|Year : 2014 | Volume
| Issue : 4 | Page : 1057-1062
A survey on anticancer effects of artemisinin, iron, miconazole, and butyric acid on 5637 (bladder cancer) and 4T1 (Breast cancer) cell lines
Amir Ali Shahbazfar1, Payman Zare1, Mehrdad Ranjbaran1, Hossein Tayefi-Nasrabadi2, Omid Fakhri1, Yashar Farshi1, Sahar Shadi1, Afsaneh Khoshkerdar1
1 Department of Pathobiology, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran
2 Department of Basic Sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran
|Date of Web Publication||9-Jan-2015|
Department of Pathobiology, Faculty of Veterinary Medicine, University of Tabriz, Tabriz
Source of Support: University of Tabriz, Faculty of Veterinary Medicine, Conflict of Interest: None
Context: Anticancer properties of artemisinin and its derivatives have been shown in many experiments.
Aims: Addition of butyric acid, miconazole, and iron to this traditional drug has been done in order to enhance its anticancer potency.
Materials and Methods: Cell lines 5637 and 4T1, were cultivated and classified into 13 groups of three each. Different doses of artemisinin with constant doses of iron, miconazole and butyric acid, were added to the cultures. At the end of exposure pathological and enzymatic studies were performed.
Results: In four groups treated with different doses of artemisinin and iron, dose-dependent changes were observed. These changes included apoptosis and necrosis with dominance of apoptosis. The supernatant lactate dehydrogenase (LDH) level was increased in a dose-dependent manner, but there was no significant increase in the cell fraction of malonyldialdehyde (MDA) or LDH. In four other groups, which received miconazole, butyric acid and iron in addition to different doses of artemisinin, necrosis was more prominent than apoptosis, and the MDA level did not show any significant change, but LDH was increased.
The groups treated with miconazole showed identical changes, with less severity compared to combination therapy groups. In butyric acid-treated groups, the only detectable changes were, mild cell swelling, few apoptosis, and rare necrosis.
Conclusions: A combination therapy with artemisinin can be more effective against cancer cells than monotherapy with that. Butyric acid was not effective on cancer cells. Miconazole deviated the nature of cell death from apoptosis to necrosis and it must be used under caution.
Keywords: 4T1 cell line, 5637 cell line, artemisinin, butyric acid, miconazole
|How to cite this article:|
Shahbazfar AA, Zare P, Ranjbaran M, Tayefi-Nasrabadi H, Fakhri O, Farshi Y, Shadi S, Khoshkerdar A. A survey on anticancer effects of artemisinin, iron, miconazole, and butyric acid on 5637 (bladder cancer) and 4T1 (Breast cancer) cell lines. J Can Res Ther 2014;10:1057-62
|How to cite this URL:|
Shahbazfar AA, Zare P, Ranjbaran M, Tayefi-Nasrabadi H, Fakhri O, Farshi Y, Shadi S, Khoshkerdar A. A survey on anticancer effects of artemisinin, iron, miconazole, and butyric acid on 5637 (bladder cancer) and 4T1 (Breast cancer) cell lines. J Can Res Ther [serial online] 2014 [cited 2020 Mar 28];10:1057-62. Available from: http://www.cancerjournal.net/text.asp?2014/10/4/1057/137975
| > Introduction|| |
Anticancer properties of pure artemisinin and its derivatives and different combination therapies with it are shown in many in vivo and in vitro experiments, ,, but there are no studies on its combination with butyric acid and miconazole. Anticancer effects of butyric acid and miconazole are suggested in a few publications.  According to the short half-life of artemisinin, higher doses must be administered or several re-doses must be given. Artemisinin, a traditional Chinese medicine that is derived from Artemisia annua, is widely used as an anti-malarial agent and its anticancer effects have been reported recently. This compound is a sesquiterpene phytolactone with an endoperoxide bridge (R-O-O-R'), which induces lipid proxidation by forming carbon-based free radicals after reacting with the iron atom.  Artemisinin acts selectively on malarial parasites or cancerous cells, because they have large quantities of iron deposit due to their metabolic activity. Many studies have shown that artemisinin has different cytotoxic effects on various cancer cell lines. This cytotoxicity includes apoptosis and necrosis in different grades.
In this study iron sulfate was added to the combination therapy in order to increase the available iron in the cell media. A high concentration of iron in the media would result in higher iron uptake by cancer cells, which need more iron as a result of increased proliferative activity.
Butyric acid, a product of bacterial anaerobic metabolism in the large intestine, is a short chain fatty acid. Prasad KN  suggested that butyric acid induces differentiation and suppresses proliferation in a wide variety of cancer cell lines.  In vitro apoptosis induction of butyric acid has been seen in cancerous colonocytes.  Regarding the high clearance rate of butyric acid and procrastinated apoptosis, the usage of this agent alone, has shown low effectiveness on neoplastic cells, hence, different derivatives or combinations are substituted. ,
Miconazole is a prominent systemic antifungal medication and used as a common treatment for superficial fungal infection. Wu et al. demonstrated that miconazole causes cell cycle arrest in different human neoplastic cell lines. This growth arrest was dose-dependent and probably related to the p53 signaling pathway. 
Transitional cell carcinoma is classified among the most lethal cancers in the United States. This neoplastic disease is highly invasive because of its metastasis and recurrence.  The 5637 cell line is a suitable model for studying bladder cancer.
The murine mammary epithelial carcinoma cell line (4T1) is a good model of metastatic breast cancer. This neoplasia metastasizes to different organs through blood recirculation in the fourth stage of breast cancer. The progressive growth of 4T1 causes a deadly situation, even after surgical removal. 
The aim of this article was to study the anticancer effects of a combination of artemisinin, miconazole, butyric acid, and iron on the human bladder and murine breast cancer cell lines.
| > Materials and methods|| |
Human 5637 transitional cell carcinoma and murine mammary carcinoma 4T1 lines were obtained from then Roswell Park Memorial Institute (RPMI 1640) without antibiotics, with 10% Fetal Bovine Serum (FBS), and non-essential amino acids from the Pasteur Institute, Tehran, Iran. Then each line was cultivated in 39 25 cm 2 flasks containing the RPMI 1640 medium and 10% FBS, which were classified into 13 groups of three each. Culture media refreshment was done every 72 hours before the cells reached confluence. Then the study began by adding the following agents and incubation for 60 hours [Table 1].
|Table 1: Drug composition and concentration in the treatment groups. Constant doses of miconazole, butyric acid, and iron sulfate are 55 μmol/ml, 3 μmol/ml, and 1 μg/ml |
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Different doses of 99% pure artemisinin, with constant doses of iron sulfate (1 μg/ml), miconazole (55 μmol/ml), and butyric acid (3 μmol/ml), were added to the culture media as follows: A combination of different doses of artemisinin (0.15, 0.3, 0.6, and 1.2 μg/ml) with iron sulfate, miconazole, and butyric acid were added to four groups, and similar doses of artemisinin in combination with iron sulfate were added to another four groups, two treatment groups received a constant dose of artemisinin (0.6 μg/ml) and iron sulfate, one containing miconazole and the other containing butyric acid.  The last two treatment groups received miconazole and butyric acid respectively. One group was considered as the control. The same procedures were conducted for both cell lines.
After 60 hours of incubation, the supernatant was harvested and centrifuged (5000 rpm for five minutes) to precipitate the floating cells. The total protein and LDH of this fluid was measured by the Lowry method,  with bovine serum albumin as the standard. Lactate dehydrogenase activity was measured by the method of Babson and Babson. 
Cell fraction extracts were prepared by repeated cycles of freeze-thawing a mixture of the centrifuged precipitate and half of the cells in the flask' s bottom, which were scraped using a stir rod. These extracts were checked for the presence of any uncleaved cells under light microscopy with a Neubauer slide. Finally, the extracts were centrifuged to precipitate cleaved cells, and the supernatants were used to measure the total protein, lactate dehydrogenase (as described above), and total lipid peroxidation product. In order to measure the lipid peroxidation, MDA was assayed by the thiobarbituric acid reactive substance (TBARs) method. 
During the 60 hours, the cellular changes were monitored and photographed under an inverted microscope every 12 hours. The remaining half of the cells on the flask's bottom were fixed with methanol and stained with hematoxylin-eosin for a microscopic assay at the end of study.
Statistical analyses were performed using SPSS 21.0 (Statistical Package for Social Sciences, SPSS Software 19, IBM Inc., NY, USA). One-way analysis of variance (ANOVA) was used to compare the quantitative data, followed by the Tukey test as post hoc. Statistical significance was accepted at P < 0.05.
| > Results|| |
In the four groups treated with different doses of artemisinin and iron sulfate, dose-dependent pathological changes were observed. These changes included apoptosis and necrosis, with a dominance of apoptosis [Figure 1]. Cell swelling and vacuolation were obvious too [Figure 2]. At the end of exposure with the highest artemisinin dose 10% of the cells were detached from the flask bottom. In four other groups that received miconazole, butyric acid, and iron sulfate, in addition to different doses of artemisinin, severe dose-dependent pathological changes were obvious. In these groups necrosis was more than apoptosis [Figure 3] and [Figure 4]. The highest dose of artemisinin caused 20 and 70% cell detachment in 12 and 60 hours, respectively [Figure 5]. After 48 hours of exposure, the rate of the pathological changes was significantly decreased. Omission of butyric acid from the combination mentioned above (0.6 μg/ml of artemisinin) caused less apoptosis and a similar grade of necrosis, but omission of miconazole caused less necrosis, although apoptosis was decreased too. The group treated with pure miconazole showed identical changes, with less severity, compared to the group that received the full combination (miconazole, iron sulfate, butyric acid, and 1.2 μg/ml artemisinin). In pure butyric acid groups, mild cell swelling, few cases of apoptosis, and rare necrosis were the only detectable changes. The control groups did not show any specific change [Figure 6], except scarce apoptosis. All treatments had approximately the same effect on both cell lines.
|Figure 1: Apoptotic figures in the group 5. Up: 5637 cell line ×800; Down: 4T1 cell line ×2000. Hour 60 ; H and E staining|
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|Figure 2: Vacuolation in the cytoplasm and severe swelling of the cells in group 6 (Artemisinin 0.6μg / ml + Iron sulfate); H and E staining; Hour 60; 5637 cell line ×2000|
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|Figure 3: Necrosis with piknosis of the nuclei in the group 3 (Artemisinin 0.3μg / ml + Miconazole + Butyric acid + Iron sulfate); H and E staining; Hour 60; 4T1 cell line ×800|
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|Figure 4: Severe necrosis with karyorrhexis of the nuclei in group 1 (Artemisinin 1.2 μg / ml + Miconazole + Butyric acid + Iron sulfate); some cells have vacuolation in cytoplasm; H and E staining; Hour 60; 5637 cell line ×800|
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|Figure 5: Swelling and detachment of cells from the bottom of the flask; Invert microscopy; Hour 48; 4T1 cell line; Group 2 (Artemisinin 0.6 ƒÊg / ml + Miconazole + Butyric acid + Iron sulfate); ×400|
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|Figure 6: Control group does not show any specific changes; 5637 cell line; Invert microscopy; Hour 60|
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In the four groups that received iron sulfate and increasing doses of artemisinin, the supernatant LDH level increased significantly in a dose-dependent manner, but there was no significant increase in the cell fraction MDA and LDH levels. In the other four groups treated with iron sulfate, miconazole, butyric acid, and different doses of artemisinin, the MDA levels did not show significant changes, but artemisinin dose enhancement in this combination increased the LDH levels in both the supernatant and cell fraction extracts. Addition of miconazole and butyric acid to artemisinin-iron combinations caused a significant increase of all analytes. Omission of miconazole from all reagent combinations caused a statistically significant decrease in concentration of the supernatant LDH, but omission of butyric acid had no significant effect on LDH. Pure miconazole treatment groups showed a significant increase in all parameters compared to the control group. Supernatant LDH had a statistical difference in all treatment groups compared to the control group except for the pure butyric acid group. Significant difference in cell fraction LDH was obvious in all groups with control, pure butyric acid, and groups with the lowest dose of artemisinin-iron combination. The MDA level increase caused a significant change in all groups that received miconazole, compared to the control group [Table 2].
|Table 2: Biochemical analysis of lactate dehydrogenase and malonyldialdehyde in cell culture medium and cell fraction. Data are displayed as mean±standard error of mean |
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| > Discussion|| |
The endoperoxide moiety in the chemical structure of artemisinin makes it capable of generating intracellular free radicals. The reaction between the artemisinin monomer and cytoplasmic free iron forms organic free radicals that disturb oxidation and reduce the equilibrium, which leads to reactive oxygen species (ROS) formation that causes caspase-dependent apoptosis or cell injury in a wide variety of cancerous cell lines. ,,, Artemisinin-selective activity on cancer cells is thought to be the result of a higher iron uptake (due to the overexpressed transferrin receptors (TfR)) to be used in the enhanced proliferation activity.  In the current research, the addition of iron sulfate to the combination was done in order to increase the cytotoxic effects of artemisinin. The synergistic effect of iron sulfate and artemisinin has been described previously.  Furthermore, artemisinin can act as an anti-angiogenic, anti-inflammatory, anti-metastasis, and growth inhibitor.  Different derivatives of artemisinin are shown to be more potent anticancer agents.  In the current study, the artemisinin monomer has been used to check its antitumor activities in different doses and combinations.
Artemisinin-derived free radicals interact with membrane fatty acids, leading to MDA formation. Hence, MDA is defined as the standard indicator of lipid peroxidation.  Another cell injury marker is lactate dehydrogenase (LDH), which was analyzed in all groups. 
No studies on artemisinin combination therapy with miconazole, iron sulfate, and butyric acid have been found, however, the results of this study show that the additional compounds mentioned above can reduce the effective dose of artemisinin and increase its cytopathic effects against 4T1 and 5637 cancer cell lines.
The accuracy and reproducibility of our histopathological results were proved by repeating the test thrice.
Both biochemical and histopathological investigations showed that the cytopathic effects of artemisinin were dose-dependent. These changes were boosted by adding miconazole and butyric acid, which was in agreement with our goal in this study. Wu et al. demonstrated that miconazole arrested various human cancer cells at the G0/G1 phase of the cell cycle. This effect was dose-dependent and they suggested that the p53-associated signaling pathway was the major mechanism in the regulation of miconazole-induced cancer cell growth inhibition. They used deoxyribonucleic acid (DNA) fragmentation and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and concluded that miconazole treatment caused apoptosis in human colon carcinoma tumor tissues.  In our study the dominance of pathological changes deviated from apoptosis to necrosis following miconazole administration. The presence or absence of miconazole or butyric acid in different groups clarified the role of these two agents and showed that miconazole played the main role in causing necrosis and of course biochemical changes.
Also, it was shown that 1 mM sodium butyrate alone caused 32% Molt-4 cell (a human lymphoblastoid leukemia cell line) death after 24 hours of exposure and when combined with 20 μM dihydroartemisinin killed all Molt-4 cells in 24 hours, but did not significantly affect the normal lymphocytes.  The results of our study declared that pure butyric acid had no significant effect on the biochemical and histopathological parameters.
| > Conclusion|| |
The results of this study showed that a combination therapy with artemisinin, iron, miconazole, and butyric acid was more effective than a therapy with artemisinin and iron alone. We did not find butyric acid to be effective on cancer cells; miconazole deviated the nature of cell death from apoptosis to necrosis and its usage should be done under caution.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2]