Viola odorata


How to cite this article:
Zeinoddini S, Nabiuni M, Jalali H. The synergistic cytotoxic effects of doxorubicin and Viola odorata extract on human breast cancer cell line T47-D. J Can Res Ther 2019;15:1073-9

How to cite this URL:
Zeinoddini S, Nabiuni M, Jalali H. The synergistic cytotoxic effects of doxorubicin and Viola odorata extract on human breast cancer cell line T47-D. J Can Res Ther [serial online] 2019 [cited 2019 Nov 13];15:1073-9. Available from: http://www.cancerjournal.net/text.asp?2019/15/5/1073/244245




 > Introduction Top


Cancer is the second most common cause of death after cardiovascular diseases worldwide [1] and was responsible for 8.8 million deaths in 2015.[2] Breast cancer accounts for one-third of cancer cases in women, in both developed and developing countries, and it is the second leading cause of cancer-related death in women after lung cancer.[3] In general, a wide variety of risk factors may influence the development of breast cancer including carcinogens, diet, lifestyle, environment, and genetics.[4],[5] In the general population, early age at menarche, late age at menopause, and late age at first full-term pregnancy may increase the risk of breast cancer; however, multiple pregnancies and breastfeeding reduce the risk of breast cancer.[6],[7] Surgery combined with chemotherapy and/or radiotherapy are the common approaches used to treat this type of cancer.[8] These methods have adverse effects on normal tissues during cancer treatment.[9] Furthermore, the increasing incidence of resistance to anticancer drugs is an important issue which is associated with the novel strategies used for breast cancer chemotherapy.[10] To reduce these adverse effects in cancer therapy, alternative medicines and the application of natural and herbal compounds have taken into consideration.[11]

Doxorubicin (Dox), as an anthracycline antitumor antibiotic, is one of the most commonly used anticancer drugs for the treatment of breast, stomach, lung, ovaries, and thyroid cancers as well as leukemia, lymphomas, and multiple myeloma.[10] Although Dox mainly acts as a topoisomerase II poison, it also exhibits the ability to form adducts with DNA. Dox binds to the double-helical DNA with a high affinity, resulting in DNA damage in cancer cells, blocking the division of them, and resulting in cell death.[11],[12] Despite its usefulness in treating cancer cells, its long-term application results in the development of resistant cells and high doses of Dox increase the risk of developing cardiac side effects.[13]

Viola odorata(Vo) Linn. is a medicinal herb of Violaceae family. It is commonly known as sweet violet, garden violet, or common violet, which grows in Western Asia, North Western Africa, and Southern Europe. The flowers of the plant are used as an herbal remedy or food additive in the above-mentioned regions. In traditional medicine, it has been used as a sedative, diuretic, antimicrobial, and anti-inflammatory agent. Moreover, it is used to treat various diseases including respiratory ailments, migraine, menstrual cramps, bronchitis, anxiety, insomnia, hypertension, and digestive disorders. Some compounds have been separated and identified from V. odorata including flavonoids, alkaloid, glycoside, saponins, methyl salicylate, mucilage, and Vitamin C; furthermore, some cyclotides have also been identified.[14],[15],[16],[17],[18],[19],[20]

Combining two or more therapeutic agents to target cancer is a complementary approach in cancer therapy. A large volume of clinical studies have reported the beneficial effects of herbal medicines on treatment and quality of life of cancer patients, when they are used in combination with conventional therapeutics.[21] The aim of this study was to determine the effect of V. odorata extract on T47-D human breast cancer cells. The synergistic effects of V. odorata extract and Dox on T47-D cells were also investigated in this study. We hypothesized that V. odorata induces antiproliferation effect on breast cancer cells and can be used as a suitable complementary herbal medicine to reduce toxic concentration of Dox as well as its undesirable effects.


 > Materials and Methods Top


Reagents

Dox was purchased from Genepharm Co., Iran. RPMI-1640 media, fetal bovine serum (FBS), penicillin-streptomycin, phosphate buffer saline (PBS), and trypsin-ethylenediaminetetraacetic acid ×10 were purchased all from Gibco. 3,(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) powder MTT, dimethyl sulfoxide (DMSO), and trypan blue solution were purchased from Sigma. Annexin V-FITC apoptosis detection kit was obtained from Abcam Company (ab14085).

Cell culture

The human breast cancer T47-D cells were obtained from Iran Pasteur Institute. Cells were cultured in RPMI-1640 supplemented with 10% (v/v) heat-inactivated FBS and 1% antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin). Cells were trypsinized when a confluence reached to 70% and incubated at 37°C in a moist atmosphere of 5% (v/v) CO2.

Morphological changes

Observation of morphological changes of the cells was monitored using phase contrast inverted microscope (at ×400 magnifications) during 48 h of treatment with Dox and V. odorata extract and changes were compared with nontreated cells.

Plant material and extraction procedure

Commercial grade aerial parts of V. odorata L. were harvested from Kurdistan, Iran and authenticated by an expert botanist. The dried materials of V. odorata weighed 100 g were soaked in 700 ml of aqueous methanol (30:70) for a total of 3 days with occasional shaking. The obtained extraction was filtered through a sterile filter paper (Whatman No. 1), and this procedure was repeated twice and the combined filtrate was concentrated by a rotary evaporator under reduced pressure at 35°C–40°C to obtain a dark brown extract with a yield of 64% (w/w). Vo was completely solubilized in sterile PBS to use in the in vitro experiments.

Migration assay

Directional cell migration was studied by an in vitro wound healing assay.[22] T47-D cells were seeded on 6-well tissue culture plates and were grown to monolayer confluence. They were then treated with Vo (1 mg/ml) for 1 h before scratching with a sterile pipette tip through the center of the plate. Images of the wounded areas were captured with an invert microscope at 0, 24, and 48 h after scratching.

3,(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

To determine the cytotoxic effects of Vo and Dox alone and in combination (Vo/Dox), the MTT assay was performed.[23] Briefly, cells were seeded in 24-well plates as 3 × 104/1 ml and incubated for 24 h before treatments of 0.01–10 mg/ml of Vo and 0.001–1 μg/ml of Dox for 24, 48, and 72 h. Up to defined time of treatment, media were removed from each well and 100 μl of MTT solution (5 mg/ml in PBS) was added to each well in the absence of light and the cells incubated for 4 h to allow metabolism of MTT by mitochondrial dehydrogenase to an insoluble formazan product. To dissolve crystals, 1 ml of DMSO was added to each well and absorbance of the dissolved solution was measured at 570 nm. For synergic experiments, the same protocol was performed with 1 and 2 mg/ml of Vo and 0.1 μg/ml of Dox for 24, 48, and 72 h. The absorbance of treated wells was compared with controls (100% survival), and percentage of cell viability was calculated and plotted.

Flow cytometry analysis

The kind of cell death was quantitatively detected by flow cytometry using the Annexin V-FITC/PI apoptosis detection kit according the manufacturer's instructions (ab14085). Briefly, cells were treated with Vo or Dox as well as combination of Vo and Dox for 48 h, washed with PBS, and harvested. Cells were suspended in binding buffer and then were stained with 5 μl of Annexin and 5 μl of PI at room temperature for 5 min in the darkness. The percentage of apoptotic cells was analyzed using a flow cytometer Becton Dickinson fluorescence-activated cell sorting caliber instrument and FCS Express 4 flow cytometry software (De Novo Software Co. Glendale, CA, USA).

Statistical analysis

Data were expressed as the mean ± standard deviation. Statistical analyses were performed with GraphPad Prism 4.0 (GraphPad Software Co. La Jolla, CA, USA) software and FCS Express 4 flow cytometry (De Novo Software Co. Glendale, CA, USA) software. Statistical differences between the means of control and treated cells were assessed using one-way ANOVA and P < 0.05 was considered statistically significant.


 > Results Top


Morphological changes of drug-treated T47-D cancer cells

The morphological changes of the T47-D cells treated with Vo, Dox, and their combination were investigated under light inverted microscope 48 h after implying the treatment. A 48 h treatment of the cells with Vo, Dox, or combination of them resulted in the development of typical features of apoptotic cells such as rounding, membrane shrinking, appearance of apoptotic bodies as well as losing contact with adjacent cells and substrate. These results indicated that Vo and Dox induced apoptotic death in T47-D cells. Cell death rate was higher in the cells treated with the combination of both drugs compared to those measured in the cells treated with each drug, individually, and the control group [Figure 1].
Figure 1: Morphological comparison of treated T47-D cancer cells with Viola odorata and doxorubicin. (a) nontreated cells; (b) treated cells with Viola odorata; (c) treated cells with doxorubicin; (d) treated cells with Viola odorata/doxorubicin. Arrows are showing cell shrinkage and apoptotic bodies (×400)

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Cytotoxicity screening using 3,(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assay

To determine the cytotoxic concentrations of Vo and Dox, MTT cell proliferation assay was performed. The results showed that Vo significantly inhibited the proliferation of the T-47D human breast cancer cells in a dose-dependent manner [Figure 2]a. This result indicated that Vo extract was a stable compound and its antiproliferative effect persisted for 72 h. The IC50 value of Vo was determined 24, 48, and 72 h after treatment [Table 1].
Figure 2: Percentage of cell viability in T47-D cells treated with (a) Viola odorata; (b) doxorubicin; (c) Viola odorata; and doxorubicin during 24, 48, and 72 h. Data are expressed as mean ± standard deviation of three independent experiments. (*P < 0.05, **P < 0.01, and ***P < 0.001)

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Table 1: Half maximal inhibitory concentration (IC50) of Vo and Dox on the human breast cancer cell line (T47-D)

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The results of MTT assay also revealed that Dox induced its effect on T47-D cells in a time- and dose-dependent manner so that higher concentrations of Dox and longer treatment time led to higher cell death rate [Figure 2]b. The IC50 value of Dox on T47-D cells was determined and presented in [Table 1].

To determine the amplifying role of Vo on antiproliferative effect of Dox, T47-D cells were simultaneously treated with Vo/Dox. For synergistic treatment, Dox was used at the concentration of 0.1 μg/ml. MTT results indicated that both 1 and 2 mg/ml concentrations of Vo had a significant multiplier effect on Dox function and combined treatment of the cells with culture medium containing 1 or 2 mg/ml of Vo and 0.1 μg/ml Dox resulted in a higher cell death rate. As presented in [Figure 2]c, cell viability decreased and reached 64% and 34.26% in 1 Vo/0.1 Dox and 2 Vo/0.1 Dox, respectively, compared to that (68.31%) detected in Dox-treated cells after 48 h of treatment. These differences were statically significant (P < 0.05). Synergistic cytotoxic effect of 1 and 2 mg/ml of Vo and 0.1 μg/ml of Dox against T47-D cells lasted longer 48 h so that the viability of the co-treated cells significantly reduced to 48.35% and 11.16%, respectively, relative to that (64%) detected in the 0.1 Dox-treated cells after 72 h of treatment [Figure 2]c. These results confirmed that Vo could increase the cytotoxic effect of Dox at the concentrations lower than its IC50 value.

Flow cytometric analysis of the apoptosis-inducing effects of Viola odorata

To investigate the rate of apoptotic cell death induced by Vo, Dox, and their combination, T47-D cells were subjected to immunohistochemical staining using Annexin V-FITC kit that targets membrane phosphatidylserine. The results indicated that the rates of viability, apoptosis, and necrosis in nontreated T47-D cells were 93.97%, 4.98%, and 1.04%, respectively. However, T47-D cells treated with 1 mg/ml of Vo showed 90.34% viability, 8.03% apoptosis, and 1.63% necrosis, while treatment of T47-D cells with 0.1 μg/ml of Dox resulted in 52.62% viability, 39.53% apoptosis, and 7.86% necrosis [Figure 3]. Co-treatment of T47-D cells with 1 mg/ml of Vo and 0.1 μg/ml of Dox revealed that Vo potentiates the cytotoxic effect of Dox so that the viability rate reduced to 48.88% which was significantly lower than that detected in Dox-treated cells. Interestingly, Vo treatment not only increased cell death but also enhanced the rate of apoptosis and declined the rate of necrosis. As it is shown in [Figure 3]d, the apoptosis and necrosis rates in Vo/Dox-treated cells were 50.8% and 0.37%, respectively [Figure 3]. These findings were comparable with the data presented in [Figure 3]b and [Figure 3]c. The apoptosis and necrosis rates of different treatment groups are illustrated as histograms in [Figure 4]. In accordance with MTT results, the flow cytometry data confirmed that Vo potentiates the anticancer effect of Dox and results in higher cell death rate while applying lower toxic concentrations. This enhancing role of Vo is mainly induced through apoptosis pathway.
Figure 3: Quantitative analysis of apoptosis and necrosis cell deaths in T47-D cells. (a) nontreated cells; (b) 0.1 μg/ml of doxorubicin; (c) 1 mg/ml of Viola odorata; (d) 1 mg/ml Viola odorata/0.1 μg/ml doxorubicin for 48 h

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Figure 4: Percentage of viable cells, apoptotic cells, and necrotic cells in groups treated with Viola odorata, doxorubicin, and combination of them for 48 h

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Viola odorata reduced the migratory and invasive abilities of breast cancer cells


The results of wound healing assay showed that treatment of T47-D cells with 1 mg/ml Vo suppressed both migration and invasion of the cells compared to the control group after the 48 h of treatment so that the scratched area was filled with nontreated cells in the control group; however, Vo-treated cells could not completely heal the wounded area throughout the 48 h of treatment [Figure 5]. These results showed that Vo not only kills cancer cells, but also it prevents them from migration and metastasis.
Figure 5: The Viola odorata decreases migration of T47-D breast cancer cells. (a) untreated cells; (b) 1 mg/ml Viola odorata-treated cells (×400)

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 > Discussion Top


Dox is one of the anticancer drugs which is widely used to treat various types of cancer. However, it has significant side effects such as cardiac toxicity and drug resistance. Current researches have mainly focused on combination therapy as well as application of natural and herbal compounds to reduce side effects of the chemical drugs.[10],[11] In the current study, we investigated the inhibitory activity of V. odorata against T47-D human breast cancer cells as well as its synergistic effect on Dox in T47-D cells. Morphological observations showed that typical features of apoptosis were clearly discerned in the cells treated with V. odorata compared to the untreated cells, suggesting the possible role of Vo in inducing apoptosis and growth arrest which was confirmed by PI staining assay. The results of MTT assay revealed that V. odorata significantly suppressed the proliferation of T47-D cells in a dose- and time-dependent manner; furthermore, it increased the antiproliferative effect of low doses of Dox. Our results confirmed that V. odorata extract not only had antiproliferation effect and could inhibit the proliferation of T47-D cell, but also it potentiated the cytotoxic effect of Dox which is important for combination therapy purposes, whose aim is to reduce the dose of chemical drugs to decrease their side effects.[24]

To clarify the mechanism underlying Vo-induced cell death, flow cytometry assay was performed. The results of flow cytometry revealed that Vo-induced cell death was mainly through apoptosis pathway; furthermore, it increased the lethal effects of lower doses of Dox mainly through apoptosis. These effects of V. odorata were comparable with those of some other herbal compounds such as PectaSol and Citrus aurantifolia. PectaSol potentiates the cytotoxic effect of Dox through inducing apoptosis and arresting the DU-145 cells cycle at sub-G1 stage.[25] Moreover, C. aurantifolia enhances the rate of apoptosis and cell accumulation at G2/M phase in MCF-7 cells in the presence of Dox.[26] Combined treatment of T-47D cells with Vo and Dox showed that Vo increased apoptosis while decreased necrosis. This result is important since apoptotic cell death is crucial in cancer treatment, especially because it inhibits inflammation and also reduces undesirable effects compared to necrotic cell death.[27]

Tumor metastasis is the main cause of cancer-related mortality worldwide.[28] The results of cell migration assay confirmed the antimetastatic effect of Vo so that the migration rate and wound healing ability of T47-D cells were significantly reduced at 1 mg/ml concentration of Vo. This effect can be explained by various mechanisms such as the role of Gβγ in preventing metastasis of breast cancer cells,[22] crosstalk of NF-κB through p53-ROS to delay the migration of breast cancer cells followed by subsequent inhibition of MMP-2 and MMP-9 gene expression,[29] the role of junctional adhesion molecule-A (JAM-A) through β1-integrin and RAP1 GTPase in reducing adhesion and migration of breast cancer cells,[30] and the role of NOTCH1 in tumor dissemination, metastasis, and proliferation,[31] which may all be related to the antimigratory effect of Vo.

Ireland et al. identified thirty cyclotides from the aerial parts and roots of V. odorata which have a wide variety of biological activities including insecticidal, uterotonic, antimicrobial, antifungal, HIV inhibition, antineurotensive, antitumor, and hemolytic activity.[10],[20] Lindholm et al. investigated the cytotoxic activities of three naturally cyclotides isolated from the two violets, Viola arvensis Murr. and V. odorata L. on breast, ovary, and leukemia cancer-related cell lines. All three cyclotides including varv A, varv F, and cycloviolacin O2 (CyO2) exhibited strong cytotoxic activities, but CyO2 was the most potent in all cell lines.[32] Gerlach et al. investigated the cytotoxic effect of CyO2 applied alone or in combination with Dox. CyO2 caused a dose-dependent cytotoxic effect on MCF-7 and MCF-7/ADR cells but not in primary human brain endothelial cells. They presumed that CyO2, by disrupting cellular membrane and forming multimeric pores with channel-like activity, increases uptake of Dox into drug-resistant MCF7/ADR cells.[10] Cytotoxic effects of CyO2 and its mode of action also were examined by Gunasekera et al. in human lymphoma U-937 GTB cell line. Their observations showed that CyO2 caused the disturbance of membrane integrity in cancerous cells.[33] As CyO2 is one of the major compounds in V. odorata extract which we tested its effects on T47-D cells, it seems that V. odorata exhibits anticancer effects and potentiates the lethal effect of Dox on T47-D cells in such ways.


 > Conclusions Top


The results of this study indicated that V. odorata has inhibitory effects against breast cancer cells. Furthermore, in the light of our findings, V. odorata may be considered as a noticeable herbal compound to be used in combination therapy to improve anticancer effects of Dox and decrease its side effects by reducing its therapeutic dose.

Acknowledgment

The authors would like to acknowledge the supports of the Faculty of Biological Sciences of Kharazmi University for this research.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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