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Anticancer effects of 1,3-dihydroxy-2-methylanthraquinone and the ethyl acetate fraction of hedyotis diffusa willd against HepG2 carcinoma cells mediated via apoptosis

PLoS ONE (2016) 11(4)
DOI: 10.1371/journal.pone.0151502
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Abstract

Hedyotis Diffusa Willd, used in Traditional Chinese Medicine, is a treatment for various diseases including cancer, owing to its mild effectiveness and low toxicity. The aim of this study was to identify the main anticancer components in Hedyotis Diffusa Willd, and explore mechanisms underlying their activity. Hedyotis Diffusa Willd was extracted and fractionated using ethyl acetate to obtain the H-Ethyl acetate fraction, which showed higher anticancer activity than the other fractions obtained against HepG2 cells with sulforhodamine B assays. The active component of the H-Ethyl acetate fraction was identified to be 1,3-dihydroxy-2-methylanthraquinone (DMQ) with much high inhibitory rate up to 48.9 ± 3.3% and selectivity rate up to 9.4 ± 4.5 folds (p<0.01) at 125 μmol/L. HepG2 cells treated with the fraction and DMQ visualized morphologically using light and fluorescence microscopy. Annexin V-fluorescein isothiocyanate/propidium iodide staining flow cytometry, DNA ladder and cell cycle distribution assays. Mechanistic studies showed up-regulation of caspase-3,-8, and-9 proteases activities (p<0.001), indicating involvement of mitochondrial apoptotic and death receptor pathways. Further studies revealed that reactive oxygen species in DMQ and the fraction treated HepG2 cells increased (p<0.01) while mitochondrial membrane potential reduced significantly (p<0.001) compared to the control by flow cytometry assays. Western blot analysis showed that Bax, p53, Fas, FasL, p21 and cytoplasmic cytochrome C were up-regulated (p<0.01), while Bcl-2, mitochondrial cytochrome C, cyclin E and CDK 2 were down-regulated dose-dependently (p<0.01). The reverse transcriptase-polymerase chain reaction showed that mRNA expressions of p53 and Bax increased (p<0.001) while that of Bcl-2 decreased (p<0.001). Pre-treatment with caspase-8 inhibitor Z-IETD-FMK, or caspase-9 inhibitor Z-LEHD-FMK, attenuated the growth-inhibitory and apoptosis-inducing effects of DMQ and the fraction on HepG2 cells. These results suggested that DMQ and the H-Ethyl acetate fraction of Hedyotis Diffusa Willd showed potential anticancer effects. Furthermore, the mechanisms of action may involve mitochondrial apoptotic and death receptor pathways.

Figures

  • Table 1. Primers Used for RT-PCR.
  • Fig 1. The Anticancer Effects of H-EtOAc Fraction and DMQ. A. The inhibitory effects of four fractions on HepG2 cells at 500 μg/mL for 24 h. B. The inhibitory effects of six indentified components on HepG2 cells at gradient concentrations (0, 5, 50, 125, 250 and 500 μmol/L) for 24 h (the inhibitory effect of geniposidic acid group was the control). C. The selectivity rates of two high anticancer components between HepG2 and HL7702 cells (the selectivity of quercetin was the control). D. HepG2 cells were treated with varying concentrations of PFT-α (0–80 μmol/L) for 24 h and cell survival was evaluated by SRB assay (0 μmol/L PFTα group was the control). E. Viability of HepG2 cells were treated with/without indicated concentration of DMQ and H-EtOAc fraction plus PFT-alpha for 24 h by SRB assay (the group of 20 μmol/L PFT-αwithout DMQ and H-EtOAc fraction treatment was the control). F. RT-PCR results of p53, p21and β-actin in HepG2 cells. G. mRNA levels of p53 and p21 in HepG2 cells. Line 0: the control (without PFT-α, DMQ and H-EtOAc fraction). Line 1: PFT-α (20 μmol/L) without DMQ and H-EtOAc fraction. Line 2: DMQ (50μmol/L) and PFT-α (0 μmol/L). Line 3: H-EtOAc fraction (100 μg/ml) and PFT-α (0 μmol/L) Line 4: DMQ (50μmol/L) and PFT-α (20 μmol/L). Line 5: H-EtOAc fraction (100 μg/ml) and PFT-α (20 μmol/L). The strength ofmRNA expression signal were analyzed by scanning densitometry using a Microtek ScanMaker 8700 (Zhongjing Inc. China) with ScanWizard 5 software. Values are means ± SD of three independent experiments. * (p<0.05), ** (p<0.01) and *** (p<0.001) represented significant differences compared to the control.
  • Table 2. In vitro antitumor activity of H-EtOAc fraction against six human tumors ( x ±SD).
  • Fig 2. The Morphology Changes and apoptosis rates of H-EtOAc fraction and DMQ treated HepG2 cells. A(a)-(c). The morphology changes of HepG2 cells treated with gradient H-EtOAc fraction for 24 h using a visible light under light microscope for 24 h. A (d)-(f). The morphology changes of HepG2 cells treated with DMQ for 24 h. A visible light was used under light microscope (20 × magnification). B(a)-(c). Morphological observation of fluorescence microscope (10 × magnification) for 24 h. B(d)-(f). Morphological observation of fluorescence microscope (40 × magnification) for 24 h. A green excitation wavelength (460—495nm) and emission wavelength 510nm filter was used by staining with AO/EB. C(a). Apoptosis was measured on HepG2 cells treated with gradient DMQ for 24 h. C(b). Apoptosis was measured on HepG2 cells treated with H-EtOAc fraction for 24 h. Values are means ± SD of three independent experiments. * (p<0.05), ** (p<0.01) and *** (p<0.001) represented significant differences compared to the control.
  • Fig 3. Apoptosis Induced by DMQ and H-EtOAc fraction of HepG2 Cells. A. Apoptosis was measured on HepG2 cells treated with gradient DMQ (a)-(d) and H-EtOAc fraction (e)-(g) by flow cytometry. B. The apoptosis rates were calculated of DMQ (a) and H-EtOAc fraction (b) on HepG2 cells. C. DNA ladder. Lane 1: Control; Lane 2–4: 79 μmol/L at 24 h, 157 μmol/L at 12 h and 157 μmol/L at 24 h for DMQ; Lane 5: 400 μg/mL at 24 h for H-EtOAc fraction; Lane 6: Negative group. D. The activities of caspase-3, -8, -9 proteases of HepG2 cells treated with DMQ (a) and H-EtOAc fraction (b). Values are means ± SD of three independent experiments. * (p<0.05), ** (p<0.01) and *** (p<0.001) represented significant differences compared to the control.
  • Fig 4. Mitochondrial Apoptotic Pathway Related to DMQ and H-EtOAc Fraction Treated HepG2 Cells. A. The MMP changes of HepG2 cells treated with DMQ (a) and H-EtOAc fraction (b). B. The production changes of intracellular ROS in HepG2 cells treated with DMQ (a) and H-EtOAc fraction (b). C. Western blot analysis of proteins expression levels of p53, Bax, Bcl-2, mitochondrial cyto C, cytoplasmic cyto C and β-actin. Lane 1: Control; Lane 2–4: 79, 157 and 315 μmol/L of DMQ for 24 h, respectively; Lane 5: 250 μg/mL of H-EtOAc fraction for 24 h. D. The protein expression levels of p53 (a) Bax (b) Bcl-2 (c) the ratio of Bax/Bcl-2 (d) mitochondrial cyto C (e) cytoplasmic cyto C (f) and the ratio of cytoplasmic cytoC/mitochondrial cyto C (g). The protein expression strength were analyzed by scanning densitometry using a Microtek ScanMaker 8700 (Zhongjing Inc. China) with ScanWizard 5 software. E. RT-PCR results
  • Fig 5. Death Receptor Pathway Related to HepG2 Cells Treated with DMQ and H-EtOAc Fraction. A. Western blot analysis of FasL and Fas. Lane 1: Control; Lane 2–4: 79, 157 and 315 μmol/L of DMQ for 24 h, respectively; Lane 5: 250 μg/mL of H-EtOAc fraction for 24 h. B. The protein expression levels of FasL (a) and Fas (b). Values are means ± SD of three independent experiments. * (p<0.05), ** (p<0.01) and *** (p<0.001) represented significant differences compared to the control.
  • Fig 6. Caspase-8 and Caspase-9 Dependent Apoptosis of HepG2 Cells Induced by DMQ and H-EtOAc Fraction. A. HepG2 cells were incubated with caspase-8 inhibitor (Z-IETD-FMK: 20 μmol/L) or caspase-9 inhibitor (Z-LEHD-FMK: 20 μmol/L) for 30 min followed by DMQ at 0, 50, 125 and 250 μmol/L for 24 h, the inhibitory rates were detected by SRB assays. B. HepG2 cells were incubated with Z-IETD-FMK or Z-LEHD-FMK for 30 min and subsequently treated with H-EtOAc fraction at 0, 12.7, 31.8 and 63.5 μg/mL for 24 h, and the inhibitory rates were determined by SRB assays. C. HepG2 cells were preincubated with Z-IETD-FMK or Z-LEHD-FMK for 30 min before treated with DMQ (79 μmol/L) or H-EtOAc fraction (100 μg/mL) for 24 h, the cellular apoptotic rates were measured by flow cytometry. Values are means ± SD of three independent experiments. * (p<0.05), ** (p<0.01) and *** (p<0.001) represented significant differences compared to the control.

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Li, Y. L., Zhang, J., Min, D., Hongyan, Z., Lin, N., & Li, Q. S. (2016). Anticancer effects of 1,3-dihydroxy-2-methylanthraquinone and the ethyl acetate fraction of hedyotis diffusa willd against HepG2 carcinoma cells mediated via apoptosis. PLoS ONE, 11(4). https://doi.org/10.1371/journal.pone.0151502

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