Nutr Res Pract.  2016 Aug;10(4):393-397. 10.4162/nrp.2016.10.4.393.

Therapeutic effects of dihydroartemisinin and transferrin against glioblastoma

• Kim SH
• Kang SH
• Kang BS

• KMID: 2390141
• DOI: http://doi.org/10.4162/nrp.2016.10.4.393

Abstract

BACKGROUND/OBJECTIVES
Artemisinin, a natural product isolated from Gaeddongssuk (artemisia annua L.) and its main active derivative, dihydroartemisinin (DHA), have long been used as antimalarial drugs. Recent studies reported that artemisinin is efficacious for curing diseases, including cancers, and for improving the immune system. Many researchers have shown the therapeutic effects of artemisinin on tumors such as breast cancer, liver cancer and kidney cancer, but there is still insufficient data regarding glioblastoma (GBM). Glioblastoma accounts for 12-15% of brain cancer, and the median survival is less than a year, despite medical treatments such as surgery, radiation therapy, and chemotherapy. In this study, we investigated the anti-cancer effects of DHA and transferrin against glioblastoma (glioblastoma multiforme, GBM).
MATERIALS/METHODS
This study was performed through in vitro experiments using C6 cells. The toxicity dependence of DHA and transferrin (TF) on time and concentration was analyzed by MTT assay and cell cycle assay. Observations of cellular morphology were recorded with an optical microscope and color digital camera. The anti-cancer mechanism of DHA and TF against GBM were studied by flow cytometry with Annexin V and caspase 3/7.
RESULTS
MTT assay revealed that TF enhanced the cytotoxicity of DHA against C6 cells. An Annexin V immune-precipitation assay showed that the percentages of apoptosis of cells treated with TF, DHA alone, DHA in combination with TF, and the control group were 7.15 ± 4.15%, 34.3 ± 5.15%, 66.42 ± 5.98%, and 1.2 ± 0.15%, respectively. The results of the Annexin V assay were consistent with those of the MTT assay. DHA induced apoptosis in C6 cells through DNA damage, and TF enhanced the effects of DHA.
CONCLUSION
The results of this study demonstrated that DHA, the derivative of the active ingredient in Gaeddongssuk, is effective against GBM, apparently via inhibition of cancer cell proliferation by a pharmacological effect. The role of transferrin as an allosteric activator in the GBM therapeutic efficacy of DHA was also confirmed.

Keyword

Gaeddongssuk; artemisia annua L.; C6 cell; glioblastoma; dihydroartemisinin

MeSH Terms

Annexin A5
Antimalarials
Apoptosis
Brain Neoplasms
Breast Neoplasms
Cell Cycle
Cell Proliferation
DNA Damage
Drug Therapy
Flow Cytometry
Glioblastoma*
Immune System
In Vitro Techniques
Kidney Neoplasms
Liver Neoplasms
Therapeutic Uses*
Transferrin*
Annexin A5
Antimalarials
Therapeutic Uses
Transferrin

Figure

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  Fig. 1 Effects of dihydroartemisinin (DHA) and holotransferrin (TF) on proliferation of C6 cells. DHA and TF efficacy in the inhibition of C6 cell proliferation were measured by MTT assay after 12 or 24 hrs from administration. It is shown that the inhibition efficacy of the combination of DHA and TF depends on exposure time as well as the concentration. P-value by paired t-test of DHA and DHA+TF.

• 

  Fig. 2 Effects of dihydroartemisinin (DHA) and holotransferrin (TF) on the cell cycle of C6 cells. Analysis of cell cycle assay with PI labeling was accomplished to quantify the percentage of C6 cells by phases. DNA fragmentation in apoptotic cells translates into the fluorescence intensity in sub-G0/G1 phase, and it is lower than that of G0/G1 cells. P-value by t-test of DHA and DHA+TF.

• 

  Fig. 3 Flow cytometry analysis of Annexin V in dihydroartemisinin (DHA) and/or holotransferrin (TF) treated C6 cells. Because the decrement in cell proliferation came with the induction of apoptosis, we investigated DHA-induced proliferation inhibition of C6 cells. The percentage of apoptotic cells was determined by FITC-labeled annexin V and PI double-staining flow cytometry. P-value by t-test of DHA and DHA+TF.

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  Fig. 4 In vitro effects of dihydroartemisinin (DHA) and holotransferrin (TF). The morphological differences in induction of mitotic catastrophe by DHA only or DHA with TF could be observed by optical microscopy. Scale bar = 86.77 µm

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  Fig. 5 Flow cytometry of caspase 3/7 of dihydroartemisinin (DHA) and/or holotransferrin (TF) treated C6 cells. The results of caspase 3/7 immunoprecipitation assay was consistent with the result of the annexin V assay. P-value by t-test of DHA and DHA+TF.

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