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Yun SH
,
Sim EH
,
Han SH
,
Han JY
,
Kim SH
,
Silchenko AS
,
Stonik VA
,
Park JI
.
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Marine triterpene glycosides are attractive candidates for the development of anticancer agents. Holotoxin A₁ is a triterpene glycoside found in the edible sea cucumber, Apostichopus (Stichopus) japonicus. We previously showed that cladoloside C₂, the 25(26)-dihydro derivative of holotoxin A₁ induced apoptosis in human leukemia cells by activating ceramide synthase 6. Thus, we hypothesized that holotoxin A₁, which is structurally similar to cladoloside C₂, might induce apoptosis in human leukemia cells through the same molecular mechanism. In this paper, we compared holotoxin A₁ and cladoloside C₂ for killing potency and mechanism of action. We found that holotoxin A₁ induced apoptosis more potently than cladoloside C₂. Moreover, holotoxin A₁-induced apoptosis in K562 cells by activating caspase-8 and caspase-3, but not by activating caspase-9. During holotoxin A₁-induced apoptosis, acid sphingomyelinase (SMase) and neutral SMase were activated in both K562 cells and human primary leukemia cells. Specifically inhibiting acid SMase and neutral SMаse with chemical inhibitors or siRNAs significantly inhibited holotoxin A₁-induced apoptosis. These results indicated that holotoxin A₁ might induce apoptosis by activating acid SMase and neutral SMase. In conclusion, holotoxin A₁ represents a potential anticancer agent for treating leukemia. Moreover, the aglycone structure of marine triterpene glycosides might affect the mechanism involved in inducing apoptosis.
Figure 2. Holotoxin A1 induces apoptosis through extrinsic pathway activation in human leukemic cells. (A) Analysis of the mechanism underlying apoptosis. Western blot of K562 cell proteins after treating cells with 0.06 μM holotoxin A1 shows changes in protein levels over time. β-actin served as a loading control. This blot is representative of three separate experiments. Densitometry values above the bands indicate the fractional changes in protein levels, compared to initial levels at time 0; (B) Functional involvement of caspases in holotoxin A1-induced apoptosis in K562 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 0.06 μM holotoxin A1 for 6 h. (Upper panel) Representative flow cytometry results indicate the extent of apoptosis (Lower panel) The mean ± SD of three independent experiments. ** p < 0.01 vs. holotoxin A1-treated cells; (C,D) Analysis of the intrinsic pathway. (C) Mitochondrial membrane potential was not affected by holotoxin A1. Left panel: K562 cells were treated with 0.06 μM holotoxin A1 for 6 h. (Upper panel) Representative flow cytometry results indicate the extent of apoptosis. (Lower panel) The mean ± SD of three independent experiments. *** p < 0.001 vs. control cells. Right panel: K562 cells were treated with 0.06 μM holotoxin A1 for 2 h. The cells were stained with DiOC6 (3,3′-dihexyloxacarbocyanine iodide). (Upper panel) Flow cytometry results show mitochondrial membrane potential (∆ϕm), determined by monitoring the DiOC6 uptake. (Lower panel) The mean ± SD of three independent DiOC6 uptake experiments; (D) Western blots show the effect of holotoxin A1 over time on the levels of mitochondrial (left) and cytosolic (right) proteins: AIF, Smac/DIABLO, cytochrome oxidase IV, and cytochrome c, in K562 cells. Cytochrome oxidase IV (COX IV) served as a mitochondrial marker. β-actin served as a loading control. These blots are representative of three separate experiments. Densitometry values above the bands indicate fractional changes from initial values at time 0; (E) Western blot shows the effect of holotoxin A1 over time on the levels of antiapoptotic proteins, Mcl-1, Bcl-2, and Bcl-xL, and proapoptotic protein, Bax, in K562 cells. β-actin served as a loading control. This blot is representative of three separate experiments. Densitometry values above the bands indicate fractional changes from initial values at time 0.
Figure 3. Holotoxin A1 induces apoptosis through activation of acid SMase and neutral SMase in human leukemic cells. (A) Immunohistochemistry images of K562 cells show that holotoxin A1 stimulated the production of ceramide (green) by activating (left) acid SMase (red) and (right) neutral SMase (red); (B) SMase inhibitors block apoptosis. K562 cells (1 × 105 cells/well) were incubated for 6 h with holotoxin A1 in the presence or absence of myriocin, fumonisin B1, desipramine, or GW4869, and the percentage of apoptotic cells was determined with annexin V-FITC/PI staining. Upper panel: Representative flow cytometry results of three experiments for each condition. Lower panel: Mean ± SD of three independent experiments. ** p < 0.01 vs. holotoxin A1-treated cells; (C,D) Pathway analysis. K562 cells were untreated (control) or exposed to 0.06 μM holotoxin A1 (HA1) for 2 h in the presence or absence of desipramine or GW4869. (C) Cells were fixed, permeabilized, and then stained with FITC-labeled ceramide antibody (green) and PE-conjugated antibodies against (left) acid SMase (red) or (right) neutral SMase (red). Images are representative of three separate experiments; (D) K562 cell lysates were analyzed on Western blots probed with the indicated antibodies. Western blots are representative of three separate experiments. β-actin was used as a loading control. Densitometry values above the bands indicate fractional changes compared to control; (E,F) Membrane translocation. K562 cells were treated with 0.06 μM holotoxin A1, then cells were fractionated to analyze membrane and cytosolic proteins. (E) After the indicated treatment times, (left) cytosolic and (right) membrane fractions were run on western blots and probed with antibodies against acid SMase (ASM) or neutral SMase (NSM). Western blots are representative of three separate experiments. β-actin was used as a loading control. Densitometry values above the bands indicate fractional changes from initial values at time 0; (F) K562 cells were untreated (control) or incubated for 2 h with holotoxin A1 in the presence or absence of (top panels) desipramine or (bottom panels) GW4869. Western blots show (left) cytosolic and (right) membrane fractions probed with the indicated antibodies. Western blots are representative of three separate experiments. β-actin was used as a loading control. Densitometry values above the bands indicate fractional changes compared to control.
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