Biochemical and Biophysical Research Communications xxx (xxxx) xxx

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Biochemical and Biophysical Research Communications

journal homepage: www.elsevier.com/locate/ybbrc

Comparative pharmacoproteomics reveals potential targets for

berberine, a promising therapy for colorectal cancer
Mingfu Tong a, 1, Haiming Liu b, 1, Jianyu Hao a, **, Daiming Fan a, c, *
a
Department of Gastroenterology, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, 100020, China
b
College of Computer Science and Technology, Jilin University, Changchun, 130012, Jilin, China
c
State Key Laboratory of Cancer Biology, National Clinical Research Center for Digestive Diseases and Xijing Hospital of Digestive Diseases, Air Force
Military Medical University, Xi’an, 710032, China

a r t i c l e i n f o a b s t r a c t

Article history: Berberine (BBR), a natural isoquinoline alkaloid, has been shown to be a promising therapeutic agent for
Received 31 January 2020 colorectal cancer (CRC), but the molecular mechanism remains unclear. Here, we used mass
Accepted 7 February 2020 spectrometry-based label-free proteomics to explore the potential targets of BBR in CRC cells. Compre-
Available online xxx
hensive proteomic profiles demonstrated that of 8051 identified proteins, 503 and 277 differentially
expressed proteins (DEPs) were screened out of CACO2 and LOVO cells, respectively. 83 DEPs were
Keywords:
overlapped and most of these were down-regulated. A pathway enrichment analysis pinpointed mito-
Colorectal cancer
chondrial translation, respiratory electron transport and the citric acid (TCA) cycle as biological effectors.
Berberine
Label-free
The data of proteomics was subsequently confirmed by citrate synthase (CS), Tu translation elongation
Proteomics factor (TUFM), pentatricopeptide repeat domain 3 (PTCD3) and mitochondrial ribosomal protein L48
Mechanism (MRPL 48) protein measurement. CS protein expression in CRC cells and tissues was higher than it was in
normal specimens. Additionally, forcible downregulation of CS led to remarkable cell proliferation in-
hibition. Taken together, we concluded that the anticancer effects of BBR are attributable to mitochon-
drial protein synthesis, TCA and respiratory electron transport inhibition and that CS might be a useful
therapeutic target in CRC treatment.
© 2020 Elsevier Inc. All rights reserved.

1. Introduction þ oxaliplatin þ leucovorin) or FOLFIRI (5Fu þ irinotecan þ leucovorin)

Human colorectal cancer (CRC) is the third most common ma-
lignant disease and causes 900,000 deaths annually worldwide [1].
Although national screening programs and the prevalence of co-
lonoscopy testing have been introduced in more than 50 countries,
the incidence and mortality are still increasing [2].
Cytotoxic agents including fluoropyrimidines, irinotecan, capeci-
tabine, leucovorin and oxaliplatin constitute the main chemothera-
peutic treatment of CRC patients. Although the choice of
chemotherapeutic approaches to the management of mCRC patients
is generalized, the best treatment regimen should be evaluated for
each patient. The most widely used cytotoxic regime FOLFOX (5Fu
* Corresponding author.; Department of Gastroenterology, Beijing Chao-Yang
Hospital, Capital Medical University, Beijing, 100020, China.
** Corresponding author.
E-mail addresses: haojianyu@ccmu.edu.cn (J. Hao), daimingfan@fmmu.edu.cn
(D. Fan).
1
These authors contributed equally to this work.
exhibits no significant differences between either regimen, while
FOLFOXIRI (5Fu þ oxaliplatin þ irinotecan þ leucovorin) achieves
better overall survival (OS) in patients who can tolerate such an
aggressive regimen [3,4]. 5-FU or capecitabine, which has been
shown to be inferior to FOLFOX and FOLFIRI, remains a choice for
patients who can’t tolerate irinotecan or oxaliplatin [5]. Given the
current complexity of chemotherapy options, the issue of the best
treatment sequence remains a challenge for mCRC patients.
The addition of recent targeted therapies to these cytotoxic
regimens has led to better outcomes, but the OS was still less than
36 months [6]. Since the mid-1990s, a total of six targeted agents
(bevacizumab, cetuximab, panitumumab, aflibercept, regorafenib,
and ramucirumab) has been approved for the treatment of mCRC
patients, and debate still revolves around the best way to sequence
these agents [7]. Drug resistance is another obstacle, resulting in
disease progression in virtually every patient, which limits the
application of targeted therapy. An emerging debate occurred that
patients with KRAS G13D mutation could benefit from EGFR
blocking [8e10]. Therefore, the status quo of targeted therapy for

https://doi.org/10.1016/j.bbrc.2020.02.052
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Please cite this article as: M. Tong et al., Comparative pharmacoproteomics reveals potential targets for berberine, a promising therapy for
colorectal cancer, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.052
2 M. Tong et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
CRC is full of intricacies and unpredictability.
Novel and more effective approaches are urgently needed for
mCRC treatment. Phytochemicals rooted in food and vegetables
constitute another important drug resource that has exhibited
inspiring therapeutic efficiency for CRC [11]. Berberine (BBR), a
principal component in Coptis chinensis Franch and Phellodendron
chinense C. K. Schneid, has been investigated as a promising ther-
apeutic agent for CRC, but the cellular targets underlying are
illegible [12e14]. Pharmacoproteomics is a useful application to
explore biological effectors of drugs by comparing the proteomic
profiles of treated and untreated samples [15]. In the present study,
we used a label-free pharmacoproteomics approach to characterize
the molecular targets and signatures affected by BBR in CRC cells.
2. Methods and materials
2.1. Reagents
BBR was purchased from Sigma (Laramie, USA), dissolved in
dimethyl sulfoxide (DMSO), stored in aliquots at 80  C. All other
reagents used were of analytical grade.
2.2. Cell lines and culture conditions
The human CACO2 and LOVO CRC cell lines were purchased
from the American Type Culture Collection (ATCC). Other cell lines
were obtained from the Cell Resource Center of the Chinese
Academy of Sciences (Shanghai, China). All cell lines were cultured
in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supple-
mented with 10% fetal bovine serum (FBS, BI), 100 U/ml penicillin,
and 100 mg/ml streptomycin and incubated in 5% CO2 at 37  C. The
CRC cells were plated in the medium with one-half the usual
amount of serum (5%) when they were treated with BBR [16].
2.3. Cell viability and colony formation assays
Cell viability was assessed with a cell counting kit (CCK; ZETA
Life, USA) according to the manufacturer’s instructions. For colony
formation, 2  103 cells were seeded in 6-well plates and allowed to
grow for 10 days in an incubator. Colonies were stained with a 1%
crystal violet solution and counted according to a defined colony
size (>50 cells).
2.4. Immunoblot assay
Protein (15e20 mg) was loaded and separated on SDS poly-
acrylamide gels and then transferred to PVDF membranes. The
membranes were blocked with 5% fat-free milk in TBST buffer and
incubated with the appropriate primary antibodies. The following
primary antibodies in the study were used: anti-CS (Abcam
#ab129095), anti-TUFM (Abcam #ab173300), anti-PTCD3 (St John’s
Labs #STJ110795), anti-MRPL 48 (Abcam #ab194826), anti-cleaved-
PARP (CST #5625), anti-caspase 3 (CST #9665), anti-cleaved-
caspase 3 (CST #9664), and anti-cyclin D1 (CST #55506T). The
corresponding secondary antibodies against the primary antibody
were used.
2.5. Label-free analysis
A label-free analysis was performed using an ultimate 3000
nano-HPLC system coupled with a QExactive mass spectrometer
(Thermo Scientific, USA). A mixture of 500 ng digested peptide was
preconcentrated and loaded onto a self-made C18 trap column
(3 mm, 0.1  20 mm). Then, the sample was analyzed by a self-made
capillary C18 column (1.9 mm, 0.15  120 mm) operating at a flow
Please cite this article as: M. Tong et al., Comparative pharmacoproteomics reveals potential targets for berberine, a promising therapy for
colorectal cancer, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.052
rate of 600 nL/min, for which the eluents were dissolved in solution
A (0.1% formic acid) and solution B (0.08% formic acid). The chro-
matographic conditions of the gradient elution with solution B
increased from 6% to 15% in 25 min, from 15% to 40% in 5 min, an
increase to 95% in 2 min, where it was maintained 6 min at 95%. The
solution B gradient decreased sharply from 95% to 6% in 1 min, then
maintained at 6% for 5 min. A Q- Exactive HF MS/MS system
(Thermo Scientific, USA) was used to identify the separated peptide
fragments. Full-scan MS spectra were acquired at a resolution of
120,000 with an automatic gain control (AGC) target value of
3  106, and the mass range set to 300e1400 m/z. The 20 most
abundant signal for the ions per cycle in the MS scan were selected
using higher energy collision-induced dissociation (HCD) frag-
mentation at a resolution of 15,000 at m/z 200 with an AGC target
value of 5  104 and a maximum injection time of 42 ms. HCD
spectra were obtained using normalized collision energy of 32%.
2.6. Protein identification and quantification
The raw data from label-free LC-MS/MS were processed by
Mascot 2.2 and Proteome discovery software 2.0. Matching
searches were performed against the UniPort database with the
following sets: a peptide mass tolerance of 15 ppm and fragment
mass tolerance of 20 mmu. High peptide confidence was used to
screen the data, and fully tryptic peptides with 2 missed were
permitted. The peptide length was set as >6, and the false discovery
rate (FDR) was set as 0.01. Protein quantification was conducted
based on the chromatographic peak intensity of the reported ions
of the only unique peptides in the MS/MS spectra.
2.7. Bioinformatics
Gene Ontology (GO) terms and Reactome pathways were
analyzed with DAVID (http://dabid.abcc.ncifcrf.gov/, ver, 6.8).
Protein-protein interaction (PPI) networks were analyzed with the
STRING (http://string-db.org/, ver.11.0).
2.8. Immunohistochemistry (IHC)
Tissue sections were deparaffinized, subjected to antigen
retrieval and endogenous peroxidase inactivation and incubated
with primary antibodies against CS (Abcam #ab129095). Then the
sections were incubated with a peroxidase-conjugated secondary
antibody (Santa Cruz), and visualized with diaminobenzidine.
2.9. Quantitative real-time polymerase chain reaction (RT-PCR)
Total RNA was extracted using a MiniBEST universal RNA
extraction kit (TaKaRa, Japan) according to the manufacturer’s in-
structions. cDNA was synthesized using a PrimeScript RT reagent
kit (Takara, Japan). SYBR Premix Ex TaqII (TaKaRa, Japan) was used
to amplify the cDNA of interest. The primers for CS and GAPDH
were as follows: CS, forward 5’ -AGAGAAGGCAGCGGTATTGG-30 and
reverse 50 -CTCATGGTCACTGTGGATGGT-3’; GAPDH, forward 50 -
GCACCGTCAAGGCTGAGAAC-30 and reverse 50 -TGGTGAA-
GACGCCAGTGGA-3’.
2.10. Lentiviral infection and gene silencing
The additional lentivirus-delivered SiRNAs against CS (Si-CS)
and negative control (Si-NS) were purchased from Gene Chem
(Shanghai, China). The targeting sequences of two Si-CS were fol-
lows: Si-CS#1, 50 - GCAGCAAAGATCTACCGAAAT-30 and Si-CS#2, 50 -
CTGGCAAATCAGGAAGTGCTT-3’. For transfection, LOVO cells were
first seeded in 6-well plates and infected with lentivirus according
 M. Tong et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx 3

to the manufacturer’s manual.
2.11. Statistical analysis
In this study, the data are presented as the mean (M) ± standard
deviation (SD) values. Student’s t-tests (two tailed) were used for
comparisons between groups. The significant differences between
proteins were identified by the Duncan’s multiple range test at the
5% level. For all analyses, a value of p < 0.05 was considered sta-
tistically significant.
3. Results
3.1. Anticancer effects of BBR in two CRC cell lines
To confirm the inhibitory effect of BBR on cell proliferation,
CACO2 and LOVO cells were treated with increasing concentrations
of BBR for 48 h and cell viability was quantified using a CCK assay.
BBR led to a dose-dependent inhibition with IC50 value of 39.87
and 23.27 mM for CACO2 and LOVO cells, respectively (Fig. 1A and
B). The colony formation assay validated the cell proliferation in-
hibition effect of BBR (Fig. 1C and D).
As the reduction in cell viability may be the explained by either
cell death or cell cycle inhibition, we explored the effects of BBR on
the known effectors of these pathways. BBR significantly induced
cleaved-PARP and caspase 3 activation, but had no reduction in
cyclin D1 expression (Fig. 1E, F, G).
3.2. Proteomic profiles of CRC cells treated with BBR
To identify the differentially expressed proteins (DEPs) in the
BBR treated cells, we used label-free quantitative shotgun prote-
omics technology (Fig. S1). To avoid biological and technical devi-
ation, three independent replicates were used for the LC-MS/MS
analysis. The accuracy of the data was verified by analyzing the
intensity values of the proteins from the treated and untreated
CACO2 and LOVO cells (r ¼ 0.984, 0.983 and 0.977, 0.975), respec-
tively (Fig. S2). The Pearson R values were consistent, and the
values of the replicates in the repeated experiments were
remarkably higher than the values across treatments (Fig. S2).
In the CACO2 and LOVO cells, 8051 proteins were identified
(Fig. 2A, Table S1), 503 and 277 of which were significantly altered,
respectively, based on the threshold FC  3 and p < 0.05 (Fig. 2B and
C, Table S2). Among these DEPs, 223 and 79 upregulated (red font)
and 280 and 198 downregulated (green font) were screened out of
the CACO2 and LOVO cells, respectively (Fig. 2B and C). Eighty-three
DEPs overlapped and most of them were down-regulated. (Fig. 2D).
However, of the 1143 and 588 DEPs were detected in the CACO2 and
LOVO cells, respectively, 493 and 180 were increased, 650 and 408
were decreased, and 203 overlapped when the thresholds were set

Fig. 1. Anticancer effect of BBR in the CRC cells. (A) CACO2 and (B) LOVO cells proliferation inhibited by berberine over 48 h. (C) Colony formation assay of CRC cells treated with
vehicle (DMSO), berberine (30 mM). (D) Quantification of the colony numbers shown in the histogram of (C). (E) Cleaved-PARP and cleaved-caspase 3 tested in CRC cells after
treatment with berberine over 48 h. (F), (H) Quantification of the western blot image of (E). ns: no significance, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Please cite this article as: M. Tong et al., Comparative pharmacoproteomics reveals potential targets for berberine, a promising therapy for
colorectal cancer, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.052
4 M. Tong et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx

Fig. 2. Proteomic profiles of CRC cells treated with BBR. (A) All proteins identified in both two CRC cells. (B), (C) Volcano plots based on the FC  3, p < 0.05. The X-axis is log2
(FC), Y-axis is -log10 (P value), the further that this value is from zero, the more significant the difference is. The upregulated proteins are on the right (red dots), the downregulated
proteins are on the left (green dots) and the unchanged proteins are presented as black dots. (D) Venn charts of the two CRC cells. (For interpretation of the references to color in this
figure legend, the reader is referred to the Web version of this article.)

at FC  2 and p < 0.05 (Fig. S3). On the basis of these comparisons, oxidoreductase subunits, which are the core subunits of complex I.
we chose the screening threshold of FC  3 and p < 0.05 to easily
pinpoint the targeted proteins in the subsequent analysis. 3.4. Mitochondrial activity is a key target of BBR in CRC cells

3.3. Functional annotation of the DEPs in both CRC cell lines
The functional annotation and classification of the DEPs were
conducted to identify the pathways regulated by BBR. The top three
GO terms from cellular component (CC) were organelle, cytoplasm,
and nucleus (Table S3). Mitochondria accounted for 7.25% and
12.10% of the total components, and 29.4% (7.25/24.64) and 44.9%
(12.10/26.93) of the organelles in the CACO2 and LOVO cells,
respectively (Table S3). For overlapping DEPs, mitochondria
accounted for 16.78%, which was equivalent to 64.1% of all organelle
(Fig. 3A). The top four GO terms from biology process (BP) were
“mitochondrial gene translation”, “cellular component disas-
sembly”, “amide biosynthetic process”, and “peptide metabolic
process” (Fig. 3B). The “structural constituent of ribosome”, “NADH
(nicotinamide adenine dinucleotide) dehydrogenase (ubiquinone)
activity”, “rRNA binding” and “electron transfer activity” were the
top four GO terms of enrichment from molecular function (MF)
(Table S4). The most specific Reactome pathways enriched were
associated with mitochondrial translation, metabolism of proteins,
complex I biogenesis, the citric acid (TCA) cycle and respiratory
electron transport (Fig. 3C).
The PPI network of the overlapping DEPs, shown in Fig. 3D,
describes the functional cluster of each DEP in detail. The proteins
enriched in mitochondrial translation cluster mainly belong to
mitoribosomal proteins (MRPs). CS is the only protein enriched in
the TCA cycle cluster. Of the 8 proteins enriched in the respiratory
electron transport pathway, 7 proteins are NADH: ubiquinone
Of the 83 overlapping DEPs, 62 and 66 proteins were down-
regulated in the CACO2 and LOVO cells, respectively, and the
upregulated proteins were rarely reported or had poorly under-
stood functions in the cell activities (Figs. 4A and 3D). Therefore, to
validate the raw proteomic data, we chose four downregulated
proteins that extensively interact with other proteins or play
important roles in mitochondrial activities. As shown in Fig. 4B, the
expression of CS, TUFM, PTCD3 and MRPL 48 was decreased by BBR
in a dose-dependent manner (Fig. 4B). Additionally, the same
inhibitory effect was induced in other CRC cell lines (Fig. 4C).
To further determine that these mitochondrial targets regulated
by BBR play important roles in cancer development, we investi-
gated CS protein levels in the CRC cell lines and tissue versus
normal samples. A higher level of CS expression was showed in the
carcinoma samples than was found in the normal samples (Fig. 4D,
E, F, G). As anticipated, forcible downregulation of CS inhibited CRC
cell proliferation (Fig. 4H, I, J, K).
4. Discussion
BBR has been shown to have anticancer effects on CRC through
various proposed mechanisms [12e14,17]. Although these mecha-
nisms were proposed, the integral molecules and pathways remain
poorly characterized. To the best of our knowledge, we demon-
strate, for the first time, the integral molecules and pathways tar-
geted by BBR through label-free proteomics.
The proteomic analysis revealed a panel of altered

Please cite this article as: M. Tong et al., Comparative pharmacoproteomics reveals potential targets for berberine, a promising therapy for
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 M. Tong et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx 5

Fig. 3. Functional annotation of the overlapping DEPs. (A) GO terms from CC with FDR<0.05. (B) The top 15 GO terms from BP with FDR<0.05. (C) The most specific (nonre-
dundant) Reactome pathways with FDR<0.05. In (B) and (C) plots, the terms are listed according to their statistical significance FDR in log scale. The colors of each bar show the
percentage of the annotations by every term in the set of DEPs. (D) The functional details of each DEP in the PPI network.

mitochondrial proteins, a finding consistent with that of previously
reports of alterations to mitochondrial dynamics upon cancer
development [18,19]. Mitochondria, the power houses of cell, are at
the crossroad of many cellular pathways, that regulate various
bioprocesses, including the quality of mitochondria, cell meta-
bolism, cell survival and cell migration. In recent years, changes in
these processes were frequently encountered in cancer initiation
and progression, like mitochondrial DNA (mtDNA) mutations, de-
letions and/or amplification [20]. Tumor regulators including
hypoxia-inducible factor 1a(HIF-1a), c-myc, tumor suppressor p53
and PCG-1a have also been reported to mediate mitochondrial
metabolism [21e23].
In particular, the DEPs detected in both cells were collectively
associated with mitochondrial translation, citric acid (TCA) cycle
and respiratory electron transport. MRPs, an assembled part of
mitochondrial ribosomes that function in mitochondrial trans-
lation, have been found to regulate cell survival functions in tumors
[24,25]. One integrated analysis showed that more than 95 gene
transcripts associated with mitochondrial biogenesis were signifi-
cantly elevated in breast cancer and nearly 40 were MRPs, including
MRPL 48 [26]. PTCD3, also known as MRPL-S39, was overexpressed
in breast cancer, prostate cancer and lymphoma and serves as an
oncogene [27e29]. TUFM, a key factor in mitochondrial protein
translation, was significantly increased and acts as a prognostic
indicator in cancer development and/or drug resistance [30e33].
The well-established metabolic perturbation in cancer is the
Warburg effect, an alteration of normal glucose metabolism in which
cancer cells use carbon source to synthesize amino acids, nucleic
acids and lipids instead of completely oxidizing them into carbon
dioxide [34]. Citrate synthase (CS), the first key rate-limiting enzyme
in the tricarboxylic acid (TCA) cycle, catalyzes the condensation re-
action of oxaloacetate with acetyl-CoA to generate citrate. Citrate in
cancer cells is then exported to the cytosol and serves as a building-
block of lipid synthesis [35e37]. Consistent with these findings, CS
was overexpressed in CRC, and downregulation of CS inhibited cell
proliferation in our study. Carbon sources experiencing the TCA cycle
produce NADH and FADH2, which transfer electrons to the electron
transport chain called oxidative phosphorylation (OXPHOS). It is now
clear that OXPHOS, that is, mitochondrial ATP generation coupled
with oxygen consumption, accelerates cancer progression in many
cancer types [38,39]. Cancer cells often exhibit indispensably upre-
gulated OXPHOS, and emerging evidences shows that cancer cells
with severe OXPHOS deficiency grow slowly unless they reconstitute
OXPHOS by mitochondrial reactivation [40,41]. The data shown in
our experiment identified that 7 subunits of altered NADH ubiqui-
none oxidoreductase belong to respiratory chain complex I. Indeed,
respiratory complexes are critical for cancer cell metabolism and
hypoxic adaptation and regarded as novel adjuvant therapeutic tar-
gets for cancer treatment [42]. Furthermore, cancer cells harboring
overexpressed mtDNA mutations in complex I subunits adapt more
readily to the altered tumor microenvironment and are more sen-
sitive to the complex I inhibitors than the cell lines lacking such
mutations [39].
Although scattered studies had suggested that BBR inhibits
cancer cells via activation of mitochondrially apoptotic pathway or
damaging mitochondria in CRC, the specific molecular targets were
unclear [12,14,43]. We present herein a proteomic analysis to sys-
tematically elucidate the specific targets of BBR in CRC cells. The

Please cite this article as: M. Tong et al., Comparative pharmacoproteomics reveals potential targets for berberine, a promising therapy for
colorectal cancer, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.052
6 M. Tong et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx

Fig. 4. Mitochondrial activity is a key target of BBR. (A) Heatmap of 83 overlapping DEPs. (B) Validation of the CS, TUFM, PTCD3 and MRPL48 protein alterations by using
immunoblot assay. (C) The alteration of CS, TUFM, PTCDS and MRPL48 induced by BBR in the SW480 and DLD-1 cells. (D) Comparison of CS protein expression between the CRC and
normal epithelial cells. (F) Comparison of CS protein expression between the CRC and adjacent normal tissues. (E), (G) Quantification of images (D), (F) respectively. (H) mRNA and
(I) protein level of CS in the LOVO cells downregulated by SiRNAs. (J), (K) LOVO cell proliferation inhibition resulted from Si-CSs measured by CCK and colony formation assay. *:
p < 0.05, **: p < 0.01, ***: p < 0.001.

results confirm that BBR impairs mitochondrial function de facto Acknowledgments

and further suggest that BBR precisely decrease cellular energy
supply and macromolecular synthesis by inhibiting mitochondrial
genome translation, OXPHOS activities and TCA cycle. Additionally,
we propose that complex I, but not the other complexes, was
inhibited by BBR in OXPHOS inhibition. Nevertheless, the limitation
of our present study is the lack of further experiments designed to
explore the specific signaling networks which will be addressed in
the next step of our research.
This work was supported by the Natural Science Foundation of
China (No. 81430072 to Daiming Fan), the Scientific Fund for
Innovative Research Groups of the National Natural Science Foun-
dation of China (No. 81421003 to Daiming Fan) and Fundamental
Research Funds for the National Center for Clinical Medicine of
Digestive Diseases (No. 2015BAI13B07 to Daiming Fan).

Declaration of competing interest

Appendix A. Supplementary data
The authors declare that we don’t have any financial or asso-
ciative interest that represents a conflict of interest corelated with Supplementary data to this article can be found online at
the work submitted. https://doi.org/10.1016/j.bbrc.2020.02.052.

Please cite this article as: M. Tong et al., Comparative pharmacoproteomics reveals potential targets for berberine, a promising therapy for
colorectal cancer, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.052
 M. Tong et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx 7

References [22] Z. Tan, X. Luo, L. Xiao, The role of PGC1alpha in cancer metabolism and its
therapeutic implications, Mol. Canc. Therapeut. 15 (2016) 774e782.
[23] F. Li, Y. Wang, K.I. Zeller, Myc stimulates nuclearly encoded mitochondrial
[1] E. Dekker, P.J. Tanis, J.L.A. Vleugels, et al., Colorectal cancer, Lancet 394 (2019)
genes and mitochondrial biogenesis, Mol. Cell Biol. 25 (2005) 6225e6234.
1467e1480.
[24] E. Cavdar Koc, A. Ranasinghe, W. Burkhart, A New Face on Apoptosis: Death-
[2] ArnoldM, SierraMS, LaversanneM, et al., Global patterns and trends in colo-
Associated Protein 3 and PDCD9 are Mitochondrial Ribosomal Proteins, vol.
rectal cancer incidence and mortality, Gut 66 (2017) 683e691.
492, 2001, pp. 166e170.
[3] A. Falcone, S. Ricci, I. Brunetti, et al., Gruppo Oncologico Nord Ovest. Phase III
[25] Y.A. Yoo, M.J. Kim, J.K. Park, et al., Mitochondrial ribosomal protein L41 sup-
trial of infusional fluorouracil, leucovorin, oxaliplatin, and irinotecan (FOL-
presses cell growth in association with p53 and p27Kip1, Mol. Cell Biol. 25
FOXIRI) compared with infusional fluorouracil, leucovorin, and irinotecan
(2005) 6603e6616.
(FOLFIRI) as first-line treatment for metastatic colorectal cancer, J. Clin. Oncol.
[26] F. Sotgia, D. Whitaker-Menezes, U.E. Martinez-Outschoorn, et al., Mitochon-
25 (2007) 1670e1676.
dria "fuel" breast cancer metabolism: fifteen markers of mitochondrial
[4] G. Masi, E. Vasile, F. Loupakis, et al., Randomized trial of two induction
biogenesis label epithelial cancer cells, but are excluded from adjacent stro-
chemotherapy regimens in metastatic colorectal cancer: an updated analysis,
mal cells, Cell Cycle 11 (2012) 4390e4401.
J. Natl. Cancer Inst. 103 (2011) 21e30.
[27] S. Madhavan, Y. Gusev, S. Singh, et al., ERRgamma target genes are poor
[5] S. Temraz, D. Mukherji, A. Shamseddine, Sequencing of treatment in meta-
prognostic factors in Tamoxifen-treated breast cancer, J. Exp. Clin. Canc. Res.
static colorectal cancer where to fit the target, World J. Gastroenterol. 20
34 (2015) 45.
(2014) 1993e2004.
[28] A. D’Andrea, I. Gritti, P. Nicoli, The mitochondrial translation machinery as a
[6] I. Catalano, L. Trusolino, The stromal and immune landscape of colorectal
therapeutic target in Myc-driven lymphomas, Oncotarget 7 (2016)
cancer progression during anti-EGFR therapy, Canc. Cell 36 (2019) 1e3.
72415e72430.
[7] K.K. Ciombor, T. Bekaii-Saab, A comprehensive review of sequencing and
[29] Y. Huang, G. Jiang, X. Liang, et al., Elevated expression of PTCD3 correlates
combination strategies of targeted agents in metastatic colorectal cancer,
with tumor progression and predicts poor prognosis in patients with prostate
Oncol. 23 (2018) 25e34.
cancer, Mol. Med. Rep. 18 (2018) 3914e3922.
[8] T. McFall, J.K. Diedrich, M. Mengistu, et al., A systems mechanism for KRAS
[30] H. Shi, M. Hayes, C. Kirana, et al., TUFM is a potential new prognostic indicator
mutant allele-specific responses to targeted therapy, Sci. Signal. 12 (2019),
for colorectal carcinoma, Pathology 44 (2012) 506e512.
eaaw8288.
[31] X. Xu, A. Huang, X. Cui, et al., Ubiquitin specific peptidase 5 regulates colo-
[9] M. Nakamura, T. Aoyama, K. Ishibashi, et al., Randomized phase II study of
rectal cancer cell growth by stabilizing Tu translation elongation factor,
cetuximab versus irinotecan and cetuximab in patients with chemo-refractory
Theranostics 9 (2019) 4208e4220.
KRAS codon G13D metastatic colorectal cancer (G13D-study), Canc. Chemo-
[32] Y. Lei, B.A. Kansy, J. Li, et al., EGFR-targeted mAb therapy modulates auto-
ther. Pharmacol. 79 (2017) 29e36.
phagy in head and neck squamous cell carcinoma through NLRX1-TUFM
[10] S. Tejpar, I. Celik, M. Schlichting, et al., Association of KRAS G13D tumor
protein complex, Oncogene 35 (2016) 4698e4707.
mutations with outcome in patients with metastatic colorectal cancer treated
[33] H.Q. Xi, K.C. Zhang, J.Y. Li, et al., Expression and clinicopathologic significance
with first-line chemotherapy with or without cetuximab, J. Clin. Oncol. 30
of TUFM and p53 for the normal-adenoma-carcinoma sequence in colorectal
(2012) 3570e3577.
epithelia, World J. Surg. Oncol. 15 (2017) 90.
[11] Y.H. Li, Y.B. Niu, Y. Sun, et al., Role of phytochemicals in colorectal cancer
[34] W.H. Koppenol, P.L. Bounds, C.V. Dang, Otto Warburg’s contributions to cur-
prevention, World J. Gastroenterol. 21 (2015) 9262e9272.
rent concepts of cancer metabolism, Nat. Rev. Canc. 11 (2011) 325e337.
[12] H. Ruan, Y.Y. Zhan, J. Hou, et al., Berberine binds RXRalpha to suppress beta-
[35] M. Peng, D. Yang, Y. Hou, Intracellular citrate accumulation by oxidized ATM-
catenin signaling in colon cancer cells, Oncogene 36 (2017) 6906e6918.
mediated metabolism reprogramming via PFKP and CS enhances hypoxic
[13] W.H. Hsu, Y.S. Hsieh, H.C. Kuo, Berberine induces apoptosis in SW620 human
breast cancer cell invasion and metastasis, Cell Death Dis. 10 (2019) 228.
colonic carcinoma cells through generation of reactive oxygen species and
[36] B. Schlichtholz, J. Turyn, E. Goyke, et al., Enhanced citrate synthase activity in
activation of JNK/p38 MAPK and FasL, Arch. Toxicol. 81 (2007) 719e728.
human pancreatic cancer, Pancreas 30 (2005) 99e104.
[14] K.N. Chidambara Murthy, G.K. Jayaprakasha, et al., The natural alkaloid
[37] L. Chen, T. Liu, J. Zhou, Citrate synthase expression affects tumor phenotype
berberine targets multiple pathways to induce cell death in cultured human
and drug resistance in human ovarian carcinoma, PloS One 9 (2014), e115708.
colon cancer cells, Eur. J. Pharmacol. 688 (2012) 14e21.
[38] P.E. Porporato, N. Filigheddu, J.M.B. Pedro, et al., Mitochondrial metabolism
[15] A. D’Alessandro, L. Zolla, Pharmacoproteomics: a chess game on a protein
and cancer, Cell Res. 28 (2018) 265e280.
field, Drug Discov. Today 15 (2010) 1015e1023.
[39] T.M. Ashton, W.G. McKenna, L.A. Kunz-Schughart, et al., Oxidative phos-
[16] S. Misale, I. Bozic, J. Tong, et al., Vertical suppression of the EGFR pathway
phorylation as an emerging target in cancer therapy, Clin. Canc. Res. 24 (2018)
prevents onset of resistance in colorectal cancers, Nat. Commun. 6 (2015)
2482e2490.
8305.
[40] M. Bajzikova, J. Kovarova, A.R. Coelho, et al., Reactivation of dihydroorotate
[17] N. Wang, H.Y. Tan, L. Li, et al., Berberine and Coptidis Rhizoma as potential
dehydrogenase-driven pyrimidine biosynthesis restores tumor growth of
anticancer agents: recent updates and future perspectives, J. Ethnopharmacol.
respiration-deficient cancer cells, Cell Metabol. 29 (2019) 399e416.
176 (2015) 35e48.
[41] A.S. Tan, J.W. Baty, L.F. Dong, Mitochondrial genome acquisition restores
[18] S. Vyas, E. Zaganjor, M.C. Haigis, et al., Mitochondria and cancer, Cell 166
respiratory function and tumorigenic potential of cancer cells without mito-
(2016) 555e566.
chondrial DNA, Cell Metabol. 21 (2015) 81e94.
[19] E.C. Koc, E. Haciosmanoglu, P.P. Claudio, Impaired mitochondrial protein
[42] R. Vatrinet, L. Iommarini, I. Kurelac, et al., Targeting respiratory complex I to
synthesis in head and neck squamous cell carcinoma, Mitochondrion 24
prevent the Warburg effect, Int. J. Biochem. Cell Biol. 63 (2015) 41e45.
(2015) 113e121.
[43] C.J. Thirupurasundari, R. Padmini, S.N. Devaraj, Effect of berberine on the
[20] Y. Yang, S. Karakhanova, W. Hartwig, et al., Mitochondria and mitochondrial
antioxidant status, ultrastructural modifications and protein bound carbohy-
ROS in cancer: novel targets for anticancer therapy, J. Cell. Physiol. 231 (2016)
drates in azoxymethane-induced colon cancer in rats, Chem. Biol. Interact.
2570e2581.
177 (2009) 190e195.
[21] C.C. Hsu, L.M. Tseng, H.C. Lee, Role of mitochondrial dysfunction in cancer
progression, Exp. Biol. Med. 241 (2016) 1281e1295.

Please cite this article as: M. Tong et al., Comparative pharmacoproteomics reveals potential targets for berberine, a promising therapy for
colorectal cancer, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.052