Zerumbone-loaded nanostructured lipid carrier induces G2/M cell cycle arrest and apoptosis via mitochondrial pathway in a human lymphoblastic leukemia cell line

Figures & data

Figure 1 Effect of ZER-NLC on normal human peripheral blood mononuclear cells assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. The cells were treated for 24, 48, and 72 hours(h). The results are shown as the mean percentage of absorbance ± standard deviation of three separate experiments. No significant (P>0.05) decreases in cell viability were observed at any concentration.

Abbreviation: ZER-NLC, zerumbone-loaded nanostructured lipid carrier.

Figure 2 Fluorescent micrograph of acridine orange/propidium iodide double-stained Jurkat cells treated with the ZER-NLC. (A) Untreated cells showing a normal structure. (B) Early apoptosis of cells after 24 hours of treatment showing intercalated bright green staining with a marginated nucleus, chromatin condensation, and blebbing. (C) Blebbing, chromatin condensation, and nuclear margination after 48 hours of treatment. (D) Late apoptosis of cells after 72 hours of treatment showing reddish-orange staining with apoptotic body formation. Secondary necrotic cells displayed a reddish nucleus with an intact structure (400× magnification).

Abbreviations: VC, viable cells; EA, early apoptotic cells; CC, chromatin condensation; MN, marginated nucleus; BL, cell membrane blebbing; AB, apoptotic body; LA, late apoptotic cells; SN, secondary necrotic cell; ZER-NLC, zerumbone-loaded nanostructured lipid carrier.

Figure 3 Ultrastructure of Jurkat cells treated with ZER-NLC. (A) Untreated control cells with typical cancer cell morphologic features with numerous MV. (B) Cells treated for 24 hours showing membrane blebbing and hole formation. (C) Cells treated for 48 hours showing cell shrinkage, increasing membrane blebbing, and hole formation. (D) Cells treated for 72 hours showing distinctive morphologic changes typical of apoptosis, including membrane blebbing and cell shrinkage with apoptotic body and hole formation.

Abbreviations: AB, apoptotic body; MB, membrane blebbing; MV, microvilli; ZER-NLC, zerumbone-loaded nanostructured lipid carrier; Mag, magnification; EMUPM, electron microscopy-university Putra Malaysia; EHT, extra high tension; WD, width.

Figure 4 Ultrastructure of Jurkat cells treated with the zerumbone-loaded nanostructured lipid carrier. (A) Untreated control cells demonstrate a structure and morphology typical of leukemic cells. The nucleus (N) contains evenly distributed chromatin and a large nucleolus (Nu). Large and numerous mitochondria (M), rough endoplasmic reticulum (RER), ribosome (R), and typical microvilli (MV) are also seen. (B) Cells treated for 24 hours showing early-stage apoptosis characterized by membrane blebbing (MB) and a reduced number of cell organelles. (C) Cells treated for 48 hours showing mid-stage apoptosis characterized by chromatin condensation (CC), formation of apoptotic micronuclei (AMN), and apoptotic bodies (AB) with vacuolization (V). Loss, rupture, and condensation of mitochondria cristae in the Jurkat cells also occurred after 48 hours of treatment (D). (E) Cells treated for 72 hours showing late-stage apoptosis characterized by distinct morphologic changes, including cell shrinkage, increased cell granularity, nuclear margination (MN), AB and V.

Figure 5 Flow cytometric analysis of Jurkat cells treated with the zerumbone-loaded nanostructured lipid carrier and after staining with FITC-conjugated Annexin V and propidium iodide. (A1–D1) Untreated Jurkat cell control at 6, 12, 24, and 48 hours, respectively. (A2–D2) Jurkat cells treated with the zerumbone-loaded nanostructured lipid carrier for 6, 12, 24, and 48 hours, respectively.

Abbreviation: FITC, fluorescein isothiocyanate.

Figure 6 Analysis of the Jurkat cell cycle after treatment with the zerumbone-loaded nanostructured lipid carrier. The DNA content was analyzed by flow cytometry. (A1–D1) Untreated Jurkat control cells after 12, 24, 48, and 72 hours. (A2–D2) Jurkat cells treated with the zerumbone-loaded nanostructured lipid carrier for 12, 24, 48, and 72 hours. G0/G1, G2/M, and S are cell phases, and sub-G1 DNA content refers to apoptotic cells.

Figure 7 Apoptosis of Jurkat cells treated with the zerumbone-loaded nanostructured lipid carrier as determined by flow cytometry after staining with rTdT. (A1–D1) Untreated Jurkat cells at 12, 24, 48, and 72 hours. (A2–D2) Jurkat cells treated for 12, 24, 48, and 72 hours. Cells in the lower quadrant (R2 and R3) are nonapoptotic and cells in the upper quadrant (R4) are apoptotic.

Abbreviations: TUNEL, Tdt-mediated dUTP nick-end labeling; FITC, fluorescein isothiocyanate.

Figure 8 Caspase activity in untreated control Jurkat cells and Jurkat cells treated with the ZER-NLC at 24, 48, and 72 hours(h). The results show statistically significant (P<0.05) differences in caspase-3 and caspase-9 activity between untreated and treated cells, but no statistically significant (P>0.05) activity for caspase-8.

Abbreviation: ZER-NLC, zerumbone-loaded nanostructured lipid carrier.

Figure 9 (A) Western blots showing the effect of the zerumbone-loaded nanostructured lipid carrier on levels of cell cycle proteins regulating apoptosis in Jurkat cells after 24, 48, and 72 hours(h). B-actin was used as the loading control. (B) Western blotting analysis of the zerumbone-loaded nanostructured lipid carrier in Jurkat cells. The level of each protein was measured and normalized to β-actin. The values are shown as the mean ± standard deviation percentage of three independent experiments. Statistically significant differences (P<0.05) were found between treated cells and control cells in each group.

Abbreviations: Cyt-c, cytochrome c; Bcl-2, B cell lymphoma 2; Bax, Bcl-2 associated X protein; PARP, poly(adenosine diphosphate-ribose) polymerase; β-actin, Beta actin.

Figure 10 Hypothetical diagram demonstrating possible apoptotic effects of the ZER-NLC in Jurkat cells in vitro.

Abbreviations: ZER-NLC, zerumbone-loaded nanostructured lipid carrier; Bcl-2, B cell lymphoma 2; Bax, Bcl-2 associated X protein; PARP, poly(adenosine diphosphate-ribose) polymerase.

Figure S1 Percentages of viable, early apoptotic, late apoptotic, and secondary necrotic cells after treatment with the ZER-NLC for 24, 48, and 72 hours using the acridine orange/propidium iodide double-staining test. Jurkat cell death via apoptosis increased significantly (P<0.05) in a time-dependent manner, but no significant difference (P>0.05) was observed in the necrotic cell count.

Abbreviations: EA, early apoptosis; LA, late apoptosis; SN, secondary necrosis; ZER-NLC, zerumbone-loaded nanostructured lipid carrier.

Figure S2 Profile of the Jurkat cell cycle in the presence of the ZER-NLC suspension. After 12, 24, 48, and 72 hours(h) of incubation with the ZER-NLC, the DNA content was evaluated with propidium iodide and acridine orange staining, and fluorescence was measured and then analyzed. The values are shown as the mean ± standard deviation percentage of three independent experiments.

Abbreviation: ZER-NLC, zerumbone-loaded nanostructured lipid carrier.

Table S1 Flow cytometric analysis of Annexin V-FITC in Jurkat cells after treatment with ZER-NLC

Cell condition	Cells (%)
	6 hours		12 hours		24 hours		48 hours
	Control	ZER-NLC	Control	ZER-NLC	Control	ZER-NLC	Control	ZER-NLC
Viable cells	96.93±0.188	79.2±0.16	96.39±0.25	21.71±0.66	93.88±0.16	15.48±0.73	93.16±0.98	28.62±0.94
Early apoptosis	1.67±0.11	12.34±0.51*	1.85±0.07	63.08±0.48*	3.45±0.11	21.37±0.42*	3.91±0.54	12.15±0.40*
Late apoptosis/necrosis	1.39±0.14	8.45±0.65**	1.76±0.2	15.19±0.12**	2.66±0.11	63.14±0.58**	2.92±0.44	59.34±1.10**

Notes: Values are expressed as the mean ± standard deviation of three different experiments. The data were analyzed using post hoc comparison test-one way analysis of variance, and the means were compared by Tukey’s b test.

* Significant (P<0.05) increase in early apoptotic cells in ZER-NLC-treated group versus untreated control.

** Significant (P<0.05) increase in late apoptotic/necrotic cells in ZER-NLC-treated group versus untreated control.

Abbreviations: FITC, fluorescein isothiocyanate; ZER-NLC, zerumbone-loaded nanostructured lipid carrier.

Table S2 Flow cytometric analysis of Jurkat cell cycle after treatment with ZER-NLC

Cell cycle phase	Cells (%)
	12 hours		24 hours		48 hours		72 hours
	Control	ZER-NLC	Control	ZER-NLC	Control	ZER-NLC	Control	ZER-NLC
G0/G1	59.81±0.82	40.44±0.58	52.81±0.68	34.09±0.5	62.73±0.57	38.28±0.32	58.7±0.59	32.42±0.83
G2/M	18.38±0.53	35.46±0.49*	21.43±0.84	40.14±0.75*	15.36±0.55	34.91±1.22*	10.64±0.31	24.59±2.5*
S	21.51±0.47	22.83±0.08	24.79±0.33	24.46±0.35	19.81±0.32	21.83±0.5	21.75±0.69	21.45±1.2
SubG0/G1	0.81±0.07	1.39±0.06	1.56±0.09	1.99±0.64	2.47±0.24	5.52±0.47	9.27±0.67	22.12±0.76

Notes: Cells were stained with propidium iodide and incubated at 37°C for 24, 48, and 72 hours. Values are expressed as the mean ± standard deviation of three different experiments. The data were analyzed using post hoc comparison test-one way analysis of variance, and the means were compared by Tukey’s b test.

* Significant (P<0.05) increase in cells in G2/M phase in ZER-NLC-treated group versus untreated control.

Abbreviation: ZER-NLC, zerumbone-loaded nanostructured lipid carrier.

Table S3 TUNEL flow cytometric analysis of Jurkat cells treated with ZER-NLC

Cell condition	Cells (%)
	24 hours		48 hours		72 hours
	Control	ZER-NLC	Control	ZER-NLC	Control	ZER-NLC
Viable	96.63±0.4	77.75±0.09	97.63±0.21	68.97±0.7	95.68±0.24	60.31±1.07
Apoptotic	3.36±0.3	22.73±0.09*	2.36±0.5	31.19±0.7*	4.32±0.28	39.68±1.1*

Notes: Values are expressed as the mean ± standard deviation of three different experiments. The data were analyzed using post hoc comparison test-one way analysis of variance, with means compared by the Tukey’s b test.

* Significant (P<0.05) increase in apoptotic cells in ZER-NLC-treated group versus untreated control.

Abbreviations: TUNEL, Tdt-mediated dUTP nick-end labeling; ZER-NLC, zerumbone-loaded nanostructured lipid carrier.

Table S4 Spectrophotometric analysis of Jurkat cell caspase activity after treatment with ZER-NLC

Caspases	Cells (%)
	24 hours		48 hours		72 hours
	Control	ZER-NLC	Control	ZER-NLC	Control	ZER-NLC
Caspase-8	0.05±0.01	0.058±0.003	0.056±0.004	0.063±0.003	0.061±0.003	0.068±0.003
Caspase-3	0.06±0.003	0.13±0.01*	0.09±0.003	0.199±0.007*	0.1±0.1	0.221±0.01*
Caspase-9	0.065±0.001	0.151±0.016*	0.071±0.002	0.211±0.012*	0.086±0.001	0.261±0.011*

Notes: Values are expressed as the mean ± standard deviation of three different experiments. The data were analyzed using post hoc comparison test-one way analysis of variance, with means compared by Tukey’s b test.

* Significant (P<0.05) increase in caspase activity in the ZER-NLC-treated group versus untreated control.

Abbreviation: ZER-NLC, zerumbone-loaded nanostructured lipid carrier.

Table S5 Western blot analysis of protein transcription in ZER-NLC-treated Jurkat cells

Proteins	Control	24 hours	48 hours	72 hours
Protein transcription level (%)
Bax	21.29	36.11*	44.32*	57.19*
Bcl-2	55.90	32.75**	25.54**	18.55**
Cyt-c	17.61	25.23*	33.45*	45.78*
PARP	22.02	31.20*	43.98*	54.11*
Cyclin B1	48.54	35.23**	24.14**	16.34**

Notes: Values are expressed as the mean ± standard deviation of three different experiments. The data were analyzed using post hoc comparison test-one way analysis of variance, with means compared by Tukey’s b test.

* Significant (P<0.05) upregulation of protein in the ZER-NLC-treated group versus untreated control.

** Significant (P<0.05) downregulation of protein in ZER-NLC-treated group versus untreated control.

Abbreviations: PARP, poly(adenosine diphosphate-ribose) polymerase; ZER-NLC, zerumbone-loaded nanostructured lipid carrier; Bcl-2, B cell lymphoma 2; Bax, Bcl-2 associated X protein; Cyt-c, cytochrome c.