Evaluating cytotoxicity potency of glucomoringin from the seeds of Moringa oleifera lam against prostate cancer cell line, PC-3
Inhibition of several protein pathways involved in cancer cell regulation is a necessary key in the discovery of cancer chemotherapy. Moringa oleifera Lam is often used in traditional medicine in several countries for the treatment of various illnesses. The plant was also found to contain bioacti...
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Abdul Razis, Ahmad Faizal |
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Moringa oleifera Cancer - Alternative treatment |
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Moringa oleifera Cancer - Alternative treatment Abdul Karim, Nurul Ashikin Evaluating cytotoxicity potency of glucomoringin from the seeds of Moringa oleifera lam against prostate cancer cell line, PC-3 |
| description |
Inhibition of several protein pathways involved in cancer cell regulation is a
necessary key in the discovery of cancer chemotherapy. Moringa oleifera
Lam is often used in traditional medicine in several countries for the
treatment of various illnesses. The plant was also found to contain bioactive
compounds with therapeutic potential against various cancer cells. This
study was carried out to determine the cancer cell inhibition of compounds
from natural origins, specifically from the seeds of M. oleifera, where
glucomoringin can be found abundantly and might carry the potential to
inhibit the proliferation of prostate cancer cells.
The crude seed extracts were obtained from water, acetone, chloroform,
ethanol, methanol, and hexane. The water extract produced the highest
crude yield (21.78%). The crude extracts were initially screened for
phytochemical content, followed by cell viability tests using MTT assay
against MCF 7, HepG2, DU 145, and PC-3 cell lines. Crude hexane,
acetone and chloroform, were found to have coumarin and fat. Crude
ethanol contains coumarin, quinone and fat. Crude methanol contains only
saponin, coumarin and quinone, and finally, crude water contains saponin,
alkaloid and coumarin. The crude water extract showed an inhibition (ICso)
value with a time-dependent manner at 24, 48, and 72 hours of treatment.
The crude water extract showed low ICso against MCF 7 (39.95 μg/ml), Hep
G2 (20.9 μg/ml), DU 145 (32.12 μg/ml), and PC-3 {4.12 μg/ml) at 72 hours
respectively. The crude water extract was chosen to be use in the
experiment because it was the most effective against the cell line that shows
the lowest ICso value as well as giving the highest crude yield All ICso values
are significant compared with control after analysis using one sample T-test
(P <0.05)
The crude water extract was used for further for compound isolation, which
yielded 9.43% of glucomoringin (GMG). Subsequently, the compound was
activated with myrosinase enzymes to yield its active compound,
glucomoringin isothiocyanate (GMG-ITC), followed by characterization
using high performance liquid chromatography (HPLC) and nuclear
magnetic resonance (NMR) spectroscopy analyses. The isolated GMG-ITC
was screened for its cytotoxicity, employing MTT assays against all the cell
lines mentioned previously, and PC-3 was found to be more selective with
ICso of the isolated compound is 3.5 μg/ml. All ICso values are significant
compared to control after analysis using one sample T-test (P <0.05). The
ICso value of GMG-ITC was then used in the following studies, including
morphological observation of apoptosis via AO/Pl and TUNEL assays on
the PC-3 cell line. The cell showed clear apoptotic body characterisation
under phase contrast analysis, as well as colour changes with membrane
blebbing and chromatin condensation in the AO/Pl staining, followed by dark
brown colour staining in the TUNEL assay due to nudear DNA
fragmentation of the cancer cells.
The cytotoxic potency study was further performed with Annexin V-FITC,
cell cycle analysis, mRNA quantification, and protein signalling. In Annexin
V-FITC, the apoptosis cell activity increased significantly (P < 0.05) from 24,
48, to 72 hours of treatment compared to the control by GMG-ITC. The
apoptotic cell percentage increased significantly (P<0.01) from 21.35% (24
hours), followed by 34.47% (48 hours) and finally 72.9% (72 hours) after
treatment. The compound was observed to arrest the cell cycle at G2/M
phase, which is an important phase in apoptosis. mRNA quantification
indicates Nrf2 expression was downregulated significantly (P<0.05), but
p53, Bax, and parp 6 were upregulated by the treatment of the GMG-ITC
against PC-3 cell lines. The expression of p53 can be linked with a blockage
of cell cycle progression at G:v'M phase and the upregulation of Bax,
caspase-3, while Bcl2 and Nrf2 were downregulated. Finally, protein
analysis employing cell signalling immunoassay studies by Multiplex
analysis revealed the modulation of apoptotic proteins after the treatment of
the cancer cells with GMG-ITC. The expression of proteins in the analysis;
JNK, Bad, Bd2, and p53 were significantly upregulated (p<0.01) compared
to control. It indicates that early apoptosis expression was triggered by the
protein signalling induced by the treatment. In this study, apoptosis induction
and DNA fragmentation processes were observed in treated PC-'3 cells.
These phenomena suggest that the bioactive compound from M. oleifera
seed could be useful for future cytotoxic agent against prostate cancer. |
| format |
Thesis |
| qualification_level |
Doctorate |
| author |
Abdul Karim, Nurul Ashikin |
| author_facet |
Abdul Karim, Nurul Ashikin |
| author_sort |
Abdul Karim, Nurul Ashikin |
| title |
Evaluating cytotoxicity potency of glucomoringin from the seeds of Moringa oleifera lam against prostate cancer cell line, PC-3 |
| title_short |
Evaluating cytotoxicity potency of glucomoringin from the seeds of Moringa oleifera lam against prostate cancer cell line, PC-3 |
| title_full |
Evaluating cytotoxicity potency of glucomoringin from the seeds of Moringa oleifera lam against prostate cancer cell line, PC-3 |
| title_fullStr |
Evaluating cytotoxicity potency of glucomoringin from the seeds of Moringa oleifera lam against prostate cancer cell line, PC-3 |
| title_full_unstemmed |
Evaluating cytotoxicity potency of glucomoringin from the seeds of Moringa oleifera lam against prostate cancer cell line, PC-3 |
| title_sort |
evaluating cytotoxicity potency of glucomoringin from the seeds of moringa oleifera lam against prostate cancer cell line, pc-3 |
| granting_institution |
Universiti Putra Malaysia |
| publishDate |
2019 |
| url |
http://psasir.upm.edu.my/id/eprint/111695/1/NURUL%20ASHIKIN%20-%20IR.pdf |
| _version_ |
1811767756652019712 |
| spelling |
my-upm-ir.1116952024-07-31T06:53:54Z Evaluating cytotoxicity potency of glucomoringin from the seeds of Moringa oleifera lam against prostate cancer cell line, PC-3 2019-11 Abdul Karim, Nurul Ashikin Inhibition of several protein pathways involved in cancer cell regulation is a necessary key in the discovery of cancer chemotherapy. Moringa oleifera Lam is often used in traditional medicine in several countries for the treatment of various illnesses. The plant was also found to contain bioactive compounds with therapeutic potential against various cancer cells. This study was carried out to determine the cancer cell inhibition of compounds from natural origins, specifically from the seeds of M. oleifera, where glucomoringin can be found abundantly and might carry the potential to inhibit the proliferation of prostate cancer cells. The crude seed extracts were obtained from water, acetone, chloroform, ethanol, methanol, and hexane. The water extract produced the highest crude yield (21.78%). The crude extracts were initially screened for phytochemical content, followed by cell viability tests using MTT assay against MCF 7, HepG2, DU 145, and PC-3 cell lines. Crude hexane, acetone and chloroform, were found to have coumarin and fat. Crude ethanol contains coumarin, quinone and fat. Crude methanol contains only saponin, coumarin and quinone, and finally, crude water contains saponin, alkaloid and coumarin. The crude water extract showed an inhibition (ICso) value with a time-dependent manner at 24, 48, and 72 hours of treatment. The crude water extract showed low ICso against MCF 7 (39.95 μg/ml), Hep G2 (20.9 μg/ml), DU 145 (32.12 μg/ml), and PC-3 {4.12 μg/ml) at 72 hours respectively. The crude water extract was chosen to be use in the experiment because it was the most effective against the cell line that shows the lowest ICso value as well as giving the highest crude yield All ICso values are significant compared with control after analysis using one sample T-test (P <0.05) The crude water extract was used for further for compound isolation, which yielded 9.43% of glucomoringin (GMG). Subsequently, the compound was activated with myrosinase enzymes to yield its active compound, glucomoringin isothiocyanate (GMG-ITC), followed by characterization using high performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) spectroscopy analyses. The isolated GMG-ITC was screened for its cytotoxicity, employing MTT assays against all the cell lines mentioned previously, and PC-3 was found to be more selective with ICso of the isolated compound is 3.5 μg/ml. All ICso values are significant compared to control after analysis using one sample T-test (P <0.05). The ICso value of GMG-ITC was then used in the following studies, including morphological observation of apoptosis via AO/Pl and TUNEL assays on the PC-3 cell line. The cell showed clear apoptotic body characterisation under phase contrast analysis, as well as colour changes with membrane blebbing and chromatin condensation in the AO/Pl staining, followed by dark brown colour staining in the TUNEL assay due to nudear DNA fragmentation of the cancer cells. The cytotoxic potency study was further performed with Annexin V-FITC, cell cycle analysis, mRNA quantification, and protein signalling. In Annexin V-FITC, the apoptosis cell activity increased significantly (P < 0.05) from 24, 48, to 72 hours of treatment compared to the control by GMG-ITC. The apoptotic cell percentage increased significantly (P<0.01) from 21.35% (24 hours), followed by 34.47% (48 hours) and finally 72.9% (72 hours) after treatment. The compound was observed to arrest the cell cycle at G2/M phase, which is an important phase in apoptosis. mRNA quantification indicates Nrf2 expression was downregulated significantly (P<0.05), but p53, Bax, and parp 6 were upregulated by the treatment of the GMG-ITC against PC-3 cell lines. The expression of p53 can be linked with a blockage of cell cycle progression at G:v'M phase and the upregulation of Bax, caspase-3, while Bcl2 and Nrf2 were downregulated. Finally, protein analysis employing cell signalling immunoassay studies by Multiplex analysis revealed the modulation of apoptotic proteins after the treatment of the cancer cells with GMG-ITC. The expression of proteins in the analysis; JNK, Bad, Bd2, and p53 were significantly upregulated (p<0.01) compared to control. It indicates that early apoptosis expression was triggered by the protein signalling induced by the treatment. In this study, apoptosis induction and DNA fragmentation processes were observed in treated PC-'3 cells. These phenomena suggest that the bioactive compound from M. oleifera seed could be useful for future cytotoxic agent against prostate cancer. Moringa oleifera Cancer - Alternative treatment 2019-11 Thesis http://psasir.upm.edu.my/id/eprint/111695/ http://psasir.upm.edu.my/id/eprint/111695/1/NURUL%20ASHIKIN%20-%20IR.pdf text en public doctoral Universiti Putra Malaysia Moringa oleifera Cancer - Alternative treatment Abdul Razis, Ahmad Faizal English |