Abstract
Combining anti-cancer agents in cancer therapies is becoming
increasingly popular due to improved efficacy, reduced toxicity and
decreased emergence of resistance. Here, we test the hypothesis that
dietary agents such as oligomeric proanthocyanidins (OPCs) and curcumin
cooperatively modulate cancer-associated cellular mechanisms to inhibit
carcinogenesis. By a series of in vitro assays in colorectal cancer
cell lines, we showed that the anti-tumorigenic properties of the
OPCs-curcumin combination were superior to the effects of individual
compounds. By RNA-sequencing based gene-expression profiling in six
colorectal cancer cell lines, we identified the cooperative modulation
of key cancer-associated pathways such as DNA replication and cell
cycle pathways. Moreover, several pathways, including protein export,
glutathione metabolism and porphyrin metabolism were more effectively
modulated by the combination of OPCs and curcumin. We validated genes
belonging to these pathways, such as HSPA5, SEC61B, G6PD, HMOX1 and
PDE3B to be cooperatively modulated by the OPCs-curcumin combination.
We further confirmed that the OPCs-curcumin combination more potently
suppresses colorectal carcinogenesis and modulated expression of genes
identified by RNA-sequencing in mice xenografts and in colorectal
cancer patient-derived organoids. Overall, by delineating the
cooperative mechanisms of action of OPCs and curcumin, we make a case
for the clinical co-administration of curcumin and OPCs as a treatment
therapy for patients with colorectal cancer.
Introduction
The development of chemoresistance presents a major challenge in the
treatment and management of colorectal cancer (CRC; Fig. [36]1A). When
treated with a chemotherapeutic agent that targets a specific
mechanism, the tumor relapses due to compensatory mutations or
activation of alternative signaling pathways in subsets of cancer
cells^[37]1,[38]2. Switching to a second agent after the tumor relapses
is usually ineffective due to ensuing compensatory mechanisms against
the second agent^[39]3. Alternatively, combining agents that
coordinately modulate multiple tumorigenic mechanisms often demonstrate
enhanced therapeutic efficacy due to the lower likelihood of resistance
emerging against both agents in the cancer cells. Moreover, the dosage
of individual drugs is often smaller in drug combinations, diminishing
the toxic effects caused by higher doses of a single therapeutic
agent^[40]4,[41]5.
Figure 1.
[42]Figure 1
[43]Open in a new tab
The combination of curcumin and OPCs has superior anti-cancer effects
than individually. (A) Top: Schematic illustration of the development
of chemo-resistance against drug(s) used as the sole agent or in
sequence with another agent. Bottom: Schematic showing the rational for
combining drugs with different mechanisms of anti-cancer action in
cancer therapy. (B) Cell proliferation assay showing the effect of
curcumin alone or in combination with OPCs at different doses in six
CRC cells. X-axis shows the doses of curcumin in ng/ul. Y-axis shows
the percentage change in cell count with respect to control cells
treated with curcumin alone at the corresponding dose. (C) Percentage
of cells undergoing apoptosis as measured by percentage that stained
positive for Annexin-V assay (D) Analysis of effect on cell cycle as
evaluated by the DNA staining of propidium iodide in HCT116 and SW480
cells treated with indicated concentrations of OPCs or curcumin.
*P < 0.05, **P < 0.01, ***P < 0.001 compared to control treatments.
Emerging evidence indicates that several dietary agents possess
anti-tumorigenic properties by targeting multiple oncogenic signaling
pathways^[44]6–[45]11. In particular, two dietary agents,
curcumin^[46]12–[47]14 and oligomeric proanthocyanidins (OPCs)^[48]15,
have been extensively studied for their anti-cancer properties in CRC.
Curcumin, a dietary polyphenol derived from the spice turmeric, has
been shown to exert chemopreventive and anti-tumor effects by
modulating transcription factors and signaling pathways, including
PI3K/mTOR, Ras/Raf/MEK and GSK-3beta pathways^[49]16–[50]21. In
addition, we recently delineated the anti-cancer mechanisms of OPCs, a
group of flavonoids from grape seeds, in CRC by genome-wide mRNA
expression profiling (unpublished results). Given their diverse
anti-cancer mechanisms, we hypothesized that co-administering OPCs and
curcumin might cooperatively modulate multiple cancer-associated
signaling pathways and, thereby be more effective against CRC.
Furthermore, a combination of OPCs and curcumin at minimum tolerable
doses might effectively decrease tumorigenesis with minimal concurring
toxicity and lower the prospect of acquiring drug resistance.
Adjunctive treatment with curcumin together with conventional cytotoxic
drugs including 5-fluorouracil, cisplatin and mitomycin, have been
shown to improve the overall chemotherapeutic response^[51]22–[52]25.
However, the underlying mechanisms by which curcumin enhances efficacy
of these compounds remain unclear. Herein, through a series of
systematic cell lines, animal model and patient-derived tumor organoid
experiments, we for the first time report that the combination of
curcumin and OPCs has a superior anti-tumorigenic effect in colorectal
cancer. Subsequently, we sought to identify the underlying mechanism(s)
by which the two agents cooperatively hinder colorectal tumor
progression. Although a comprehensive analysis of all the genes
regulated by curcumin has been investigated in a variety of cancers,
including melanoma^[53]26, breast^[54]27 and lung^[55]28 cancers, such
an assessment in CRC has not been accomplished to date. Therefore, we
performed genome-wide RNA-sequencing in a panel of CRC cell lines
treated with curcumin and/or OPCs, and interrogated signaling pathways
modulated by these two compounds. By comparing the genes regulated by
OPCs in CRC cell lines, we discovered that though curcumin and OPCs
cooperatively affect several critical mechanisms of carcinogenesis,
each treatment also affects distinctive signaling pathways, making them
a promising combination in cancer therapy.
Overall, this study reveals that curcumin and OPCs potently inhibit
colorectal carcinogenesis by cooperatively modulating multiple
cancer-associated mechanisms. The anti-tumor response to the
combination of curcumin and OPCs in mice xenograft tumors and organoids
derived from primary human CRC tumors correlated with the altered gene
expressions of HSPA5, IHH, PDE3B, cyclin D1 and SEC61B. Collectively,
our data presents the possibility of combining curcumin and OPCs to
curb colorectal carcinogenesis, which has the potential for leveraging
as a safe and inexpensive therapeutic option, on its own or in
combination with conventional chemotherapeutic drugs.
Results
Curcumin and OPCs cooperatively inhibit cellular growth in colorectal cancer
cells
The anti-tumorigenic properties of OPCs and curcumin in CRC are
well-established^[56]15,[57]29–[58]36. In order to study the effect of
the combination of curcumin and OPCs, we used six colorectal cancer
cell lines that have been well-studies and broadly represent the common
mutational and microsatellite statuses found in colorectal cancer. We
found that the combination was more effective in inhibiting cell
proliferation compared to the individual agents (Fig. [59]1B). To
determine any evidence of the cooperativity between OPCs and curcumin,
we calculated the Combination Index (CI) for OPCs-curcumin combinations
by two methods. The first method, called the ‘Highest Single Agent’
method (Supplemental Fig. [60]1, shown for OPCs at 25 ng/ul and
curcumin at 0.5 ng/ul), measures if the resulting effect of the
combination is greater than the individual agents. Our second method
utilized the ‘Bliss Independence’ model (Supplemental Fig. [61]2, shown
for OPCs at 25 ng/ul and curcumin at 0.5 ng/ul), which is the
multiplicative probability assuming the agents act independently toward
a common result. The Combination Indices (CI) calculated by both
methods were less than 1 for most cell lines, suggesting strong
cooperative action between curcumin and OPCs.
Additionally, to measure the extent to which the doses of curcumin or
OPCs can be reduced in the combination to derive comparable efficacy as
the individual agents, we calculated the ‘Dose Reduction Index (DRI)’
(Supplemental Table [62]3). The values of the DRI were above 1 for most
cell lines, highlighting a beneficial combination.
We then assessed whether the reduced cellular growth observed in the
cell proliferation assay by OPCs-curcumin combination was due to
apoptosis using a flow cytometry-based assay in two representative cell
lines: MSI cell line HCT116 and MSS cell line SW480. Both OPCs
(100 ng/ul) and curcumin (1 ng/ul) induced apoptosis in HCT116 and
SW480 cells, and their combination at full doses (OPCs at 100 ng/ul and
curcumin at 1 ng/ul) further significantly enhanced apoptosis rates in
these cells (p = 0.0002 for SW480 cells, p = 0.0014 for HCT116 cells;
Fig. [63]1C). Interestingly, combining OPCs at a much lower dose of
25 ng/ul with curcumin at 0.5 ng/ul still induced apoptosis in HCT116
and SW480 cells at levels comparable to those of the full doses of OPCs
and curcumin, supporting the rationale for combined treatment with
these two botanicals.
Additionally, we found that while both OPCs (100 ng/ul) and curcumin
(0.5 ng/ul) individually caused cell cycle arrest in HCT116 (p = 0.0016
for OPCs, 0.0016 for curcumin) and SW480 (p = 0.028 for OPCs, 0.02 for
curcumin) cells, a combination of low doses of OPCs at only 25 ng/ul
and curcumin of 0.5 ng/ul induced a comparable and significant cell
cycle arrest (p = 0.01 for SW480 cells; p = 0.02 HCT116 cells)
(Fig. [64]1D). Taken together, these data suggest the superiority of
the anti-cancer properties of the combination of OPCs and curcumin over
the individual agents.
Curcumin and OPCs modulate multiple cancer-associated pathways
Encouraged by the results of our in vitro assays, we wanted to examine
the underlying molecular mechanisms by which OPCs and curcumin
cooperatively function against CRC. Thus, we looked at the genome-wide
changes in gene expression induced by either OPCs or curcumin and their
combination in six CRC cell lines SW480, SW620, HT29, HCT116, RKO and
LoVo using RNA sequencing. KEGG pathway analysis of the differentially
expressed genes relative to untreated cells revealed 30 pathways that
were commonly regulated by both OPCs and curcumin, 25 pathways that
were uniquely regulated by OPCs and 28 pathways uniquely regulated by
curcumin (Fig. [65]2A). Consistent with our previously unpublished data
on OPCs, the top pathways that were commonly regulated by both OPCs and
curcumin were DNA replication (p = 1.7e-11 for curcumin, p = 0.003 for
OPCs) and cell cycle (p = 4.7e-11 for curcumin, p = 8.2e-07 for OPCs;
Fig. [66]2B). We validated the RNA-sequencing data experimentally by
measuring changes in mRNA levels of well-recognized key genes
associated with DNA replication, PCNA and CCND1 (cyclin D1), and the
cell cycle, namely E2F1 and CDKN1a (P21) in HCT116 and SW480 cells
(Fig. [67]2D). While OPCs and curcumin both decreased the mRNA levels
of proliferation markers PCNA and cyclin D1, the OPCs-curcumin
combination further decreased their mRNA levels in both HCT116 and
SW480 cells (Fig. [68]2D, top). Additionally, the combination of OPCs
and curcumin at lower doses of 25 ng/ul and 0.5 ng/ul respectively,
were as effective as the individual doses in decreasing the levels of
PCNA and cyclin D1. The expression of certain genes, such as the
transcription factor E2F1, was decreased by both OPCs and curcumin
individually, and the OPCs-curcumin combination did not further
decrease their expression (Fig. [69]2D, bottom left). Other genes, such
as p21, were better regulated by OPCs than curcumin (Fig. [70]2D,
bottom right).
Figure 2.
[71]Figure 2
[72]Open in a new tab
OPCs and curcumin affect several molecular pathways. (A) Venn diagram
showing the number of common and unique KEGG pathways affected by
curcumin or OPCs in six CRC cell lines. (B) The top 10 pathways
affected by both OPCs and curcumin. (C) Top pathways cooperatively
regulated by OPCs and curcumin. (D) Levels of mRNA of genes belonging
to pathways commonly affected by both OPCs and curcumin, normalized to
mRNA levels of β-actin. (E) Levels of mRNA of genes belonging to top
pathways cooperatively affected by OPCs and curcumin, namely (top)
HSPA5 and SEC. 61 from protein export pathway, (middle) G6PD and GCLC
from glutathione metabolism, and (bottom) HMOX1 and BDNF from porphyrin
metabolism pathways, normalized to mRNA levels of β-actin. (F) mRNA
levels of genes from pathways activated only by OPCs-curcumin
combination and not by the individual agents, normalized to mRNA levels
of β-actin. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control
treatments.
Moreover, based on the pathway analysis, steroid biosynthesis
(p = 8.8e-05) and axon guidance (p = 0.3 e-04) were among the top
pathways regulated exclusively by OPCs (Supplemental Fig. [73]3A). The
top pathways regulated exclusively by curcumin were glutathione
metabolism (p = 1.2e-06) and spliceosome (p = 6.8e-06) pathways. Taken
together, these data illustrate the diverse mechanisms by which OPCs
and curcumin impede carcinogenesis.
The OPCs-curcumin combination cooperatively modulates cancer-associated
pathways
Next, we were curious to identify the pathways that were cooperatively
modulated by the combination of OPCs and curcumin. To do this, we
identified all the genes differentially altered by the OPCs-curcumin
combination and compared to those altered by the compounds individually
in at least three cell lines by RNA-sequencing. By KEGG pathway
analysis, we identified protein export (p = 1e-04), glutathione
metabolism (p = 0.001) and porphyrin-chlorophyll metabolism (p = 0.012)
as the top pathways that were altered more by the combination of
curcumin and OPCs than by these compounds alone (Fig. [74]2C).
We next validated alterations in these pathways experimentally by
measuring the mRNA levels of key representative genes belonging to
these pathways. The expression level of HSPA5, which forms the binding
immunoglobulin portion of the chaperone protein Hsp70, and plays a
critical role in protein folding and translocation machinery^[75]37,
was decreased by both curcumin and OPCs in HCT116 and SW480 cells, and
was further down-regulated significantly when treated with a
combination of both compounds (p = 0.025 for SW480 cells, p = 0.049 for
HCT116 cells; Fig. [76]2E, top left). Similarly, the mRNA level of
Sec61b, a protein involved in modulating sensitivity to platinum-based
chemotherapeutic agents such as oxaliplatin and cisplatin^[77]38,
showed a greater increase with the combination of OPCs and curcumin,
vis-à-vis, individual agents (Fig. [78]2E, top right). Interestingly,
while the expression levels of G6PD, a key enzyme in the pentose
phosphate pathway and linked to colon carcinogenesis^[79]39,[80]40,
were not altered by curcumin or OPCs alone: the combination of the two
compounds significantly decreased its expression (p = 0.0054 for SW480
cells, p = 0.039 for HCT116 cells; Fig. [81]2E, middle). Likewise, the
expression of GCLC, a gene notorious for driving chemoresistance in
several cancers^[82]41–[83]43, was only significantly downregulated by
a combination of curcumin and OPCs (p = 0.005 for SW480 cells), not
individually (Fig. [84]2E, middle). We then validated genes from the
porphyrin and chlorophyll metabolism pathway, namely HMOX1 and BDNF,
both of which were better modulated by the combination of agents than
individually (Fig. [85]2E, lower).
Furthermore, by comparing the KEGG pathways modulated by the OPCs and
curcumin combination vs. those modulated by OPCs or curcumin alone, we
identified that several molecular pathways, such as hedgehog signaling,
PPAR signaling and insulin signaling pathways were uniquely regulated
by the combination of OPCs and curcumin, but not by the individual
agents (Supplemental Fig. [86]2B). We confirmed modulation of these
pathways by evaluating the levels of IHH and PDE3B by RT-qPCR
(Fig. [87]2F). Curcumin and OPCs in combination suppressed the
expression of IHH in both SW480 and HCT116 cells, whereas individual
doses of these compounds did not. While the expression of PDE3B was
suppressed only by curcumin and not by OPCs, the combination of OPCs
and curcumin completely abolished the expression of PDE3B. These
results confirm the cooperative modulation of molecular mechanisms by
OPCs and curcumin.
Combination of curcumin and OPCs more effectively decreases tumor growth in
mice xenografts
To evaluate the effect of the combination of curcumin and OPCs in vivo,
we followed the tumor growth in athymic mice with subcutaneous
xenografts of HCT116 cells that were orally administered OPCs or
curcumin alone, or in combination (Fig. [88]3A). There was no
significant change in weight of the mice during the course of the
treatment (Supplemental Fig. [89]4). While the tumors continued to grow
in mice that were administered vehicle, tumor-growth in
OPCs-administered mice (p = 0.005 for tumor volume, p = 0.005 for tumor
weight) or curcumin-administered mice (p = 0.006 for tumor volume,
p = 0.017 for tumor weight) was significantly attenuated
(Fig. [90]3A,B). Interestingly, the combination of OPCs (100 mg/kg) and
curcumin (100 mg/kg) was significantly more effective in decreasing
tumor growth than the individual agents (p = 6 e-04 for tumor volume,
p = 0.000214 for tumor weight; Fig. [91]3A–C). Interestingly, the
combination of OPCs and curcumin at even lower doses of 50 mg/kg
attenuated tumor growth as effectively as the higher doses of
individual agents. Furthermore, in accordance with the results obtained
in the cell lines, the combination of OPCs and curcumin decreased the
expression of IHH, PDE3B, cyclin D1 and HSAP5, and increased HMOX1 and
SEC61B more effectively than treatment with the singular compounds
(Fig. [92]3D).
Figure 3.
[93]Figure 3
[94]Open in a new tab
OPCs-curcumin combination effectively inhibits tumor growth in mice
xenografts. (A) Representative images of mice with subcutaneous tumors
15 days after administering orally with curcumin, OPCs or in
combination. (B) Progressive tumor volume in mice orally gavaged with
OPCs and curcumin, individually and in combination. (C) Left: Xenograft
tumors collected from sacrificed mice at the end of the 15-day
treatments. Right: Quantification of tumor weights from different
treatment groups. (D) qPCR analysis of mRNA levels of genes normalized
to control group. mRNA levels of β-actin was used as the internal
normalizing control. Indicated amounts of curcumin or OPCs is in mg/kg.
*P < 0.05, **P < 0.01, ***P < 0.001 compared to control treatments.
OPCs inhibit tumor growth in patient-derived organoids
Tumor organoid model allows the maintenance and expansion of cells
derived from patients in a 3D culture, and is physiologically superior
to the conventional monolayer of cultured cells to study anti-cancer
agents^[95]44. Therefore, we next used a tumor organoid model derived
from CRC patients to further confirm our in vitro and in vivo
observations. In line with our results in cell lines and mice
xenografts, the OPCs-curcumin combination significantly decreased
patient-derived tumor organoid formation and growth (Fig. [96]4A, top:
representative images of organoids; bottom: organoid counts, p = 0.016
for patient 1, p = 0.003 for patient 2, p = 0.002 for patient 3). In
line with our data from in vitro and in vivo experiments, the
expression levels of IHH (p = 1.54 e-05), PDE3B (p = 0.04), cyclin D1
(p = 2.45 e-06), HSPA5 (p = 0.0003), SEC61B (p = 0.039) and BDNF
(p = 0.0005) were significantly down-regulated by OPCs-curcumin
combination in the patient-derived tumor organoids (Fig. [97]4B).
Figure 4.
[98]Figure 4
[99]Open in a new tab
OPCs-curcumin combination effectively suppresses growth of organoids
derived from human colorectal tumors. (A) Top: Images showing tumor
organoid cultures derived from 3 different CRC patients, treated with
OPCs and curcumin in combination or individually. Bottom: Bar graph
showing decrease in spheroid count with treatments. (B) mRNA levels of
genes in patient-derived organoids treated with OPCs and curcumin in
combination or individually. mRNA levels of β-actin was used as the
internal normalizing control. *P < 0.05, **P < 0.01, ***P < 0.001
compared to control treatments.
Discussion
The cellular heterogeneity and the constant evolution of colorectal
cancer complicates the design of effective treatment regimens. Drug
combination therapies are becoming increasingly popular as they improve
clinical outcomes^[100]45. When selecting drugs for combination
therapies, it is beneficial to choose agents that work by targeting
multitude of pathways with the potential likelihood of having additive
or synergistic effects on the tumor and decreased chances of acquired
resistance. In this study, by illuminating the cooperative molecular
mechanisms of action of OPCs and curcumin, we contend that a
therapeutic regimen combining OPCs and curcumin could effectively
control colorectal cancer growth.
Several successful combinations of curcumin with other anti-cancer
agents, including chemotherapeutic drugs and other natural compounds,
have previously been explored and established. Curcumin considerably
improves tumor-suppressive properties of chemotherapeutic agents, such
as 5-fluorouracil, cisplatin and mitomycin^[101]22–[102]25 and other
phytochemicals, such as epigallocatechin-3-gallate from green
tea^[103]46, boswellic acid from frankincense plant^[104]47 and
quercetin from fruits and vegetables^[105]48. In this study, we not
only showed an improvement in the anti-tumor properties of curcumin
when combined with OPCs from grape seeds, but also delineated the
molecular pathways through which curcumin and OPCs function
cooperatively. Our data indicate that both OPCs and curcumin affect
cell cycle and DNA replication, which are fundamental to cancer cell
proliferation and progression. Cell cycle regulators, PCNA and cyclin
D1, are hubs for interaction with other cancer-associated
proteins^[106]49. Given the addiction of cancer cells to PCNA and
cyclin D1, their targeted inhibition has been shown to successfully
suppress cancer growth without affecting normal cells^[107]50–[108]52.
Likewise, targeting HSPA5 using small molecule drugs has proven to
induce endoplasmic reticulum stress and apoptosis selectively in cancer
cells, and not normal cells^[109]53. In our study, we observed that the
expression of cyclin D1, PCNA and HSPA5 were attenuated by the OPCs and
curcumin combination in CRC cell lines, mice xenograft tumors and
patient-derived organoids. The inhibition of PCNA, cyclin D1 and HSPA5
expression by this combination is encouraging as a means to
preferentially target cancer cells without affecting normal cells.
Our RNA-sequencing data revealed that while curcumin and OPCs commonly
affect several genes, they also affect different genes within a
pathway, thus effectively shutting off the entire signaling circuitry.
By measuring mRNA levels using qRT-PCR, we confirmed that while certain
genes, such as p21, were affected only by OPCs, other genes, such as
SEC61B and PDE3B, were affected solely by curcumin. Intriguingly
several key genes, such as G6PD and IHH, and pathways, such as protein
export, notch and hedgehog signaling, were effectively regulated only
by the combination of curcumin and OPCs, and not by the individual
compounds. All these data demonstrate the potential of the
OPCs-curcumin combination to block redundant cancer-associated
pathways, which a main reason for the failure of conventional
drugs^[110]1,[111]2.
Several genes that were identified and validated in our study to be
cooperatively modulated by OPCs and curcumin are also implicated in the
development of chemo-resistance. SEC61B is essential for platinum drug
accumulation, and its knockdown desensitizes cancer cells to
oxaliplatin, carboplatin and cisplatin^[112]38, while G6PD and PDE3B
enhance chemoresistance to oxaliplatin and cisplatin^[113]39,[114]54.
OPCs and curcumin together influenced several ABC transporters
(Supplemental Fig. [115]3B) that impart resistance to multiple drugs by
expelling drug molecules out of the cell. The intimation of the data
that curcumin and OPCs could sensitize cells to chemotherapeutic agents
remains to be studied systematically.
Collectively, in this study, we discovered that both curcumin and OPCs
cooperatively affect several critical mechanisms of carcinogenesis such
as DNA replication, cell cycle and mismatch repair. Apart from the
commonly regulated pathways, our results highlighted the distinctive
modes of actions of the two dietary agents; several pathways, such as
Wnt-signaling and TGF-β signaling, were found to be uniquely modulated
by OPCs, and not curcumin. Likewise, cellular mechanisms such as
homologous recombination and nucleotide excision repair were modulated
specifically by curcumin. The results from this study are important in
the pre-clinical development of the OPCs-curcumin combination in
colorectal cancer.
Materials and Methods
Cell culture and materials
Colorectal cancer cell lines, HCT116, SW480, SW620, HT29, RKO and LoVo
were purchased from the American Type Culture Collection (Manassas,
VA). All cell lines were tested and authenticated using a panel of
genetic and epigenetic markers and tested for mycoplasma on a regular
basis. The cells were grown in Dulbecco’s Medium Eagle’s medium (DMEM;
Gibco, Carlsbad, CA), supplemented with 10% fetal bovine serum, 1%
penicillin and streptomycin and maintained at 37 °C in a humidified
incubator at 5% CO[2].
Grape seed-OPCs (VX1 extract, EuroPharma, USA) and curcumin
(BCM-95, Arjuna Natural Extracts, India) were dissolved in DMSO and
diluted to appropriate experimental concentrations in culture medium.
Cell viability and proliferation
Cells were plated in 96-well dishes at a density of 2000 cells/well in
DMEM supplemented with 5% FBS and antibiotics, and allowed to attach
overnight. Cell proliferation was measured in cells treated with a
combination of OPCs (10, 100, 500, 1000 ng/ul) and curcumin (0.01,
0.05, 0.1, 0.5, 1, 2.5, 5) for 72 hours using WST-1 assay
(Sigma-Aldrich) per manufacturer’s instructions. Each experiment was
performed in triplicates.
Cell cycle and apoptosis analysis
Cells plated in 24-well dishes were treated with OPCs or curcumin for
48 hours in triplicates. Cell cycle and apoptosis assays were performed
using Muse Cell Cycle Assay kit (MCH100106, Millipore) and Muse Annexin
V and Dead Cell Assay kit (MCH100105, Millipore) respectively, on Muse
Cell Analyzer (Millipore) per manufacturer’s instructions.
Patient-derived tumor organoids
Fresh tumor tissues were obtained from CRC patients enrolled at the
Baylor University Medical Center, Dallas, and clinicopathologic
characteristics of the patients from whom the CRC tissues were obtained
is listed in Supplemental Table [116]1. The study was approved by the
Institutional Review Board of Baylor Scott & White Research Institute,
Dallas, TX. Written informed consent was obtained from all patients
providing tissue specimens, and all experiments were performed in
accordance with relevant guidelines and regulations proposed in the
Declaration of Helsinki. CRC tumor organoids were cultured using a
modified protocol described previously^[117]55. Briefly, following
excision, tumors were maintained in a medium containing Advanced
DMEM-F12 (Gibco) supplemented with 1% HEPES (Sigma-Aldrich), 1%
L-glutamine (Gibco), 10% FBS (Gibco), 2% penicillin/streptomycin
(Sigma-Aldrich) and 10 uM Y-27632 (R&D Systems). Tissues were minced
and digested with collagenase solution (5 ml of above medium with 75 ul
collagenase, 124 ug/ml dispase type II and 0.2% Primocen) for 30 min,
then filtered through a 70 um filter (Corning). Cells were pelleted by
centrifugation (200 g for 10 min), then suspended in Matrigel (BD
Biosciences, Franklin Lake, NJ). Fifteen microliters of the
cell-Matrigel suspension was placed in the center of 24-well plate and
polymerized. A 1:1 mixture of L-WRN conditioned medium and DMEM/F12
medium (Gibco) supplemented with 20% FBS (Gibco), 2 mM L-glutamine
(Gibco), 0.2% Primocen, 10 uM Y-27632 (R&D Systems), 10 uM SB431542
(R&D Systems) and 5% penicillin/streptomycin (Sigma-Aldrich) were added
to the well and replaced every two days. For treatments, appropriate
concentration of OPCs or curcumin or a combination of OPCs and curcumin
were added to the culture medium and tumor organoids were allowed to
grow for 1 week. The experiment was performed in triplicates.
The organoids were observed under a bright-field microscope. All 3D
cell structures that were about 150–300 microns in diameter and that
had a distinct epithelial cell layer were counted. Organoids in a
certain plane of field were counted, while leaving out the ones that
were out of focus.
mRNA expression analysis
RNA from HCT116, SW480, SW620, RKO, LoVo and HT29 cells treated for
18 hours or tumor organoids treated for 7 days with DMSO (vehicle),
OPCs (100 ng/ul), curcumin (1 ng/ul),
OPCs(100 ng/ul) + curcumin(1 ng/ul) and
OPCs(25 ng/ul) + curcumin(0.5 ng/ul), were isolated using mRNeasy kit
(Qiagen). RNA from mice xenograft tumors collected in RNAlater solution
(Qiagen) were extracted using mRNeasy Kit (Qiagen) following the
manufacturer’s instructions. Extracted RNA was used as a template for
cDNA synthesis using High Capacity cDNA Reverse Transcription Kit
(ThermoFisher Scientific) according to manufacturer’s protocol. RT-qPCR
was performed using SensiFAST SYBR mix (Bioline, London, UK) using the
primer sequences listed in Supplemental Table [118]2. All RT-qPCR
target genes were calculated using ΔΔCt method normalized to β-actin.
Genomewide RNA Sequencing analysis
RNA from cell lines treated with DMSO or 100 ng/ul of OPCs or 1 ng/ul
of curcumin or combination of OPCs (100 ng/ul) and curcumin (1 ng/ul)
in duplicates were single-end sequenced. NGS library construction was
performed using the TruSeq RNA Library Kit (Illumina) with up to 1 ug
of total RNA input according to manufacturer’s protocol. The quality of
individual libraries was assessed using the High Sensitivity DNA Kit
(Agilent). Libraries were pooled together using a Pippin HT instrument
(Sage Science). Efficiency of size selection was assessed using a High
Sensitivity DNA Kit (Agilent). Pooled libraries were quantitated via
qPCR using the KAPA Library Quantification Kit, Universal (KAPA
Biosystems) prior to sequencing on an Illumina HighSeq 2500 with
single-end 75 base read lengths. For the analysis of RNA-sequencing,
Fastq files were trimmed using Flexbar to remove 3′ bases with quality
scores lower than 30 before alignment, as described previously^[119]56.
The trimmed reads were mapped to human genome version GRCH38 downloaded
from GENCODE^[120]57 using HISAT2^[121]58 to generate alignment files
in bam format. Samtools name-sorted bam files^[122]59 were processed
using htseq-count to summarize gene level counts as described
previously^[123]60. DESeq. 2 was used for differential gene expression
analysis of RNA-sequencing read counts^[124]61. All sequencing data has
been deposited to the GEO database ([125]GSE109607).
Meta-analysis was performed using Stouffer’s p-value combination
method^[126]62 to identify genes that are homogenously up or down
regulated independently in OPCs-, curcumin- and OPCs + curcumin-treated
cells. Fisher’s enrichment test on KEGG pathways was performed and the
cellular pathways commonly regulated by both OPCs and curcumin with a
p < 0.05 were identified. A similar meta-analysis approach was used to
identify the cellular pathways that were uniquely regulated by OPCs or
curcumin. Genes that were uniquely regulated by OPCs or curcumin or the
combination of OPCs and curcumin were identified by Venn comparison.
KEGG pathway analysis was done with these unique genes for OPCs,
curcumin, OPCs-curcumin combination and pathways with p < 0.05 were
plotted.
Additionally, KEGG pathway enrichment analysis was performed on genes
whose fold change expression (with respect to untreated controls) in
cells treated with OPCs-curcumin combination were higher than curcumin-
or OPCs-only treated cells.
Xenograft animal experiments
Seven week-old male athymic nude mice (Envigo, Houston, TX) were housed
under controlled conditions of light and fed ad libitum. Approximately
1 × 10^6 HCT116 cells were suspended in Matrigel matrix (BD
Biosciences) and subcutaneously injected into mice using 27-gauge
needle (n = 10 per group). Mice were randomly assigned to different
treatment groups and orally gavaged with vehicle (glycerol:water, 1:1)
or OPCs or curcumin (100 mg/kg body weight dissolved in vehicle) or
OPCs-curcumin combination (100 mg/kg each and 50 mg/kg each) on
alternative days for 15 days. Tumor size was measured each day by
calipers. Tumor volume was calculated using the following formula:
1/2(length × width × width). The investigator was not blinded to the
group allocation during the experiment and/or when assessing the
outcome. The animal protocol was approved by the Institutional Animal
Care and Use Committee, Baylor Scott & White Research Institute,
Dallas, Texas and all experiments were conducted strictly in accordance
to the National Institute of Health Guide for the Care and Use of
Laboratory Animals (8th Edition Institute for Laboratory Animal
Research).
Calculation of cooperativity between agents
The following two methods described in the review article by Foucquier
and Guedj^[127]63 were used to calculate cooperativity between curcumin
and OPCs in cell proliferation assays:
1. ‘Highest single agent’ (HSA) is the higher of the effects produced
by each component in the combination at the same concentration as
in the combination^[128]64. The agents are considered cooperative
if the combined effect is in excess over the HSA. The Combination
Index (CI) was calculated by dividing the maximum of the effect
caused by the individual agents curcumin (E[cur]) or OPCs (E[OPCs])
by the effect by the combination of the agents (E[cur+OPCs]), i.e.,
[MATH: CI=[max(Ecur,EOPCs)]/Ecur+OPCs :MATH]
2. ‘Bliss Independence’ model predicts the combined response for the
individual agents. The agents are considered cooperative if the
empirical effect of the combination is higher than the predicted
effect^[129]65,[130]66. To calculate the CI by this method, the
product of the effects caused by the individual agents is
subtracted from their sum, which is then divided by the effect
caused by the combination of the two agents, i.e.,
[MATH: CI=(Ecur+EOPCs−Ecur⋅EOPCs)/Ecur+OPCs :MATH]
Two drugs with a CI value of <1 were considered to be cooperative.
Calculation of dose reduction index (DRI)
DRI[50] is a measure of the extent to which the concentration of OPCs
or curcumin can be reduced at 50% inhibition of cell proliferation
compared with the concentration of OPCs or curcumin
individually^[131]67.
DRI[50] for OPCs = IC50 for OPCs/Concentration of OPCs in combination
for 50% inhibition.
DRI[50] for curcumin = IC50 for curcumin/Concentration of curcumin in
combination for 50% inhibition
In general, DRI > 1 is considered beneficial.
Statistical analysis
All experiments were repeated three times. All data are expressed as
mean ± SD with statistical significance indicated when P < 0.05.
Statistical comparisons between control and treatment groups were
determined using paired t-test.
Electronic supplementary material
[132]Supplementary Information^ (149.4KB, pdf)
Acknowledgements