Zainuddin, Nik Aina Syazana Nik
(2020)
Characterisation of standardised fraction from clinacanthus nutans (SF1) and its anti tumor effects on in vitro and xenograft model.
PhD thesis, Universiti Sains Malaysia.
Abstract
Cervical cancer is a leading cause of death in women, worldwide, and third in
Malaysia. Nowadays, medicinal plants become an alternative treatment due to side effects
of conventional treatment and easily available. Clinacanthus nutans has been locally
recognized and was claimed for cancer treatment. From our pilot study, a fraction of C.
nutans shown a potent inhibition on human cervical cancer cells, in vitro. Therefore, this
study was conducted to characterize active anticancer compounds from C. nutans and
determine tumor suppression effect with its mechanism in order to evaluate the potential
of C. nutans as an alternative treatment for cervical cancer. Initially, optimization
methods of bioassay-guided fractionation was carried out. All collected fractions of C.
nutans were examined for cytotoxic activity towards human cervical cancer cell lines,
HeLa and SiHa, and normal cell line, NIH by MTT Assay. The most potent standardized
fraction, SF1 was characterised and identified by Fourier Transform Infrared (FTIR),
Liquid Chromatography Mass Spectrometer (LCMS) and quantitative phytochemical
analysis. The anticancer mechanism of SF1 was evaluated by flowcytometric analysis for
cell cycle progression, mode of cell death and protein expression using Annexin-
V/propidium iodide double staining and specific antibody conjugated fluorescent dye;
Bax, Bcl-2, p53 and cytochrome C. Further investigation of SF1 on tumor suppression
effect was conducted using xenografted nude mice as a model of living subject. The nude
mice were subcutaneously inoculated with SiHa cells. Tumor volume and body weight
were recorded at 3 days intervals. When tumor volume reached 100 mm3, SF1 was
administered once daily via intraperitoneal injection for 28 days. Tumor and liver were
surgically removed and fixed for hematoxylin and eosin (H&E) staining and
immunohistochemistry (IHC) using caspase-3. Blood was collected by cardiac puncture
for assessment of aspartate aminotransferase (AST) and alanine aminotransferase (ALT)
level. The findings suggested that the major constituent of SF1 was identified as alkaloid
with functional group, amines. SF1 exhibited better cytotoxicity with best growth
inhibition against SiHa (IC50 = 9.98±1.24 μg/ml) compared to HeLa (IC50 = 81.21±1.17
μg/ml) and showed cytoselectivity with no IC50 detected on NIH cells. SF1 induced early
apoptosis in SiHa cells with arrested cell cycle at G1/S checkpoint. Up-regulation of p53
followed by increasing of pro-apoptotic Bax and decreasing of anti-apoptotic Bcl-2 as
well as increment of cytochrome C levels upon treatment with SF1 were also shown. The
results showed that tumor volume (60.18±2.17 mm3) in SF1-treated mice were reduced
compared to negative control (139.16±12.97 mm3). Upon SF1 treatment, 43.74± 2.27%
of relative tumor growth ratio (T/C) and 0.64±0.03 of relative tumor volume (RTV) were
calculated. SF1 showed a good inhibition rate with more than 50% of tumor were
suppressed. ALT and AST level in SF1-treated mice were remained in normal ranges
compared to cisplatin group indicating no sign of toxicity effects. The H&E analysis
revealed no abnormal toxicity condition on liver and reduced number of mitosis on tumor
upon treatment with SF1. The IHC analysis confirmed an increased expression of a
crucial mediators of apoptosis, caspase-3, in SF1-treated mice. In conclusion, SF1
demonstrated anticancer activity by inducing apoptosis through arrested G1/S cell cycle
checkpoint via p53 mediated mitochondrial pathway. The tumor suppression effect of
SF1 was demonstrated on the growth of xenografted human cervical cancer in nude mice.
Thus, these findings provided a data for a potential of SF1 as future therapeutic drug for
cervical cancer treatment
Actions (login required)
 |
View Item |
