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ORIGINAL ARTICLE
Year : 2014  |  Volume : 10  |  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


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 Publication9-Jan-2015

Correspondence Address:
Payman Zare
Department of Pathobiology, Faculty of Veterinary Medicine, University of Tabriz, Tabriz
Iran
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Source of Support: University of Tabriz, Faculty of Veterinary Medicine, Conflict of Interest: None


DOI: 10.4103/0973-1482.137975

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 > Abstract 

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.

 > Abstract in Chinese 

青蒿素、铁、咪康唑和丁酸对5637(膀胱癌)和4T1(乳腺癌)细胞株抗癌作用的调查

摘要

背景:青蒿素及其衍生物的抗癌特性,已在许多实验中被证明。

目的:将丁酸、咪康唑和铁加入到(青蒿素)这一传统药物,以提高其抗癌效力。

材料与方法:将5637和4T1细胞株进行培养,分为13组,每组3株。不同剂量青蒿素加上恒定剂量的铁、咪康唑和丁酸,被加入到培养基。在暴露结束时进行病理和酶的研究。

结果:四组用不同剂量的青蒿素和铁处理,观察剂量依赖性变化,这些变化还包括细胞凋亡和坏死。上清液中乳酸脱氢酶(LDH)水平呈剂量依赖性增加,但细胞碎片中丙二醛(MDA)或LDH没有显著增加。在其他四组,接受咪康唑、丁酸,加入不同剂量铁的青蒿素,见细胞坏死较凋亡更为显著,而MDA水平没有明显改变,但LDH升高。咪康唑处理组表现出相同的变化,但比联合治疗组的严重程度小。丁酸处理组,检测到的变化只有轻度细胞肿胀,很少的细胞凋亡和难得的坏死。

结论:青蒿素联合疗法可以比单独用更有效的对抗癌细胞。丁酸对癌细胞不是很有效。咪康唑可使天然的细胞由凋亡到坏死而死亡,应谨慎使用。


关键词:4T1细胞株,5637细胞株,青蒿素,丁酸,咪康唑


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 Top


Anticancer properties of pure artemisinin and its derivatives and different combination therapies with it are shown in many in vivo and in vitro experiments, [1],[2],[3] 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. [4] 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. [2] 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 [5] suggested that butyric acid induces differentiation and suppresses proliferation in a wide variety of cancer cell lines. [5] In vitro apoptosis induction of butyric acid has been seen in cancerous colonocytes. [6] 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. [7],[8]

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. [9]

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. [10] 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. [11]

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 Top


Cell culture

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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Drug

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. [12] 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.

Biochemical analysis

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, [13] with bovine serum albumin as the standard. Lactate dehydrogenase activity was measured by the method of Babson and Babson. [14]

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. [15]

Light microscopy

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 analysis

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 Top


Pathology

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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Biochemistry

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 Top


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. [16],[17],[18],[19] 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. [20] 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. [12] Furthermore, artemisinin can act as an anti-angiogenic, anti-inflammatory, anti-metastasis, and growth inhibitor. [3] Different derivatives of artemisinin are shown to be more potent anticancer agents. [3] 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. [21] Another cell injury marker is lactate dehydrogenase (LDH), which was analyzed in all groups. [22]

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. [9] 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. [8] The results of our study declared that pure butyric acid had no significant effect on the biochemical and histopathological parameters.


 > Conclusion Top


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.

 
 > References Top

1.
Lombard MC, N'Da DD, Breytenbach JC, Kolesnikova NI, Tran Van Ba C, Wein S, et al. Antimalarial and anticancer activities of artemisinin-quinoline hybrid-dimers and pharmacokinetic properties in mice. Eur J Pharm Sci 2012;47:834-41.  Back to cited text no. 1
    
2.
Zhang YJ, Gallis B, Taya M, Wang S, Ho RJ, Sasaki T. pH-Responsive artemisinin derivatives and lipid nanoparticle formulations inhibit growth of breast cancer cells In Vitro and induce down-regulation of HER family members. PloS one 2013;8:e59086.  Back to cited text no. 2
    
3.
Lai HC, Singh NP, Sasaki T. Development of artemisinin compounds for cancer treatment. Invest New Drugs 2013;31:230-46.  Back to cited text no. 3
    
4.
Nudelman A, Ruse M, Aviram A, Rabizadeh E, Shaklai M, Zimrah Y, et al. Novel anticancer prodrugs of butyric acid. 2. J Med Chem 1992;35:687-94.  Back to cited text no. 4
    
5.
Prasad KN. Butyric acid: A small fatty acid with diverse biological functions. Life Sci 1980;27:1351-8.  Back to cited text no. 5
    
6.
Roediger WE, Millard S. Colonocyte metabolism. Gut 1996;38:792-3.  Back to cited text no. 6
    
7.
Niitsu N, Kasukabe T, Yokoyama A, Okabe-Kado J, Yamamoto-Yamaguchi Y, Umeda M, et al. Anticancer derivative of butyric acid (Pivalyloxymethyl butyrate) specifically potentiates the cytotoxicity of doxorubicin and daunorubicin through the suppression of microsomal glycosidic activity. Mol Pharmacol 2000;58:27-36.  Back to cited text no. 7
    
8.
Singh NP, Lai HC. Synergistic cytotoxicity of artemisinin and sodium butyrate on human cancer cells. Anticancer Res 2005;25:4325-31.  Back to cited text no. 8
    
9.
Wu CH, Jeng JH, Wang YJ, Tseng CJ, Liang YC, Chen CH, et al. Antitumor effects of miconazole on human colon carcinoma xenografts in nude mice through induction of apoptosis and G0/G1 cell cycle arrest. Toxicol Appl Pharmacol 2002;180:22-35.  Back to cited text no. 9
    
10.
Monami G, Gonzalez EM, Hellman M, Gomella LG, Baffa R, Iozzo RV, et al. Proepithelin promotes migration and invasion of 5637 bladder cancer cells through the activation of ERK1/2 and the formation of a paxillin/FAK/ERK complex. Cancer Res 2006;66:7103-10.  Back to cited text no. 10
    
11.
duPre SA, Redelman D, Hunter KW Jr. Microenvironment of the murine mammary carcinoma 4T1: Endogenous IFN-gamma affects tumor phenotype, growth, and metastasis. Exp Mol Pathol 2008;85:174-88.  Back to cited text no. 11
    
12.
Shahbazfar AA, Zare P, Mohammadpour H, Tayefi-Nasrabadi H. Effects of different concentrations of artemisinin and artemisinin-iron combination treatment on Madin Darby Canine Kidney (MDCK) cells. Interdiscip Toxicol 2012;5:30-7.  Back to cited text no. 12
    
13.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-75.  Back to cited text no. 13
    
14.
Babson AL, Babson SR. Kinetic colorimetric measurement of serum lactate dehydrogenase activity. Clin Chem 1973;19:766-9.  Back to cited text no. 14
    
15.
Buege JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol 1978;52:302-10.  Back to cited text no. 15
    
16.
Xiao F, Gao W, Wang X, Chen T. Amplification activation loop between caspase-8 and -9 dominates artemisinin-induced apoptosis of ASTC-a-1 cells. Apoptosis 2012;17:600-11.  Back to cited text no. 16
    
17.
Gao W, Xiao F, Wang X, Chen T. Artemisinin induces A549 cell apoptosis dominantly via a reactive oxygen species-mediated amplification activation loop among caspase-9, -8 and -3. Apoptosis 2013;18:1201-13  Back to cited text no. 17
    
18.
Cheng R, Li C, Wei L, Li L, Zhang Y, Yao Y, et al. The artemisinin derivative artesunate inhibits corneal neovascularization by inducing ROS-dependent apoptosis in vascular endothelial cells. Invest Ophthalmol Vis Sci 2013;54:3400-9.  Back to cited text no. 18
    
19.
Liu JJ, Fei AM, Nie RM, Wang J, Li Y, Wang ZY, et al. A new artemisinin derivative SM1044 induces apoptosis of Kasumi-1 cells and its mechanism. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2011;19:607-11.  Back to cited text no. 19
    
20.
Nakase I, Gallis B, Takatani-Nakase T, Oh S, Lacoste E, Singh NP, et al. Transferrin receptor-dependent cytotoxicity of artemisinin-transferrin conjugates on prostate cancer cells and induction of apoptosis. Cancer Lett 2009;274:290-8.  Back to cited text no. 20
    
21.
Thamilselvan S, Byer KJ, Hackett RL, Khan SR. Free radical scavengers, catalase and superoxide dismutase provide protection from oxalate-associated injury to LLC-PK1 and MDCK cells. J Urol 2000;164:224-9.  Back to cited text no. 21
    
22.
Thamilselvan V, Menon M, Thamilselvan S. Selective Rac1 inhibition protects renal tubular epithelial cells from oxalate-induced NADPH oxidase-mediated oxidative cell injury. Urol Res 2012;40:415-23.  Back to cited text no. 22
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1], [Table 2]



 

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