α-Tomatine significantly inhibited HL60 xenograft tumor growth and affected AIF and survivin expression <i>in vivo</i>.

<p>HL-60 cells were ectopically implanted into SCID mice and when the tumor size reached 100 mm³, the mice were injected with 5 mg/kg (q2d, i.p.) doses of α-tomatine. (<b>A</b>) Effects of α-tomatine on tumor volume and the body weights of mice were studied. The growth curves are the means of the tumor sizes measured for each group (n = 5). (<b>B</b>) The tumors were then excised and processed for immunohistochemical staining. The upper lanes (a.c.e.g and i) are the control, and the down lanes (b.d.f.h and j) are the treated group, with α-tomatine (5 mg/kg). a,b: Hematoxylin and eosin staining; c,d,e and f staining for AIF (brown) ; g,h,i and j staining for survivin (brown). c,d,g and h are under 200× magnification; a,b,e,f and j are under 1000× magnification. (<b>C</b>) Western blot analysis was performed for AIF and survivin expressions together with actin as a loading control from randomly selected tumor in each of the control and 5 mg/kg α-tomatine treatment groups.</p

α-Tomatine induced cell death independent of caspase activation in both K562 and HL-60 cell lines.

<p>(<b>A</b>) HL60 cells were treated with α-tomatine (5 µM) or paclitaxel (3 µM) for 24 hr and caspase-3, -6, -7, -8, and -9 activations were detected. The proteins were separated and evaluated using Western blot analysis. Paclitaxel (3 µM) was used as a positive control. (<b>B</b>) HL60 cells were pretreated with 100 µM z-VAD-fmk for 30 min and then treated with α-tomatine (5 µM) for 24 hr. The cytotoxicity was determined by MTT assay. (<b>C</b>) K562 cells were treated with α-tomatine (5 µM) and caspase-3, -6, -7, -8, and -9 activations were detected. (<b>D</b>) K562 cells were pretreated with 100 µM z-VAD-fmk for 30 min and then treated with α-tomatine (5 µM) for 24 hr.</p

α-Tomatine-induced apoptosis in human leukemia cell lines.

<p>(<b>A</b>) The chemical structure of α-tomatine. (<b>B</b>) Cells were treated with or without α-tomatine for 24 hr, and cell viability was measured by using the mitochrondrial MTT reduction activity assay. Data are expressed as the means ± SEM of at least three determinations. * <i>P</i><0.05, ** <i>P</i><0.01, and *** <i>P</i><0.001 compared with the control. (<b>C</b>) Flow cytometry analysis of plasma membranes with Annexin V-FITC/PI double staining. Cells were incubated with DMSO for 12 hr or in the presence of 5 µM α-tomatine for 12 and 24 hr. In the following experiments, 0.1% DMSO was used as control. Undamaged cells were stained negative by Annexin V-FITC/PI (bottom left quadrant). After incubation with 5 µM of α-tomatine for 12 hr, there were a significant number of apoptotic cells that stained positive with Annexin V-FITC and negative with PI (bottom right quadrant). Data are expressed from at least three separate determinations.</p

α-Tomatine induced nuclear translocation of AIF and survivin down-regulation in both HL60 and K562 cell lines.

<p>(<b>A</b>) HL60 and (<b>B</b>) K562 cells were treated with and without α-tomatine (5 µM) for 12 hr, 18 hr, 24 hr, and 30 hr. Cells were then fractionated into nuclear components, and the protein expressions of AIF and nucleolin (nuclear loading control) were evaluated by Western blot analysis. (<b>C</b>) HL60 and (<b>D</b>) K562 cells were treated with α-tomatine at the indicated concentrations and time. Survivin and actin protein levels were detected by Western blot analysis. Data are expressed from at least three separate determinations.</p

α-Tomatine affected mitochondrial apoptotic or anti-apoptotic protein levels.

<p>(<b>A</b>) α-Tomatine induced Mcl-1s and Bak up-regulations (pro-apoptotic) but did not affect Bcl-2 and Bid protein levels in the HL60 cells. (<b>B</b>) In the K562 cells, α-tomatine significantly enhanced the activation of Bak and up-regulated Mcl-1s; however α-tomatine did not influence Bcl-2 and Bid protein expressions. Both cell lines were treated with 5 µM α-tomatine for the indicated intervals. Data are expressed from at least three separate determinations.</p

α-Tomatine-Mediated Anti-Cancer Activity <em>In Vitro</em> and <em>In Vivo</em> through Cell Cycle- and Caspase-Independent Pathways

<div><p>α-Tomatine, a tomato glycoalkaloid, has been reported to possess antibiotic properties against human pathogens. However, the mechanism of its action against leukemia remains unclear. In this study, the therapeutic potential of α-tomatine against leukemic cells was evaluated <em>in vitro</em> and <em>in vivo</em>. Cell viability experiments showed that α-tomatine had significant cytotoxic effects on the human leukemia cancer cell lines HL60 and K562, and the cells were found to be in the Annexin V-positive/propidium iodide-negative phase of cell death. In addition, α-tomatine induced both HL60 and K562 cell apoptosis in a cell cycle- and caspase-independent manner. α-Tomatine exposure led to a loss of the mitochrondrial membrane potential, and this finding was consistent with that observed on activation of the Bak and Mcl-1 short form (Mcl-1s) proteins. Exposure to α-tomatine also triggered the release of the apoptosis-inducing factor (AIF) from the mitochondria into the nucleus and down-regulated survivin expression. Furthermore, α-tomatine significantly inhibited HL60 xenograft tumor growth without causing loss of body weight in severe combined immunodeficiency (SCID) mice. Immunohistochemical test showed that the reduced tumor growth in the α-tomatine-treated mice was a result of increased apoptosis, which was associated with increased translocation of AIF in the nucleus and decreased survivin expression <em>ex vivo</em>. These results suggest that α-tomatine may be a candidate for leukemia treatment.</p> </div

Effects of α-tomatine on the mitochondrial membrane potential in both HL60 and K562 cell lines.

<p>The mitochondrial membrane potential was quantitated by flow cytometric analysis with rhodamine 123. The (<b>A</b>) HL60 and (<b>B</b>) K562 cell lines were treated with 10 µM rhodamine 123 and incubated at 37°C for 30 min in the presence of 5 µM α-tomatine. The horizontal axis shows the relative fluorescence intensity, and the vertical axis indicates the cell number. The green curve indicates the control. The blue curve indicates the α-tomatine-treated cells. A shift from the green curve to the blue curve indicates a loss of mitochondrial membrane potential. Data are expressed from at least three separate determinations.</p

Aciculatin inhibits NF-κB activation in IL-1β-stimulated FLS.

<p>(A) FLS (1×10⁶ cells) were treated with 0–10 µM aciculatin or 20 µM PDTC for 30 min, and were then incubated for 30 min with 10 ng/mL of IL-1β in the continued presence of aciculatin or PDTC. The cells were then harvested, and whole cell extracts were subjected to western blot analysis for the indicated proteins. The bar graphs represent the ratio of phosphorylation of IKKα/β and of p65 proteins expression to the relative levels of GAPDH protein that were quantitated using a densitometer scanner and Image-Pro plus software. (B) FLS were incubated with 0 or 10 µM aciculatin for 30 min, and then for 1 h with 10 ng/mL of IL-1β in the continued presence of aciculatin. The DNA binding activity of the nuclear extracts was then examined in an electrophoretic mobility shift assay using a biotinylated NF-κB DNA probe; controls included 10 µM aciculatin alone and the use of the unlabeled probe (“cold”) to compete for the binding of the labeled probe. (C) Cells (1×10⁵ cells) were transiently transfected with 1 µg of pGL4.32[<i>luc2P/</i>NF-κB-RE<i>/</i>Hygro] for 24 h and incubated with 0–10 µM aciculatin for 30 min prior to stimulation for 6 h with 10 ng/mL of IL-1β in the continued presence of aciculatin, and then luciferase activity was measured. A control with only 10 µM aciculatin was used. In (A) and (C), the results are expressed as the mean ± SEM, with n = 3. *<i>p</i><0.05 and **<i>p</i><0.01 compared with the control group; #<i>p</i><0.05 and ##<i>p</i><0.01 for the comparisons of the groups indicated.</p

Aciculatin inhibits IL-1β-induced G-CSF production in a concentration-dependent manner.

<p>(A) 1×10⁶ fibroblast-like synoviocytes (FLS) were incubated with or without 10 µM aciculatin for 30 min and then for 24 h with or without 10 ng/mL of IL-1β in the continued presence of aciculatin; thereafter, cell culture supernatants were assayed for cytokine levels by using a Milliplex® assay. (B) FLS were incubated with 0–30 ng/mL of IL-1β for 24 h, and then the culture supernatants were assayed for G-CSF by using ELISA. (C) FLS were incubated with 0, 1, or 10 µM aciculatin for 30 min, and then for 5 h with 10 ng/mL of IL-1β in the continued presence of aciculatin. The G-CSF mRNA levels in the cells were measured using RT-PCR, and a control with only 10 µM aciculatin was included. (D) Cells were incubated for 30 min with 0–10 µM aciculatin and then for 24 h with 10 ng/mL of IL-1β in the continued presence of aciculatin, before the G-CSF in the culture supernatants were measured using ELISA. (E) The viability of the FLS was determined after 24 h treatment with 1–10 µM of aciculatin compared to the control group by using the MTT assay. Data are represented as mean ± SEM, with n = 3. *<i>p</i><0.05 and **<i>p</i><0.01 compared with the control group; #<i>p</i><0.05 and ##<i>p</i><0.01 for the comparisons of the groups indicated.</p

Aciculatin inhibits the phosphorylation of JAK, STAT, and Akt in FLS and the differentiation of 32Dcl3 cells.

<p>(A, C, and D) FLS were incubated with 0, 1, 3, or 10 µM aciculatin (A1, A3, and A10) for 30 min, and then for 24 h with 10 ng/mL of IL-1β in the continued presence of aciculatin. Whole cell extracts were then prepared for western blot analysis for the indicated proteins (A and C); equal amounts of cell culture media (“conditioned medium”) were collected and concentrated 10-fold (v/v) (lanes 1–4) or PBS only (lane 5), and then immunoprecipitated with 1 µg of anti-G-CSF antibody, followed by immunoblot analysis using anti-G-CSF antibody or anti-β-actin antibody (as an internal control) (D). (B) FLS were incubated with 0 or 10 µM aciculatin for 30 min, and then for 1 h with 10 ng/mL of IL-1β in the continued presence of aciculatin. The DNA binding activity of the nuclear extracts was then examined in an electrophoretic mobility shift assay using a specific STAT3 DNA probe. (E) Ten-fold concentrated conditioned medium was prepared from FLS incubated with or without aciculatin, and then with IL-1β as in (D), or with IL-1β plus an anti-G-CSF antibody. 32Dcl3 cells were incubated for 10 days with a medium containing 50% of these different conditioned mediums. The cells were then were subjected to Wright-Giemsa staining to detect neutrophils (top row) or washed twice with PBS, incubated at 4°C for 45 min with anti-CD11b FITC-conjugated and anti-CD11a/CD18 PE-conjugated antibodies, and their fluorescence was analyzed by FACScan flow cytometry (bottom row). Magnification  = ×100; scale bar  = 20 µm. In (A) and (C), the extents of indicated proteins expression were quantitated using a densitometer with the Image-Pro plus software, and the relative levels were calculated as the ratios of proteins to GAPDH or β-actin protein levels. The results are expressed as the mean ± SEM, with n = 3. *<i>p</i><0.05 and **<i>p</i><0.01 compared with the control group; #<i>p</i><0.05 and ##<i>p</i><0.01 for the comparisons of the groups indicated.</p