|Year : 2022 | Volume
| Issue : 2 | Page : 273-277
Cytotoxicity and apoptosis assay of novel cyclomyrsinol diterpenes against breast cancer cell lines
Faezeh Rabbani1, Zeinab Yazdiniapour2, Mustafa Ghanadian2, Behzad Zolfaghari2, Melika Maleki1, Fatemeh Shafiee3
1 Pharmacy Student's Research Committee, School of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran
2 Department of Pharmacognosy, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran
3 Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran
|Date of Submission||07-Jun-2020|
|Date of Acceptance||09-Oct-2020|
|Date of Web Publication||21-Jun-2021|
Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Hezar Jarib Ave., Isfahan
Source of Support: None, Conflict of Interest: None
Background: Cyclomyrsinane diterpenes especially those extracted from various Euphorbia species have shown interesting biological properties in recent years. Because of the high prevalence of breast cancer and the challenges ahead in its treatment, the use of these compounds as potential anti cancer agents seem reasonable. Objectives: The aim of the present study was to evaluate the cytotoxic effects of some myrsinane type diterpenoids extracted from Euphorbia sogdiana Popov and determine their induced cell death mechanism. Methods: MTT assay was used to determine the cytotoxicity of six various myrsinane compounds on MCF 7 and 4T1 breast cancer cell lines. Human umbilical vein endothelial cells were used as the normal cell line too. The apoptotic effects of the structure with the most cytotoxic effects were determined using flow cytometry assay in IC50 concentration for 24 h of incubation. Results: Compound (6) showed the most cytotoxic effects with IC50 of about 8 ± 4 and 24 ± 4 μg/mL for MCF 7 and 4T1 cell lines, respectively. Furthermore, the cells treated with 5 and 10 μg/mL of compound (6) for 24 h, showed 37 and 55% of apoptotic cells. Conclusions: These surveyed compounds have the potential to be considered as useful anti-breast cancer agents due to the great cytotoxicity and apoptotic effects against these cancer cells and the fact that there was no significant cytotoxicity on normal cells.
Keywords: Apoptosis, breast cancer, cyclomyrsinol diterpenoids, cytotoxicity, Euphorbia sogdiana Popov
|How to cite this article:|
Rabbani F, Yazdiniapour Z, Ghanadian M, Zolfaghari B, Maleki M, Shafiee F. Cytotoxicity and apoptosis assay of novel cyclomyrsinol diterpenes against breast cancer cell lines. World J Tradit Chin Med 2022;8:273-7
|How to cite this URL:|
Rabbani F, Yazdiniapour Z, Ghanadian M, Zolfaghari B, Maleki M, Shafiee F. Cytotoxicity and apoptosis assay of novel cyclomyrsinol diterpenes against breast cancer cell lines. World J Tradit Chin Med [serial online] 2022 [cited 2022 Aug 8];8:273-7. Available from: https://www.wjtcm.net/text.asp?2022/8/2/273/318904
| Introduction|| |
Approximately 1.67 million new breast cancer cases have been diagnosed in 2012 around the world. Among these, it is interesting that about 1970 cases have been diagnosed in men, so the disease is not just for women.
In recent years, the treatment of breast cancer has challenged due to the lack of effective and safe drugs and for this reason, attempts to find new drugs for its treatment are essential. Various herbal compounds have been evaluated for the treatment of breast cancer and some of them have shown excellent effects. For example, Etoposide extracted from Podophyllum peltatum, vinblastine extracted from Vinca rosea and camptothecin derived from Camptotheca acuminate are chemotherapeutic agents widely used in the treatment of breast cancer and other malignancies. Biological compounds extracted from various species of Euphorbia showed antitumor, antiviral, and cytotoxic activities and can be used as a drug candidate for breast cancer.
Euphorbia species have long been used as traditional treatments for skin diseases, gastrointestinal parasites, and migraine headaches. Furthermore, these species have been used in traditional Iranian medicine for gout and back pain as well as wound healing. Plants belonging to this family showed their biological activity due to the presence of active compounds, especially diterpenes. The significant anticancer effects of diterpenes derived from Euphorbia species have also been shown.
Cyclomyrsinane diterpenes have shown interesting biological properties in recent studies. For example, Euphorbia sogdiana cyclomersinols have shown cytotoxic effects against Jurkat and EJ-138 cells. Furthermore, myrsinane derivatives of E. connata have shown significant cytotoxic effects on breast cancer cell lines such as MDA-MB-231 and MCF-7.
On the other hand, the cytotoxic effects of Euphorbia kopetdaghi cyclomyrsinane against OVCAR-3 and EJ-138 have been demonstrated.
According to the results of recent studies, it seems that myrsinane diterpenes have cytotoxic effects against different cell lines regardless of the type of cancerous tissue. Therefore, investigation the novel six cyclomyrsinane compounds for their cytotoxic and apoptotic effects against 4T1 and MCF-7 was the aim of the present study.
| Materials and Methods|| |
Cells, compounds, and reagents
purified myrsinane structures including 3-Nicotinyl-5, 10, 14,15-tetraacetyl-8-(2-methyl butanoyl)-cyclomyrsinol (1), 3-Nicotinyl-5, 10, 14,15-tetraacetyl-8-isobutanoyl-cyclomyrsinol (2), 3-O-isobutanoyl-5, 8, 10, 14,15-O-pentaacetyl-cyclomyrsinol (3), 3, 5, 10, 14–O-tetraacetyl-8-O isobutanoyl–cyclomyrsinol (4), 3, 5, 10, 14–O-tetraacetyl-8-O-(2'-methyl-butanoyl)–cyclomyrsinol (5), and 14-Desoxo-3 β,5α,7 β, 10, 15 β-O-pentaacetyl-14α-O-benzoyl-10,18-dihydromyrinsol (6) were provided from previous studies as explained below [Figure 1]. 4T1, MCF-7, and human umbilical vein endothelial cell (HUVEC) cell lines were purchased from the National Bank of Iran (Pasteur Institute, Tehran, Iran). Annexin-V-FLUOS Staining Kit was obtained from (Massachusetts, USA) Invitrogen® Company. Finally, cell culture instruments were provided from Biosera, France.
|Figure 1: Chemical structure of various cyclomyrsinane compound investigated in this project|
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Sample preparation for in vitro assays
The extraction of air-dried samples of E. sogdiana and isolation of various compounds was explained previously. Each pure compound was dissolved in 1 μL (10% in water) DMSO and diluted by phosphate-buffered saline (PBS) to produce various concentrations used for biological tests after filtration.
MTT was used to assay the cytotoxic effects of the mentioned structures. Cell suspension with 5 × 104 cells/mL concentration of 4T1, MCF-7, and HUVEC cells in RPMI 1640 (180 μL) was seeded to each well of a 96 well-plate and incubated at 37°C in a CO2 incubator for 24 h and then 20 μL of various concentrations (50, 25, 12.5, 6.25, and 3.125 μg/mL) of compounds was added to wells. After 72 h of incubation, 20 μL of MTT solution (5 mg/mL) was added to each well, and the plate was further incubated for 3 h. Finally, the formazan crystals were dissolved in 150 μl DMSO, and the plate was subjected to absorbance read at 570 nm using a microplate reader.
Flow cytometry analysis
About 5 × 105 cells/well of MCF-7 cells were cultured in a 6-well microplate. After 12 h, the cells were incubated with the IC50 and half of the IC50 concentration of structure with the most cytotoxicity for 24 h and subsequently subjected to flow cytometry analysis. Briefly, all cells were collected via trypsinization, washed with binding buffer (1X), and then incubated with annexin-V-FITC and propidium iodide according to the manufacturer's instructions (Invitrogen, US) for 15-20 min at dark. Finally, the cells were centrifuged at 300 × g and washed using binding buffer (1X), suspended in 200 μl of binding buffer (1X), and analyzed by flow cytometry on a BD FACSCalibur (BD, USA).
At least, three independent experiments and four replicate wells for every structure with various concentrations was performed for cytotoxicity assay. SPSS inc, South Wacker Drive, Chicago, US 23 software was used for statistical analysis. Analysis of variance followed by a suitable post hoc test, according to the result of the homogeneity test, was used to distinguish the differences between groups. PBS-treated cells were considered as negative control and results were expressed as cell viability % ± standard deviation. The significance was assumed as P < 0.05. The IC50 of each structure was determined by drawing the graph of cell survival percent against concentration using GraphPad Prism 7.0 GraphPad Software inc, San Diego, Canada software. Finally, the selectivity index for each compound against two types of cancerous cell lines was calculated by dividing the IC50 value against HUVEC cells to the IC50 of MCF-7 or 4T1 cells.
| Results|| |
Cytotoxicity of myrsinane terpenoids against breast cancer cells
Cytotoxic effects of six myrsinane compounds were evaluated by MTT assay. Analyzing data using SPSS showed that the cytotoxic effects for all compounds are concentration-dependent; increasing in concentration led to more toxic effects either for MCF-7, 4T1, or even the normal cells (HUVECs).
According to the calculated IC50 for each compound against 4T1 and MCF-7, it was shown that the cytotoxicity of all compounds was statistically different for MCF-7 and 4T1. For all myrsinane diterpenes, MCF-7 showed more cytotoxic effects than 4T1; the difference between the cytotoxic of compound (1) to (6) against two cell lines was comparable according to the P values determined as 0.002, 0.038, 0.006, 0.006, 0.007, and 0.021, respectively.
For the 4T1 cell line, there was a significant difference between the cytotoxic effects of compound (6) with other compounds (P = 0.004) and this cyclomyrsinol flavonoid showed the most cytotoxic effects against the 4T1 cell line [Figure 2]a.
|Figure 2: Cytotoxic effects of various cyclomyrsinane diterpenoid against different cell lines. (a) Treatment of 4T1 cells showed significant toxicity for all concentrations of compounds 1–4 and concentrations of 25 and 50 μg/mL for compound 6 toward the negative control (PBS treated cells). (b) Treatment of MCF-7 cells showed significant cytotoxic effects in concentrations of 6.25, 12.5, 25, and 50 μg/mL for compound 5, and all concentrations of compound 6. For other compounds, the significant cytotoxic effects were shown in concentrations of 25 and 50 μg/mL. (c) Treatment of HUVEC cells showed significant cytotoxic effects in concentrations of 12.5, 25, and 50 μg/mL for compound 6 in comparison to the negative control. Data represent the mean percent of three independent experiments of triplicates. Error bars represent SD. Stars showed significant differences of compound 6 toward the negative control. HUVEC: Human umbilical vein endothelial cells, PBS: Phosphate-buffered saline, SD: Standard deviation|
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For the MCF-7 cell line, on the other hand, there was no significant difference between the cytotoxic effects of all compound (P = 0.57); except for compound (6) with the most cytotoxicity which showed statistically significant cytotoxicity in comparison to the other compounds (P = 0.001) [Figure 2]b.
Furthermore, the cytotoxic effects of compound (6) against both cell lines were statistically more than PBS treated cell in all concentration even in the lowest concentration, 3.125 μg/mL (P > 0.05).
Lastly, for HUVEC cells, structures (6) showed the most cytotoxic effects in comparison to the other structure and there was a significant difference between the cytotoxic effects of compound (6) and others in concentrations above 12.5 μg/mL (P < 0.05) [Figure 2]c. However, it was showed significant differences between the cytotoxicity of compound (6) for MCF-7 and HUVECs in similar concentrations.
Finally, based on drawn graphs of concentrations versus cell survival percent, the IC50 for compounds (1) to (6) for MCF-7 was determined as 40 ± 1.5, 39 ± 8, 27 ± 2, 31 ± 4, 31 ± 9, and 8 ± 4 μg/mL, respectively. These data for the 4T1 cell line, on the other hands, for all six compounds were calculated as 22.5 ± 1.5, 32 ± 4, 35 ± 5, 18 ± 8, 19 ± 9, and 12 ± 4 μg/mL, respectively. For the HUVEC cell line, the IC50 value was calculated as 75 ± 5, 98 ± 2, 100 ± 1, 90 ± 4, 75 ± 1.5, and 67 ± 3.5 μg/mL, for compound (1) to (6), respectively. The calculated selectivity index for each breast cancer cell line is summarized in [Table 1].
Cell death mechanism detection of myrsinane terpenoids using flow cytometry
The results of flow cytometry analysis of MCF-7 cells exposed with the concentrations of 5 and 10 μg/mL of compound (6) for 24 h showed that the percent of apoptotic cells increased with increasing concentrations of this myrsinane. In fact, the untreated cells showed 69% of live cells, 30% of apoptotic cells, and approximately 0.01% of necrotic cells. In the case of the cells treated with 5 μg/mL of compound (6) for 24 h, results showed 44% of apoptotic cells, and 0.32% necrotic cells. The percent of live cells was calculated as 55%. Furthermore, the apoptotic and necrotic percent of the cells treated with the IC50 of compound (6) (10 μg/mL) after 24 h of incubation, was showed to be 55 and 2.5%, respectively [Figure 3]. Finally, the percent of live cells in the sample treated with the IC50 concentration of this compound was calculated as 42. These data confirmed the apoptosis of the compound with the most cytotoxic effects [Figure 3].
|Figure 3: Determination of cell death mechanism of compound 6 by flow cytometry after 24 h of incubation. (a) untreated control cells. (b) cells treated with 5 μg/mL of compound 6. (c) cells treated with 10 μg/mL of compound 6. Lower left chamber: Live cells (annexin V-/PI-); Lower right chamber: Early apoptotic cells (annexin V+/PI-); Upper left chamber: Dead cells (annexin V-/PI+); Upper right chamber: Late apoptotic cells (annexin V+/PI+)|
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| Discussion|| |
The aim of the present study was to evaluate the cytotoxic effects of some cyclomyrsinol terpenoids against two breast cancer cell lines, MCF-7, and 4T1. Our results showed that compound (6), has the most cytotoxic effects against both cells and so subjected to determine the cell death mechanism as a good candidate in this category.
Cyclomyrsinol compounds were extracted from other species of Euphorbia. For example, in a study conducted by Ghanadian et al., two new cyclomyrsinol compounds with the similar backbone to compounds (1) to (5) were identified from E. kopetdaghi Prokh and their inhibitory effects against human lymphocyte were investigated. The finding of this study confirmed the anti-proliferative effects of one compound with an isobutanoyl and benzoyl group in R1 and R2 situation on T-cells with an IC50 of 1.83 μg/mL. Furthermore, it was shown that this cyclomyrsinol compound suppressed the IL-2 production significantly in 19 μg/mL concentration. However, the cytotoxic effects of this cyclomyrsinol compound against CC-1 and 3T3-L1 cell lines were ignorable. In another study, three cyclomyrsinol diterpenes which one of them was the compound (4), were extracted from Euphorbia aellenii and their effects on the proliferation of human peripheral blood lymphocytes were investigated. The results of the mentioned study showed that all these three compounds were found to inhibit lymphocyte proliferation at 50 μg/mL concentration. The immunomodulatory effects of compound (4) was more than other compounds.
In Zolfaghari et al. study, the cytotoxic effects of myrsinane (1) to (3) extracted from Euphorbia sogdiana Popov were investigated against Jurkat and EJ-138 cell lines. The results of the mentioned study confirmed the cytotoxicity of these compounds with the IC50 value of about 25–36 and 19–25 μM, for EJ-138 and Jurkat cell lines, respectively. The cytotoxicity of these compounds against breast cancer cells was calculated as similar results, 16–40 μM for both surveyed cell lines.
Our study confirmed the apoptotic effects of compound (6) as a compound with the most cytotoxicity against the MCF-7 cell line. Compound (6) was recognized for the first time by Yazdiniapour et al., but the potential anticancer effects were determined in our study for the first time.
In Shekofteh et al. study, it was shown that the hexane extract of three Euphorbia species including Euphorbia microciadia Boiss, Euphorbia osyridea Boiss, and Euphorbia heteradenia Jaub, induced apoptosis in more than 60% of treated cells in the concentration of 50 μg/mL of all the extract. However, they did not identify the compounds responsible for the induction of apoptosis by the extracts.
In another study, it was shown that Jolkinolide B diterpenoid, isolated from the roots of Euphorbia fischeriana Steud, showed apoptotic effects against various cancer cells. In Wang et al. study, enhancing the apoptosis of U937 cells in the concentration of 50 μg/mL of Jolkinolide was established via downregulation of PI3K/Akt and IAP proteins. Furthermore, the activation of caspase-3 and-9 was also seen in B16F10 cells treated with the same concentration of Jolkinolide B. However, in our study, the apoptosis was seen in concentrations of 5 and 10 μg/mL of (6) diterpenoid against MCF-7 cells.
| Conclusion|| |
This project was the first cytotoxicity evaluation and determination of the cell death mechanism of myrosinase type diterpenoids against two breast cancer cell lines. The cytotoxic effects of compound (6) with different structure to other surveyed compounds was more and this compound showed apoptotic effects against MCF-7 cell line. On the other hand, the suitable selectivity index was calculated especially for compound (6) has the potential to be considered as useful anticancer agents with low adverse effects against normal tissue and cells.
This work was supported by the Pharmacy Student's Research Committee, School of Pharmacy, Isfahan University of Medical Sciences (grant number: 198018). The authors also would like to appreciate the valuable technical assistance of laboratory experts in cell culture labs.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 1]