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

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 ICvalue of 7.11 ± 0.240 g/mL, which did not reveal cytotoxic effects towards normal human blood mononuclear cells (IC> 50 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

Induction of selective cytotoxicity and apoptosis in human T4-lymphoblastoid cell line (CEMss) by boesenbergin a isolated from boesenbergia rotunda rhizomes involves mitochondrial pathway, activation of caspase 3 and G2/M phase cell cycle arrest

Background Boesenbergia rotunda (Roxb.) Schlecht (family zingiberaceae) is a rhizomatous herb that is distributed from north-eastern India to south-east Asia, especially in Indonesia, Thailand and Malaysia. Previous research has shown that the crude extract of this plant has cytotoxic properties. The current study examines the cytotoxic properties of boesenbergin A isolated from Boesenbergia rotunda. Methods MTT assay was used to check the cytotoxicity of boesenbergin A. The morphological assessment of apoptosis was monitored using normal and fluorescence microscopy. The early and late phase of apoptosis was investigated using annexin V and DNA laddering assays, respectively. The mitochondrial membrane potential (MMP) was assessed by fluorescence microscopy. Human apoptosis proteome profiler assays were performed to investigate the mechanism of cell death. In addition, the protein levels of Bax, Bcl2 and HSP 70 were also analyzed using western blot. Assays of caspase =-3/7, -8 and =-9 were carried out in order to test for induction during treatment. Lastly, cell cycle progression was analyzed using flow cytometry. Results Boesenbergin A was found to have the highest toxicity towards CEMss cancer cells (IC50 = 8 μg/ml). The morphology of CEMss cells after treatment showed evidence of apoptosis that included blebbing and chromatin condensation. The annexin V assay revealed that early apoptosis is induced after treatment. The DNA laddering assay confirmed that DNA fragmentation had occurred during late apoptosis. The cell cycle analysis indicated that boesenbergin A was able to induce G2/M phase arrest in CEMss cells. The activity of caspases -3/7, -8 and -9 was increased after treatment which indicates both intrinsic and extrinsic pathways are induced during apoptosis. The involvement of mitochondria was established by increased mitochondrial membrane potential and up and down regulation of Bcl2 and Bax proteins as well as HSP70. Conclusion In conclusion, the results demonstrated that boesenbergin A induced apoptosis of CEMss cells through Bcl2/Bax signaling pathways with the involvement of caspases and G2/M phase cell cycle arrest. The current findings warrant further research on boesenbergin A as a novel chemotherapeutic agent for leukemia intervention including studies in animal models

Induction of apoptosis by cis-3-(3',4'-dimethoxyphenyl)-4-[(E)-3"',4"'-dimethoxystyryl]cyclohex-1-ene isolated from the rhizome of Zingiber cassumunar roxb. on human T-lymphoblastic leukemia cell line, CEMss

Zingiber cassumunar Roxb. is one of the most widely cultivated species of Zingiberaceae family and commonly known as ‘plai’ in Thailand and ‘bonglai’ in Malaysia. cis-3-(3',4'-Dimethoxyphenyl)-4-[(E)-3''',4'''-dimethoxystyryl]cyclohex-1-ene (ZC-B11) is a phenylbutenoid dimer isolated from the rhizomes of Z.cassumunar. The objective of this study is to investigate the antiproliferative activities of this compound on human T-lymphoblastic cell line, CEMss and the mechanism by which apoptosis is triggered. In vitro cytotoxic effect of ZC-B11 was determined using MTT assay in several human cancer cell lines including leukemia (CEMss). ZC-B11 showed selectivity towards CEMss with an IC50 value of 7.11 ± 0.24 μg/ml. The antiproliferative activity of ZC-B11 was also tested against nontumorigenic human blood mononuclear cells and ZC-B11 does not show cell growth inhibition of human blood mononuclear cells (IC50 > 50 μg/ml). Various microscopy techniques used in this study showed distinctive morphological changes corresponding to typical apoptosis. Cell cycle analysis revealed significant (p < 0.05) S phase arrest in a time-depended manner whilst DNA fragmentation of ZC-B11 treated CEMss cells was detected using 1.2% agarose gel. Decrement of mitochondrial membrane potential was also observed in treated CEMss cells in timedepended manner using the Rh123 staining. To evaluate further the mechanisms of apoptosis induction by ZC-B11 towards CEMss cells, screening of several proteins implicated to apoptosis induction were done using the human apoptosis proteome profiler array, in which, proteins such as Bax, caspase 3, cytochrome c and SMAC showed significant increase (p < 0.05) compared to untreated control cells, whilst proteins such as Bcl-2, HSP70 and XIAP decreased significantly. On the other hand, caspase 8, p53 and BID remain unaffected (p > 0.05). Caspase bioluminescent assay and Western blot analysis were done to further confirm these results. Collectively, results presented in this study demonstrate that ZC-B11 isolated from the rhizome of Z. cassumunar inhibited the proliferation of CEMss selectively, leading to the programmed cell death via mitochondrial signaling pathway and has the potential to be developed as an antileukemic and chemotherapy agent

Acute Toxicity Study of Zerumbone-Loaded Nanostructured Lipid Carrier on BALB/c Mice Model

Zerumbone- (ZER-) loaded nanostructure lipid carrier (NLC) (ZER-NLC) prepared for its antileukemia effect in vitro was evaluated for its toxicological effects by observing changes in the liver, kidney, spleen, lung, heart, and brain tissues, serum biochemical parameters, total haemogram, and bone marrow stem cells. The acute toxicity study for ZER-NLC was conducted by orally treating BALB/c mice with a single dose with either water, olive oil, ZER, NLC, or ZER-NLC for 14 days. The animals were observed for clinical and behavioral abnormalities, toxicological symptoms, feed consumption, and gross appearance. The liver, kidney, heart, lung, spleen, and brain tissues were assessed histologically. Total haemogram was counted by hemocytometry and microhematocrit reader. Bone marrow examination in terms of cellular morphology was done by Wright staining with bone marrow smear. Furthermore, serum biochemical parameters were determined spectrophotometrically. Grossly all treated mice, their investigated tissues, serum biochemical parameters, total haemogram, and bone marrow were normal. At oral doses of 100 and 200 mg/kg ZER-NLC there was no sign of toxicity or mortality in BALB/c mice. This study suggests that the 50% lethal dose (LD50) of ZER-NLC is higher than 200 mg/kg, thus, safe by oral administration