Yun SH et al. (2017), In vitro and in vivo anti-leukemic effects of c...

ECB-ART-46120

Oncotarget 2017 Dec 08;91:495-511. doi: 10.18632/oncotarget.23069.

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In vitro and in vivo anti-leukemic effects of cladoloside C2 are mediated by activation of Fas/ceramide synthase 6/p38 kinase/c-Jun NH2-terminal kinase/caspase-8.

Yun SH , Sim EH , Han SH , Kim TR , Ju MH , Han JY , Jeong JS , Kim SH , Silchenko AS , Stonik VA , Park JI .

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We previously demonstrated that the quinovose-containing hexaoside stichoposide C (STC) is a more potent anti-leukemic agent than the glucose-containing stichoposide D (STD), and that these substances have different molecular mechanisms of action. In the present study, we investigated the novel marine triterpene glycoside cladoloside C2 from Cladolabes schmeltzii, which has the same carbohydrate moiety as STC. We assessed whether cladoloside C2 could induce apoptosis in K562 and HL-60 cells. We also evaluated whether it showed antitumor action in mouse leukemia xenograft models, and its molecular mechanisms of action. We investigated the molecular mechanism behind cladoloside C2-induced apoptosis of human leukemia cells, and examined the antitumor effect of cladoloside C2 in a HL-60 and K562 leukemia xenograft model. Cladoloside C2 dose- and time-dependently induced apoptosis in the analyzed cells, and led to the activation of Fas/ceramide synthase 6 (CerS6)/p38 kinase/JNK/caspase-8. This cladoloside C2-induced apoptosis was partially blocked by specific inhibition by Fas, CerS6, and p38 siRNA transfection, and by specific inhibition of JNK by SP600125 or dominant negative-JNK transfection. Cladoloside C2 exerted antitumor activity through the activation of Fas/CerS6/p38 kinase/JNK/caspase-8 without showing any toxicity in xenograft mouse models. The antitumor effect of cladoloside C2 was reversed in CerS6 shRNA-silenced xenograft models. Our results suggest that cladoloside C2 has in vitro and in vivo anti-leukemic effects due to the activation of Fas/CerS6/p38 kinase/JNK/caspase-8 in lipid rafts. These findings support the therapeutic relevance of cladoloside C2 in the treatment of human leukemia.

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Species referenced: Echinodermata
Genes referenced: aifm1 irak1bp1 jun LOC115919910 LOC115929340 LOC577219 LOC590297 LOC590751 mapk9

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	Figure 1. Cladoloside C2 induces apoptosis through extrinsic pathway activation in human leukemic cells(A) Structures of cladoloside C2 and stichoposides C and D. (B) Left panel: K562 and HL-60 cells were seeded, cultured for 4 h, and then treated for 6 h with various concentrations of cladoloside C2 (0, 0.1, 0.3, or 0.5 µM). Right panel: K562 and HL-60 cells were seeded, cultured for 4 h, and then treated for the indicated times with 0.2 or 0.1 µM cladoloside C2. The percentage of apoptotic cells was determined by annexin V-FITC/PI staining. Upper panels: Representative of three separate experiments. Lower panels: Mean ± SD of three independent experiments. *P < 0.001 vs. control cells. (C) K562 and HL-60 cells were treated with 0.2 or 0.1 µM cladoloside C2 for the indicated times. Protein lysates were prepared and used for western blot analysis with the corresponding antibodies. β-actin was used as a loading control. The blot is representative of three separate experiments. (D) Functional involvement of caspases in cladoloside C2-induced apoptosis of K562 and HL-60 cells. Cells were pretreated for 1 h with the pan-caspase inhibitor Z-VAD-FMK (25 μM), the caspase-8 inhibitor Z-IETD-FMK (20 μM), the caspase-9 inhibitor Z-LEHD-FMK (20 μM), or the caspase-3 inhibitor Z-DEVD-FMK (50 μM), followed by treatment with 0.2 or 0.1 μM cladoloside C2 for 6 h. The extent of apoptosis was measured by flow cytometry after annexin V staining. These data represent the mean ± SD of three independent experiments. P < 0.01; *P < 0.001 vs. cladoloside C2-treated cells. (E) Left panel: K562 and HL-60 cells were treated with 0.2 or 0.1 μM cladoloside C2 for 6 h. The extent of apoptosis was measured by flow cytometry after annexin V staining. These data represent the mean ± SD of three independent experiments. *P < 0.001 vs. control cells. Right panel: K562 and HL-60 cells were treated with 0.2 or 0.1 mM cladoloside C2 for 4 h or 2 h. The cells were stained with DiOC6, and the reduction in Δφm was determined by monitoring the DiOC6 uptake using flow cytometry. Low Δφm values are expressed as the percentage of cells exhibiting diminished mitochondrial potential. The values obtained from the DiOC6 assays represent the mean ± SD of three independent experiments. (F) Western blot for the mitochondrial proteins AIF, Smac/DIABLO, cytochrome oxidase IV, and cytochrome c. Cytochrome oxidase IV (COX IV) was used as a mitochondrial marker. Protein lysates were prepared and subjected to western blot analysis using corresponding antibodies. β-actin was used as a loading control. The blot is representative of three separate experiments.
	Figure 2. Cladoloside C2 induces apoptosis of K562 and HL-60 cells through the activation of ceramide synthase 6 (CerS6)(A) K562 and HL-60 cells treated with cladoloside C2 exhibited increased ceramide generation. (B) K562 and HL-60 cells (1 × 105 cells/well) were incubated for 6 h with cladoloside C2 in the presence or absence of myriocin, fumonisin B1, desipramine, or GW4869. After treatment, the percentage of apoptotic cells was determined by annexin V-FITC/PI staining. Upper panel: Representative of three experiments in each cell line. Lower panel: Mean ± SD of three independent experiments. P < 0.01; P < 0.001 vs. cladoloside C2-treated cells. (C) K562 and HL-60 cells were incubated for 6 h with cladoloside C2 in the presence or absence of myriocin or fumonisin B1. Protein lysates were prepared and subjected to western blot analysis using corresponding antibodies. Western blots are each representative of three separate experiments. β-actin was used as a loading control. Densitometry results are expressed above the bands. (D–F) K562 and HL-60 cells were transiently transfected for 48 h by electroporation with CerS6 siRNA, nonspecific control (NC) siRNA, or no siRNA (shock). (D) Western blot analysis of protein lysates. (E) Transfected K562 cells were exposed to 0.2 μM cladoloside C2 for 2 h, and then fixed and permeabilized. Samples were then stained with PE-conjugated antibodies against Fas, CerS6, ceramide, cleaved caspase-8, p-p38 kinase, p38 kinase, p-ERK, ERK, p-JNK, or JNK, and with Alexa 488-labeled cholera toxin B antibody. The pictures are representative of three separate experiments. (F) Upper panel: The culture medium was changed, and K562 and HL-60 cells were incubated for 6 h with or without 0.2 or 0.1 μM cladoloside C2. The percentage of apoptotic cells was determined by annexin V-FITC/PI staining. Results are representative of three independent experiments in each cell line. Lower panel: Mean ± SD of three independent experiments. P < 0.01, **P < 0.001, cells treated with cladoloside C2 alone versus cells transfected with CerS6 siRNA and treated with cladoloside C2.
	Figure 3. Fas knockdown inhibits cladoloside C2-induced apoptosis in K562 and HL-60 cellsK562 and HL-60 cells were transiently transfected for 48 h by electroporation with Fas siRNA, nonspecific control (NC) siRNA, or no siRNA (shock). (A) Western blot analysis of protein lysates. (B) Transfected K562 cells were treated with cladoloside C2 for 2 h, and then fixed and permeabilized. The samples were then stained with PE-conjugated antibodies against Fas, CerS6, ceramide, cleaved caspase-8, p-p38 kinase, p38 kinase, p-ERK, ERK, p-JNK, or JNK, as well as Alexa 488-labeled cholera toxin B antibody. The pictures are representative of three separate experiments. (C) The culture medium was changed, and cells were incubated for 6 h with or without cladoloside C2. The percentage of apoptotic cells was determined by annexin V-FITC/PI staining. Upper panels: Representative of three independent experiments in each cell line. Lower panels: Mean ± SD of three independent experiments. **P < 0.01, cells treated with cladoloside C2 versus cells transfected with Fas siRNA and treated with cladoloside C2.
	Figure 4. Cladoloside C2 induces apoptosis of K562 and HL-60 cells through activation of p38 kinase and JNK(A) K562 and HL-60 cells were treated with cladoloside C2 for the indicated times. Protein lysates were prepared and subjected to western blot analysis. β-actin was used as a loading control. The blot is representative of three separate experiments. (B) K562 and HL-60 cells (1 × 105 cells/well) were pretreated with the p38 kinase inhibitor SB203580, the JNK inhibitor SP600125, or the ERK inhibitor PD98059, followed by 6 h of treatment with 0.2 or 0.1 μM cladoloside C2. After treatment for the indicated times, the percentage of apoptotic cells was determined by annexin V-FITC/PI staining. Upper panel: Representative of three independent experiments. Lower panel: Mean ± SD of three independent experiments. P < 0.05; P < 0.01; **P < 0.001, versus cells treated with cladoloside C2 in the absence of SB203580 or SP600125. (C and D) K562 and HL-60 cells (1 × 105 cells/well) were pretreated with the p38 kinase inhibitor SB203580 (C) or the JNK inhibitor SP600125 (D), followed by treatment with 0.2 or 0.1 μM cladoloside C2 for 6 h. Protein lysates were prepared and subjected to western blot analysis using corresponding antibodies. Western blots are each representative of three separate experiments. β-actin was used as a loading control. Densitometry results are expressed above the bands.
	Figure 5. p38 kinase knockdown and DN-JNK transfection inhibit cladoloside C2-induced apoptosis in K562 and HL-60 cells(A–C) K562 and HL-60 cells were transiently transfected by electroporation for 48 h with p38 siRNA, nonspecific control (NC) siRNA, or no siRNA (shock). (A) Transfected K562 and HL-60 cells were incubated for 6 h with or without cladoloside C2. Protein lysates were prepared and subjected to western blot analysis. (B) The culture medium was changed, and cells were incubated for 6 h with or without cladoloside C2. The percentage of apoptotic cells was determined by annexin V-FITC/PI staining. Upper panel: Representative of three independent experiments in each cell line. Lower panel: Mean ± SD of three independent experiments. *P < 0.001, cells treated with cladoloside C2 versus cells transfected with p38 kinase siRNA and treated with cladoloside C2. (C) Transfected K562 cells were treated with cladoloside C2 for 2 h, and then fixed and permeabilized. The samples were then stained with PE-conjugated antibodies against Fas, CerS6, ceramide, cleaved caspase-8, p-p38 kinase, p38 kinase, p-ERK, ERK, p-JNK, or JNK, as well as Alexa 488-labeled cholera toxin B antibody. The pictures are representative of three separate experiments. (D and E) K562 cells were transiently transfected by electroporation for 48 h with pcDNA or DN-JNK plasmid. (D) Transfected K562 cells were incubated for 6 h with or without cladoloside C2. Protein lysates were prepared and subjected to western blot analysis. (E) The culture medium was changed, and cells were incubated for 6 h with or without cladoloside C2. The percentage of apoptotic cells was determined by annexin V-FITC/PI staining. Upper panel: Representative of three independent experiments in K562 cells. Lower panel: Mean ± SD of three independent experiments. *P < 0.001, cells treated with cladoloside C2 versus cells transfected with DN-JNK and treated with cladoloside C2.
	Figure 6. Clustering of Fas and its downstream molecules in lipid rafts during cladoloside C2-induced apoptosis of K562 and HL-60 cells(A) K562 and HL-60 cells were pretreated for 1 h with MβCD (20 μg/mL) and nystatin (20 μg/mL), and then cultured for 6 h in medium containing 0.2 or 0.1 μM cladoloside C2. After treatment for the indicated times, the percentage of apoptotic cells was determined by annexin V-FITC/PI staining. Upper panel: Representative of three independent experiments. Lower panel: Mean ± SD of three independent experiments. P < 0.01; *P < 0.001 versus cladoloside C2-treated cells. (B) Whole cell lysates were prepared from K562 and HL-60 cells incubated for 6 h with cladoloside C2 in the presence or absence of MβCD, and were subjected to western blot analysis. (C) K562 cells were treated for 2 h with cladoloside C2 in the presence or absence of MβCD or nystatin, and then fixed and permeabilized. These samples were then stained with PE-conjugated antibodies against Fas, CerS6, ceramide, cleaved caspase-8, p-p38 kinase, p38 kinase, p-ERK, ERK, p-JNK, or JNK, as well as Alexa 488-labeled cholera toxin B antibody. The pictures are representative of three separate experiments.
	Figure 7. Cladoloside C2 inhibits the growth of K562 and HL-60 xenograft tumors and induces apoptosis through Fas/CerS6/p38 kinase/JNK activation in vivo(A) K562 (1.5 × 107 cells/100 μL) and HL-60 cells (2 × 107 cells/100 μL) were subcutaneously injected into Balb/c nude mice. After the cells formed palpable tumors, the mice were randomized into two groups (n = 5 mice/group) and treated with vehicle control or cladoloside C2 (7.19 μg/kg). Tumor size was measured daily using a caliper: calculated volume = shortest diameter2 × longest diameter/2. P < 0.05, P < 0.01, and *P < 0.001, versus respective controls. (B) Left panel: NC-shRNA-transfected and CerS shRNA-transfected stable K562 (1.5 × 107 cells/100 mL) were subcutaneously injected into Balb/c nude mice. Right panel: NC-shRNA-transfected and CerS shRNA-transfected stable HL-60 (2 × 107 cells/100 mL) were subcutaneously injected into Balb/c nude mice. After the cells formed palpable tumors, the mice were randomized into two groups (n = 5 mice/group), and treated with vehicle control or cladoloside C2 (7.19 μg/kg). Tumor size was measured daily using a caliper. P < 0.05, P < 0.01, and *P < 0.001, versus respective controls. (C and D) Tumor tissues obtained from the above-described experiment on Day 14 were subjected to western blot analysis (C) and immunohistochemistry (D) using antibodies against Fas, CerS6, ceramide, p-p38, p38 kinase, p-JNK, or JNK. The sections were lightly counterstained with hematoxylin, and photographed with a ScanScope. K562 and HL-60 leukemia xenografts from cladoloside C2-treated mice exhibited apoptosis and extensive necrosis (200×).
	Figure 8. Hypothetical molecular mechanisms of cladoloside C2-induced apoptosis in human leukemia cells

References [+] :

Azmi, Emerging Bcl-2 inhibitors for the treatment of cancer. 2011, Pubmed

	Figure 1. Cladoloside C2 induces apoptosis through extrinsic pathway activation in human leukemic cells(A) Structures of cladoloside C2 and stichoposides C and D. (B) Left panel: K562 and HL-60 cells were seeded, cultured for 4 h, and then treated for 6 h with various concentrations of cladoloside C2 (0, 0.1, 0.3, or 0.5 µM). Right panel: K562 and HL-60 cells were seeded, cultured for 4 h, and then treated for the indicated times with 0.2 or 0.1 µM cladoloside C2. The percentage of apoptotic cells was determined by annexin V-FITC/PI staining. Upper panels: Representative of three separate experiments. Lower panels: Mean ± SD of three independent experiments. *P < 0.001 vs. control cells. (C) K562 and HL-60 cells were treated with 0.2 or 0.1 µM cladoloside C2 for the indicated times. Protein lysates were prepared and used for western blot analysis with the corresponding antibodies. β-actin was used as a loading control. The blot is representative of three separate experiments. (D) Functional involvement of caspases in cladoloside C2-induced apoptosis of K562 and HL-60 cells. Cells were pretreated for 1 h with the pan-caspase inhibitor Z-VAD-FMK (25 μM), the caspase-8 inhibitor Z-IETD-FMK (20 μM), the caspase-9 inhibitor Z-LEHD-FMK (20 μM), or the caspase-3 inhibitor Z-DEVD-FMK (50 μM), followed by treatment with 0.2 or 0.1 μM cladoloside C2 for 6 h. The extent of apoptosis was measured by flow cytometry after annexin V staining. These data represent the mean ± SD of three independent experiments. P < 0.01; *P < 0.001 vs. cladoloside C2-treated cells. (E) Left panel: K562 and HL-60 cells were treated with 0.2 or 0.1 μM cladoloside C2 for 6 h. The extent of apoptosis was measured by flow cytometry after annexin V staining. These data represent the mean ± SD of three independent experiments. *P < 0.001 vs. control cells. Right panel: K562 and HL-60 cells were treated with 0.2 or 0.1 mM cladoloside C2 for 4 h or 2 h. The cells were stained with DiOC6, and the reduction in Δφm was determined by monitoring the DiOC6 uptake using flow cytometry. Low Δφm values are expressed as the percentage of cells exhibiting diminished mitochondrial potential. The values obtained from the DiOC6 assays represent the mean ± SD of three independent experiments. (F) Western blot for the mitochondrial proteins AIF, Smac/DIABLO, cytochrome oxidase IV, and cytochrome c. Cytochrome oxidase IV (COX IV) was used as a mitochondrial marker. Protein lysates were prepared and subjected to western blot analysis using corresponding antibodies. β-actin was used as a loading control. The blot is representative of three separate experiments.
	Figure 2. Cladoloside C2 induces apoptosis of K562 and HL-60 cells through the activation of ceramide synthase 6 (CerS6)(A) K562 and HL-60 cells treated with cladoloside C2 exhibited increased ceramide generation. (B) K562 and HL-60 cells (1 × 105 cells/well) were incubated for 6 h with cladoloside C2 in the presence or absence of myriocin, fumonisin B1, desipramine, or GW4869. After treatment, the percentage of apoptotic cells was determined by annexin V-FITC/PI staining. Upper panel: Representative of three experiments in each cell line. Lower panel: Mean ± SD of three independent experiments. P < 0.01; P < 0.001 vs. cladoloside C2-treated cells. (C) K562 and HL-60 cells were incubated for 6 h with cladoloside C2 in the presence or absence of myriocin or fumonisin B1. Protein lysates were prepared and subjected to western blot analysis using corresponding antibodies. Western blots are each representative of three separate experiments. β-actin was used as a loading control. Densitometry results are expressed above the bands. (D–F) K562 and HL-60 cells were transiently transfected for 48 h by electroporation with CerS6 siRNA, nonspecific control (NC) siRNA, or no siRNA (shock). (D) Western blot analysis of protein lysates. (E) Transfected K562 cells were exposed to 0.2 μM cladoloside C2 for 2 h, and then fixed and permeabilized. Samples were then stained with PE-conjugated antibodies against Fas, CerS6, ceramide, cleaved caspase-8, p-p38 kinase, p38 kinase, p-ERK, ERK, p-JNK, or JNK, and with Alexa 488-labeled cholera toxin B antibody. The pictures are representative of three separate experiments. (F) Upper panel: The culture medium was changed, and K562 and HL-60 cells were incubated for 6 h with or without 0.2 or 0.1 μM cladoloside C2. The percentage of apoptotic cells was determined by annexin V-FITC/PI staining. Results are representative of three independent experiments in each cell line. Lower panel: Mean ± SD of three independent experiments. P < 0.01, **P < 0.001, cells treated with cladoloside C2 alone versus cells transfected with CerS6 siRNA and treated with cladoloside C2.
	Figure 3. Fas knockdown inhibits cladoloside C2-induced apoptosis in K562 and HL-60 cellsK562 and HL-60 cells were transiently transfected for 48 h by electroporation with Fas siRNA, nonspecific control (NC) siRNA, or no siRNA (shock). (A) Western blot analysis of protein lysates. (B) Transfected K562 cells were treated with cladoloside C2 for 2 h, and then fixed and permeabilized. The samples were then stained with PE-conjugated antibodies against Fas, CerS6, ceramide, cleaved caspase-8, p-p38 kinase, p38 kinase, p-ERK, ERK, p-JNK, or JNK, as well as Alexa 488-labeled cholera toxin B antibody. The pictures are representative of three separate experiments. (C) The culture medium was changed, and cells were incubated for 6 h with or without cladoloside C2. The percentage of apoptotic cells was determined by annexin V-FITC/PI staining. Upper panels: Representative of three independent experiments in each cell line. Lower panels: Mean ± SD of three independent experiments. **P < 0.01, cells treated with cladoloside C2 versus cells transfected with Fas siRNA and treated with cladoloside C2.
	Figure 4. Cladoloside C2 induces apoptosis of K562 and HL-60 cells through activation of p38 kinase and JNK(A) K562 and HL-60 cells were treated with cladoloside C2 for the indicated times. Protein lysates were prepared and subjected to western blot analysis. β-actin was used as a loading control. The blot is representative of three separate experiments. (B) K562 and HL-60 cells (1 × 105 cells/well) were pretreated with the p38 kinase inhibitor SB203580, the JNK inhibitor SP600125, or the ERK inhibitor PD98059, followed by 6 h of treatment with 0.2 or 0.1 μM cladoloside C2. After treatment for the indicated times, the percentage of apoptotic cells was determined by annexin V-FITC/PI staining. Upper panel: Representative of three independent experiments. Lower panel: Mean ± SD of three independent experiments. P < 0.05; P < 0.01; **P < 0.001, versus cells treated with cladoloside C2 in the absence of SB203580 or SP600125. (C and D) K562 and HL-60 cells (1 × 105 cells/well) were pretreated with the p38 kinase inhibitor SB203580 (C) or the JNK inhibitor SP600125 (D), followed by treatment with 0.2 or 0.1 μM cladoloside C2 for 6 h. Protein lysates were prepared and subjected to western blot analysis using corresponding antibodies. Western blots are each representative of three separate experiments. β-actin was used as a loading control. Densitometry results are expressed above the bands.
	Figure 5. p38 kinase knockdown and DN-JNK transfection inhibit cladoloside C2-induced apoptosis in K562 and HL-60 cells(A–C) K562 and HL-60 cells were transiently transfected by electroporation for 48 h with p38 siRNA, nonspecific control (NC) siRNA, or no siRNA (shock). (A) Transfected K562 and HL-60 cells were incubated for 6 h with or without cladoloside C2. Protein lysates were prepared and subjected to western blot analysis. (B) The culture medium was changed, and cells were incubated for 6 h with or without cladoloside C2. The percentage of apoptotic cells was determined by annexin V-FITC/PI staining. Upper panel: Representative of three independent experiments in each cell line. Lower panel: Mean ± SD of three independent experiments. *P < 0.001, cells treated with cladoloside C2 versus cells transfected with p38 kinase siRNA and treated with cladoloside C2. (C) Transfected K562 cells were treated with cladoloside C2 for 2 h, and then fixed and permeabilized. The samples were then stained with PE-conjugated antibodies against Fas, CerS6, ceramide, cleaved caspase-8, p-p38 kinase, p38 kinase, p-ERK, ERK, p-JNK, or JNK, as well as Alexa 488-labeled cholera toxin B antibody. The pictures are representative of three separate experiments. (D and E) K562 cells were transiently transfected by electroporation for 48 h with pcDNA or DN-JNK plasmid. (D) Transfected K562 cells were incubated for 6 h with or without cladoloside C2. Protein lysates were prepared and subjected to western blot analysis. (E) The culture medium was changed, and cells were incubated for 6 h with or without cladoloside C2. The percentage of apoptotic cells was determined by annexin V-FITC/PI staining. Upper panel: Representative of three independent experiments in K562 cells. Lower panel: Mean ± SD of three independent experiments. *P < 0.001, cells treated with cladoloside C2 versus cells transfected with DN-JNK and treated with cladoloside C2.
	Figure 6. Clustering of Fas and its downstream molecules in lipid rafts during cladoloside C2-induced apoptosis of K562 and HL-60 cells(A) K562 and HL-60 cells were pretreated for 1 h with MβCD (20 μg/mL) and nystatin (20 μg/mL), and then cultured for 6 h in medium containing 0.2 or 0.1 μM cladoloside C2. After treatment for the indicated times, the percentage of apoptotic cells was determined by annexin V-FITC/PI staining. Upper panel: Representative of three independent experiments. Lower panel: Mean ± SD of three independent experiments. P < 0.01; *P < 0.001 versus cladoloside C2-treated cells. (B) Whole cell lysates were prepared from K562 and HL-60 cells incubated for 6 h with cladoloside C2 in the presence or absence of MβCD, and were subjected to western blot analysis. (C) K562 cells were treated for 2 h with cladoloside C2 in the presence or absence of MβCD or nystatin, and then fixed and permeabilized. These samples were then stained with PE-conjugated antibodies against Fas, CerS6, ceramide, cleaved caspase-8, p-p38 kinase, p38 kinase, p-ERK, ERK, p-JNK, or JNK, as well as Alexa 488-labeled cholera toxin B antibody. The pictures are representative of three separate experiments.
	Figure 7. Cladoloside C2 inhibits the growth of K562 and HL-60 xenograft tumors and induces apoptosis through Fas/CerS6/p38 kinase/JNK activation in vivo(A) K562 (1.5 × 107 cells/100 μL) and HL-60 cells (2 × 107 cells/100 μL) were subcutaneously injected into Balb/c nude mice. After the cells formed palpable tumors, the mice were randomized into two groups (n = 5 mice/group) and treated with vehicle control or cladoloside C2 (7.19 μg/kg). Tumor size was measured daily using a caliper: calculated volume = shortest diameter2 × longest diameter/2. P < 0.05, P < 0.01, and *P < 0.001, versus respective controls. (B) Left panel: NC-shRNA-transfected and CerS shRNA-transfected stable K562 (1.5 × 107 cells/100 mL) were subcutaneously injected into Balb/c nude mice. Right panel: NC-shRNA-transfected and CerS shRNA-transfected stable HL-60 (2 × 107 cells/100 mL) were subcutaneously injected into Balb/c nude mice. After the cells formed palpable tumors, the mice were randomized into two groups (n = 5 mice/group), and treated with vehicle control or cladoloside C2 (7.19 μg/kg). Tumor size was measured daily using a caliper. P < 0.05, P < 0.01, and *P < 0.001, versus respective controls. (C and D) Tumor tissues obtained from the above-described experiment on Day 14 were subjected to western blot analysis (C) and immunohistochemistry (D) using antibodies against Fas, CerS6, ceramide, p-p38, p38 kinase, p-JNK, or JNK. The sections were lightly counterstained with hematoxylin, and photographed with a ScanScope. K562 and HL-60 leukemia xenografts from cladoloside C2-treated mice exhibited apoptosis and extensive necrosis (200×).
	Figure 8. Hypothetical molecular mechanisms of cladoloside C2-induced apoptosis in human leukemia cells