Taken together, the work proved that teroxirone induced extrinsic
pathway-mediated apoptotic cell death in Huh7 monolayer cells. Teroxirone up-regulates FADD and caspase-8. Activated caspase-8 then cleaves the
downstream procaspases-3 and -6. The cleaved and activated caspases triggered ultimate apoptotic cell death by fragmenting PARP. The findings validated the potency of teroxirone to eliminate spheroids composing of Huh7 CSCs. The induced apoptotic cell death accounts for the decreased stemness, EMT and drug resistance of the CSCs-enriched spheroids.
Figures
Figure 1. Chromatographic fingerprint analysis of the aqueous BJ extract Twelve major components in BJ extract were recognized by LC/MS chromatogram. The peak ESI (-) mode as identified including: 1) Bruceoside D (10.6 min); 2) Bruceine E (15.7 min); 3) Bruceine F (17.5 min); 4) Bruceine D (18.6 min); 5) Bruceine B (37.0 min); 6) Bruceine I (39.2 min); 7) Bruceine J (44.2 min); 8) Yadanzioside F (44.7 min); 9) Bruceantinol B (47.7 min); 10) Brusatol (49.2 min); 11) Bruceine A (50.8 min), and 12) Bruceoside E (57.2 min).
(Courtesy of Brion Research Institute of Taiwan, New Taipei City, Taiwan.)
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Figure 2. The aqueous BJ extract induces apoptosis and reduces EGFR in H1975 cells
(A) Determination of cell proliferation by MTT assay. NSCLC cells treated with BJ at different concentrations (1, 2 and 5 mg BJ/mL for 12 h) were incubated with MTT in duplicates and the absorbance measured at 570 nm. Low concentrations of aqueous extracts of BJ inhibited growth of NSCLC cells bearing double mutation of EGFR (L858R/T790M). The absorbance was converted into percentages with those of vehicle control as 100%. (B)
Histograms of one-dimensional flow cytometry. The appearance of sub-G1
populations was measured by FACS cytometry. Dose-dependent appearance of sub-G1 cells of BJ Human NSCLC cells H1975 cultured in 1%
serum-supplemented DMEM were incubated with various concentrations of BJ (1, 2 and 5 mg/mL) or water control for 12 h before being labeled with PI and followed by flow cytometry analysis. (C) Quantitative analysis of the cell populations. The phase distribution was analyzed by FlowJo software and converted into percentage relative to water control. The results were expressed as mean values from three independent experiments. *p<0.05; **p<0.01 (D) Histograms of two-dimensional flow cytometry. H1975 cells were treated with various concentrations (1, 2 and 5 mg/mL) of BJ for 12 h and the trypsinized cells labeled with Annexin V and PI were analyzed by flow
cytometry. (E) Quantitative analysis of the apoptotic cell populations. The early (dark) and late (light) apoptotic population distributions were expressed as mean values from three independent experiments. *p<0.05; **p<0.01
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Figure 3. Effects of teroxirone on apoptotic features in H1975 cells
(A) The protein lysates from H1975 as treated with 1, 2 and 5 mg/mL of BJ extract for 12 h were collected and used for western blot analysis as
described in Materials and Methods. The blots were incubated with various primary antibodies including EGFR, pEGFRY1068, Akt, pAkt473, caspase-3 and PARP as specified that were followed by horseradish peroxidase -conjugated secondary antibodies. GAPDH was used as loading control. The blots were visualized by ECL (enhanced chemiluminescence) detection system. (B) Densitometry determination of EGFR and pEGFRY1068 amelioration. The densitometry ratios of EGFR and pEGFRY1068 in H1975 cells from Western blot analysis were obtained by first normalizing individual band intensity at each concentration to that of the loading control and compared with those of water treatment. The results were expressed as mean values of three independent experiments (*p<0.05; ** p<0.01, unpaired Student’s t-test as compared with control water).
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Figure 4. EGFR shRNA suppresses BJ sensitivities by inhibiting apoptosis (A) Cell viability determination. H1975 cells transfected with EGFR shRNA or non-specific control for 24 h were treated with 2 mg/mL of aqueous BJ extract for 12 h and the collected cells counted by trypan blue exclusion assay. Symbol (-) meant no transfection. The results were expressed as mean values from three independent experiments. *p<0.05 and **p<0.01 (B) Western blot analysis.
Protein lysates H1975 cells of transfected with EGFR shRNA and non-specific control of H1975 proteins before being treated with BJ extract (2 mg/mL) or water were determined to Western blot analysis. The antibodies included EGFR, pEGFRY1068, PARP and loading control GAPDH. Symbol (-) indicated no
transfection.
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Figure 5. Oral administration of aqueous BJ extract inhibited xenograft tumor growth
(A) BJ extract suppressed the growth of xenograft tumors in animal models.
Nude BAL B/c mice with established xenograft tumors of H1975 cells were fed orally with 1 and 2 g BJ/kg daily for 7 consecutive days (arrows). The graph represented tumor growth variation (y-axis) from the start of feeding (x-axis).
*p<0.05 indicated significant difference in the measured tumor volumes between mice fed with 2 g BJ/kg and those with water from three individual experiments with two or three mice in each group. (B) The body weights of the mice. Mice with established xenograft H1975 tumors orally administered water, 1 and 2 g BJ/kg daily for 7 consecutive days (arrow). No significant difference was observed in the average body weight. The graph represented variations of mice weight (y-axis) from the start of feeding in days (x-axis). (C) The
decreased resected tumor weights. Tumor weights of the resected xenograft tumors were reduced in mice with gavage feeding of BJ compared with those of water. The horizontal bars represented mean values of tumor mass as collected following different treatments. *p<0.05 indicated significant weight difference between mice fed with 2 g BJ/kg and those with water. Each graph is
representative of three independent experiments.
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Figure 6. Histological and fluorescence examinations of the suppressed tumors
(A) Investigation by H&E staining. The H1975 tumors treated with BJ (1 and 2 g/kg) and water control were dissected, stained by H&E and analyzed by light microscopy (left, scale bar = 100 µm). The rectangular inset of the image were zoomed in to the right of the panel for each treatment (right, scale bar =25 µm).
The white arrow indicates apoptotic body location. (B) Images of
immunofluorescent EGFR and pEGFRY1068 H1975 tumor sections from mice fed with 2 g/kg of BJ and water control were incubated with EGFR and
pEGFRY1068 antibody (green) followed by FITC-conjugated secondary antibody treatment before being counter-stained with DAPI (blue) (scale bar =50 µm). (C) Release of mitochondrial cytochrome c in H1975 tumors treated with BJ.
The dissected H1975 tumors as treated with BJ (1 and 2 g/kg) and water control were fixed and incubated with antibody against cytochrome c followed by staining with secondary antibody conjugated with TRITC (red). The slides were counter-stained with mitotracker (green) and DAPI (blue) before being analyzed by confocal microscopy. The merged images of red color cytochrome c and green color mitochondria shown the appearance of puncta (yellow), while blue color indicates nucleus (scale bar =50 µm).
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Figure 7. BJ reduces tumor growth by apoptosis
(A) Dose-dependent decrease of fluorescent PCNA in nucleus. Tumor sections were incubated with antibody against PCNA followed by FITC-conjugated secondary antibody. The slides with immunofluorescent PCNA (green) were counter-stained with DAPI (blue) before being taken by confocal microscopy (scale bar =50 µm). The images of rectangular inset of tumor sections in mice treated with water were zoomed-in to the top of the panel (scale bar =15 µm). (B) BJ inhibited mitotic index PCNA signals in nucleus.
Quantitative number of PCNA- positive cells was counted total positive cells in 100 H1975 tumor cells at each concentration. The numbers at various BJ
concentrations were the average of at least three different fields. The data were expressed as the mean of three individual experiments (**p<0.01). (C) Dose-dependent increase of TUNEL fluorescent. The tumors from the mice fed with water or 1 and 2 g BJ/kg were frozen, fixed, resected, and subjected to TUNEL experiment for confocal microscopy analysis. TUNEL-positive cells (green) were counter-stained with DAPI (blue) and visualized (scale bar =50 µm). The image of rectangular inset of tumor sections in mice treated with 2 g BJ/kg was zoomed in to the bottom of the panel (scale bar =15 µm). (D) BJ induced TUNEL-positive intensities in nucleus. The numbers of fluorescent TUNEL-positive cells counted in each field were 100, as marked by DAPI staining. The numbers of TUNEL-positive cells at various BJ concentrations were the average of at least three different fields. The data were expressed as the mean of three individual experiments (**p<0.01).
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Figure 8. BJ suppresses H1975 spheroids growth
(A) Effects of BJ on the growth of H1975 multicellular spheroids. The representative image of H1975 spheroids were treated at each condition with water or 5 and 10 mg BJ/mL for 12 h. The spheroids were taken by inverted microscope (scale bar =150 μm). (B) Soft agar colony forming assay. H1975 spheroids were treated with 5, 10 and 15 mg BJ/mL, respectively, for 12 h. BJ treated spheroids were seeded and grown on agar for 25-28 days and stained with 0.002% crystal violet. Each representative image of colony grown on agar at different concentrations and water control was taken from an inverted
microscope. The numbers of colonies on agar were counted and plotted.
*p<0.05 and **p<0.01 indicates significant difference between treatment groups and water control as determined from three independent experiments.
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Figure 9. BJ reduces expression of stemness markers of H1975 spheres (A) The fluorescent CD133 of H1975 spheroids. Immunofluorescent images were detected by CD133 antibody (green) of H1975 spheroids after BJ
treatment at water or 5, 10 and 15 mg BJ/mL for 12 h. The spheroids were counter-stained with DAPI (blue) (scale bar =100 μm). (B) The fluorescent Nanog of H1975 spheroids. Immunofluorescent images were detected by Nanog antibody (green) of H1975 spheroids after BJ treatment at water or 5, 10 and 15 mg BJ/mL for 12 h. The spheroids were counter-stained with DAPI (blue) (scale bar =100 μm). (C) Western blot analysis. Protein lysates from H1975 spheroids treated with water or 5, 10 and 15 mg BJ/mL for 12 h were collected and resolved by SDS-PAGE gels. The blots were transferred to nitrocellulose membranes and incubated with antibodies against different stemness markers, including ALDH1A1, Nanog and CD133 and loading control GAPDH.
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Figure 10. BJ induces apoptotic characteristics of H1975 spheroids
(A) The decreased of BrdU incorporation. H1975 spheroids were treated with 5 and 15 mg BJ/mL for 12 h followed by evaluation with BrdU incorporation assay. The images of BrdU (green) and nucleus marker DAPI (blue) were taken by a fluorescence microscope (scale bar =100 μm). (B) BJ reduced BrdU incorporation. All colored images were converted to black and white by Photoshop software before being quantitated with Multi Gauge software (version 2.1, FUJIFILM). The green fluorescence at each concentration was obtained and compared with that of water control as BrdU intensity ratio. The numbers of fluorescence BrdU positive spheroid cells were counted in each field of different concentrations. The numbers as counted were the average of at least three different fields. The data are expressed as mean ± SD of three
individual experiments (*p<0.05). (C) Dose-dependent increase of TUNEL positive spheroid cells. The spheroids were treated with 5 and 15 mg BJ/mL for 12 h followed by TUNEL staining evaluation. The images of TUNEL (green) and nucleus marker DAPI (blue) were taken by a fluorescence microscope (scale bar =100 μm). (D) BJ increased TUNEL positive spheroid cells. The numbers of fluorescent TUNEL positive spheres in each field of BJ
concentration of 5 and 15 mg BJ/mL were counted. All fluorescent TUNEL images were converted to black and white by Photoshop software before being quantitated with Multi Gauge software (version 2.1, FUJIFILM). The TUNEL intensities were converted into percentages as average intensity ratios by comparing with those of DAPI in at least five spheres. Three independent experiments were carried out. (*p<0.05, **p<0.05).
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Figure 11. Oral administration of BJ suppresses growth of tumor spheroid xenograft
(A) BJ extract inhibited the growth of xenograft tumors Nude BALB/c mice with established xenograft spheroid tumors of H1975 cells were administered orally with 2 and 4 g BJ/kg daily for 6 consecutive days (arrow). The graph represented tumor growth variation (y-axis) from the day at the start of feeding (x-axis). Mice fed with aqueous BJ extract reduced tumor growth compared with those with water. *p< 0.05 and **p< 0.01 means significant difference in the measured tumor volumes between mice fed with 2 and 4 g BJ/kg and those with water from three individual experiments with two or three mice in each group. (B) The body weight of nude mice. There data showed no significant difference of the average body weight in mice with established xenograft tumors orally administered with water or 2 and 4 g BJ/kg daily for 6 consecutive days (arrow). The graph represented variations of mice weight (y-axis) from the start of feeding in days (x-axis). (C) The decrease tumor weights. Tumor weights of the xenograft tumors were measured in mice with gavage feeding of BJ
compared with those of water. The horizontal bars represented mean values of tumor weight as collected following different treatments. *p< 0.05 meant significant weight difference between mice fed with 4g BJ/kg and those with water. The graph is a representative of three independent experiments. (D) H&E staining. The spheroid tumors as treated with BJ (2 and 4 g/kg) and water
control were dissected, stained by H&E and analyzed by light microscopy (scale bar = 100 μm). The image of rectangular inset of spheroid tumor
sections in mice treated with 2 and 4 g BJ/kg were zoomed-in to the right of the panel (scale bar =25 µm). The white arrow signified apoptotic body location.
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Figure 12. BJ reduces spheroid tumor growth by apoptosis
(A) Dose-dependent decrease of stained PCNA in spheroid tumor nucleus.
Spheroid tumor sections were incubated with antibody against PCNA followed by FITC-conjugated secondary antibody. The slides with immunofluorescent PCNA (green) were counter-stained with DAPI (blue) before being taken by confocal microscopy (scale bar =50 µm). The images of rectangular inset of spheroid tumor sections in mice treated with water were zoomed in to the top of the panel (scale bar =15 µm). (B) BJ inhibited mitotic index PCNA positive signals in spheroid tumor nucleus. The numbers of fluorescent nucleus PCNA-positive cells counted in each field were 100, as marked by DAPI
staining. The numbers at various BJ concentrations were the average of at least three different fields. The data were expressed as the mean of three individual experiments (**p<0.01). (C) Dose-dependent increase of TUNEL staining.
The spheroid tumors from the mice fed with water or 2 and 4 g BJ/kg were frozen, fixed, resected, and subjected to TUNEL experiment for confocal microscopy analysis. TUNEL-positive cells (green) were counter-stained with DAPI (blue) and visualized (scale bar =50 µm). The image of rectangular inset of spheroid tumor sections in mice treated with 4 g BJ/kg was zoomed-in to the bottom of the panel (scale bar =15 µm). (D) BJ induced TUNEL-positive intensities in spheroid tumor nucleus. The numbers of fluorescent TUNEL-positive cells counted in each field were 100, as marked by DAPI staining. The numbers of TUNEL-positive cells at various BJ concentrations were the average of at least three different fields. The data were expressed as the mean of three individual experiments (**p<0.01).
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Figure 13. Fluorescence examinations of the suppressed spheroid tumors (A) Images of immunofluorescent EGFR H1975 spheroid tumor sections from mice fed with 2 and 4 g BJ/kg and water control were incubated with EGFR antibody (green) followed by FITC-conjugated secondary antibody treatment before being counter-stained with DAPI (blue) (scale bar =50 µm). (B) Images of immunofluorescent pEGFRY1068 H1975 spheroid tumor sections from mice fed with 2 and 4 g BJ/kg and water control were incubated with pEGFRY1068 antibody (green) followed by FITC-conjugated secondary antibody treatment before being counter-stained with DAPI (blue) (scale bar =50 µm). (C) Release of mitochondrial cytochrome c in H1975 spheroid tumors treated with BJ. The dissected H1975 spheroid tumors as treated with BJ (2 and 4 g/kg) and water control were fixed and incubated with antibody against cytochrome c followed by staining with secondary antibody conjugated with TRITC (red). The slides were counter-stained with mitotracker (green) and DAPI (blue) before being analyzed by confocal microscopy. The merged images of red color cytochrome c and green color mitochondria shown the appearance of puncta (yellow), while blue color indicates nucleus (scale bar =50 µm). (D) Images of
immunofluorescent ALDH1A1 H1975 spheroid tumor sections from mice fed with 2 and 4 g BJ/kg and water control were incubated with ALDH1A1 antibody (green) followed by FITC-conjugated secondary antibody treatment before being counter-stained with DAPI (blue) (scale bar =50 µm).
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Figure 14. Fluorescence examination of the collected adherent cells and spheroid tumors
(A) Images of immunofluorescent CD133 and Nanog of H1975 spheroid tumor sections from mice fed with 2 and 4 g/kg of BJ and water control were
incubated with CD133 antibody (green) followed by FITC-conjugated
secondary antibody and Nanog antibody (red) followed by TRITC-conjugated secondary antibody before being counter-stained with DAPI (blue) and merged (scale bar = 50 μm). (B) Images of immunofluorescent CD133 and Nanog of adherent H1975 cell tumor sections from mice fed with 1 and 2 g/kg of BJ and water control were incubated with CD133 antibody (green) followed by FITC-conjugated secondary antibody and Nanog antibody (red) followed by TRITC-conjugated secondary antibody before being counter-stained with DAPI (blue) and merged (scale bar = 50 μm). (C) Images of immunofluorescent SOX2 and Nanog of spheroid tumors treated with 2 and 4 g/kg of BJ. (D) Images of
immunofluorescent SOX2 and Nanog of adherent H1975 cell tumors treated with 1 and 2 g/kg of BJ. (E) Images of immunofluorescent ABCG2 and Nanog of spheroid tumors treated with 2 and 4 g/kg of BJ. (F) Images of
immunofluorescent ABCG2 and Nanog of adherent H1975 cell tumors treated with 1 and 2 g/kg of BJ.
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Figure 15. Fluorescence examination of the collected tumors
(A) Images of immunofluorescent c-Met of spheroid tumors. H1975 spheroid tumor sections from mice fed with 2 and 4 g/kg of BJ and water control were incubated with β- catenin (green) followed by FITC-conjugated secondary with DAPI (blue) and merged (scale bar = 50 μm). (B) Images of immuno-fluorescent β-catenin in spheroid tumors. (B) Images of
immunofluorescent Vimentin in spheroid tumors.
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(A) Liver
(B) Lung
(C) Brain
(D) Kidney
Figure 16. Histological examinations by H&E staining of the collected specimens
(A) Liver. The liver as treated with BJ (2 and 4 g/kg) and water control were dissected, stained by H&E and analyzed by light microscopy (scale bar = 200 μm). (B) Lung. The lung as treated with BJ (2 and 4 g/kg) and water control were dissected, stained by H&E and analyzed by light microscopy (scale bar = 200 μm). (C) Brain. The brain as treated with BJ (2 and 4 g/kg) and water control were dissected, stained by H&E and analyzed by light microscopy
(scale bar = 200 μm). (D) Kidney. The kidney as treated with BJ (2 and 4 g/kg) and water control were dissected, stained by H&E and analyzed by light
microscopy (scale bar = 200 μm).
Figure 17. Dose-response growth of Huh7, HepG2 and Hep3B cells treated teroxirone
Determination of cell proliferation by MTT assay. Huh7, HepG2 and Hep3B cells were treated with teroxirone at different concentrations (2, 5 and 20 μM for 48 h) then incubated with MTT in duplicates and the absorbance measured at 570 nm. The absorbance was converted into percentages with those of vehicle control as 100%. The data are the average of quadruplicate for each experiment.
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Figure 18. Teroxirone increases apoptotic population of Huh7 cells
(A) Dose-dependent appearance of sub-G1 cells. Huh7, HepG2 and Hep3B cells were cultured in 1% serum-supplemented DMEM then incubated with various concentrations of teroxirone (2, 5, and 10 μM) or DMSO for 48 h before being labeled with PI followed by flow cytometry analysis. (B) Cell cycle distribution. The percentages of cell cycle distribution in Huh7, HepG2 and Hep3B cells following treatment were analyzed by flow cytometry analysis.
The analysis of the phase distribution was done by FlowJo software and converted into percentages. *p<0.05; **p<0.01 (C) Histograms of
two-dimensional flow cytometry. Huh7 and HepG2 cells were treated with various concentrations of teroxirone (2, 5, and 10 μM) or DMSO for 48 h and the trypsinized cells were labeled with Annexin V and PI were analyzed by flow cytometry. Early apoptosis (right down), Late apoptosis (right up) (D)
Quantitative analysis of the apoptotic cell populations. Populations of early (dark) and late (light) apoptotic distributions in Huh7 and HepG2 cells treated with various concentrations of teroxirone were analyzed. The results were expressed as mean values from three independent experiments.
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Figure 19. Effects of teroxirone on apoptotic features in Huh7 cells
(A) Western blot analysis. Cell lysates of Huh7 as treated with 2, 5 and 10 μM of teroxirone for 48 h were analyzed by western blot and incubated with
antibodies against markers including p53, caspase-9 and loading control GAPDH. (B) Western blot analysis. Cell lysates of Huh7 were analyzed by western blot and incubated with antibodies against markers including FADD, caspase-3, -6, -8, PARP and loading control GAPDH.
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Figure 20. Teroxirone’s effects are reverted by z-DEVD-FMK in Huh7 cells
(A) Determination of cell proliferation by MTT assay. Hepatocellular
carcinoma Huh7 cells were incubated with z-DEVD-FMK (caspase-3 inhibitor, 20 μM for 24 h), then treated with teroxirone at different concentrations (2, 5 and 20 μM for 48 h). After that, HCC Huh7 cells were incubated with MTT in duplicates and the absorbance measured at 570 nm. The absorbance was converted into percentages with those of vehicle control as 100%. **p<0.01 w/o; without z-DEVD-FMK w; with z-DEVD-FMK (B) The appearance of sub-G1 populations in Huh7 cells was measured by FACS cytometry.
Comparing with dose-dependent appearance of teroxirone treated (2, 5 and 20 μM for 48 h) sub-G1 cells in Huh7 cells pre-treated with or without z-DEVD-FMK (inhibitor caspase-3, 20 μM for 24 h). The analysis of the phase
distribution was done by FlowJo software and converted into percentages.
distribution was done by FlowJo software and converted into percentages.