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. 2017 Oct-Dec;13(52):702-706.
doi: 10.4103/0973-1296.218113. Epub 2017 Nov 13.

Induction of Apoptosis by Tithonia diversifolia in Human Hepatoma Cells

Min-Ren Lu  1 , Huey-Lan Huang  2 , Wen-Fei Chiou  1   3 , Ray-Ling Huang  4
Affiliations

Induction of Apoptosis by Tithonia diversifolia in Human Hepatoma Cells

Min-Ren Lu et al. Pharmacogn Mag. 2017 Oct-Dec.

Abstract

Background: Traditional Chinese herb Tithonia diversifolia, belonging to the Compositae family, has long been applied for the treatment of liver diseases. In recent years, many reports also indicated that it possesses hepato-protective, anti-inflammatory, and anti-cancer activities.

Objective: In this study, we evaluated whether T. diversifolia is an effective therapy for hepatocellular carcinoma (HCC).

Materials and methods: Dry leaves of T. Diversifolia were first extracted in ethyl acetate, then further fractionated by different ratio of n-hexane-ethyl acetate (8:2→0:1) or methanol as fractions 1-6 (Td-F1 to Td-F6), respectively. We first showed that the ethyl acetate extracts of T. diversifolia leaves (Td-L-EA) exhibits growth inhibition on human hepatoma HepG2 cells. To further check the extracts-induced apoptosis, microscopic observation, fragmented chromosomal DNA electrophoresis, apoptotic DNA-detection ELISA assay, flow cytometry, and Western blot analysis were performed.

Results: After isolating the effective fractions from Td-L-EA, we found strong cytotoxic effects of fraction-2 (Td-F2). By further analyzing the mechanisms of cytotoxic activities using microscopic observation, fragmented chromosomal DNA electrophoresis, apoptotic DNA-detection ELISA assay, and flow cytometry, we found that induction of apoptosis such as DNA fragmentation increased the apoptosis rate and the apoptosis sub-G1 populations in Td-F2-treated HepG2 cells. In addition, we also confirmed Td-F2-induced degradation of caspase-8, caspase-9, caspase-3, and caspase-3 substrate PARP. Besides, Td-F2 also increased the Bcl-2 proapoptotic family protein Bax expression.

Conclusion: In short, our results clearly showed the induction of apoptosis by ethyl acetate extracts of T. diversifolia leaves in human hepatoma HepG2 cells, suggesting its potential application as an antitumor agent.

Summary: T. Diversifolia leaves were first extracted in ethyl acetate, then further fractionated by different ratio of n-hexane/ethyl acetate (8:2→0:1) or methanol.These extracts exhibit growth inhibition on human hepatoma (HCC) HepG2 cells.n-Hexane/ethyl acetate (6:4) extract (Td-F2) induces apoptosis of HCC. Abbreviations used:T. diversifolia, Tithonia diversifolia; HCC, Hepatocellular carcinoma DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; HRP, horseradish peroxidase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; OD, optical density; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; PARP, Poly (ADP-ribose) polymerase; PBS, phosphate buffered saline; PI, propidium iodide.

Keywords: Tithonia diversifolia; apoptosis; caspase; hepatocellular carcinoma.

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Conflict of interest statement

There are no conflicts of interest.

Figures

Figure 1
Figure 1
Preparation of T. diversifolia extracts. Dry leaves of T. diversifolia were first extracted with ethyl acetate as Td-L-EA fraction after subjecting to freeze-drying process. To fractionate, Td-L-EA powder was then dissolved in n-hexane-ethyl acetate (8:2→0:1) or methanol (Td-F1 to Td-F6) and chromatographed as described in “Materials and Methods” section.
Figure 2
Figure 2
Cytotoxic activity of Td-F2 extracts in human hepatoma cells. HepG2 hepatoma cells were left untreated, treated with 0.25% DMSO (solvent control), various concentrations of Td-F2 (A) or Td-L-EA extracts (B) as indicated or 10 μM camptothecin (positive control) for 48 h, and the viability of cells was determined by MTT assay. Mean OD values of untreated control were calculated as 100% viability. ***P < 0.001 (t-test) as compared to untreated control
Figure 3
Figure 3
Morphology of Td-F2 extracts-treated human hepatoma cells. HepG2 hepatoma cells were left untreated, treated with 0.25% DMSO (solvent control), various concentrations of Td-F2 extracts as indicated for 24 h or 10 μM camptothecin (positive control) for 48 h. Cell morphology was observed by microscopy
Figure 4
Figure 4
Induction of fragmented chromosomal DNA by Td-F2 extracts in hepatoma cells. HepG2 hepatoma cells were left untreated, treated with DMSO (solvent control), various concentrations of Td-F2 extracts as indicated, positive control podophyllotoxin 20 μg/mL (Pod) or 10 μM camptothecin (Cam) for 24 h. Cells were trypsinized, fixed, and stained with PI. Cell cycle distribution was analyzed by flow cytometry as described in “Material and Methods” section. Percent G0/G1 cells include cells with 2N chromosomal DNA and with DNA less than 2N (sub-G1)
Figure 5
Figure 5
Induction of apoptosis by Td-F2 extracts in hepatoma cells. HepG2 cells were left untreated, treated with DMSO, various concentrations of Td-F2 extracts, or positive control 10 μM camptothecin (Cam) podophyllotoxin 20 μg/mL (Pod) for 24 h. After cells were lyzed, chromosomal DNA inside cells was purified and analyzed by agarose electrophoresis (A) or cells with apoptotic DNA (cytosolic histone-bound DNA fragments) were analyzed by the Cell Death Detection ELISAPLUS Kit (B). **P < 0.01 and ***P < 0.001 (t-test) as compared with untreated control
Figure 6
Figure 6
Involvement of apoptosis-related proteins in Td-F2-induced apoptosis in human hepatoma cells. After treatment with DMSO or various concentrations of Td-F2 extracts for 24 h, HepG2 cell lysates were collected and subjected to 12.5% SDS-PAGE. Immunoblotting was done using antibodies against caspase-8, -9, -3, PARP, Bax, Bcl-xL, and actin as indicated
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