Arsenic trioxide inhibits viability of pancreatic cancer stem cells in culture and in a xenograft model via binding to SHH-Gli

Figures & data

Figure 1 Arsenic trioxide induced apoptosis in CD24⁺CD44⁺ SW1990 cells.

Notes: (A) Time dependent effects of arsenic trioxide (ATO) on pancreatic cancer cell viability. (B) ATO induced apoptosis in cells expressing CD24⁺CD44⁺. The blue points indicate the CD24⁺CD44⁺ cells in the whole population. Cells were treated with 2.5 or 5 μm ATO for 72 hours.
Abbreviation: PI, propidium iodide.

Table 1 Apoptosis in SW1990 cells treated with arsenic trioxide (percentage, mean ± standard deviation)

SW1990 cells	ATO concentrations
	0 μm	2.5 μm	5 μm
Total	1.4 ± 0.3	3.7 ± 0.8*	4.6 ± 1.7*
CD24^+CD44^+	0.7 ± 0.2	10.7 ± 2.4*	15.5 ± 2.8*

Notes: Data are from flow cytometry analysis of apoptotic cells. SW1990 cells were treated with vehicle, or 2.5, or 5 μm arsenic trioxide for 72 hours. Percentage values indicate the proportion of apoptotic cells.

* Denotes P < 0.01 versus 0 μm.

Abbreviation: ATO, arsenic trioxide.

Figure 2 Arsenic trioxide inhibited tumorigenesis of SW1990 cells in vivo.

Notes: (A) Tumor growth chart showing the effects of each management arm. Points represent the mean values of tumor volumes in each group. The bars represent standard deviation. (B) Pictures of tumors.
Abbreviations: ATO, arsenic trioxide; gem, gemcitabine hydrochloride.

Figure 3 Alterations in apoptosis signaling in xenograft tumor samples and illustration of the potential mechanism.

Notes: Tumors were collected after a 3-week treatment and the prepared tissue slides were analyzed by immunohistochemistry (IHC) using anti-CD24, anti-CD44, and anti-aldehyde dehydrogenase 1 family, member A1 (AlDH1A1) antibodies, accompanied by hematoxylin and eosin and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. (A) representative figures of hematoxylin and eosin, The black bars are scale bars, arrows indicate positive cells. TUNEL, and IHC assays using treated tumor samples. Scale bars are 20 μm. (B) Apoptosis and relative expression of CD24, CD44, and AlDH1A1 in tumors. Apoptosis in tumor tissues were detected by TUNEL assay. Relative expression of CD24, CD44, and AlDH1A1 was detected by IHC, and analyzed with image-Pro Plus software (Media Cybernetics, Inc, Maryland, USA). in the three lower panels, MOD, mean optical density, the columns are mean values of MOD of each group, the bars represent standard deviation. *denotes P < 0.05 and **denotes P < 0.01.
Abbreviations: AlDH1A1, aldehyde dehydrogenase 1 family, member A1; ATO, arsenic trioxide; Gem, gemcitabine hydrochloride; H&E, hematoxylin and eosin; MOD, mean optical density; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Table 2 Apoptosis in tumors of each group analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling assay (percentage, mean ± standard deviation)

	Groups
	Control	Gem	ATO	ATO + Gem
Apoptosis	8.6 ± 4.1	12.9 ± 3.5*	13.8 ± 4.5*	46.9 ± 5.6**

Notes: Tumor tissues were from mice in each group and apoptosis analysis was performed manually.

* Denotes P < 0.05

** denotes P < 0.01 versus the control.

Abbreviations: ATO, arsenic trioxide; gem, gemcitabine hydrochloride ; dUTP, 2’-deoxyuridine-5’-triphosphate.

Table 3 Mean optical density analysis on the immunohistochemistry slides (×10⁻², mean ± standard deviation)

Markers	Groups
	Control	Gem	ATO	ATO + Gem
CD24	1.07 ± 0.33	1.79 ± 0.54*	1.11 ± 0.28	1.04 ± 0.37
CD44	1.05 ± 0.36	2.08 ± 0.48**	0.73 ± 0.22	0.56 ± 0.25
ALDH1A1	0.06 ± 0.05	0.37 ± 0.11**	0.06 ± 0.04	0.07 ± 0.04

Notes: Immunohistochemistry slides were prepared with the tumors from mice in each treatment group and the mean optical density of label color (brown) in immunohistochemistry were analyzed with image-Pro Plus software (Media Cybernetics, Inc, Maryland, USA).

* Denotes P < 0.05

** denotes P < 0.01 versus the control. No significant difference was observed among other groups for the same marker.

Abbreviations: AlDH1A1, aldehyde dehydrogenase 1 family, member A1; ATO, arsenic trioxide; Gem, gemcitabine hydrochloride.

Figure 4 Arsenic trioxide binds to Gli1.

Notes: (A) Near-ultraviolet absorbance spectrometry assay of the Gli1 zinc finger peptides titrated with arsenic trioxide (ATO). The absorbance decreased with various molar equivalents of ATO added, indicating a binding action of ATO to the peptide of the Gli1 zinc finger domain. (B) Colocalization of GFP-labeled Gli1 with Reash could be weakened by ATO treatment in cultured SW1990 cells. Scale bars are 10 μm.
Abbreviations: equ, molar equivalents versus the concentration of recombinant Gli1 zinc finger peptides in the solution examined; GFP, green fluorescent protein.

Figure S1 Gli1 zinc finger peptide synthesis.

Notes: (A) The amino acid sequence of the five zinc finger motifs of the Gli1 protein. The “–” symbol indicates a gap. (B) Restriction enzyme analysis of the recombinant plasmid pMAL-C2X-Gli1-Zf by EcoRI and HindIII. Lane M1, Dl-2000 DNA marker; lane 1, products of the recombinant pMAL-C2X-Gli1-Zf plasmid digested with EcoRI and HindIII. (C) Polymerase chain reaction analysis of the cloned strains. Lane M2, DL-2000 DNA marker, lanes 1–6, products of the six clones. (D) SDS-PAGE: Lane M3, TaKaRa protein marker (broad); lane 1, uninduced cell pellet; lane 2, induced cell pellet containing the Gli1 zinc finger motifs as indicated by the arrow.

Abbreviations: Zfs, zinc finger motifs; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; Bp, base pair; KD, kilo-Dalton.