Chemotherapeutics potential of matcha green tea(Camellia sinensis (L.) Kuntze) on weri-Rb-1 retinoblastoma cancer cells
Retinoblastoma is a childhood eye cancer that affects approximately 8,200 children each year. There is a paucity of published studies for retinoblastoma, specifically in Malaysia. Natural plants are widely used as alternative medicine in developed and developing countries. Camellia sinensis (matc...
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| Format: | Thesis |
| Language: | English |
| Published: |
2022
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/105897/1/NOR%20HAFIZA%20BINTI%20SAYUTI%20-%20IR.pdf http://psasir.upm.edu.my/id/eprint/105897/ |
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| Summary: | Retinoblastoma is a childhood eye cancer that affects approximately 8,200
children each year. There is a paucity of published studies for retinoblastoma,
specifically in Malaysia. Natural plants are widely used as alternative medicine
in developed and developing countries. Camellia sinensis (matcha green tea)
has been used as a traditional medicine to treat various diseases in Southeast
Asia. However, their efficacy against retinoblastoma cancer has not been
thoroughly investigated and characterized. The main objective of this study was
to reveal the new insight on chemotherapeutics potential of matcha green tea
extract (MGTE) against WERI-Rb-1, human retinoblastoma cells. Firstly, the
optimized extraction condition of MGTE was determined through Response
Surface Methodology (RSM) that aimed high amount of polyphenol compounds.
The identification and quantification of polyphenol were performed via High-
Performance Liquid Chromatography (HPLC). The highest polyphenol and
antioxidant content yield were reached at a temperature of 80 °C, an extraction
time of 20 min, a liquid-to-solid ratio of 100 mL/g. The HPLC analysis at the
optimum extraction condition revealed 14 polyphenol compounds in MGTE.
The chemotherapeutics potential of optimized MGTE was assessed in
vitro through two different types of cultures: two dimensional (2D) and three
dimensional (3D) cultures of WERI-Rb-1. Normal epithelial retina, ARPE-19 cell
lines are used as a positive control. In vitro analysis for 2D cultured cells was
performed on cytotoxicity assay, morphological studies, cell cycle, apoptosis
analysis and gene expression studies. MGTE extract showed promising
chemotherapeutics effect on WERI-Rb-1 cells. The treatment of MGTE showed
low cytotoxicity (IC50 > 200 μg/mL) toward ARPE-19 cells but high cytotoxicity on
WERI-Rb-1 with IC50 13.3 ± 1.40 μg/mL after 72 hours. MGTE induced apoptosis
cell death rather than necrosis and caused arrest in the sub G0 phase, probably
due to DNA fragmentation. Cell cycle analysis also proved that MGTE induced
apoptotic cell death in the sub G0 phase. The chemotherapeutics effect of MGTE
occurred via extrinsic and intrinsic apoptosis pathway by the activation of
caspase 3, caspase 8, caspase 9, Bad and Bax that culminated in the apoptosis
of WERI-Rb-1 cells. The chemotherapeutics potential of MGTE was further
assessed in vitro through the 3D culture of WERI-Rb-1. The 3D collagen WERIRb-
1 cells were successfully developed using collagen type I as their
extracellular matrix. The chemotherapeutics effect of MGTE on WERI-Rb-1 3D
culture was investigated through cytotoxicity assay, morphological, gene and
protein expression studies. The IC50 of MGTE on 3D collagen WERI-Rb-1 cells
at 72 hours was higher in 3D culture cell with 64.41 ± 2.5 μg/mL. The treatment
of MGTE cause a decrease in cell viability as the concentration increased as
observed with DAPI/PI staining. The Scanning Electron Microscope (SEM)
images showed the distinct morphological surface of 2D and 3D WERI-Rb-1
cells. MGTE and cisplatin-treated cells showed characteristics of apoptotic cell
death. Treatment with MGTE on WERI-Rb-1 cells caused upregulation of proapoptotic
proteins, thus resulting in apoptosis cell death. The gene and protein
expression revealed the induction of extrinsic and intrinsic apoptosis pathways
mediated by MGTE through expression of bax, caspase 3, caspase 8 and
caspase 9 protein in 3D collagen WERI-Rb1 cells. However, 72 hours of MGTE
treatment also induced the expression of Nrf2, HO-1, and SOD1 proteins, this
likely decreased the sensitivity of WERI-Rb-1 cells toward MGTE treatment in
the 3D culture system of WERI-Rb-1 cells. The upregulation of antioxidant
proteins may provide cryoprotection for 3D collagen WERI-Rb-1 cells towards
MGTE treatment. Despite the observed expression of Nrf2, HO-1, and SOD1
proteins, MGTE showed the ability to activate apoptosis cell death in 3D collagen
WERI-Rb-1 cells. In conclusion, the findings suggest the new chemotherapeutic
potential of MGTE in inducing apoptosis cell death in both 2D and 3D culture
systems. This finding can be used as the fundamental understanding and new
knowledge of the chemotherapeutics potential of matcha green tea extract
specifically on retinoblastoma cancer cells. |
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