Mitochondrial Apoptotic Pathway Related to DMQ and H-EtOAc Fraction Treated HepG2 Cells.

<p>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 of p53, Bax, Bcl-2 and β-actin. Lane 1: Control; Lane 2–4: 79 μmol/mL for 12 h, 157 μ/L for 12 h and 157 μmol/L for 24 h of DMQ, respectively; Lane 5: 100 μg/mL for 24 h of H-EtOAc fraction. F. The calculated mRNA expression levels of p53 (a) Bax (b) Bcl-2 (c) and the ratio of Bax/Bcl-2 (d). The mRNA expression strength 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. * (<i>p</i><0.05), ** (<i>p</i><0.01) and *** (<i>p</i><0.001) represented significant differences compared to the control.</p

Caspase-8 and Caspase-9 Dependent Apoptosis of HepG2 Cells Induced by DMQ and H-EtOAc Fraction.

<p>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 pre-incubated 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. * (<i>p</i><0.05), ** (<i>p</i><0.01) and *** (<i>p</i><0.001) represented significant differences compared to the control.</p

<i>In vitro</i> antitumor activity of H-EtOAc fraction against six human tumors (±SD).

<p><i>In vitro</i> antitumor activity of H-EtOAc fraction against six human tumors (±SD).</p

Cell Cycle Arrest of HepG2 Cells Treated with DMQ and H-EtOAc Fraction.

<p>A. The cell cycle distribution of treated HepG2 cells. B. Contents of each phase of HepG2 cells treated with DMQ (a), H-EtOAc fraction (b). C. Western blot analysis of p21, CDK 2 and cyclin E. 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 p21 (a), CDK 2 (b) and cyclin E (c). The protein expression strength 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. * (<i>p</i><0.05), ** (<i>p</i><0.01) and *** (<i>p</i><0.001) represented significant differences compared to the control.</p

Death Receptor Pathway Related to HepG2 Cells Treated with DMQ and H-EtOAc Fraction.

<p>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. * (<i>p</i><0.05), ** (<i>p</i><0.01) and *** (<i>p</i><0.001) represented significant differences compared to the control.</p

The Morphology Changes and apoptosis rates of H-EtOAc fraction and DMQ treated HepG2 cells.

<p>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. * (<i>p</i><0.05), ** (<i>p</i><0.01) and *** (<i>p</i><0.001) represented significant differences compared to the control.</p

The Anticancer Effects of H-EtOAc Fraction and DMQ.

<p>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 HL-7702 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 PFT-α 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-α without 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. * (<i>p</i><0.05), ** (<i>p</i><0.01) and *** (<i>p</i><0.001) represented significant differences compared to the control.</p

Apoptosis Induced by DMQ and H-EtOAc fraction of HepG2 Cells.

<p>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. * (<i>p</i><0.05), ** (<i>p</i><0.01) and *** (<i>p</i><0.001) represented significant differences compared to the control.</p

Primers Used for RT-PCR.

<p>Primers Used for RT-PCR.</p