A Phenylbutenoid Dimer, cis-3-(3',4'-Dimethoxyphenyl)-4-[(E)-3''',4'''-Dimethoxystyryl] Cyclohex-1-ene, Exhibits Apoptogenic Properties in T-Acute Lymphoblastic Leukemia Cells via Induction of p53-Independent Mitochondrial Signalling Pathway

Abstract

The current study was designed to evaluate the in vitro cytotoxicity effect of a phenylbutenoid dimer, cis-3-(3',4'-dimethoxyphenyl)-4-[(E)-3 (‴) ,4 (‴) -dimethoxystyryl]cyclohex-1-ene (ZC-B11) isolated from the rhizome of Zingiber cassumunar on various cancer cell line, and normal human blood mononuclear cells, and to further investigate the involvement of apoptosis-related proteins that leads, to the probable pathway in which apoptosis is triggered. Cytotoxicity test using MTT assay showed selective inhibition of ZC-B11 towards T-acute lymphoblastic leukemia cells, CEMss, with an IC50 value of 7.11 ± 0.240 μ g/mL, which did not reveal cytotoxic effects towards normal human blood mononuclear cells (IC50 > 50 μ g/mL). Morphology assessments demonstrated distinctive morphological changes corresponding to a typical apoptosis. ZC-B11 also arrested cell cycle progression at S phase and causes DNA fragmentation in CEMss cells. Decline of mitochondrial membrane potential was also determined qualitatively. In the apoptosis-related protein determination, ZC-B11 was found to significantly upregulate Bax, caspase 3/7, caspase 9, cytochrome c, and SMAC and downregulate Bcl-2, HSP70, and XIAP, but did not affect caspase 8, p53, and BID. These results demonstrated for the first time the apoptogenic property of ZC-B11 on CEMss cell line, leading to the programmed cell death via intrinsic mitochondrial pathway of apoptosis induction.

Figure 1

The chemical structure of ZC-B11.

Figure 2

Normal phase contrast inverted micrograph of CEMss cells treated withZC-B11 (IC₅₀) for 24, 48, and 72 h. (a) Control, (b) most of the cells exhibit normal morphology while some cells show cytoplasmic protrusions (24 h), (c) clear apoptogenic morphology such as blebbing and cell shrinkage observed (48 h), and (d) prominent growth inhibition, blebbing of the cell membrane, and shrinkage of cells observed (72 h). BL: blebbing of the cell membrane; CS: cell shrinkage (400x magnification).

Figure 3

Confocal micrograph of acridine orange and propidium iodide double-stained CEMss cells after 24, 48, and 72 h treatment with ZC-B11 (IC₅₀). (a) Control, (b) cells exhibit blebbing of the cell membrane and bright green nucleus showing condensation of chromatin (24 h), (c) blebbing was observed with some orange-coloured cells which denotes late apoptosis (48 h), and (d) more blebbing and late apoptosis, orange colour represents the hallmark of late apoptosis while red color represents secondary necrosis or dead cells (72 h). VI: viable cells; BL: blebbing of the cell membrane; CC: chromatin condensation; AB: apoptotic body; LA: late apoptosis; SN: secondary necrosis (400x magnification).

Figure 4

Flow cytometric analysis of cell cycle phase distribution of CEMss cells treated with ZC-B11 (IC₅₀) in a time-dependent manner. (a) Control, (b) 24 h, (c) 48 h, and (d) 72 h. Region I is “sub-G0/G1” peak denoting apoptotic cells with hypodiploid DNA content, Region II is “G0/G1” phase, Region III is S phase, and Region IV is “G2/M” phase.

Figure 5

Graphical presentation of cell cycle phase distribution analysis. Induction of S phase arrest in the cell cycle progression of CEMss cells treated with ZC-B11 (IC₅₀). Results were represented as means ± SD of three independent experiments. “∗” indicates a significant difference from the control (P < 0.05).

Figure 6

Electrophoresis separation of fragmented DNA of untreated and treated CEMss cells for 48 hours with ZC-B11 (IC₅₀). Lane A: negative control (untreated CEMss cells); Lane B: 48 hours treatment; Lane C: DNA marker; Lane D: positive control.

Figure 7

Fluorescent micrograph of CEMss cells treated with ZC-B11 (IC₅₀) for 12, 24, 48 and 72 h, stained with Rh123 dye. (a) Control, (b) 12 h, (c) 24 h, (d) 48 h, and (e) 72 h. (400x magnification).

Figure 8

Human apoptosis proteome profiler array in CEMss cells treated with ZC-B11 (IC₅₀) for 48 hours. Graph shows the difference between treated and untreated control cells. Results were represented as means ± SD for three independent experiments. “*” indicates a significant difference from the control (P < 0.05).

Figure 9

The bioluminescent assay of caspases 3/7 (a), 8 (b), and 9 (c) in CEMss cells treated with ZC-B11 (IC₅₀) after 24, 48, and 72 hours of treatment. Untreated cells serve as negative control. (a), (c) Caspase 3/7 and 9 activities increased significantly (*P < 0.05) compared to untreated control; (b) Caspase 8 activity remained at the basal level throughout the treatment period. Results were represented as means ± SD for three independent experiments.

Figure 10

Western blot analysis of Bax, Bcl-2, and HSP70 levels in CEMss cells treated with ZC-B11 (IC₅₀) for 3, 6, 12, and 24 hours and compared with negative control (0 hours). The expression of Bax increased while Bcl-2 and HSP70 decreased after treatment in a time-dependent manner. β-actin was used as the internal control to confirm equal sample loading.