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Journal of Thai Traditional & Alternative Medicine Vol. 7 No. 2 May-August (Supplement) 2009 ¯Ò

OP-20

Antiproliferation and Apoptosis of Crude Extract of Andrographis paniculata

Nees. on Human Breast Adenocarcinoma Cells (MCF-7) In Vitro

Chantarawan Saengkhae, Keitisak Raksapol

Department of Medical Science, Faculty of Science, Burapha University, Chonburi 20131

Rationale: Cancer is one of the leading causes of death. This has led to the search for more medicinal
plants that might improve the therapeutic efficacy and safety. Andrographis paniculata Nees. has a wide
spectrum of pharmacological effects for centuries and contributes for both anticancer and immunostimulatory
activities. The Andrographis paniculata extract (APE) and its main diterpenoid components have been found to
be able to inhibit cancer cell proliferation, induce cell-cycle arrest and promote apoptosis in different types of
human cancer cell lines. However, there have been very limited studies on the anti-proliferative activities and
the underlying mechanisms of APE in breast cancer.
Objective: The aim of this study is to evaluate the molecular mechanisms for the anti-proliferative effects
of Andrographis paniculata extract in human breast adenocarcinoma cells.
Methodology: The antiproliferative effect of APE against breast cancer cells was determined by colori-
metric assay, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and compared with
untreated control (0.21 % DMSO) and positive control (Doxorubicin). The morphology of apoptotic nuclei was
quantified using double fluorescent staining, DAPI and propidium iodide (PI). Cells were then visualized with
fluorescence microscope. For each treatment group, 200-400 nuclei were counted. Data were expressed as
percentages of fragment nuclei in viable cells. DNA fragmentation was qualitatively analyzed by agarose gel
electrophoresis. After various treatments, floating and adherent cells were incubated with a lysis buffer, then
DNA was extracted. The degree of fragmentation was analyzed using a 1.5% agarose gel electrophoresis
followed by an SYBER gold staining.
Results: APE inhibited cell proliferation in a dose-dependent manner but had less toxicity than doxorubi-
cin. Further, APE-induced cell death was associated with round cells, lost of cell-to-cell contract and fewer
adherent cells when compared with control group. The IC values for APE and Doxorubicin were 75 ± 4.21 μg
50
/ ml and 0.5 ± 0.02 μg / ml respectively. Nuclear morphology of APE-treated cells exhibited chromatin conden-
sation, and nuclear fragmentation as compared to control. Quantitative estimation of apoptotic nuclei in APE-
treated cells (75 μg / ml for 48 hr) was 16.71 ± 4.65 % (normal cell), 65.07 ± 7.18 % (viable cells with apoptotic
nuclei) and 18.21 ± 2.53 % (necrosis or late apoptotic nuclei). The oligonuclesomal DNA fragmentation in
agarose gel was observed in a dose-dependent manner when cells were treated with 30, 75 and 150 μg/ml APE
for 48 hr.
Conclusion: These findings indicated that the APE exhibited the anti-proliferative effects via morphologi-
cal changes, typical demonstration of apoptosis, including membrane blebbing, chromatin condensation, nuclear
and DNA fragmentation. As apoptosis has become a therapeutic target in cancer research, the APE may be a
potential natural remedies or used in combination with synthetic chemotherapeutic compounds to improve
efficacy and decrease side effects in the treatment of breast cancer. However, further in vivo studies are
necessary.