Article

Curcumin-Induced Molecular Mechanisms in U-87 MG

Glioblastoma Cells: Insights from Global Gene
Expression Profiling
Nicole Tendayi Mashozhera 1,† , Chinreddy Subramanyam Reddy 1,† , Yevin Nenuka Ranasinghe 1 ,
Purushothaman Natarajan 1,2 , Umesh K. Reddy 1, * and Gerald Hankins 1, *

1 Department of Biology, West Virginia State University, Institute, WV 25112, USA;

nmashozhera@wvstateu.edu (N.T.M.); subramanyam.chinreddy@wvstateu.edu (C.S.R.);
yevin.n.ranasinghe@gmail.com (Y.N.R.); pnatarajan@wvstateu.edu (P.N.)
2 Department of Agriculture, Food, and Resource Sciences, University of Maryland Eastern Shore,
Princess Anne, MD 21853, USA
* Correspondence: ureddy@wvstateu.edu (U.K.R.); ghankins@wvstateu.edu (G.H.)
† These authors contributed equally to this work.

Abstract: Curcumin, a major phytochemical derived from Curcuma longa, has been shown
to enhance the efficacy of chemotherapeutic agents such as doxorubicin, 5-fluorouracil, and
cisplatin by overcoming drug resistance, making it a promising adjunct in the treatment
of glioblastoma. However, the global gene-expression changes triggered by curcumin in
glioblastoma remain underexplored. In this study, we investigated the effects of curcumin
on human glioblastoma (U87 MG) cells, where it significantly reduced cell viability and
proliferation in a dose- and time-dependent manner and induced apoptosis without af-
fecting senescence. Transcriptomic analysis revealed 5036 differentially expressed genes,
with pathway enrichment identifying 13 dysregulated cancer-associated pathways. No-
tably, curcumin modulated several key regulators involved in MAPK, Ras, TGF-β, Wnt,
Cytokine, and TNF signaling pathways. Several apoptosis and cell cycle-associated genes,
Academic Editors: Marcus including PRKCG, GDF7, GDF9, GDF15, GDF5, FZD1, FZD2, FZD8, AIFM3, TP53AIP1,
Tullius Scotti and Luciana Scotti CRD14, NIBAN3, BOK, BCL2L10, BCL2L14, BNIPL, FASLG, GZMM, TNFSF10, TNFSF11,
Received: 3 April 2025 and TNFSF4, were significantly altered. Several pro-apoptotic and anti-BCL, cell-cycle-
Revised: 6 May 2025 regulated genes were modulated following curcumin treatment, emphasizing its potential
Accepted: 8 May 2025 role in curcumin-mediated anti-tumor effects. This study provides insight into the molecu-
Published: 9 May 2025
lar mechanisms underlying curcumin’s action against glioblastoma.
Citation: Mashozhera, N.T.; Reddy,
C.S.; Ranasinghe, Y.N.; Natarajan, P.; Keywords: glioblastoma-U87 cells; natural compound; curcumin; apoptosis; RNA-seq
Reddy, U.K.; Hankins, G.
Curcumin-Induced Molecular
Mechanisms in U-87 MG
Glioblastoma Cells: Insights from
Global Gene Expression Profiling.
1. Introduction
Molecules 2025, 30, 2108. Glioblastoma multiforme is the most common primary malignancy of the brain, with
https://doi.org/10.3390/ an incidence of 3.2 in 100,000 [1]. Glioblastoma remains a formidable challenge, with
molecules30102108
a five-year survival rate below 10% despite aggressive treatment [2]. The hallmarks of
Copyright: © 2025 by the authors. glioblastoma include aggressive and diffuse proliferation, and resistance to apoptosis-
Licensee MDPI, Basel, Switzerland. inducing drugs [3]. Tumor drug resistance is a major cause of treatment failure, driven by
This article is an open access article
cellular heterogeneity, diverse molecular signatures, and variable drug responsiveness [4].
distributed under the terms and
Conventional chemotherapy fails to achieve complete remission and induces toxicity,
conditions of the Creative Commons
Attribution (CC BY) license
harming normal cells and causing severe side effects [5].
(https://creativecommons.org/ Plant-derived compounds, known for their pleiotropic anti-cancer effects, have gained
licenses/by/4.0/). interest in overcoming multi-drug resistance [6,7]. Natural products like resveratrol,

Molecules 2025, 30, 2108 https://doi.org/10.3390/molecules30102108

Molecules 2025, 30, 2108 2 of 16

quercetin, EGCG, and curcumin enhance chemotherapy by sensitizing tumor cells through
antioxidant, anti-inflammatory, immune-modulatory, and apoptosis-inducing mechanisms,
boosting efficacy without added toxicity [8].
Curcumin, a yellow pigment from Curcuma longa, is a well-studied anti-cancer
agent with low toxicity and therapeutic potential [9]. Preclinical studies show that cur-
cumin enhances the efficacy of chemotherapeutic drugs like sulfinosine, 5-FU, doxorubicin,
oxaliplatin, and cisplatin, often sensitizing drug-resistant cells [10]. In prostate cancer,
curcumin with docetaxel improved anti-proliferative and apoptotic effects [11]. It also
boosted 5-FU efficacy in colon, breast, and gastric cancers [12,13]. In a pancreatic cancer
xenograft, curcumin with gemcitabine significantly reduced tumor volume by enhancing
anti-proliferative and anti-angiogenic pathways [14]. Clinical trials suggest daily curcumin
supplementation exerts anti-inflammatory and immune-modulatory effects in various
cancers [15].
Curcumin modulates key pathways in tumor initiation, promotion, and progression.
It suppresses carcinogenesis by inducing apoptosis, arresting the cell cycle, and inhibit-
ing metastasis, invasion, and angiogenesis [16]. In glioblastoma, curcumin suppresses
growth and chemoresistance via AP-1 and NFκB transcription factors, which regulate
cell proliferation, apoptosis, and inflammation. In lung cancer, it inhibits metastasis by
targeting matrix metalloproteinases (MMP-2 and MMP-9) and vascular endothelial growth
factor (VEGF), crucial factors in tumor invasion. For hepatocellular carcinoma, curcumin’s
suppression of proliferation and induction of apoptosis occurs through modulation of the
Wnt signaling pathway. Similarly, in non-small-cell lung cancer, it impedes migration and
invasion through the up-regulation of miR-206 and suppression of the PI3K/AKT/mTOR
pathway. Across these studies, curcumin’s multifaceted role in regulating critical molecular
signaling pathways and ncRNAs make it a potential multi-targeted therapeutic agent that
can be leveraged in cancer prevention and treatment, enhancing efficacy and overcoming
resistance mechanisms [17–21].
Although extensive research has revealed curcumin’s anti-carcinogenic properties and
its potential to support cancer therapy, studies examining its impact on global gene expres-
sion via transcriptome profiling in various cancer cells remain limited. High-throughput
RNA sequencing (RNA-seq) of curcumin-treated cells can elucidate the anti-proliferative
and cell-death pathways associated with curcumin [22]. Recent studies have employed
RNA-seq to profile the most significantly up- and down-regulated genes following cur-
cumin treatment, followed by protein–protein interaction analysis and in silico molecular
docking to identify novel curcumin targets [23]. Compared with targeted proteomic meth-
ods, integrating global pathway enrichment analyses with systemic studies yields a more
comprehensive insight into the regulatory and functional dynamics driving curcumin-
induced cytotoxicity in cancer, thereby enhancing its therapeutic application [24]. In two
breast cancer cell lines, transcriptome profiling showed that curcumin primarily induced
cell death via ferroptosis rather than apoptosis, suggesting that ferroptosis may be a more
promising therapeutic target [25]. In contrast, in adrenocortical carcinoma cells and their
xenograft model, curcumin induced apoptosis mainly through ER stress pathways with
accompanying up-regulation of p38 and JNK/MAPK signaling [24].
Given the limited number of transcriptome profiling studies on curcumin, further
research could enhance our understanding of its cytotoxic mechanisms in cancer [25].
Global gene expression analyses can identify novel genetic targets of curcumin, guiding
future research efforts [26]. Accordingly, this study evaluated the impact of curcumin on
global gene expression in the U87-MG human glioblastoma cell line using RNA-seq, with
its cytotoxic effects confirmed via CCK-8 proliferation, apoptosis, and senescence assays.
 its cytotoxic effects confirmed via CCK-8 proliferation, apoptosis, and senescence

Molecules 2025, 30, 2108 2. Results 3 of 16

2.1. Curcumin Treatment Decreases U87 MG Cell Viability and Proliferation

2. Results
The growth-inhibitory effects of curcumin on human glioblastoma cells wer
2.1. Curcumin Treatment Decreases U87 MG Cell Viability and Proliferation
ated. Cell viability was assessed using the CCK-8 assay. Results demonstrated tha
minThe growth-inhibitory effects of curcumin on human glioblastoma cells were evaluated.
reduced the viability and proliferation of U87 MG cells in a dose- and time-dep
Cell viability was assessed using the CCK-8 assay. Results demonstrated that curcumin
manner (Figure 1). The median IC50 for curcumin was determined to be 20 µmol
reduced the viability and proliferation of U87 MG cells in a dose- and time-dependent
plementary
manner (FigureFigure S1).
1). The Based
median onfor
IC50 this, subsequent
curcumin experiments
was determined to be were conducted
20 µmol/L
treatment concentration
(Supplementary of 20
Figure S1). Based onµmol/L.
this, subsequent experiments were conducted using a
treatment concentration of 20 µmol/L.

Figure 1. Effect of curcumin on glioblastoma cells. The anti-proliferative effects of increasing

Figure 1. Effect
concentrations of curcumin
of curcumin on glioblastoma
on viability of U87 MG cellscells. The anti-proliferative
was assessed effects
over a three-day period of increas
using
centrations
the of curcumin
CCK-8 assay. on viability
Vehicle-treated of used
cells were U87 as
MG thecells was control
negative assessed overinterval
at each a three-day
(100% period u
viability). The viability is presented relative to the negative control. Values are
CCK-8 assay. Vehicle-treated cells were used as the negative control at each interval means ± SD; n = 3; (100% v
* p < 0.05 and ** p < 0.01, *** p < 0.001 (as compared with control).
The viability is presented relative to the negative control. Values are means ± SD; n = 3; *
2.2.
andCurcumin Reduced
** p < 0.01, *** p <Migration
0.001 (asofcompared
U87 MG Cells
with control).
The effects of curcumin treatment on the migratory ability of U87 MG cells were
assessed using the
2.2. Curcumin scratch Migration
Reduced wound healing assay.
of U87 MGThe potential for cells to migrate into
Cells
the wound in vitro represents their metastatic ability in vivo. A scratch wound was made
in theThe effectsofofU87
monolayer curcumin
MG cells treatment
using a 20 µLonpipette
the migratory ability
tip, and cells of U87with
were treated MG cells w
sessed using
curcumin the scratch
or vehicle. wound
Quantitatively, the healing
inhibition assay. The was
of migration potential
assessedfor
by cells to migrate
measuring
wound
gap in after
closure vitro24represents
h of treatment their
withmetastatic
curcumin orability
vehicle.in vivo. A scratch wound was m
Treatment of of
the monolayer theU87
cellsMG
withcells
10 and 20 µmol/L
using a 20 µL curcumin
pipettesignificantly
tip, and cellsreduced
werethetreated w
number of cells that migrated into the wound after 24 h compared with vehicle-treated cells
cumin or vehicle. Quantitatively, the inhibition of migration was assessed by me
(p-value < 0.0001) (Figure 2). Furthermore, the rate of gap closure was reduced when the
gap closure
curcumin after 24 h
concentration wasof increased
treatment with
from curcumin
10 to 20 µmol/Lor vehicle.
(p-value < 0.01). The results
of thisTreatment of thethat
study demonstrate cells with 10
curcumin and 20
exhibits µmol/L curcumin
anti-migratory significantly
effects in U87 MG cells, redu
and this effect
number is dose-dependent.
of cells that migrated into the wound after 24 h compared with vehicle
cellsCurcumin
2.3. (p-value < 0.0001)
Induced (Figure
Apoptosis 2).Senescence
but Not Furthermore, the Cells
in U87 MG rate of gap closure was reduce
the curcumin concentration was increased from 10 to 20 µmol/L (p-value < 0.01).
To investigate the effects of curcumin on apoptosis and senescence in human glioblas-
sultscells,
toma of this studywas
curcumin demonstrate
administered that curcumin
to cells, and these exhibits
cells were anti-migratory
incubated for 24 h. effects
Fol- in U
cells, and
lowing this effect
incubation, is dose-dependent.
apoptotic cells were identified through poly-caspase activity using the
FAM-FLICA poly-caspase assay. When compared with untreated cells, curcumin-treated
cells had significantly higher poly-caspase activity (p-value < 0.0001) (Figure 3A). Increas-
ing curcumin concentration from 10 to 20 µmol/L also increased the rate of poly-caspase
activity, indicating that curcumin-induced apoptosis in U87 MG cells was dose-dependent.
Molecules 2025,
Molecules 2025, 30,
30, 2108
x FOR PEER REVIEW 44 of 16
of 16

Figure 2. Effect of curcumin on migration of U87 MG cells. (A) The cell monolayer was scratched
with a 20 µL pipette tip, photographed at 0 h, and treated for 24 h. (B) Gap closure was quantified
by comparing gap width at 0 h and 24 h post-treatment with vehicle or curcumin. Data are mean
gap width of six replicates ± SD. One-way ANOVA was used to examine statistical differences fol-
lowed by Tukey’s honestly significant difference test for post hoc testing for differences between
treatment groups. **** Significant at p < 0.0001.

2.3. Curcumin Induced Apoptosis but Not Senescence in U87 MG Cells

To investigate the effects of curcumin on apoptosis and senescence in human glio-
blastoma cells, curcumin was administered to cells, and these cells were incubated for 24
h. Following incubation, apoptotic cells were identified through poly-caspase activity us-
ing the FAM-FLICA poly-caspase assay. When compared with untreated cells, curcumin-
treated cells had significantly higher poly-caspase activity (p-value < 0.0001) (Figure 3A).
Increasing curcumin concentration from 10 to 20 µmol/L also increased the rate of poly-
caspase activity, indicating that curcumin-induced apoptosis in U87 MG cells was dose-
dependent.
Figure 2.2. Effect
Effect ofof curcumin
curcumin on on migration
migration ofof U87 MG MG cells.
cells. (A)
(A) The
The cell
cell monolayer
monolayer was was scratched
Figure
Senescent cells are identified throughU87 elevated lysosomal activity, which canscratched
be bio-
with aa 20
with 20µLµLpipette
pipettetip,
tip,photographed
photographedatat0 0h,h,and
and treated
treated forfor
2424h. h.
(B)(B)
GapGap closure
closure waswas quantified
quantified by
chemically assayed through the activity of β-galactosidase enzyme [27]. In this study, cur-
by comparing gap width at 0 h and 24 h post-treatment with vehicle or curcumin.
comparing gap width at 0 h and 24 h post-treatment with vehicle or curcumin. Data are mean gap Data are mean
cumin-induced senescence was assayed using the SA-β-galactosidase assay kit. Curcumin
width of sixofreplicates
gap width ± SD.± One-way
six replicates SD. One-wayANOVAANOVA was used to examine
was used statistical
to examine differences
statistical followed
differences fol-
treatment did not significantly induce senescence in U87 MG cells (p > 0.05 when com-
by Tukey’s honestly significant difference test for post hoc testing for differences between
lowed by Tukey’s honestly significant difference test for post hoc testing for differences between treatment
pared
groups.to****
control) (Figure
Significant at p 3B).
< 0.0001.
treatment groups. **** Significant at p < 0.0001.

2.3. Curcumin Induced Apoptosis but Not Senescence in U87 MG Cells

To investigate the effects of curcumin on apoptosis and senescence in human glio-
blastoma cells, curcumin was administered to cells, and these cells were incubated for 24
h. Following incubation, apoptotic cells were identified through poly-caspase activity us-
ing the FAM-FLICA poly-caspase assay. When compared with untreated cells, curcumin-
treated cells had significantly higher poly-caspase activity (p-value < 0.0001) (Figure 3A).
Increasing curcumin concentration from 10 to 20 µmol/L also increased the rate of poly-
caspase activity, indicating that curcumin-induced apoptosis in U87 MG cells was dose-
dependent.
Senescent cells are identified through elevated lysosomal activity, which can be bio-
chemically assayed through the activity of β-galactosidase enzyme [27]. In this study, cur-
cumin-induced
Figure 3. Effects ofsenescence
curcumin onwas assayed
apoptosis using
and the SA-β-galactosidase
senescence in U87MG cells. (A)assay kit. Curcumin
Apoptosis induction
Figure 3. Effects of curcumin on apoptosis and senescence in U87MG cells. (A) Apoptosis induction
treatment
was did not
determined significantlyactivation
by poly-caspase induce senescence in U87 treatment
following curcumin MG cells for
(p 24
> 0.05 when
h. (B) com-
Curcumin-
was determined by poly-caspase activation following curcumin treatment for 24 h. (B) Curcumin-
induced senescence was measured
pared to control) (Figure 3B). using the SA-β-galactosidase assay. Data are means ± SD of
three independent replicates. One-way ANOVA tests were performed to determine the statistical
significance of differences between control and treatment means. Tukey’s honestly significant dif-
ference tests were used for post hoc analysis of multiple comparisons. **** Significant at p < 0.0001.
*** Significant at p < 0.001. ** Significant at p < 0.01.

Senescent cells are identified through elevated lysosomal activity, which can be bio-
chemically assayed through the activity of β-galactosidase enzyme [27]. In this study,
curcumin-induced senescence was assayed using the SA-β-galactosidase assay kit. Cur-
cumin treatment did not significantly induce senescence in U87 MG cells (p > 0.05 when
compared to control) (Figure 3B).

2.4. Curcumin Induced Differential Gene Expression in U87 MG Cells

The impact of curcumin on global gene expression in U87 MG cells was an-
alyzed using RNA sequencing (RNA-seq), including principal component analysis
(Supplementary
Figure 3. Effects ofFigure S2).onTreatment
curcumin apoptosis with 20 µmol/L
and senescence incurcumin resulted
U87MG cells. in the differential
(A) Apoptosis induction
was determined by poly-caspase activation following curcumin treatment for 24 h. (B) Curcumin-
 The impact of curcumin on global gene expression in U87 MG cells was analyzed
using RNA sequencing (RNA-seq), including principal component analysis (Supplemen-
Molecules 2025, 30, 2108 tary Figure S2). Treatment with 20 µmol/L curcumin resulted in the differential5 of expression
16
of 5036 genes, with 3418 genes up-regulated and 1618 down-regulated. Kyoto Encyclope-
dia of Genes and Genomes (KEGG) pathway analysis of the top 50 up-regulated genes
expressionenrichment
identified of 5036 genes,
in with 3418 genes
pathways up-regulated
related and 1618
to extracellular down-regulated.
matrix Kyotointerac-
(ECM)–receptor
Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the top 50 up-regulated
tion, focal adhesion, cell adhesion molecules, Notch signaling, NF-κB signaling, Jak-STAT
genes identified enrichment in pathways related to extracellular matrix (ECM)–receptor
signaling, and choline metabolism in cancer (Figure 4A). Gene Ontology (GO) analysis
interaction, focal adhesion, cell adhesion molecules, Notch signaling, NF-κB signaling, Jak-
revealed significant enrichment in molecular functions, including metal ion transmem-
STAT signaling, and choline metabolism in cancer (Figure 4A). Gene Ontology (GO) analysis
brane transport, ion
revealed significant channel activity,
enrichment channel
in molecular activity,
functions, and metal
including gatedion
channel activity (Figure
transmembrane
4B).
transport, ion channel activity, channel activity, and gated channel activity (Figure 4B).

Figure 4. Enrichment analysis of up-regulated genes. (A) Bubble plot for KEGG pathway enrichment
Figure 4. Enrichment analysis of up-regulated genes. (A) Bubble plot for KEGG pathway enrich-
of the top 50 up-regulated genes. Each circle represents an enriched function. The gene enrichment
ment
ratio of the top 50
is assigned to up-regulated
the x-axis, andgenes. Each circle
the descriptions of represents
the pathways anare
enriched
assignedfunction. The gene
to the y-axis. The enrich-
ment ratio
area of the is assigned
circles to the x-axis,
is proportional and
to the the descriptions
number of thetopathways
of genes assigned are assigned
the term, and tothe
the color to the y-axis.
The area p-value.
adjusted (B) GOisof
of the circles three ontologies
proportional enriched
to the number in curcumin-induced
of genes assigned up-regulated
to the term,genes.
and The
the color to
width of each bar represents enrichment score of genes ( − log 10 (p-value)). BP = biological
the adjusted p-value. (B) GO of three ontologies enriched in curcumin-induced up-regulated genes. process,
CC = cellular component, MF = molecular function.
The width of each bar represents enrichment score of genes (−log10(p-value)). BP = biological process,
CC = cellular component,
Conversely, MF = molecular
KEGG pathway function.
analysis of the top 50 down-regulated genes revealed
involvement in pathways including O-glycan biosynthesis; Notch signaling; glycine, serine,
and threonine metabolism; glycolysis and gluconeogenesis; and cytokine–cytokine receptor
interaction (Figure 5A). GO analysis further highlighted enrichment in molecular functions,
including DNA-binding transcription repressor activity, DNA-binding transcription factor
binding, transcription coactivator activity, and catalytic activity acting on RNA (Figure 5B).
 receptor interaction (Figure 5A). GO analysis further highlighted enrichment in molecular
functions, including DNA-binding transcription repressor activity, DNA-binding tran-
Molecules 2025, 30, 2108 scription factor binding, transcription coactivator activity, and catalytic activity
6 ofacting
16 on
RNA (Figure 5B).

Figure 5. Enrichment analysis of down-regulated genes. (A) Bubble plot for KEGG pathway en-
Figure 5. Enrichment analysis of down-regulated genes. (A) Bubble plot for KEGG pathway enrich-
richment of the top down-regulated genes. Each circle represents an enriched function. The gene
ment of the ratio
enrichment top down-regulated
is assigned to thegenes. Eachthecircle
x-axis and represents
description of theanpathways
enrichedtofunction.
the y-axis.The
Thegene en-
richment ratio
area of the is assigned
circles to thetox-axis
is proportional and the
the number description
of genes of to
assigned the
thepathways
term, andto
thethe y-axis.
color to theThe area
ofadjusted p-value.
the circles (B) GO of three
is proportional ontologies
to the number enriched
of genesin curcumin-induced
assigned to the term,down-regulated genes.
and the color to the ad-
The width of each bar represents enrichment score of genes (−log10 (p-value)). BP = biological process,
justed p-value. (B) GO of three ontologies enriched in curcumin-induced down-regulated genes. The
CC = cellular component, MF = molecular function.
width of each bar represents enrichment score of genes (−log10(p-value)). BP = biological process, CC
= cellular component,
Further analysisMFof=the
molecular
50 mostfunction.
significantly altered KEGG pathways identified
13 pathways strongly linked to tumorigenesis, cancer progression, and immune evasion.
TheseFurther
includedanalysis of the 50 most
cytokine–cytokine significantly
receptor interaction; altered
pathways KEGG pathways
in cancer; identified 13
MAPK signal-
pathways strongly
ing; regulation linked
of stem to tumorigenesis,
cell pluripotency; TGF-β cancer
signaling;progression, and
breast cancer; immune
Ras evasion.
signaling;
IL-17 signaling; PD-L1 expression and PD-1 checkpoint in cancer; proteoglycans
These included cytokine–cytokine receptor interaction; pathways in cancer; MAPK sig- in cancer;
Wnt signaling;
naling; gastric
regulation cancer;
of stem celland viral proteinTGF-β
pluripotency; interaction with cytokines
signaling; and cytokine
breast cancer; Ras signaling;
receptors. Among these, several tumor-suppressive pathways stood out. Genes within
IL-17 signaling; PD-L1 expression and PD-1 checkpoint in cancer; proteoglycans in cancer;
MAPK, Ras, TGF-β, and Wnt pathways, and cytokine–cytokine receptor signaling, such
Wnt signaling; gastric cancer; and viral protein interaction with cytokines and cytokine
as PRKCG (+8.81), GDF7 (+7.78), GDF5 (−2.68), FZD1 (+2.62), FZD2 (+2.95), and FZD8
receptors. Among these, several tumor-suppressive pathways stood out. Genes within
(−3.18) are hypothesized to have contributed to curcumin’s anti-tumorigenic effects in
MAPK, Ras,
this study. TGF-β, and
Additionally, theWnt pathways,
well-known andsuppressor
tumor cytokine–cytokine
gene RUNX3 receptor signaling, such
was significantly
up-regulated (+8.93) in curcumin-treated cells, further supporting its potential role in
curcumin-mediated tumor suppression.
Molecules 2025, 30, 2108 7 of 16

3. Discussion
Curcumin, a polyphenol from Curcuma longa, is a well-studied natural compound
that modulates tumor initiation, progression, and promotion in various cancers [28]. In
glioblastoma, curcumin suppresses oncogenic signaling, induces multimodal cell death,
and modulates immune components within the tumor microenvironment (TME) [29].
This study examines the impact of curcumin on global gene expression and its effects on
proliferation, migration, apoptosis, and senescence in U87MG glioblastoma cells.

3.1. Curcumin Suppresses Viability and Migration of U87 MG Cells

Curcumin inhibited U87 MG cell proliferation in a dose- and time-independent man-
ner, aligning with previous studies. RNA sequencing suggested that curcumin sup-
presses malignancy by inhibiting oncogenic pathways, including the Wnt/β-catenin,
PI3K/Akt/mTOR, NF-κB, and TGF-β pathways [30,31]. Also, in our study, crucial path-
ways for cell survival—the PI3K-Akt (CDK6 (−1.55) CSF3 (−1.55) LPAR1 (−1.46) EFNA5
(−1.13) PHLPP2(−1.17) PHLPP1 (−1.11) GHR (−1.12) IRS1 (−1.71) KDR (−1.16) KRAS
(−1.65) MCL1 (−1.80) PDGFA (−1.59) PDGFRA (−1.66) PIK3R1 (−1.88) PRKAA1 (−1.09)
BDNF (−1.85) SGK1 (−1.44 fold) SOS2 (−1.05) TLR2 (−1.03) TLR4 (−2.69) CCNE1 (−1.27)
CCNE2 (−1.73)), Wnt (FRAT2 (−1.12) FZD2 (−2.95) APC (−1.06) SMAD3 (−1.67) WNT5A
(−1.82) WNT7B (−1.07) FZD5 (−1.99) FZD1 (−2.62) FZD7 (−1.93) FZD8 (−3.18) ROCK2
(−1.17) mTOR, (FZD2 IRS1 KRAS PIK3R1 PRKAA1 SGK1 SKP2 (−2.19) SOS2 WNT5A
WNT7B FZD5 FZD1 FZD7 FZD8)), TNF signaling (TAB1 (−1.13) CEBPB (−1.5) MAPK14
(−1.49) JAG1 (−1.04) IL1B (−1.07) LIF (−1.49) PIK3R1 BCL3 (−1.14) TRAF5 (−1.59) RIPK1
(−1.68) FADD (−2.88) BAG4 (−1.04)), and TGF-β signaling (RGMB (−1.99) ID1 (−1.86)
ID2 SMAD3 (−1.67) SMAD5 (−1.03) PITX2 (−1.7) SMURF2 (−1.04) BMP2 (−1.62) SP1
(−1.7) TGFBR1 (−1.02) TGFBR2 (−1.98) GDF5 (−2.68) ACVR1B (−1.48) ACVR2A (−1.2)
NOG(−3.61)) pathways—were down-regulated, which is essential for cancer cell sur-
vival, metastasis, and drug resistance. Additionally, curcumin induces G2/M cell cycle
arrest by modulating cell cycle regulators, thereby curbing tumor growth both in vitro
and in vivo [32]. The current study also demonstrated cell-cycle-arrest-related cyclin-
dependent kinase (CDK6 (−1.56), CCNE1 (−1.27), CCNE2 (−1.73), KRAS (−1.65), PIK3R1
(−1.88) PDGFA (−1.59), KDR (−1.16)) anti-angiogenic effects and TLR4 (−2.4) and IRS1
(−1.71) inflammation reduction effects. Targeting these pathways effectively induces
apoptosis and prevents cancer progression.
Scratch wound assays revealed a significant (p < 0.0001) reduction in U87 MG cell
migration following curcumin treatment, with greater inhibition observed at 20 µmol/L
(p < 0.05). Curcumin’s anti-migratory effects involve the PI3K/Akt, NF-κB, ERK, and JAK-
STAT signaling pathways, which down-regulate MMPs, VEGF, and ICAM-1 [18,32–34]. In
glioblastoma, curcumin inhibited STAT3 phosphorylation, reducing MMP-9, Snail, and
Twist expression [35], and suppressed MMPs via MAPK inhibition [36]. A study by
Abdullah Thani et al. reported that decreased levels of MMP-2, -9, -14, -15, -16, -17, -24,
and -25, further hindering glioblastoma migration [37]. Given glioblastoma’s aggressive
migration at diagnosis, these findings underscore the therapeutic potential of curcumin.
In our study, JAK STAT (CSF3, GHR, LIF, MCL1, PDGFA, PDGFRA, PIK3R1, SOS2) and
Nf-kB (TAB1, IL1B, TLR4, TRAF5, TRAF6, RIPK1)-related genes were also down-regulated.

3.2. Curcumin Triggers Apoptosis in U87 MG Glioblastoma Cells

Resistance to apoptosis is a major challenge in glioblastoma treatment, making
apoptosis-inducing compounds crucial [31]. Curcumin-induced apoptosis was assessed via
poly-caspase activity using the FAM-FLICA probe, which irreversibly binds active caspase-
3, -6, -7, -8, -9, and -10. After 24 h, curcumin significantly increased poly-caspase activity,
 3.2. Curcumin Triggers Apoptosis in U87 MG Glioblastoma Cells
Resistance to apoptosis is a major challenge in glioblastoma treatment, making apop-
tosis-inducing compounds crucial [31]. Curcumin-induced apoptosis was assessed via
Molecules 2025, 30, 2108 8 of 16
poly-caspase activity using the FAM-FLICA probe, which irreversibly binds active
caspase-3, -6, -7, -8, -9, and -10. After 24 h, curcumin significantly increased poly-caspase
activity, withactivation
with higher higher activation at 20 compared
at 20 µmol/L µmol/L compared with 10 µmol/L,
with 10 µmol/L, consistentconsistent with
with previous
previous findings [38]. Curcumin promotes apoptosis by modulating apoptosis-related
findings [38]. Curcumin promotes apoptosis by modulating apoptosis-related proteins,
proteins,
activatingactivating intrinsic (mitochondrial)
intrinsic (mitochondrial) (AIFM3 (5.3),(AIFM3 (5.3),BCL2L10,
BOK (2.5), BOK (2.5),andBCL2L10,
(BCL2L14)andand
(BCL2L14) and extrinsic (death-receptor) (FASLG (4.6), TNFSF10 (2.9), and
extrinsic (death-receptor) (FASLG (4.6), TNFSF10 (2.9), and TNFSF4 (1.7)) pathways, TNFSF4 (1.7))
and
pathways,
mediating and mediating
ER stress ER stress
(TP53AIP1 (TP53AIP1
(4.2), CRD14, (4.2), (3.8),
NIBAN3 CRD14, NIBAN3
BCL2L14, (3.8),FASLG
BNIPL, BCL2L14,
(4.6),
BNIPL, FASLG (4.6), GZMM, and TNFSF11 (6.4)) [31,39–42], Figure 6.
GZMM, and TNFSF11 (6.4)) [31,39–42], Figure 6. It also enhances cleaved caspase-3, It also enhances
-8, and
cleaved caspase-3,
-9 expression -8, and -9 expression
in glioblastoma in glioblastoma cells [38,43,44].
cells [38,43,44].

Figure Thematic illustration.

Figure6.6. Thematic illustration. Curcumin
Curcumintreatment
treatmentinin
U87
U87 glioblastoma
glioblastomacells modulates
cells global
modulates gene
global
expression,
gene leading
expression, to cell
leading to cycle arrest
cell cycle and and
arrest apoptosis through
apoptosis the the
through coordinated
coordinatedup-regulation and
up-regulation
down-regulation
and down-regulationof multiple signaling
of multiple pathways
signaling andand
pathways theirtheir
associated genes.
associated genes.

Theeffect
The effectof of curcumin
curcumin on senescence
on senescence inMG
in U87 U87cells
MGwascells was examined
examined using
using β-galac-
tosidase activity. While glioma senescence can both suppress and promote malignancyma-
β-galactosidase activity. While glioma senescence can both suppress and promote in
lignancy in vivo [27], curcumin (10–20 µmol/L) did not induce senescence
vivo [27], curcumin (10–20 µmol/L) did not induce senescence (p > 0.05). Prior studies(p > 0.05). Prior
studiescurcumin’s
suggest suggest curcumin’s effectson
effects depend depend on concentration—low
concentration—low doses (≤10doses (≤10
µmol/L) µmol/L)
induce se-
induce senescence without cell death, while higher doses trigger apoptosis due
nescence without cell death, while higher doses trigger apoptosis due to excessive stress to excessive
stress
[45]. [45].
The The
lack oflack of senescence
senescence in thisinstudy
this study suggests
suggests cytotoxicity
cytotoxicity at these
at these concentrations,
concentrations, as
as confirmed by poly-caspase and viability
confirmed by poly-caspase and viability assays. assays.

3.3. Curcumin Treatment Altered Gene Expression in U87 MG Cells

3.3. Curcumin Treatment Altered Gene Expression in U87 MG Cells
Despite extensive research into curcumin’s role in glioblastoma, its impact on global
Despite extensive research into curcumin’s role in glioblastoma, its impact on global
gene expression remains underexplored. Given its pleiotropic effects in tumorigenesis,
gene expression remains underexplored. Given its pleiotropic effects in tumorigenesis,
studying global expression could reveal novel genetic targets and enhance our understand-
studying global expression could reveal novel genetic targets and enhance our under-
ing of its therapeutic potential. This study highlights the multifaceted role of curcumin
standing of its therapeutic potential. This study highlights the multifaceted role of curcu-
in cancer therapy by modulating genes involved in growth, survival, and immune re-
min in cancer therapy by modulating genes involved in growth, survival, and immune
sponses. The up-regulation of genes like C1orf116 and uncharacterized loci (LOC124902513,
responses. The up-regulation of genes like C1orf116 and uncharacterized loci
LOC107985667, LOC100506358, LOC112268263, and LOC105375676) suggests unexplored
(LOC124902513, LOC107985667, LOC100506358, LOC112268263, and LOC105375676)
pathways in cancer regulation. METTL21C (10.29), a methyltransferase, enhances protein
suggests unexplored pathways in cancer regulation. METTL21C (10.29), a methyltransfer-
modifications related to cell cycle control and apoptosis, aligning with curcumin’s epige-
ase, enhances protein modifications related to cell cycle control and apoptosis, aligning
netic modulation [46]. PNLDC1 (10.14) and AMPD1 (10.05-fold), involved in RNA process-
with curcumin’s epigenetic modulation [46]. PNLDC1 (10.14) and AMPD1 (10.05-fold),
ing and purine metabolism, may disrupt cancer cell homeostasis, impairing growth [47,48].
involved in RNA processing and purine metabolism, may disrupt cancer cell homeostasis,
The up-regulation of TBR1 (9.66), a transcription factor involved in neuronal development,
suggests that curcumin may influence differentiation pathways—which is highly relevant
in glioblastoma, where differentiation is often impaired [49].
A key finding in this study was the up-regulation of RUNX3 (8.81), a runt-related
transcription factor with tumor-suppressive roles in multiple cancers. RUNX3 is frequently
down-regulated in gastric, breast, colorectal, renal, and hepatocellular cancers through
Molecules 2025, 30, 2108 9 of 16

point mutations, promoter hypermethylation, and cytoplasmic translocation [50,51]. It

also resides in a genomic region often deleted in various tumors, resembling classic tumor
suppressor genes [52].
RUNX3 up-regulation in curcumin-treated cells likely contributed to reduced viability,
migration, and increased apoptosis, aligning with its known tumor-suppressive function in
gliomas. Glioma patient studies indicate that RUNX3 is progressively inactivated through
promoter hypermethylation, with reduced protein expression associated with disease
progression [53]. Mei et al. reported lower RUNX3 expression in glioblastoma tissue
compared with normal brain, with re-expression suppressing the migration of U251 and
U87 MG cells via MMP-2 inhibition [54]. Additionally, RUNX3 overexpression induced Bim-
caspase-dependent apoptosis and G0/G1 cell cycle arrest, and suppressed Wnt/β-catenin
signaling, thereby improving survival in a xenograft model [55,56].
Protein kinase C gamma (PRKCG), a gene identified in this study as involved in
several signaling pathways, was up-regulated by a fold change of 8.81 compared with
control cells following curcumin treatment. Pathway analysis revealed that PRKCG could
be involved in focal adhesion molecules, tyrosine kinase inhibition, and cancer-related
pathways, as well as choline metabolism in cancer. PRKCG, is a member of the protein
kinase C family of enzymes whose activation mediates various signaling pathways that
regulate the balance between cell survival and cell death [57]. This particular iso-enzyme,
while previously accepted to be expressed solely in the brain, was found to be aberrantly
expressed in some colon and breast cancer cells, where a role for it as a putative tumor
suppressor has been suggested [58].
Emerging evidence highlights the tumor-suppressive roles of protein kinase C (PKC)
isoenzymes, with most PKC mutations in cancers being not just loss-of-function but also
dominant negative [57]. Their frequent inactivation suggests that restoring PKC activity
could be a viable cancer treatment strategy. Satow et al. reported the expression of PKCγ
(PRKCG) in normal colonic epithelia, with its down-regulation linked to poor patient
outcomes [59].
In gliomas, PRKCG expression and methylation patterns correlate with tumor pro-
gression, with glioblastomas exhibiting the lowest expression and highest methylation [60].
Though PRKCG’s role in glioblastoma remains unclear, its up-regulation following cur-
cumin treatment in this study suggests a potential anti-tumorigenic effect, possibly influ-
encing key dysregulated signaling pathways in cancer.
Curcumin has been shown to modulate Wnt signaling, a pathway frequently over-
activated in glioblastoma and other cancers [61]. As key receptors for frizzled proteins,
transmembrane frizzled receptors drive canonical and non-canonical signaling, influencing
proliferation, angiogenesis, migration, and invasion [62]. High expression of FZD1, 2, 5, 6,
7, and 8 correlates with oncogenic mTOR signaling and poor prognosis in gliomas [63].
In this study, curcumin treatment down-regulated the expression of five frizzled
receptors: FZD2 (2.95), FZD8 (3.18), FZD1 (2.62), FZD7 (1.93), and FZD5 (1.99). Among
these, FZD2 is a known prognostic marker for glioma progression, with its expression
linked to tumor grade [64]. Ran et al. found that FZD2 suppression reduced glioblastoma
cell stemness, proliferation, migration, and invasion, coinciding with the inhibition of the
Notch and NF-κB pathways [65].
High expression of FZD1 and FZD8 has been linked to poor overall survival in glioblas-
toma patients [66,67]. In low-grade glioma, FZD8 was identified as a key driver of tumor re-
currence [67]. Interestingly, its repression via bivalent modification in glioma tumorigenesis
suggests a potential tumor-suppressive role [68]. While its function in glioblastoma remains
unclear, aberrant FZD8 expression has been implicated in gastric, prostate, lung, pancreatic,
and renal cancers [69,70]. Additionally, FZD8 activation of Wnt signaling contributes to
Molecules 2025, 30, 2108 10 of 16

chemoresistance in triple-negative breast cancer [71]. Whether FZD down-regulation in

curcumin-treated cells suppressed tumorigenesis warrants further investigation.
Curcumin treatment influenced TGF-β family protein expression in this study. Growth
differentiation factor (GDF) proteins, part of the TGF-β family, regulate cell differentia-
tion, proliferation, and apoptosis [72]. Loss of differentiation, a hallmark of cancer, fuels
unchecked proliferation, often driven by cancer stem cells [73]. Inducing differentiation lim-
its proliferation, shifting cells to a therapy-sensitive, less metastatic state [74]. While GDF
proteins have both tumorigenic and tumor-suppressive roles, their exact functions in cancer
remain unclear [72]. GDF2 suppresses breast and ovarian cancer metastasis [75], while
GDF9 (up-regulated 3.75-fold by curcumin) exhibits tumor-suppressive effects in bladder
cancer [76]. Conversely, elevated GDF15 levels in glioblastoma patients are correlated with
a poor prognosis, immune suppression, and increased invasion [77,78]. Interestingly, Kad-
owaki et al. reported that GDF15 overexpression in glioblastoma cells with low basal levels
induced apoptosis [79]. In this study, GDF15 mRNA increased 1.52-fold with curcumin
treatment, highlighting the complex and context-dependent roles of GDF and other TGFβ
superfamily members.

4. Materials and Methods

4.1. Curcumin
Curcumin (95% purity) [(E, E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-
3,5-dione] was obtained from Sigma-Aldrich (St. Louis, MO, USA) and stored as a 5 mM
stock solution in ethanol at −20 ◦ C, protected from light.

4.2. Cell Culture

The human glioblastoma cell line (U87 MG) was obtained from the American Type
Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in T75 or T175 flasks
(Greiner, Monroe, NC, USA) using high-glucose Dulbecco’s modified Eagle medium
(DMEM) (ATCC) supplemented with 10% fetal bovine serum (Atlas Biologicals, Fort
Collins, CO, USA) and 1× antibacterial/antimycotic solution (Gibco, Grand Island, NY,
USA). Cultures were maintained in a humidified incubator at 37 ◦ C with a gas mixture of
5% CO2 and 95% air.

4.3. Cell Viability Assay

The impact of increasing curcumin concentrations on the viability of U87 MG cells was
evaluated. A total of 5 × 103 cells were seeded per well in a 96-well microplate with 100 µL
of media and incubated overnight. Cells were then treated with curcumin at concentrations
of 10, 20, 30, 40, and 50 µmol/L, along with ethanol as the vehicle control (0.1%), for 24,
48, and 72 h. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; Dojindo
Molecular Technologies, Inc., Kumamoto, Japan) assay. Briefly, after treatment, 10 µL of
CCK-8 solution was added to each well containing 100 µL of treatment media, followed by
a 4 h incubation. Optical density was then measured at 450 nm using the SpectraMax iD3
Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA). The cytotoxicity of
curcumin was determined by calculating the IC50 from the dose-response curve obtained
through the cell viability assay at 24 h.

4.4. Poly-Caspase Assay

To examine curcumin-induced apoptosis in U87 MG cells, caspase enzyme activity
was measured using the poly-caspase assay (FAM FLICA, ImmunoChemistry Technologies,
Davis, CA, USA). Cells were cultured in T25 flasks with phenol red-free Dulbecco’s mod-
ified Eagle’s medium (Gibco, Grand Island, NY, USA) supplemented with 10% FBS and
Molecules 2025, 30, 2108 11 of 16

1× antibiotic/antimycotic solution. Once cells reached 80% confluence, they were treated
with ethanol (vehicle control) or 10 or 20 µmol/L curcumin. Staurosporine (5 µmol/L)
(ImmunoChemistry Technologies, Bloomington, MN, USA) served as a positive control.
After 24 h of treatment, cells were collected, stained with FLICA, and incubated for one
hour at 37 ◦ C. Following washing, a 100 µL aliquot of the cell suspension, at a density of
greater than 2 × 106 cells/mL, was transferred to a 96-well flat-bottom microplate. Fluores-
cence was measured at an excitation wavelength of 488 nm and an emission wavelength of
520 nm using a microplate reader.

4.5. SA-Beta Galactosidase Assay

To determine whether curcumin treatment induced or inhibited senescence, β-galacto-
sidase—a pH-dependent enzyme marker of senescent cells—activity was measured using the
SA-β-galactosidase assay (Cell Biolabs, San Diego, CA, USA). Cells were cultured in 6-well
plates with phenol red-free Dulbecco’s modified Eagle’s medium supplemented with 10% FBS
and 1× antibiotic/antimycotic solution. Once cells reached 80% confluence, they were treated
with ethanol (vehicle control) or 10 or 20 µmol/L curcumin. After 24 h of treatment, cells
were collected, stained, and transferred to 96-well flat black-bottom microplates. Fluorescence
(optical density) was measured at 360/465 nm (excitation/emission) using a microplate reader,
and the results were expressed as relative fluorescence units (RFU).

4.6. RNA Isolation and Library Preparation

The effect of curcumin treatment on global gene expression in U87-MG cells was as-
sessed using RNA-seq. Three replicates each of two experimental controls were set up: neg-
ative control (vehicle-treated cells) and treated cells. Cells were cultured in 60 mm × 15 mm
cell culture dishes (Wuxi NEST Biotechnology Co., Ltd., Wuxi, Jiangsu, China) until 80–90%
confluence and treated with 20 µmol/L curcumin. After incubation for 6 h, RNA was
isolated using the RNeasy Plus Mini Kit (QIAGEN, Germantown, MD, USA) and stored at
−20 ◦ C until sequencing. Further library preparation followed protocol in [6].

4.7. RNA-Seq Analysis

Sequencing reads were processed by trimming adapters and removing low-quality
bases (Phred score < 30) using Trimmomatic v0.39 [80]. The filtered reads were then
aligned to the human reference genome (GRCh38.p13) using the STAR RNA-Seq aligner
v2.7.11a [81], generating BAM alignment files. Read quantification was performed with the
HTSeq R package (version 2.0.3) [82] using genome annotations in GFF format. Differential
gene expression (DEG) analysis was conducted using DESeq2 [83], considering genes with
a log2FoldChange ≥ 1 and a false discovery rate (FDR) of ≤0.05. Pathway enrichment
analysis was carried out using KOBAS [84].

4.8. Statistical Analysis

All statistical analyses were conducted using GraphPad Prism v10.4.0.621 for Windows
(GraphPad Software, Boston, MA, USA, www.graphpad.com, accessed on 1 December
2024). A p-value of <0.05 was considered statistically significant.

5. Conclusions
In conclusion, this study elucidated that curcumin acts as a promising therapeutic
agent against U87 glioblastoma cells by targeting multiple cancer-related processes, in-
cluding regulation of the cell cycle, suppression of cell viability, induction of pro-apoptotic
genes, down-regulation of anti-apoptotic genes, and activation of anti-metastatic genes.
Curcumin exerts these effects through modulation of several critical signaling pathways,
such as the calcium signaling pathway; the PI3K-Akt signaling pathway; TNF signaling;
Molecules 2025, 30, 2108 12 of 16

MAPK, Ras, TGF-β, Wnt, and cytokine signaling, and regulation of the actin cytoskele-
ton. Furthermore, curcumin influences the expression of several pivotal genes involved
in apoptosis, metastasis, and stem cell dynamics, including AIFM3, TP53AIP1, CRD14,
NIBAN3, BOK, BCL2L10, BCL2L14, BNIPL, FASLG, GZMM, TNFSF10, TNFSF11, TNFSF4,
FOS, PRKCG, GDF7, GDF9, GDF15, GDF5, FZD1, FZD2, FZD8, SMAD5, APC, WNT7B,
and HOXA1. By modulating these interconnected molecular networks, curcumin impairs
the survival of cancer stem cells, reduces therapy resistance, inhibits metastatic potential,
and sensitizes tumor cells to conventional treatments. Collectively, these findings highlight
curcumin’s multifaceted role in cancer prevention and therapy, emphasizing its potential
as a powerful adjunct in glioblastoma management.

Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/molecules30102108/s1, Figure S1: Cell viability was assessed at
24 h following treatment with various concentrations of curcumin, and the corresponding IC50 value
was determined based on the dose–response relationship. Figure S2: Principal Component Analysis
(PCA) of RNA-seq data. PCA shows the variance in normalized gene expression profiles across
samples. The first two principal components, PC1 and PC2, explain 85% and 5% of the total variance,
respectively. Samples are color- and shape-coded by group: U87C (control) in red circles and U87T
(treated) in cyan triangles.

Author Contributions: Conceptualization, G.H. and U.K.R.; experimentation, N.T.M., Y.N.R. and
C.S.R.; RNA-seq analysis, P.N.; writing—original draft preparation, N.T.M., C.S.R., G.H. and U.K.R.;
writing—review and editing, C.S.R. and U.K.R. All authors have read and agreed to the published
version of the manuscript.

Funding: This material is based on work supported by the National Science Foundation (NSF) under
award number 2242771.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: No applicable.

Data Availability Statement: The raw paired-end Illumina RNA-sequencing reads generated in
the current study are available in the Sequence Read Archive (SRA) at NCBI under the Bio project
accession numbers PRJNA1243709.

Conflicts of Interest: The authors have no conflicts of interest.

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