| Summary: | Glioblastoma (GB) is a highly aggressive primary brain tumour characterized
by a high mortality rates and poor prognosis. The first-line chemotherapy for GB is
temozolomide (TMZ). However, drug resistance, unwanted side effects and GB
recurrence become the major drawbacks of chemotherapy. Hence, medicinal plants
are used as alternative treatments which offer wide accessibility and lesser side
effects. From a previous study, Quercus infectoria gall methanol extract (QIM) has
demonstrated its potent in vitro antiproliferative activity against human glioma cells.
Thus, this study aims to elucidate the anticancer compounds within QIM and explore
the underlying anticancer mechanisms. Additionally, QIM's potential to enhance the
therapeutic effects of TMZ in combination treatments, particularly against TMZresistant
(DBTRG-05MG) and TMZ-sensitive (U-87MG) glioma cell lines was
conducted. Phytochemicals screening, phytoconstituents characterization by FT-IR
and LC-MS and DPPH assay were done to evaluate the bioactivity of QIM.
Antiproliferative activity of QIM and TMZ treatment against glioma and glial (SVG
p12) cells were determined using MTT assay. IC50 values were then determined from
a dose-response curve and employed for combination treatment. The combined effect
of the treatment was determined based on their combination index (CI) using
CompuSyn software. Anticancer mechanisms of single and combined treatment were
evaluated by AO/PI staining for determination of the mode of cell death, clonogenic
assay for reproductive ability, wound scratch assay for anti-invasion and RT-qPCR
to evaluate the gene expression profiles associated with apoptosis, autophagy, antiinvasion, angiogenesis, antioxidant responses and drug resistance mechanisms. The
findings suggested that the major phytoconstituent in QIM was gallotannin with a
functional group, named phenols. QIM showed robust DPPH radical scavenging
activity (EC50 = 16.90 μg/mL) comparable to gallic acid (EC50 = 17.33 μg/mL). QIM
exhibited better cytotoxicity against U-87MG (IC50 = 20.5 μg/mL) compared to
DBTRG-05MG (IC50 = 21.4 μg/mL) and cytoselective against SVG p12 cells (no
IC50 detected). Notably, TMZ-resistant was shown by DBTRG-05MG (IC50 = 480.30
μg/mL) which require higher concentration compared to U-87MG (IC50 = 56.14
μg/mL). Remarkably, combination treatment significantly reduced the proliferation
of both glioma cell lines when compared to TMZ alone and it demonstrated a
synergistic effect (CI<1). Moreover, both single and combined treatments induced
apoptosis in glioma cells, characterized by apoptotic morphological changes and
upregulation of apoptosis-related genes (BAX and CASP3). QIM also inhibited
autophagy by downregulating the LC3B gene. Additionally, the suppression of
wound healing and the downregulation of the invasion gene (ADAM17) indicated
that the combined treatment inhibited cell migration and metastasis. The significant
downregulation of angiogenesis-related genes (VEGFA and ANGPT1), which
restricted the availability of nutrients and oxygen to cancer cells, further supported
these effects. Combination treatment also significantly increased TMZ's therapeutic
efficacy by downregulating drug-resistant genes (MGMT, MRP and PI3KA),
resulting in greater cell proliferation inhibition. In conclusion, QIM exhibits selective
anticancer activity through apoptosis and autophagy pathways, and the combination
treatment of QIM and TMZ exerted a synergistic effect in glioma cells.
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