Abstract

   Gemcitabine (Gem)-based chemotherapy is one of the first-line
   treatments for pancreatic ductal adenocarcinoma (PDAC). However, its
   clinical effect is limited due to development of chemoresistance.
   Various naturally occurring compounds, including Berberine (BBR),
   provide an anti-cancer efficacy with time-tested safety, individually
   and in combination with chemotherapeutic drugs. Accordingly, we
   hypothesized that BBR might enhance the chemosensitivity to Gem in
   PDAC. In this study, cell culture studies using MIA PaCa-2 and BxPC-3
   cells, followed by analysis in patient-derived organoids were performed
   to evaluate the anti-cancer effects of BBR in PDAC. Considering that
   cancer is a significant manifestation of increased chronic inflammatory
   stress, systems biology approaches are prudent for the identification
   of molecular pathways and networks responsible for
   phytochemical-induced anti-cancer activity, we used these approaches
   for BBR-mediated chemosensitization to Gem. Firstly, Gem-resistant
   (Gem-R) PDAC cells were established, and the combination of BBR and Gem
   revealed superior anti-cancer efficacy in Gem-R cells. Furthermore, the
   combination treatment induced cell cycle arrest and apoptosis in Gem-R
   PDAC cells. Transcriptomic profiling investigated the Rap1 and PI3K-Akt
   signaling pathway as a key regulator of Gem-resistance and was a key
   mediator for BBR-mediated chemosensitization in PDAC cells. All cell
   culture-based findings were successfully validated in patient-derived
   organoids. In conclusion, we demonstrate that BBR-mediated reversal of
   chemoresistance to Gem manifests through Rap1/PI3K-Akt signaling in
   PDAC.

   Keywords: pancreatic ductal adenocarcinoma, Berberine, Gemcitabine,
   chemoresistance, Rap1/PI3K-Akt signaling pathway

1. Introduction

   Pancreatic cancer is one of the lethal malignancies and currently ranks
   as the seventh leading cause of cancer deaths worldwide, with 495,733
   new cases and 466,003 deaths reported in 2021 worldwide [[38]1]. The
   incidence and death rates for pancreatic cancer have also risen in the
   United States (US), with 60,430 new cases and 48,220 deaths in 2021
   [[39]2]. Although recent progresses in newer treatment regimens have
   somewhat improved the prognosis of patients with pancreatic cancer, the
   overall prognosis remains quite dismal, with 5-year overall survival
   (OS) rates as low as 8–10% [[40]2,[41]3,[42]4]. Furthermore, pancreatic
   cancer is expected to become the second leading cause of cancer death
   in the US by 2030, and this trend is believed to continue even into
   2040 when mortality is expected to continuously reduce for other
   cancers [[43]5,[44]6]. Since the initial randomized clinical trial in
   1997, Gemcitabine (Gem) has been the key treatment regimen for patients
   with pancreatic ductal carcinoma (PDAC); however, the median OS with
   this single-arm treatment ranges from only ~6–7 months
   [[45]7,[46]8,[47]9,[48]10].

   Accumulating evidence suggests that single agent treatments in such a
   fatal malignancy have been inadequate. Hence, a treatment regimen
   consisting of multiple drugs is likely going to be more effective as it
   will impact multiple pathways, will improve overall therapeutic
   efficacy, and is unlikely to result in the development of
   chemoresistance—the Achilles heel for most cancer drugs
   [[49]11,[50]12,[51]13]. Accordingly, in order to improve the prognosis
   of patients with PDAC, several clinical trials have attempted to
   examine the efficacy of Gem-containing doublet regimens by using
   chemotherapeutic or targeted agents; however, most of these trials have
   yielded largely negative results [[52]14,[53]15,[54]16,[55]17]. The
   nab-paclitaxel doublet treatment has improved the OS vs. single-Gem
   regimen [[56]10], as well as more recently, the triplet combination of
   5-FU, irinotecan, and oxaliplatin (FOLFIRINOX) has shown improved
   survival outcomes in patients with PDAC [[57]9]. However, the
   therapeutic benefits of these combination treatments are often
   compromised by the simultaneous drug toxicity, as well as added
   expense, which often limits their overall clinical efficacy
   [[58]9,[59]10,[60]18,[61]19,[62]20,[63]21,[64]21,[65]22]. Therefore,
   developing an optimal combination of safe and cost-effective
   therapeutic modalities is a major clinical challenge for PDAC
   treatment, which can facilitate improved survival outcomes in patients
   suffering from a lethal malignancy such as PDAC.

   Since naturally occurring compounds provide an anti-cancer efficacy
   with time-tested safety, they are frequently used as an adjunctive
   supplementary therapy in various cancers, including PDAC
   [[66]23,[67]24,[68]25,[69]26,[70]27,[71]28,[72]29,[73]30,[74]31,[75]32,
   [76]33]. Inflammation and stress affect cancer progression, and these
   compounds are also considered to be effective for inflammation and
   stress-induced disorders. During the last decade, numerous preclinical
   research studies have revealed that a combination of natural compounds
   and conventional chemotherapy not only enhances the anti-cancer
   therapeutic efficacy but also reduces the toxicity in PDAC
   [[77]23,[78]34]. In particular, several clinical trials and
   laboratory-based research have demonstrated such anti-cancer efficacy
   of various natural compounds including Escin, Boswellic acids, and
   Andrographis, when used in combination with Gem
   [[79]35,[80]36,[81]37,[82]38]. Similarly, our recent research has also
   revealed that Curcumin enhanced the chemosensitivity of PDAC cells to
   Gem by inhibiting PRC2-PVT1-c-Myc axis [[83]39].

   Berberine (BBR) is a natural plant alkaloid, which has been used
   traditionally to treat various conditions including bacterial diarrhea
   among the Chinese and native Americans [[84]40,[85]41]. Interestingly,
   studies in the past decade in laboratory experiments have highlighted a
   plethora of additional medicinal activities of BBR including its
   anti-cancer properties in various malignancies, including PDAC—all of
   which are orchestrated through multiple molecular mechanisms and
   pathways, such as cell cycle arrest, induction of apoptosis,
   caspase-independent cell death, and alterations in citrate metabolism
   [[86]42,[87]43,[88]44,[89]45,[90]46,[91]47,[92]48]. Furthermore, more
   recent studies have also unveiled that BBR treatment also enhances
   chemosensitivity or reverses chemotherapeutic drug resistance in
   several cancers [[93]49,[94]50,[95]51,[96]52,[97]53]. In view of this
   evidence, we hypothesized that BBR might enhance chemosensitivity to
   Gem through regulation of multiple oncogenic cellular signaling
   pathways to exert its anti-cancer activity in PDAC.

   Herein, we performed systematic experiments using multiple
   Gem-resistant PDAC cell lines, followed by their study in
   patient-derived tumor organoids to evaluate the ability of BBR to
   enhance the chemosensitivity of Gem in PDAC. Furthermore, using systems
   biology approaches for cell line-based transcriptomic profiling
   datasets, we identified potential molecular mechanism(s) and key growth
   regulatory pathways that enhanced BBR’s anti-tumorigenic effects in
   attenuating the Gem-resistance in PDAC.

2. Results

2.1. Berberine Enhances the Chemosensitivity to Gemcitabine in
Gemcitabine-Resistant PDAC Cells

   First, to examine the combinatorial effects of BBR and Gem, the cell
   viability assays were performed using the parental cells with various
   concentrations of BBR, Gem, and their combination. The 50% inhibitory
   concentrations (IC[50]) of BBR were 16.5 μg/mL in MIA PaCa-2 and 19.0
   μg/mL in BxPC-3, while IC[50] of Gem were 588 nM in MIA PaCa-2 and 618
   nM in BxPC-3 ([98]Supplementary Figure S1). Next, to decide dose ratio
   of BBR and Gem in combination treatment, dose ratio of IC[25], IC[50],
   and IC[75] of BBR vs. IC[50] of Gem (BBR: Gem = 10: 600, 20: 600, 40:
   600, respectively) was examined. In these treatments, the ratio of
   IC[75] of BBR vs. IC[50] of Gem (BBR: Gem = 40: 600) revealed the
   synergistic anti-cancer effect, with 0.82 and 0.45 combination index
   (CI) values, which were calculated by the Chou-Talalay equation at 50%
   inhibitory concentrations [[99]54], in MIA PaCa-2 and BxPC-3. Because a
   CI < 1.0 value was conceived as a synergistic interaction, these data
   suggested a synergistic anti-cancer effect of BBR and Gem in PDAC
   cells. The IC[50] of combined treatment with BBR and GEM were BBR: 12.1
   μg/mL, Gem: 181 nM in MIA PaCa-2; and BBR: 9.2 μg/mL, Gem: 138 nM in
   BxPC-3 ([100]Supplementary Figure S1), therefore, following experiments
   were performed at a concentration of BBR: 10 μg/mL, Gem: 150 nM, which
   is approximately the median IC[50] of combination treatment in both
   PDAC cells.

   To focus on BBR’s chemosensitization to Gem, Gemcitabine-resistant
   (Gem-R) PDAC cells were established as described in a previous study
   [[101]39]. The cell viability assays using Gem-R PDAC cells revealed
   that no growth inhibition was observed in Gem-R PDAC cells up to 900 nM
   concentration range of Gem ([102]Figure 1A). These data confirmed
   successful establishment of Gem-R PDAC cells (Gem-R MIA PaCa-2 and
   Gem-R BxPC-3) for subsequent experiments. Next, the anti-cancer
   activity of BBR was investigated in the growth inhibition of Gem-R PDAC
   cells, using Cell Counting Kit-8 (CCK-8) assays. BBR hampered the cell
   viability of Gem-R PDAC cells in a dose-dependent manner, and the
   IC[50]of BBR in these cells were 14.6 μg/mL in Gem-R MIA PaCa-2 and
   24.8 μg/mL in Gem-R BxPC-3, which were similar doses to each parental
   cell ([103]Figure 1B). To further evaluate whether BBR enhanced
   chemosensitivity to Gem, the cell viability was examined in the
   combined treatment with BBR and Gem. The IC[50] values of the
   combination treatment were BBR: 8.2 μg/mL, Gem: 123 nM in Gem-R MIA
   PaCa-2; and BBR: 17.1 μg/mL, Gem: 257 nM in Gem-R BxPC-3 ([104]Figure
   1C). In support of our hypothesis, for the proliferation assays with
   treatment of BBR and Gem individually, and their combination, the
   combination of BBR and Gem was significantly more effective in the
   inhibition of cell viability in Gem-R MIA PaCa-2 (fold change [FC] =
   0.61 vs. BBR, p < 0.01; FC = 0.31 vs. Gem, p < 0.01) and Gem-R BxPC-3
   (FC = 0.64 vs. BBR, p < 0.01; FC = 0.34 vs. Gem, p < 0.01; [105]Figure
   1D).

Figure 1.

   [106]Figure 1
   [107]Open in a new tab

   Berberine enhances the chemosensitivity to Gemcitabine-resistant PDAC
   cells in inhibiting cell proliferation. (A) Drug dose–response curves
   comparing cell viability following treatment with Gem in parental and
   Gem-R PDAC cells. Error bars are the mean ± SD; (B) Drug dose–response
   curves comparing cell viability following treatment with BBR in Gem-R
   PDAC cells. Error bars are the mean ± SD; (C) Drug dose–response curves
   comparing cell viability following treatment with combination of BBR
   and Gem in Gem-R PDAC cells. Error bars are the mean ± SD; (D) Cell
   proliferation assays in Gem-R PDAC cells with BBR, Gem, and their
   combination. Total viable cells were measured by CCK-8 assays on the
   indicated days. Error bars are the mean ± SD (* p < 0.05 vs. control,
   ^# p < 0.05 vs. BBR, ^$ p < 0.05 vs. Gem). Gem-R,
   Gemcitabine-resistant; PDAC, pancreatic ductal adenocarcinoma; BBR,
   Berberine; Gem, Gemcitabine; CCK-8, Cell Counting Kit-8; SD, standard
   deviation.

   Next, the BBR’s ability to inhibit cellular clonogenicity, migration,
   or invasion was assessed—which are critical phenotypic features for
   cancer progression and metastases. The colony formation assays
   demonstrated that the combination treatment of BBR and Gem resulted in
   a significantly larger reduction of clonogenicity compared to either
   treatment individually in Gem-R MIA PaCa-2 (FC = 0.23 vs. BBR, p <
   0.05; FC = 0.10 vs. Gem, p = 0.04) and Gem-R BxPC-3 cells (FC = 0.33
   vs. BBR, p < 0.05; FC = 0.18 vs. Gem, p < 0.05; [108]Figure 2A).
   Likewise, with regards to the wound healing and invasion assays, the
   combination of BBR and Gem significantly hampered cell migration (p <
   0.05 vs. BBR; p = 0.01 vs. Gem in Gem-R MIA PaCa-2; p < 0.01 vs. BBR; p
   < 0.01 vs. Gem in Gem-R BxPC-3; [109]Figure 2B) and invasion (p = 0.06
   vs. BBR; p < 0.01 vs. Gem in Gem-R MIA PaCa-2; p = 0.01 vs. BBR; p <
   0.01 vs. Gem in Gem-R BxPC-3; [110]Figure 2C). Taken together, these
   data confirmed that BBR could significantly enhance the anti-cancer
   potential of Gem and even reverse Gem-resistance in PDAC cells.

Figure 2.

   [111]Figure 2
   [112]Open in a new tab

   Berberine enhances the chemosensitivity of Gemcitabine-resistant PDAC
   cells by inhibiting colony formation, migration, and invasion. (A)
   Colony formation assays of Gem-R PDAC cells following treatment. The
   average (column) ± SD is indicated (* p < 0.05 vs. control, ^# p < 0.05
   vs. BBR, ^$ p < 0.05 vs. Gem); (B) Wound healing assays following in
   Gem-R PDAC cells. Images show representative areas (marked by red
   lines). Scale bar = 250 μm. The average (column) ± SD is indicated (* p
   < 0.05 vs. control, ^# p < 0.05 vs. BBR, ^$ p < 0.05 vs. Gem); (C)
   Invasion assays following treatment in Gem-R PDAC cells. Scale bar =
   100 μm. The number of cells was randomly counted at four fields, and
   then relative invasion ratios were calculated. The average (column) ±
   SD is indicated (* p < 0.05 vs. control, ^# p < 0.05 vs. BBR, ^$ p <
   0.05 vs. Gem). Images show representative fields on the membrane
   (magnification x100). Gem-R, Gemcitabine-resistant; PDAC, pancreatic
   ductal adenocarcinoma; BBR, Berberine; Gem, Gemcitabine; SD, standard
   deviation.

2.2. Berberine and Gemcitabine Induce the G1 Phase Cell Cycle Arrest and
Apoptosis in Gemcitabine-Resistant PDAC Cells

   To clarify the underlying mechanism for BBR-mediated reversal of
   Gem-resistance in PDAC cells, its impact on the cell cycle phase
   distribution was first evaluated by flow cytometry using propidium
   iodide as a probe. The cell cycle analyses showed that BBR increased
   the percentage of cells in G0/G1 phase in both Gem-R PDAC cells (p =
   0.23 in Gem-R MIA PaCa-2; p = 0.04 in Gem-R BxPC-3; [113]Figure 3A),
   whereas the combined treatment with BBR and Gem significantly induced
   the percentage of cells in G0/G1 phase in Gem-R MIA PaCa-2 (BBR vs.
   Combination, 59.9% vs. 67.0%, p < 0.05; Gem vs. Combination, 55.4% vs.
   67.0%, p < 0.01) and Gem-R BxPC-3 (BBR vs. Combination, 63.7% vs.
   70.1%, p < 0.05; Gem vs. Combination, 51.8% vs. 70.1%, p = 0.03;
   [114]Figure 3A). Consistent with the results from the cell viability
   assays, BBR treatment significantly improved the anti-cancer potential
   by inducing cell cycle arrest in Gem-R PDAC cells.

Figure 3.

   [115]Figure 3
   [116]Open in a new tab

   Berberine and Gemcitabine induce the G0/G1 phase cell cycle arrest and
   enhance cell apoptosis in Gemcitabine-resistant PDAC cells. (A)
   Representative images of cell cycle assays following treatment in Gem-R
   PDAC cells. The graph indicated the average ratio (column) ± SD of
   cells at each stage of cell cycle (* p < 0.05 vs. control, ^# p < 0.05
   vs. BBR, ^$ p < 0.05 vs. Gem). (B) Representative images of apoptotic
   cells that stained for annexin V assays following treatment in Gem-R
   PDAC cells. The average ratio (column) ± SD of cells undergoing
   apoptosis is indicated (* p < 0.05 vs. control, ^# p < 0.05 vs. BBR, ^$
   p < 0.05 vs. Gem). Gem-R, Gemcitabine-resistant; PDAC, pancreatic
   ductal adenocarcinoma; BBR, Berberine; Gem, Gemcitabine; SD, standard
   deviation.

   Next, Annexin V binding assays were performed to evaluate the impact of
   combined treatment with BBR and Gem on apoptotic rates. BBR
   significantly induced the cellular apoptosis compared to the untreated
   group in both Gem-R cells (p = 0.03 in Gem-R MIA PaCa-2; p = 0.03 in
   Gem-R BxPC-3; [117]Figure 3B), while the combined treatment further
   enhanced the potential of apoptosis in both Gem-R MIA PaCa-2 (BBR vs.
   Combination, 21.6% vs. 35.5%, p < 0.05; Gem vs. Combination, 11.1% vs.
   35.5%, p < 0.05) and Gem-R BxPC-3 (BBR vs. Combination, 18.7% vs.
   26.3%, p < 0.05; Gem vs. Combination, 10.3% vs. 26.3%, p = 0.01;
   [118]Figure 3B). Furthermore, when the expression of apoptosis-related
   genes was evaluated by quantitative reverse transcription polymerase
   chain reaction (qRT-PCR) the combined treatment significantly
   up-regulated the gene expression of Bax and Cyclin D1, and
   down-regulated Bcl-2 and Survivin ([119]Supplementary Figure S2).
   Collectively, these data confirm that BBR reversed Gem-resistance
   through the cell cycle arrest and increased cellular apoptosis in Gem-R
   PDAC cells.

2.3. Rap1/PI3K-Akt Signaling Pathway Correlated with Gem-Resistance in PDAC
Cells

   To elucidate the key regulatory pathways associated with Gem-resistance
   in PDAC cells, the gene expression profiling results were compared
   between parental and Gem-R PDAC cells from transcriptomic profiling
   datasets ([120]GSE148200 and [121]GSE140077). This analysis revealed
   1354 up- and 1066 down-regulated (log[2]FC > ±1.0, p < 0.01) genes in
   MIA PaCa-2, and 2281 up- and 1690 down-regulated genes in BxPC-3
   ([122]Figure 4A). Among them, there were 241 up-regulated and 174
   down-regulated genes that overlapped in both PDAC cells ([123]Figure
   4B).

Figure 4.

   [124]Figure 4
   [125]Open in a new tab

   Rap1/PI3K-Akt signaling pathway correlates with Gemcitabine-resistance
   in PDAC cells. (A) Schematic of the differentially expressed gene
   discovery in Gem-R PDAC cells using [126]GSE148200 and [127]GSE140077.
   (B) Venn-diagram of up- and down-regulated expression (log[2]FC > ±1.0
   and p < 0.01) of the genes in Gem-R PDAC cells. (C,D) Scatter plot of
   KEGG pathway enrichment analysis of up- (C) and down-regulated (D)
   genes in Gem-R PDAC cells. The number of differentially expressed genes
   in the pathway is indicated by the circle area, and the circle color
   represents the range of p value. (E) qRT-PCR analysis of key
   differentially expressed genes of Rap1/PI3K-Akt signaling pathway in
   parental and Gem-R PDAC cells. β-Actin mRNA expression was used as an
   internal control. The average (column) ± SD is indicated (* p < 0.05
   vs. parental). Gem-R, Gemcitabine-resistant; PDAC, pancreatic ductal
   adenocarcinoma; BBR, Berberine; Gem, Gemcitabine; FC, fold change; SD,
   standard deviation.

   Next, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes
   (KEGG) pathway enrichment analyses of these genes were performed using
   DAVID database ([128]https://david.ncifcrf.gov/, accessed on 28 August
   2022) [[129]55,[130]56] ([131]Figure 4C,D). In the enrichment pathways,
   we focused on Rap1/PI3K-Akt signaling pathway as of the key pathways
   related to Gem-resistance in PDAC cells, because Rap1 and PI3K-Akt
   signaling interacted closely, and both of these pathways were
   significantly enriched among up-regulated genes (Rap1 signaling
   pathway, fold enrichment: 3.0, p = 0.02; PI3K-Akt signaling pathway,
   fold enrichment: 2.3, p = 0.03; [132]Figure 4C). Thereafter, the
   expression of up-regulated genes included in the Rap1/PI3K-Akt
   signaling pathway was validated using qRT-PCR assays. The results of
   qRT-PCR assays revealed that the expressions of approximately 60% of
   genes (8/14 genes) were consistent with public transcriptomic profiling
   data ([133]Figure 4E and [134]Supplementary Figure S3). Collectively,
   these results suggested that Rap1/PI3K-Akt signaling pathway plays an
   important role in Gem-resistance in PDAC cells. Therefore, we
   hypothesized Rap1/PI3K-Akt signaling pathway as one of the key targets
   of the combined treatment with BBR and Gem and selected this pathway
   for subsequent experiments.

2.4. The Combined Treatment with Berberine and Gemcitabine Regulated the
Activity of Rap1/PI3K-Akt Signaling Pathway in Gemcitabine-Resistant PDAC
Cells

   To examine whether Rap1/PI3K-Akt signaling pathway is a key target
   pathway of the combined treatment with BBR and Gem, we next performed
   Western blotting (WB) of key genes in the Rap1/PI3K-Akt signaling
   pathway following treatment with BBR, Gem, and their combination. The
   results of WB assays revealed Rap1 was significantly down-regulated by
   the combined treatment in both Gem-R MIA PaCa-2 (FC = 0.55 vs. BBR, p =
   0.04; FC = 0.26 vs. Gem, p = 0.03) and Gem-R BxPC-3 (FC = 0.68 vs. BBR,
   p < 0.01; FC = 0.31 vs. Gem, p < 0.01; [135]Figure 5A,B). Furthermore,
   PI3K expression was also significantly down-regulated by the combined
   treatment in both Gem-R MIA PaCa-2 (FC = 0.26 vs. BBR, p = 0.04; FC =
   0.07 vs. Gem, p = 0.02) and Gem-R BxPC-3 (FC = 0.18 vs. BBR, p < 0.01;
   FC = 0.04 vs. Gem, p = 0.02; [136]Figure 5A,B). For the protein
   expression level of Akt, we observed a significant down-regulation by
   the combined treatment in Gem-R BxPC-3; and this combined treatment
   attenuated phosphorylation of Akt at Ser473 (p-Akt) in both Gem-R MIA
   PaCa-2 (FC = 0.58 vs. BBR, p < 0.01; FC = 0.37 vs. Gem, p = 0.03) and
   Gem-R BxPC-3 (FC = 0.50 vs. BBR, p = 0.03; FC = 0.17 vs. Gem, p = 0.04;
   [137]Figure 5A,B). Taken together, these results indicate that the
   Rap1/PI3K-Akt signaling pathway is potentially down-regulated by the
   combination treatment of BBR and Gem and potentially play a role in
   reversing Gem-resistance in Gem-R PDCA cells.

Figure 5.

   [138]Figure 5
   [139]Open in a new tab

   The combination treatment of Berberine and Gemcitabine regulates the
   activity of Rap1/PI3K-Akt signaling pathway in Gemcitabine-resistant
   PDAC cells. (A,B) Western immunoblotting of Rap1, PI3K, Akt, and
   phospho-Akt (ser473) (p-Akt) expression in Gem-R MIA PaCa-2 (A) and
   BxPC-3 (B) following treatment. The protein of β-Actin was used as an
   internal control. The average (column) ± SD is indicated (* p < 0.05
   vs. control, ^# p < 0.05 vs. BBR, ^$ p < 0.05 vs. Gem). Gem-R,
   Gemcitabine-resistant; PDAC, pancreatic ductal adenocarcinoma; BBR,
   Berberine; Gem, Gemcitabine; SD, standard deviation.

2.5. The Combination of Berberine and Gemcitabine Suppresses Spheroid
Formation and Growth of Patient-Derived Organoids

   Three-dimensional (3D) cultures, such as 3D spheroids and tumor
   organoids, are conceived as physiologically superior to the
   conventional monolayer cell cultures for the study of anti-cancer
   agents [[140]57,[141]58]. Therefore, 3D culture models were finally
   used to validate our cell culture findings. First, the sphere formation
   assays were performed using Gem-R PDAC cells with treatment of BBR,
   Gem, and their combination. With regards to the sphere formation
   assays, the combined treatment with BBR and Gem significantly inhibited
   the sphere forming ability of both Gem-R MIA PaCa-2 (p = 0.04 vs. BBR;
   p < 0.01 vs. Gem) and Gem-R BxPC-3 (p = 0.10 vs. BBR; p = 0.02 vs. Gem;
   [142]Figure 6A).

Figure 6.

   [143]Figure 6
   [144]Open in a new tab

   The combination of Berberine and Gemcitabine suppressed spheroid
   formation and growth of human PDAC tumor-derived organoids. (A)
   Representative images of sphere forming Gem-R PDAC cells following
   treatment. Scale bar = 250 μm (Magnification ×40). The average (column)
   ± SD is indicated (* p < 0.05 vs. control, ^# p < 0.05 vs. BBR, ^$ p <
   0.05 vs. Gem). (B) Representative images of tumor organoids following
   treatment. Scale bar = 100 μm (Magnification ×100). The average
   (column) ± SD is indicated (* p < 0.05 vs. control, ^# p < 0.05 vs.
   BBR, ^$ p < 0.05 vs. Gem). Gem-R, Gemcitabine-resistant; PDAC,
   pancreatic ductal adenocarcinoma; BBR, Berberine; Gem, Gemcitabine; SD,
   standard deviation.

   For further validation, tumor-derived organoid models from patients
   with PDAC were next utilized. PDAC patient-derived organoids (IDIT5 and
   6) were generated as described in a previous study [[145]59]. For these
   experiments, the viability of the tumor-derived organoids was evaluated
   following individual treatments with BBR and Gem, and their
   combination. The results revealed that BBR significantly inhibited the
   formation and growth of patient-derived organoids compared to untreated
   controls in both IDIT5 (number, p = 0.01; size, p < 0.01) and IDIT6
   organoids (number, p = 0.03; size, p = 0.01; [146]Figure 6B).
   Furthermore, the combined treatment with BBR and Gem significantly
   enhanced the anti-cancer activity in both the number of organoids
   (IDIT5, FC = 0.72 vs. BBR, p = 0.01; FC = 0.46 vs. Gem, p < 0.01;
   IDIT6, FC = 0.33 vs. BBR, p < 0.01; FC = 0.36 vs. Gem, p < 0.01;
   [147]Figure 6B) and the size of organoids (IDIT5, FC = 0.53 vs. BBR, p
   = 0.03; FC = 0.32 vs. Gem, p < 0.01; FC = 0.44 vs. BBR, p < 0.01; FC =
   0.41 vs. Gem, p = 0.01; [148]Figure 6B). Collectively, these data
   highlighted that BBR significantly enhanced the anti-cancer potential
   of Gem, and yet again successfully validated our cell culture-based
   findings in these patient-derived tumor organoids.

3. Discussion

   The current state of treatment for patients with PDAC involves
   combination chemotherapeutic regimens involving drug combinations such
   as FOLFIRINOX or gemcitabine with nab-paclitaxel, which are often
   accompanied by severe simultaneous drug toxicity and added expense
   [[149]9,[150]10,[151]18,[152]19,[153]20,[154]21,[155]21,[156]22].
   Therefore, the development of optimal combination of safe and
   cost-effective therapeutic modalities is a major clinical challenge for
   treating patients with PDAC, which can help improve their survival
   outcomes. In this context, to overcome this clinical challenge,
   naturally occurring compounds provide an anti-cancer efficacy with time
   tested safety and cost effectiveness. In fact, more than 40% of
   anti-cancer drugs developed between 1981 and 2019 can be traced back in
   origin to naturally occurring compounds [[157]60]. In addition, there
   are hundreds and thousands of studies published in the last two decades
   that show that the combination of chemotherapeutic drugs and such
   compounds can help enhance the anti-cancer therapeutic efficacy of
   conventional therapy, and at the same time reduce the simultaneous drug
   toxicity [[158]27,[159]61,[160]62,[161]63].

   During the last few decades, a better understanding of the mechanisms
   responsible for Gem-resistance have led to the discovery of new
   therapeutic strategies for improving the survival and prognosis of
   patients with PDAC [[162]64,[163]65]. In this context, naturally
   occurring compounds have played a significant role and have been
   extensively studied for their chemosensitizing potential to various
   chemotherapeutic drugs [[164]28,[165]66,[166]67]. In particular, BBR
   has recently gained increasing attention due to its anti-cancer
   potential by enhancing chemosensitivity and reversing chemotherapeutic
   drug resistance in cancer treatment. In breast cancer, BBR reversed
   chemoresistance through inhibition of the efflux function of ABC
   transporters [[167]49] and autophagy through PTEN/Akt/mTOR signaling
   pathway [[168]52]. Additionally, in glioblastoma, BBR reduced
   Temozolomide-resistance by inducing autophagy through ERK1/2 signaling
   [[169]51]. For gastrointestinal cancers, BBR enhanced chemosensitivity
   to irinotecan via inhibition of NF-κB in colon cancer [[170]53], and
   improved chemosensitivity to Cisplatin by enhancing cell apoptosis and
   repressing PI3K-AKt/mTOR signaling in gastric cancer [[171]50].
   Consistent with these previous reports, in this study, we found
   BBR-mediated reversal of chemoresistance to Gem on cell viability,
   clonogenicity, migration, and invasion in PDAC. Furthermore, in line
   with our findings, the enhanced anti-tumor effect of combined treatment
   with BBR and Gem was also confirmed in patient-derived tumor organoids.
   Overall, these findings indicate that BBR might be a promising
   anti-cancer compound for improving the sensitivity of Gemcitabine
   through multiple mechanisms. Our findings proposed the fundamentals of
   a new therapeutic modality for patients with PDAC, as not only
   adjunctive supplementary therapy of current chemotherapeutic regimens
   but also alternative therapy for patients who struggle to receive
   current combination chemotherapeutic therapies.

   Rap1/PI3K-Akt signaling pathway plays a significant role in mediating
   the resistance of various chemotherapeutic drugs [[172]68,[173]69]. In
   particular, several previous studies have demonstrated that PI3K-Akt
   signaling pathway plays a pivotal role in Gem-resistance in various
   cancers [[174]70,[175]71,[176]72]. Furthermore, recent studies revealed
   that PI3K-Akt signaling pathway is closely associated with
   tumorigenesis, tumor progression, and metastases in PDAC
   [[177]73,[178]74,[179]75]. In line with these findings, in the present
   study, Rap1/PI3K-Akt signaling pathway was revealed as one of the key
   pathways related to Gem-resistance in PDAC cells. On the other hand,
   BBR’s properties on the regulation of PI3K-Akt signaling have been
   illustrated in other cancers [[180]50,[181]76,[182]77]. Furthermore,
   interestingly, BBR also acted synergistically with PI3K inhibitor in
   colon cancer cells [[183]78]. For the Rap1 signaling pathway,
   previously, it has been shown that BBR inhibits Rap1 signaling pathway
   which results in the suppression of platelet activation and thrombosis
   [[184]79]. In line with these observations, the present study
   demonstrated that BBR induced cell cycle arrest and enhanced cellular
   apoptosis in Gem-R PDAC cells through the regulation of Rap1/PI3K-Akt
   signaling. More interestingly, we now report that the combined
   treatment with BBR and Gem enhanced this phenomenon and dysregulation
   of Rap1/PI3K-Akt pathway, which means BBR could improve the anti-cancer
   potential of Gem and even reverse Gem-resistance in PDAC cells.

   The dose of BBR (10 μg/mL) in this study was similar to those in
   previous studies about the BBR’s chemosensitization in cancers
   [[185]49,[186]50,[187]51,[188]52,[189]53]. In previous studies, 40–100
   mg/kg oral dose of BBR was used in mice experiments [[190]80,[191]81].
   When we translated it into a human equivalent dose using the Km factor
   [[192]82], the practical dose for a 60 kg human might be 200–500 mg,
   which was an acceptable daily dose according to previous human clinical
   trials [[193]83].

   In this study, we investigated the BBR’s anti-tumor effect to enhance
   chemosensitivity to Gem and reverse Gem-resistance in PDAC cells,
   however, we would like to acknowledge some of the potential limitations
   of this study. First, although our RNA sequencing results identified
   many important molecular pathways, pathway analyses in this study
   included no data about PDAC cells treated with BBR and combination. The
   primary goal of this study was the first insight into the anti-tumor
   effects of BBR in PDAC, as well as its ability to enhance
   chemosensitivity to Gem in this malignancy. Therefore, in this study,
   we focused on Rap1/PI3K-Akt pathway, which has repeatedly been
   recognized as one of the key pathways associated with Gem-resistance in
   various cancers. Second, we were able to analyze organoids from only
   two patients. Taken together, future studies are warranted to
   investigate the further molecular mechanisms in terms of the BBR’s
   anti-tumorigenic effect to enhance chemosensitivity to Gem against PDAC
   cells, including the validation in the Gem-R tumor-derived organoids
   and animal models.

4. Materials and Methods

4.1. Cell Culture and Materials

   Human PDAC cell lines, MIA PaCa-2 and BxPC-3, were obtained from the
   American Type Culture Collection (Manassas, VA, USA). The cell lines
   were cultured in RPMI medium (Gibco, Carlsbad, CA, USA) containing 10%
   fetal bovine serum (Gibco), and 1% penicillin and streptomycin (Gibco).
   Gemcitabine (Sigma-Aldrich, St Louis, MO, USA) was dissolved in
   dimethyl sulfoxide (DMSO), and then diluted to appropriate
   concentrations in the culture medium. Gemcitabine-resistant cell lines
   (Gem-R MIA PaCa-2 and Gem-R BxPC-3) were established by continuous
   culturing with increasing doses of Gem, as described in a previous
   study [[194]39]. All cell lines were tested for mycoplasma
   contamination and authenticated using a panel of genetic and epigenetic
   markers for their genomic authenticity.

4.2. Herbal Preparations

   Berberine used in the present study was Indian Barberry (Berberis
   aristate DC.) bark and root extract (DER 40:1, extraction solvent:
   methanol) standardized for the content of Berberine (97%), (EuroPharma,
   Green Bay, WI, USA). The herbal preparation quality of Berberine was
   authenticated using high-performance liquid chromatography in
   accordance with specifications. All analytical methods were validated
   for accuracy, selectivity, and precision. DMSO was used in the dilution
   of BBR to the stock concentrations, and before use, BBR was further
   diluted to appropriate concentrations in the culture medium.

4.3. Cell Counting Kit-8 Assays

   For cell viability assays, 5 × 10^3 cells were treated with various
   concentrations of BBR, Gem, and their combination for 48 h. Uniform
   DMSO concentrations were used in each treatment group. Following 48 h
   treatment, 10 μL of the CCK-8 solution (Dojindo, Kumamoto, Japan) was
   added and the cells were incubated for an additional 1 h, followed by
   measurement using a microplate reader (Tecan Trading AG, Männedorf,
   Switzerland). For cell proliferation assays, 3 × 10^3 cells were grown
   for 24 h, and thereafter, cells were treated with various
   concentrations of BBR, Gem, and their combination. Their proliferation
   was evaluated at each time point using the CCK-8 assay kit.

4.4. Colony Formation, Wound Healing, and Invasion Assays

   For the colony formation assays, cells (5 × 10^2 cells/well) were
   seeded, followed by each treatment for 48 h. After culturing for 1 week
   in the complete medium, the number of colonies was counted following
   staining with 1% crystal violet and analyzing the colony counts by the
   Image-J 1.53q software ([195]http://imagej.nih.gov/ij/index.html,
   accessed on 28 August 2022). For wound healing assays, after cells were
   grown to 80–90% confluency, wounds were created by scraping the cell
   monolayers, and subsequently treated with BBR, Gem, and their
   combination. Cells were observed under a microscope (magnification
   ×40). Twenty-four hours after scratching, the percentage wound closure
   was calculated using the Image-J 1.53q software. For the invasion
   assays, 3 × 10^4 cells were grown in 24-well 8-μm pore size transwell
   chambers coated with Matrigel (BD Biosciences, Franklin Lakes, NJ,
   USA), followed by treatment. After 48 h, Diff-Quik staining was used to
   detect invading cells.

4.5. Cell Cycle and Apoptosis Assays

   Cell cycle and apoptosis assays were performed using the Muse Cell
   Analyzer (EMD Millipore Corp, Hayward, CA, USA) according to the
   manufacturer’s instructions
   [[196]25,[197]26,[198]33,[199]84,[200]85,[201]86,[202]87,[203]88,[204]8
   9,[205]90,[206]91]. For the cell cycle assays, cells were seeded in
   6-well plates and treated with BBR, Gem, and their combination for 48
   h. Thereafter, cells were rinsed with ice-cold phosphate-buffered
   saline (PBS), fixed with 200 μL of ice-cold 70% ethanol and incubated
   at −20 °C for more than 3 h. Two hundred microliters of Muse^TM Cell
   Cycle Reagent (EMD Millipore Corp) were used in staining cells. The
   results were measured using the Muse Cell Analyzer. For the apoptosis
   assays, after 48 h treatment, 100 μL of Muse Annexin V & Dead Cell
   Reagent (Luminex Corp, Austin, TX, USA) were added into 100 μL of cell
   suspension by PBS. The apoptotic cell fraction was analyzed using the
   Muse Cell Analyzer.

4.6. Real-Time qRT-PCR and Primers

   Total RNA extraction was performed using the Qiagen miRNeasy Kit
   (Qiagen, Hilden, Germany). The real-time qRT-PCR assays were performed
   using a SensiFAST SYBR Lo-ROX Kit (Bioline, London, UK) and the
   QuantStudio 6 Flex RT-PCR System (Applied Biosystems, Foster City, CA,
   USA). Using the β-actin gene as an internal control, the relative
   expression was quantified by the delta-delta Ct method [[207]92]. The
   primers used in the present study are described in [208]Supplementary
   Table S1.

4.7. Gene and Enrichment Pathway Discovery in Gemcitabine Resistant PDAC
Cells

   To discover differentially expressed genes in Gem-R PDAC cells, systems
   biology approaches were performed by analyzing gene expression
   profiling differences between the parental and Gem-R PDAC cells, from
   two publicly available datasets ([209]GSE148200 and [210]GSE140077).
   These datasets were downloaded from the Gene Expression Omnibus
   database in their processed form
   ([211]https://www.ncbi.nlm.nih.gov/geo/, accessed on 28 August 2022). A
   gene defined as differentially expressed if it had p < 0.01 and a
   Log[2]FC > ±1.0. Subsequently, enrichment analysis of GO and KEGG
   pathways was carried out using DAVID bioinformatic database
   ([212]https://david.ncifcrf.gov/, accessed on 28 August 2022)
   [[213]55,[214]56]. A pathway was defined as enriched if the fold
   enrichment was more than 2.0, and scatter plots were generated based on
   enrichment pathway analysis using the DAVID database.

4.8. Protein Extraction and Western Blotting

   Total proteins were extracted from Gem-R PDAC cells using RIPA Lysis
   and Extraction Buffer (Thermo Fischer Scientific) with a protease
   inhibitor cocktail (Thermo Fischer Scientific). Proteins were separated
   by electrophoresis in a 10% Mini-PROTEAN TGXTM Precast Gel (BIO-RAD,
   Hercules, CA, USA), subsequently electro-transferred onto a 0.45 µm
   PVDF blotting membrane (Cytiva, Marlborough, MA, USA), followed by
   blocking in 5% bovine serum albumin (Sigma-Aldrich) solution for 1 h.
   Membranes were incubated overnight at 4 °C with diluted primary
   antibodies. Primary antibodies against Rap1 (1:1000, #2399; Cell
   Signaling Technology [CST], Danvers, MA, USA), PI3K (1:500, #4249;
   CST), Akt (1:1000, #4691; CST), and phospho-Akt (Ser473) (1:1000,
   #4060; CST) were used. The membranes were incubated with secondary
   antibodies (#7074 or #7076; CST) for 1 h. Immunoblots were visualized
   using an HRP-based chemiluminescence kit (Thermo Fisher Scientific)
   using Gel Imaging Systems (BIO-RAD). β-actin (1:1000, #58169; CST) was
   used as an internal control. The relative protein levels were evaluated
   using the Image-J 1.53q software.

4.9. Sphere Formation Assays

   For sphere formation assays, 5 × 10^2–1 × 10^3 cells, following
   treatment for 48 h, were seeded in ultra-low attachment plates in
   serum-free DMEM-F12 medium (STEMCELL Technologies, Vancouver, BC,
   Canada) with B27 supplement (Gibco), basic fibroblast growth factor
   (bFGF; Gibco), and epidermal growth factor (EGF; STEMCELL
   Technologies). After 7–10 days, the spheroids were observed under a
   bright-field microscope (magnification ×40) and counted using the
   Image-J 1.53q software.

4.10. Patient-Derived Tumor Organoids

   Tumor-derived organoids from PDAC patients were generated as described
   previously [[215]59]. Following approval by the ethics committees of
   the institution, written informed consent was obtained from all
   patients. Patients were anonymously coded in accordance with ethical
   guidelines, as instructed by the Declaration of Helsinki. The tumor
   organoids were suspended in 40 μL of Matrigel (Corning, Tehama County,
   CA, USA) with 500 μL of PancreaCultTM Organoid Growth Medium (Catalog
   #100–0781, STEMCELL Technologies) containing EGF (STEMCELL
   Technologies) and prostaglandin E2 (STEMCELL Technologies) according to
   the manufacturer’s instructions. The organoids were treated with BBR
   (10 μg/mL), Gem (150 nM), and their combination (BBR: 10 μg/mL, Gem:
   150 nM). After five days, the PDAC organoids that were more than 50
   microns in diameter were observed under a microscope (magnification
   ×100). The number and size of organoids were measured using the Image-J
   1.53q software.

4.11. Statistical Analysis

   All statistical analyses were conducted using EZR version 1.55, which
   is a graphical user interface for R (R Foundation for Statistical
   Computing, Vienna, Austria, version 4.1.2) [[216]93]. All experiments
   were performed as independent technical triplicates, and all data were
   expressed as mean ± standard deviation (SD). A two-sided Student’s
   t-test was used in the analysis of differences between continuous
   values of each group. p < 0.05 was considered statistically
   significant.

5. Conclusions

   We firstly demonstrate BBR-mediated enhanced chemosensitization to Gem
   in PDAC cells using a systematic series of Gem-resistant cell culture
   and patient-derived tumor organoids experiments. We observed that
   Rap1/PI3K-Akt signaling pathway plays a critical role for
   Gem-resistance in PDAC, and BBR could overcome Gem-resistance via
   inhibition of key genes in Rap1/PI3K-Akt signaling pathway. Our
   findings could offer essential evidence of the combination treatment of
   BBR and Gem as a new safe and cost-effective therapeutic modality in
   this fatal malignancy.

Acknowledgments