Sinonim : Leea sambucina L Willd. Bunga bintang, mahkota bentuh torong,kepala sari putih, hijau. Buah Akar : Buni, bulat, hitam. Bulat, putih.
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MTT assay measured the cell viability based on the reduction of yellow tetrazolium MTT to a purple formazan dye by mitochondrial dehydrogenase enzyme. Hence, the amount of formazan produced reflected the number of metabolically active viable cells [ 20 ].
MTT results showed that Leea indica possessed cytotoxic effect against Ca Ski cells whereby all the extract and fractions significantly reduced formazan accumulation in a dose-dependent manner Figure 1 a.
The Ca Ski cells varied in their sensitivity to the extract and fractions and were found most susceptible to LIEAF which demonstrated the strongest growth inhibitory effect lowest IC50 value. Therefore, Ca Ski cervical cancer cell line was selected for further studies to determine the mechanism of cell death underlying the observed growth inhibitory action.
Since apoptotic cells exhibited some characteristic morphological features, nuclear morphological changes were assessed using DAPI staining. In order to analyze whether the growth inhibition was accompanied by any alterations in cell cycle distribution, PI staining was applied to Ca Ski cells treated without and with LIEAF at different doses and time periods. The appearance of sub-G1 peak in the DNA histogram represented cells with hypodiploid DNA content and this sub-G1 population was considered as apoptotic fraction [ 21 ].
Annexin V is a phosphatidylserine- PS- binding protein while PI is a DNA-binding dye and this dual staining analyzes the externalization of phosphatidylserine PS from the inner to the outer leaflet of membranes during the early phase of apoptosis.
Mitochondrial dysfunction such as loss of MMP is an early apoptotic event that occurs following induction of apoptosis [ 23 ]. JC-1 is a widely used dye to detect mitochondrial depolarization which occurs in the early stage of apoptosis.
During the onset of apoptosis, the MMP decreases and the JC-1 remains in the monomeric form and emits green fluorescence. Thus, the ratio of red to green fluorescence measures the ratio of high-to-low MMP [ 24 ].
In the control cells, JC-1 was in aggregate forms within the mitochondria, resulting in higher levels of red fluorescence, which corresponded to a polarized MMP. We further investigated the involvement of oxidative stress in cell death by looking at the alterations of total glutathione GSH level. It has been reported that depletion in intracellular GSH can contribute to the onset of apoptosis, by rendering the cells more sensitive and susceptible to apoptotic agents [ 26 ].
Our results are consistent with this observation. Caspases-3, a member of the cysteine proteases family, plays an important role in the execution of apoptosis. They proteolytically cleave many cellular proteins which lead to loss of cellular structure and functions, and ultimately cell death [ 27 — 29 ].
In the present study, a quantitative assay was performed for caspase-3 protease activity using a commercial assay kit.
Next, we investigated the possibility that the LIEAF-induced apoptosis might be due to activation of caspase However, these apoptotic responses seem to increase with time. It is possible that the occurrence of DNA fragmentation sub-G1 analysis and DNA strand breaks TUNEL assay that were prominent at 72 hours might be attributed to the activation of intracellular caspase-3 which peaked at 72 hours.
Our data on MMP depolarization and caspase-3 induction suggested that LIEAF-induced apoptosis in Ca Ski cells might be mediated by mitochondria death pathway involving the caspase cascade. At first, LIEAF depolarized the mitochondria, and this mitochondrial dysfunction possibly contributed to the activation of caspase-3 enzyme.
At the same time, the PS residues were externalized across the plasma membrane. Then the sequential activation of caspase-3 eventually cleaved the cellular and nuclear components which caused the DNA fragmentation and DNA strands breaks. Despite the phytochemical studies on Leea indica, the biological activities of this plant and its active constituents isolated from this plant have not been examined to a large extent.
The evidences presented here have shown for the first time that LIEAF could inhibit the growth of cancer cells and induce apoptosis in Ca Ski cells.
This warrants the need for bioactivity-guided isolation of the bioactive compound s in LIEAF, which is now in progress. Elaborate studies of the detailed mechanism underlying its apoptotic action are necessary. The obtained findings will definitely provide valuable information for its possible application in cervical cancer treatment in the future. Acknowledgment The authors would like to thank Dr. This study was supported by grant from the Institute of Research Management and Consultancy of University of Malaya through postgraduate research fund no.
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