Abstract

   Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with
   poor survival rates, primarily due to the limited effectiveness of
   gemcitabine (Gem)-based chemotherapy, as well as the acquisition of
   chemotherapeutic resistance. Aronia berry extracts (ABEs), abundant in
   phenolic constituents, have been recently recognized for their
   anticancer properties as well as their encouraging potential to help
   overcome chemoresistance in various cancers. In the present study, we
   explored ABE’s potential to overcome Gem resistance in PDAC and
   identify specific growth regulatory pathways responsible for its
   anticancer activity. Through a series of in vitro experiments in
   gemcitabine-resistant (Gem-R) cells, we elucidated the synergistic
   interactions between Gem and ABE treatments. Using advanced
   transcriptomic analysis and network pharmacology, we revealed key
   molecular pathways linked to chemoresistance and potential therapeutic
   targets of ABE in Gem-R PDAC cells. Subsequently, the findings from
   cell culture studies were validated in patient-derived 3D tumor
   organoids (PDOs). The combination treatment of ABE and Gem demonstrated
   significant synergism and anticancer effects on cell viability,
   proliferation, migration, and invasion in Gem-R cells. Transcriptomic
   analysis revealed a correlation between the NF-Κb signaling pathway and
   Gem-R (p < 0.05), exhibiting a marked upregulation of MYD88.
   Additionally, MYD88 exhibited a significant correlation with the
   overall survival rates in patients with PDAC patients in the TCGA
   cohort (HR = 1.58, p < 0.05). The MYD88/NF-Κb pathway contributes to
   chemoresistance by potentially upregulating efflux transporters like
   P-glycoprotein (P-gp). Our findings revealed that the combined
   treatment with ABE suppressed the NF-Κb pathway by targeting MYD88 and
   reducing P-gp expression to overcome Gem resistance. Lastly, the
   combination therapy proved highly effective in PDOs in reducing both
   their number and size (p < 0.05). Our study offers previously
   unrecognized insights into the ability of ABE to overcome Gem
   resistance in PDAC cells through its targeting of the MYD88/NF-κb/P-gp
   axis, hence providing a safe and cost-effective adjunctive therapeutic
   strategy to improve treatment outcomes in PDAC.

   Keywords: pancreatic ductal adenocarcinoma, aronia berry extracts,
   gemcitabine resistance, MYD88, anticancer effect, chemoprevention

1. Introduction

   Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of
   cancer-related deaths. Despite the 5-year overall survival (OS) rates
   in the United States having improved from less than 5% in 1990 to as
   high as ~10% in 2023, the prognosis for patients with PDAC remains
   distressingly low. This high rate of mortality is primarily attributed
   to the difficulty in early diagnosis of pancreatic cancer, as well as
   the inadequate efficacy of the currently available treatment options
   [[34]1,[35]2]. Gemcitabine (Gem)-based chemotherapy has played a
   crucial role in advancing the treatment of patients with PDAC since its
   initial endorsement as a primary therapeutic option in 1997.
   Nab-paclitaxel combined with Gem hydrochloride is a commonly used
   therapeutic regimen in PDAC [[36]3,[37]4,[38]5,[39]6,[40]7]. However,
   its efficacy is notably limited by the emergence of drug resistance in
   most patients, highlighting a primary challenge for the effective
   management of this lethal malignancy [[41]8,[42]9]. The newer
   combination therapeutic regimens, such as FOLFIRINOX, while resulting
   in slightly improved treatment outcomes, exhibit significant toxicity
   and are expensive. Collectively, these findings highlight the critical
   importance of gaining deeper insights into the molecular mechanisms
   underlying Gem resistance and investigating novel therapeutic
   approaches to overcome chemoresistance in PDAC and improve patient
   outcomes.

   Previous studies have demonstrated the pivotal role of ABC drug
   transporters in conferring therapeutic resistance to Gem in PDAC
   [[43]10,[44]11]. Among these, P-glycoprotein (P-gp) is the most
   recognized and established regulator contributing to chemoresistance
   [[45]12]. Also known as ABCB1 or MDR1, P-gp plays a vital role in the
   emergence of multidrug resistance (MDR) and was the first human ABC
   transporter to be characterized [[46]13]. P-gp has been shown to affect
   the uptake, distribution, and elimination of compounds through
   modulation of cellular efflux function [[47]14]. Furthermore, the
   efficacy of several chemotherapeutic agents, including paclitaxel
   (PTX), cisplatin (CP), and doxorubicin (DOX), can be affected by P-gp
   overexpression. However, P-gp inhibitors such as quinidine and
   verapamil have been plagued by their toxicity and poor selectivity, in
   turn limiting their clinical use [[48]14,[49]15,[50]16]. Thus,
   exploring safer and more effective P-gp inhibitors remains highly
   valuable in various cancers.

   More recently, there is growing enthusiasm to explore the possibility
   of using various natural products and their derivatives as
   complementary and alternative therapeutic approaches, given their
   multi-targeted efficacy and safety in various cancers
   [[51]17,[52]18,[53]19,[54]20,[55]21,[56]22,[57]23,[58]24,[59]25,[60]26,
   [61]27,[62]28,[63]29]. These natural compounds include curcumin
   [[64]22,[65]30,[66]31,[67]32,[68]33,[69]34,[70]35,[71]36], Andrographis
   [[72]29,[73]37,[74]38,[75]39], resveratrol [[76]40,[77]41], and ginseng
   [[78]42], all of which have been studied for their adjunctive and
   synergistic efficacy in various cancers, including PDAC. Additionally,
   several of these natural remedies have been identified as inhibitors of
   P-gp, MRP1, MRP2, and BCRP and can thus sensitize cancer cells to
   various chemotherapeutic drugs. A range of natural compounds such as
   flavonoids, coumarins, resins, saponins, and terpenoids have been
   explored for their ability to combat drug resistance in cancer by
   inhibiting P-gp [[79]43]. In particular, aronia berries (black
   chokeberries) are thought to have anticancer potential due to their
   richness in phenolic compounds that possess potent antioxidant and
   anti-inflammatory properties, such as procyanidins, anthocyanins,
   phenolic acids, and their analogs [[80]44]. Previous studies also
   indicated that derivatives of aronia berry extract (ABE) can impede the
   formation of breast cancer stem cells, making it a promising candidate
   for cancer chemoprevention [[81]45]. Furthermore, Aronia Melanocarpa
   extract has been reported to impede the growth of PDAC through
   induction of apoptosis in pancreatic cancer cell lines, such as AsPC-1
   [[82]46]. While recent research has suggested the anticancer effects of
   ABE in PDAC, its specific impact on chemosensitivity, especially when
   combined with chemotherapeutic agents, is largely unexplored. Given
   that most natural medicines are used as adjuncts to conventional
   chemotherapy, we hypothesized that ABE might have the potential to
   inhibit tumor growth and overcome chemoresistance in PDAC.

   In our current study, we conducted a comprehensive set of experiments
   in PDAC cell lines and patient-derived 3D organoids (PDOs) to gain
   additional insights into the molecular mechanisms of ABE’s anticancer
   effects and its potential in overcoming Gem resistance in PDAC.

2. Results

2.1. The Treatment of ABE and Gemcitabine Shows Synergistic Inhibition in
Gem-R Pancreatic Cancer Cells

   In our present investigation, we initially utilized the two
   Gem-resistant (Gem-R) cell lines as described in our prior study
   [[83]34]. Notably, the half-maximal inhibitory concentration (IC[50])
   of Gem was substantially higher in Gem-R BxPC-3 and Gem-R MIA-PaCa-2
   cells compared to their respective parental cell lines ([84]Figure
   1A,E). Specifically, the IC[50] of Gem in the parental cells was
   determined to be 680.35 nM and 728.81 nM, respectively. These values
   were significantly lower than those observed in the Gem-R counterparts,
   Gem-R BxPC-3 and Gem-R MIA-PaCa-2, which exhibited IC[50] values
   exceeding 1200 nM. Next, we determined that the ABE could reduce Gem
   resistance in Gem-R PDAC cells. Subsequently, we administered
   concurrent treatments with varying concentrations of ABE (0, 30, 60,
   90, and 120 μg/mL) and Gem (0, 400, 800, and 1200 nM), demonstrating a
   synergistic reduction in cell viability. Next, we investigated the
   anticancer effects of ABE on Gem-R BxPC-3 and Gem-R MIA-PaCa-2 cell
   lines. These resistant cell lines were treated with ABE for 48 h, and
   cell viability was assessed using the CCK-8 assay. The results
   demonstrated a dose-dependent inhibition of cell viability by ABE, with
   IC[50] values of 110.97 μg/mL for Gem-R BxPC-3 and 89.17 μg/mL for
   Gem-R MIA-PaCa-2 ([85]Supplementary Figure S1). Ultimately, the
   criteria for the optimal dosage ratio of ABE and Gem in the combination
   treatment was established by both dose–response curves needing to
   achieve over 50% inhibition and the Bliss synergy score exceeding 10
   points. In these treatments, the ABE/Gem ratio of 90:800 exhibited a
   synergistic anticancer effect in both cell lines. In the Gem-R BxPC-3
   cell line, under this ratio, the inhibitory rate was 64.36%, in
   contrast to −9.23% with only Gem treatment and 45.92% with only ABE
   treatment, resulting in a synergy score of 23.43 ([86]Figure 1B–D).
   Similarly, in the Gem-R MIA-PaCa-2 cell line, under the same ratio, the
   inhibitory rate was 67.02%, compared to 9.53% with only Gem treatment
   and 49.6% with only ABE treatment, yielding a synergy score of 12.61
   ([87]Figure 1F–H). Hence, all subsequent experiments were conducted
   using a concentration of 90 μg/mL ABE and 800 nM Gem.

Figure 1.

   [88]Figure 1
   [89]Open in a new tab

   Identification of drug resistance in Gem-R PDAC cells and the treatment
   ABE with Gem synergistically inhibits the growth of Gem-R PDAC cells.
   The IC[50] of parental BxPC-3/Gem-R BxPC-3 (A) and parental
   MIA-PaCa-2/Gem-R MIA-PaCa-2 (E) to Gem were calculated using the CCK8
   assay. Error bars are the mean ± SD. Percentage of growth inhibition of
   Gem-R BxPC-3 cell lines (B) and Gem-R MIA-PaCa-2 (F) cell lines treated
   in multiple combinations of ABE (0, 30, 60, 90, and 120 μg/mL) and Gem
   (0, 400, 800, and 1200 nM). The dose–response matrix was measured using
   the CCK8 assay. Bliss synergy values were calculated from data in B and
   F, accessed as a 2D-contour (C,G) and 3D-contour (D,H) drug
   dose–response model evaluating cell viability after treatment with ABE
   in Gem-R PDAC cells.

2.2. The Combination of ABE and Gemcitabine Inhibits Cell Proliferation,
Colony Formation, Migration, and Invasion in Gem-R PDAC Cell Lines

   To evaluate the potential effects of ABE in enhancing chemosensitivity
   to Gem in Gem-R PDAC cells, we conducted a series of functional
   experiments, including colony formation, wound healing (scratch), and
   invasion assays. To evaluate the impact of the combination of ABE and
   Gem on PDAC cell proliferation, we treated Gem-R BxPC-3 and Gem-R
   MIA-PaCa-2 cells with Gem (800 nM) and ABE (90 µg/mL), either
   separately or in combination for 48 h. We performed CCK-8 assays, which
   revealed that the combination of Gem and ABE significantly outperformed
   the individual treatments (p < 0.01 vs. Gem; p < 0.01 vs. ABE in Gem-R
   MIA-PaCa-2 cells; p < 0.01 vs. Gem; p < 0.01 vs. ABE in Gem-R BxPC-3
   cells; [90]Figure 2A). Furthermore, the colony formation assay
   demonstrated a significant reduction in clonogenicity with the
   combination of Gem and ABE compared to individual treatments in BxPC-3
   cells (combination vs. Gem: fold change [FC] = 0.49, p < 0.01;
   combination vs. ABE: FC = 0.66, p < 0.01, [91]Figure 2B) and MIA-PaCa-2
   cell lines (combination vs. Gem: FC = 0.22, p < 0.01; combination vs.
   ABE: FC = 0.37, p < 0.01, [92]Figure 2B). To determine whether the
   combination of Gem and ABE had a more significant impact on the
   motility and invasive potential of PDAC cells, we conducted
   scratch–wound and transwell assays. The scratch–wound assay revealed
   that the combination of Gem and ABE significantly inhibited cell
   migration to a greater extent than individual treatments in both BxPC-3
   (combination vs. Gem: FC = 0.22, p < 0.01; combination vs. ABE: FC =
   0.37, p < 0.01, [93]Figure 2C) and MIA-PaCa-2 cell lines (combination
   vs. Gem: FC = 0.14, p < 0.01; combination vs. ABE: FC = 0.37, p < 0.01,
   [94]Figure 2C). The transwell assay showed that a combination of Gem
   and ABE shows a significantly more significant reduction in invasion
   than individual treatment in both BxPC-3 (combination vs. Gem: FC =
   0.53, p < 0.01; combination vs. ABE: FC = 0.68, p < 0.01, [95]Figure
   2D) and MIA-PaCa-2 cell lines (combination vs. Gem: FC = 0.20, p <
   0.01; combination vs. ABE: FC = 0.27, p < 0.01, [96]Figure 2D).
   Furthermore, epithelial-to-mesenchymal transition (EMT) and matrix
   metalloproteinase-related proteins are closely linked to tumor
   metastasis and invasion. We examined the expression of these proteins
   following treatments with gemcitabine, ABE, and their combination. Our
   findings revealed that the combination treatment significantly reduced
   the expression of proteins associated with migration and metastasis
   (e.g., MMP9, vimentin) while significantly increasing the expression of
   those that promote cell adhesion (e.g., E-cadherin; [97]Supplementary
   Figure S2).

Figure 2.

   [98]Figure 2
   [99]Open in a new tab

   Aronia berry extract exhibits anti-proliferation, migration, and
   invasion effects in Gem-R PDAC cells. (A) Cell proliferation was
   compared in Gem-R cell lines treated with Gem, ABE, and their
   combination. Cell viability was assessed using a CCK-8 assay at 24, 48,
   and 72 h time points. (B) A colony formation assay was conducted to
   evaluate the clonogenicity of each group after a 48 h exposure to the
   treatments. Representative images of colonies were taken, and the
   number of colonies was counted. (C) A wound healing assay was performed
   on Gem-R PDAC cells treated with Gem, ABE, and their combination for 48
   h. Representative images of the wound and the recovering areas (marked
   by black lines) were taken, and the percentage of wound closure was
   measured. (D) A transwell assay was carried out on Gem-R PDAC cells
   treated with Gem, ABE, and their combination for 48 h. The number of
   invading cells was counted in three randomly selected fields on the
   membrane. * p indicates p < 0.05 vs. control group; # p indicates p <
   0.05 vs. combination group. ** p indicates p < 0.01; *** p indicates p
   < 0.001.

   Consistent with the previous results, our findings demonstrated that
   the combination of Gem and ABE significantly reduced the migration and
   invasion abilities of PDAC cells.

2.3. ABE, in Combination with Gemcitabine, Promotes Cell Apoptosis

   To evaluate the apoptotic effects of the combination of Gem and ABE on
   PDAC cells, we initially conducted an annexin V binding assay. The
   analysis indicated that the combination of Gem and ABE increased the
   rate of apoptosis in both Gem-R cell lines ([100]Figure 3A) compared to
   the individual administrations of Gem and ABE (BxPC-3 cell line:
   combination vs. Gem: 26.55% vs. 8.095%, p < 0.01; combination vs. ABE:
   26.55% vs. 17.33%, p < 0.01, [101]Figure 3A; and MIA-PaCa-2 cell line:
   combination vs. ABE: 25.60% vs. 11.29%, p < 0.01; combination vs. ABE:
   25.60% vs. 14.8%, p < 0.01, [102]Figure 3A). Next, we conducted Western
   blotting (WB) to analyze changes in apoptotic proteins following
   treatment. The WB analysis demonstrated that the combination of Gem and
   ABE increased the expression of cleaved caspase-9 and Bax,
   pro-apoptotic proteins, while simultaneously reducing the expression of
   total PARP, an anti-apoptotic protein, in comparison to the control and
   individual treatments ([103]Figure 3B)
   [[104]47,[105]48,[106]49,[107]50,[108]51]. Collectively, these data
   confirm that the combination of Gem and ABE induced more Gem-R PDAC
   cell apoptosis compared to individual treatments in vitro.

Figure 3.

   [109]Figure 3
   [110]Open in a new tab

   Aronia berry extract induces cell apoptosis in Gem-R PDAC cells. (A)
   Annexin V assay was conducted to measure the percentage of apoptotic
   cells on Gem-R PDAC cells treated with Gem, ABE, and their combination
   for 48 h. (B) Western blot analysis was performed on Gem-R PDAC cells
   treated with Gem, ABE, and their combination for 48 h. The relative
   levels of PARP, caspase-9, and Bax were quantitatively analyzed by
   comparing them to the control GAPDH expression. * p indicates p < 0.05
   vs. control group; # p indicates p < 0.05 vs. combination group.

2.4. The MYD88/NF-κB Signaling Pathway Is Aviated in Gem-R PDAC Cells

   To investigate the mechanism of Gem resistance in PDAC cells, we
   conducted gene expression profiling analysis by comparing parental and
   Gem-R PDAC cells using publicly available Gene Expression Omnibus (GEO)
   datasets ([111]GSE148200 and [112]GSE140077). We identified 2520
   upregulated and 1292 downregulated genes in MIA-PaCa-2 cells, and 1958
   upregulated and 1984 downregulated genes in the BxPC-3 cell line. These
   genes were selected based on specific criteria: a log2FC > ±0.5 and a
   p-value < 0.05. ([113]Figure 4A). Subsequently, we focused on the genes
   that were consistently regulated in the same direction in both cell
   lines, identifying 276 upregulated and 111 downregulated genes. We
   performed KEGG pathway analysis to gain further insights into the
   biological pathways associated with these significantly dysregulated
   genes. This analysis was carried out utilizing the DAVID database
   ([114]https://david.ncifcrf.gov/, accessed on 28 August 2022)
   [[115]52,[116]53]. The top 15 enrichment pathways were prioritized
   ([117]Figure 4B). Among these, of particular interest was the
   MYD88/NF-κB signaling pathway, which ranked as the top pathway and has
   been the subject of numerous previous studies highlighting its
   potential involvement in PDAC and Gem resistance
   [[118]54,[119]55,[120]56,[121]57]. Additionally, previous research has
   shown that MYD88 expression is increased in cancer tissues and
   correlated with paclitaxel resistance in other cancers such as breast,
   ovarian, and lung [[122]58,[123]59,[124]60]. MYD88 is a significant
   target for innovative therapies in pancreatic cancer due to its crucial
   molecular role in linking various upstream ligand–receptor complexes
   [[125]61]. We evaluated the expression level of MYD88 in 179 primary
   tumor tissues and 171 normal tissues from the TCGA database. Our
   findings revealed a significantly higher expression of MYD88 in cancer
   tissues compared to normal tissues (p < 0.001, [126]Figure 4C).
   Furthermore, Kaplan–Meier survival analyses identified a significant
   association between MYD88 expression and overall survival in PDAC
   patients from the TCGA cohort (HR = 1.58, p = 0.045, [127]Supplementary
   Figure S3). Finally, we validated the higher mRNA expression of MYD88
   in Gem-R PDAC cell lines compared to parental cell lines through
   qRT-PCR assays (BxPC-3 cell line: p < 0.05, [128]Figure 4D and p <
   0.05, [129]Figure 4D). Consequently, our findings suggest that the
   combination of ABE and Gem could potentially target the MYD88/NF-κB
   signaling pathway.

Figure 4.

   [130]Figure 4
   [131]Open in a new tab

   The MYD88/NF-κB signaling pathway is associated with Gem resistance in
   PDAC cells. (A) A schematic illustrates the differentially expressed
   genes in the BxPC-3-R vs. BxPC-3-Parental cell line (GSE 140077) and
   MIA-PaCa-2-R vs. MIA-PaCa-2-Parental cell line (GSE 148200). The Venn
   diagram represents the upregulated and downregulated expression of
   genes selected using Log2FC > 0.5 and p < 0.05. (B) Scatter plots of
   KEGG pathway enrichment analysis of up- and downregulated genes in
   Gem-R PDAC cell lines. The top 15 significantly enriched KEGG pathways
   were prioritized. The circle represents the number of differentially
   expressed genes, while the circle color represents the range of
   p-values. (C) The expression level of the selected genes, MYD88, was
   analyzed using PDAC cases from the TCGA dataset. (D) qRT-PCR assays
   were undertaken to measure the mRNA expression levels of selected genes
   in Gem-R and parental PDAC cell lines, with β-actin expression as the
   internal control. * p indicates p < 0.05 vs. control group; *** p
   indicates p < 0.001.

2.5. Combined Treatment with Gemcitabine and ABE Downregulates P-gp through
the MYD88/TLR3/NF-κB Signaling Pathway

   The Toll-like receptor (TLR) family, which includes TLR1-10, comprises
   transmembrane glycoproteins primarily expressed in immune cells. In
   particular, TLR3 and TLR4 can activate transcription factor NF-κB
   through both MYD88-dependent pathways and MYD88-independent pathways
   [[132]62]. To investigate whether the MYD88/TLR3/NF-κB signaling
   pathway is a key target pathway of the combined treatment of Gem and
   ABE, WB was performed to assess the expression levels of key genes
   involved in the MYD88/TLR3/NF-κB axis when treated with Gem, ABE, and
   their combination. Our results revealed that TLR3, MYD88, and p65 were
   significantly downregulated by the combined treatment in both Gem-R
   BxPC-3 and Gem-R MIA-PaCa-2 cell lines ([133]Figure 5A). In addition,
   the MYD88/NF-κB signaling pathway is linked to chemoresistance, as it
   has the ability to upregulate efflux transporters such as P-gp
   [[134]56,[135]63]. To evaluate the protein expression level of P-gp, we
   conducted immunofluorescence analysis (excitation: 488 nm; emission:
   564 nm). Compared to the parental PDAC cell lines, the expression of
   P-gp in the Gem-R cell lines was significantly higher, and the
   treatment with ABE reduced the expression of P-gp in both PDAC cell
   lines after treatment ([136]Figure 5B).

Figure 5.

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

   The combination treatment of Gem and ABE has been shown to regulate the
   MYD88/TLR3/NF-κB/P-gp complex in Gem-R PDAC cell lines. (A) Western
   blot analysis was performed on Gem-R PDAC cells treated with Gem, ABE,
   and their combination for 48 h. The relative levels of TLR3, MYD88, and
   p65 were quantitatively analyzed by comparing them to the control GAPDH
   expression. (B) Representative images for an immunofluorescence assay
   were obtained to evaluate P-gp expression in parental PDAC cell lines,
   Gem-R PDAC cell lines, and Gem-R PDAC cell lines treated with ABE for
   48 h. * p indicates p < 0.05 vs. control group; # p indicates p < 0.05
   vs. combination group.

2.6. The Combination of Gemcitabine and ABE Suppressed the Growth of PDOs

   Three-dimensional organoid culture systems have emerged as valuable
   tools for modeling various aspects of cancer biology, particularly in
   investigating drug responses and evaluating treatment efficacy. In this
   study, we used tumor organoid models derived from two patients to
   examine the anti-tumor effects of Gem and ABE. As anticipated, our
   results demonstrated that the combination of Gem and ABE significantly
   suppressed both the size and number of organoids ([139]Figure 6A). In
   terms of the number of organoids, when compared to individual
   treatments (Patient1: combination vs. Gem: FC = 0.48, p < 0.05;
   combination vs. ABE: FC = 0.56, p < 0.05, [140]Figure 6B; Patient2:
   combination vs. Gem: FC = 0.65, p < 0.05; combination vs. ABE, FC =
   0.62, p <0.05; [141]Figure 6B), the combination was more effective.
   Similarly, the combination of Gem and ABE significantly reduced
   organoid sizes compared to individual treatments (Patient1: combination
   vs. Gem: FC = 0.52, p < 0.05; combination vs. ABE: FC = 0.64, p < 0.05,
   [142]Figure 6C; Patient2: combination vs. Gem: FC = 0.65, p < 0.05;
   combination vs. ABE, FC = 0.73, p <0.05; [143]Figure 6C). These
   findings support the notion that the combination of Gem and ABE
   effectively inhibit the growth of PDOs, corroborating the results of
   our cell culture experiments.

Figure 6.

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

   The combination of Gem and ABE demonstrates effective anticancer
   activity in PDAC patient-derived 3D organoid (PDO) models. (A)
   Representative images of PDOs treated with Gem, ABE, and their
   combination for 48 h. (B) The average number and size of PDOs treated
   with Gem, ABE, and their combination for 48 h. (C) A schematic
   illustration of ABE-induced reversal of Gem resistance in PDAC. This
   illustration reveals that Gem can activate the MYD88/NF-Κb axis,
   resulting in chemoresistance by upregulating the efflux transporters
   (P-gp) and hindering cellular apoptosis in PDAC cells (left). In
   contrast, ABE helps counteract chemoresistance by downregulating MYD88
   and downstream signaling pathways in PDAC cells (right). * p indicates
   p < 0.05 vs. control group; # p indicates p < 0.05 vs. combination
   group.

3. Discussion

   Since the FDA’s approval in 1996, Gem-based chemotherapy has been used
   extensively as a standard treatment regimen in multiple kinds of solid
   cancers, including ovarian, breast, and non-small cell lung cancer
   [[146]64]. Combination chemotherapeutic strategies incorporating Gem
   remain the preferred treatment for advanced PDAC [[147]65]. However,
   chemotherapeutic resistance often occurs in PDAC patients. In
   particular, the development of resistance to Gem presents an important
   clinical challenge in the effective therapeutic management of patients
   with PDAC [[148]66]. Several studies have demonstrated the significant
   role of ABC transporters like P-gp, BCRP, and MRP1 in mediating
   resistance to Gem chemotherapy [[149]12], and the ability of ABC
   transporter inhibitors to sensitize tumor cells to chemotherapeutic
   agents has been researched extensively. While some positive outcomes
   have been observed in preclinical studies and early-phase clinical
   trials, no effective MDR-reversing agents targeting ABC transporters
   have been approved for clinical use thus far [[150]67].

   As such, natural products have been explored for their potential
   anticancer activity and low toxicity in healthy tissues and have
   recently been reported to overcome drug resistance [[151]68,[152]69].
   In our study, we aimed to combine Gem with ABE to investigate whether
   ABE could enhance the sensitivity of Gem-R cells to Gem. Following the
   administration of a combination of Gem and ABE to Gem-R cells, our
   results revealed that ABE synergistically increased the sensitivity of
   Gem-R cells to Gem. Moreover, the combination of ABE and Gem
   effectively inhibited cell viability, clonogenicity, migration, and
   invasion while inducing a higher apoptosis rate in PDAC cells. We
   further found that the combined ABE and Gem treatment could
   significantly strengthen the anticancer activity in inhibiting PDAC
   PDOs, supporting our cell culture-based findings.

   To investigate the mechanism by which ABE enhances sensitivity to Gem,
   we analyzed gene expression profiles from two publicly available GEO
   datasets and compared parental and Gem-R PDAC cells. Our analysis
   revealed that MYD88, an adaptor molecule for Toll-like receptors
   [[153]70,[154]71,[155]72,[156]73], was upregulated in Gem-R PDAC cells.
   Furthermore, we found that TLR3 can activate the transcription factor
   NF-κB through both the MYD88-dependent pathway and the
   MYD88-independent pathway [[157]62]. The MYD88/NF-κB signaling pathway
   is linked to chemoresistance as it can elevate the expression of efflux
   transporters such as P-gp [[158]56,[159]74,[160]75]. In the current
   study, we revealed that MYD88 is upregulated in tumor tissues and that
   high expression levels of MYD88 correlate with poor prognosis in PDAC.
   Second, we discovered that MYD88 is also upregulated in Gem-R cell
   lines. Therefore, we hypothesized that ABE could target MYD88,
   potentially restoring sensitivity to Gem in Gem-R cell lines. Our WB
   results showed that co-treatment of Gem and ABE inhibited the
   expression of MYD88, TRL3, and P65 expression. These findings provide
   further evidence for MYD88/TRL3/NF-κB as a critical pathway in the
   ability of ABE to overcome Gem resistance. However, the present study
   lacks definitive evidence to establish whether alterations in TLR3
   expression are MYD88-dependent. Additionally, the co-treatment of Gem
   and ABE suppressed the expression of P-gp, as shown by
   immunofluorescence staining. These findings provide compelling evidence
   for the involvement of the MYD88/TRL3/NF-κB/P-gp axis as the target
   mechanism through which ABE counteracts Gem resistance in PDAC cells
   ([161]Figure 6C).

   Finally, we confirmed the anti-tumor efficacy of Gem and ABE by
   utilizing tumor organoid models derived from two PDAC patients.
   However, we did not generate the Gem-R organoids, which may limit our
   finding validation in vivo. Based on these, we propose that we can
   validate these findings in animal models in the future.

   In summary, our study provides valuable insights into the potential of
   ABE to overcome Gem resistance in PDAC. Our data reveal a crucial
   mechanism underlying the synergistic interaction between ABE and Gem.
   These findings contribute to the growing body of evidence supporting
   the potential of ABE as a novel therapy for PDAC, particularly for
   patients confronting Gem resistance.

4. Materials and Methods

4.1. Cell Culture

   The PDAC cell lines BxPC-3 and MIA-PaCa-2 were sourced from the
   American Type Culture Collection (ATCC, Manassas, VA, USA). The cell
   lines were cultured in RPMI medium (Gibco, Carlsbad, CA, USA) with 1%
   penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA) and 10%
   fetal bovine serum (Gibco, Waltham, MA, USA). The cells were cultured
   at 37 °C with 5% CO2 in a humidified environment. The adherent cells
   were harvested using 0.05% trypsin (Invitrogen, Carlsbad, CA, USA).
   Gem-R MIA-PaCa-2 and Gem-R BxPC-3 were established by continuously
   culturing the cells with increasing doses of Gem, as described
   previously [[162]3].

4.2. Herbal Preparations

   The aronia berry extract utilized in this study (Aronia Berry Complex,
   EuroPharma USA, Green Bay, WI, USA) was a powdered product
   characterized by colors ranging from purple to dark red. It was derived
   from the fruit of the Aronia Melanocarpa plant, extracted in 70%
   ethanol, and standardized to a 40% polyphenol content. The extract was
   initially dissolved in dimethyl sulfoxide to create stock
   concentrations and then further diluted to suitable levels in the
   culture media.

4.3. Reagents

   Gemcitabine (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in
   dimethyl sulfoxide (DMSO, Sigma-Aldrich). The stock solutions of Gem
   (10 μM) and ABE (100 mg/mL) were carefully stored at −20 °C in the
   dark, ensuring their stability and reliability. These stock solutions
   were diluted with a complete culture medium to the necessary
   experimental concentrations before each application.

4.4. Cell Counting Kit-8 Assays

   For the Cell Counting Kit-8 (CCK-8) assay, a CCK-8 kit (Dojindo,
   Kumamoto, Japan) was used to measure cell viability according to the
   instructions as follows: Initially, to confirm the Gem-resistant
   properties, the proliferation rates were compared between Gem-R and
   their parental counterparts by subjecting them to escalating doses of
   Gem. Cells were seeded in 96-well flat plates at a density of 5 × 10^3
   cells per well and incubated for 24 h. Thereafter, cells were treated
   with increasing concentrations of Gem (0–1200 nM). After 48 h of
   treatment, 10 μL of CCK-8 solution was added to each well and incubated
   for 2 h. The absorbance of the product was measured at a wavelength of
   450 nm (OD450) using a microplate reader from Tecan Trading AG (Tecan
   Trading AG, Männedorf, Switzerland).

   Subsequently, to investigate the influence of ABE and Gem combination
   on PDAC cell proliferation, cells were seeded in 96-well plates at a
   density of 2 × 10^3 cells/well in 100 µL of complete culture medium and
   incubated for 24 h. Subsequently, the cells were exposed to appropriate
   concentrations of Gem (800 nM), ABE (90 μg/mL), and their combination.
   The cell proliferation rates were evaluated at different time points.

4.5. Drug Response Testing

   The Gem-R PDAC cell lines were plated at a density of 5 × 10^3 cells
   per well in 96-well plates and incubated for 24 h before drug
   administration. Subsequently, the cells were exposed to varying
   concentrations of Gem (0, 400, 800, and 1200 nM), ABE (0, 30, 60, 90,
   and 120 μg/mL), and twenty different combinations of these drugs for a
   duration of 48 h, to identify the synergistic concentrations. Cell
   viability was assessed using the CCK8 assay. Synergy scores were
   subsequently calculated using SynergyFinder 3.0, a freely accessible
   tool specifically designed for interactive analysis and visualization
   of combination response outcomes [[163]76].

4.6. Colony Formation Assay

   For the cell colony formation assay, 5 × 10^2 cells were seeded per
   well in 6-well flat plates and then treated with Gem, ABE, and their
   combination for 48 h. Following this treatment, colony formation was
   allowed to proceed for a duration of 7 to 10 days, with the culture
   medium refreshed every three days. At the end of this incubation
   period, the cell colonies were fixed with 4% paraformaldehyde in PBS
   (Thermo Fisher Scientific, Fair Lawn, NJ, USA) for 30 min and stained
   with 1% crystal violet (Thermo Fisher Scientific). The number of
   colonies was quantified using ImageJ 1.53q software.

4.7. Wound Healing Assay

   In the cell wound healing assay, we seeded 5 × 10^5 cells per well in
   6-well flat plates following a 48 h treatment with Gem, ABE, or their
   combination. After the cells reached 80% to 90% confluence, we utilized
   a sterile 20 μL micropipette tip to create a controlled scratch in the
   monolayer. This was followed by a wash with a serum-free medium to
   eliminate detached cells. Subsequently, the cells were cultured in a
   complete medium. Photographs of the cells were captured 24 h after the
   formation of the wound, and the percentage of wound closures was
   determined using ImageJ software.

4.8. Invasion Assays

   To conduct cell invasion assays, BioCoat Matrigel Invasion Chambers
   with 8.0 μm Pore Polyester Membrane (BD Biosciences, Franklin Lakes,
   NJ, USA) were utilized. For the invasion assay, 5 × 10^4 cells per well
   were seeded in 24-well flat plates after treatment with Gem, ABE, and
   their combination for 48 h. The cells were then transferred onto
   inserts in serum-free medium and subsequently moved to wells containing
   culture medium supplemented with 10% FBS. After a 48 h incubation
   period, the cells that had invaded the bottom surface of the membrane
   were fixed and stained using a Diff-Quick staining kit (Thermo Fisher
   Scientific). The stained cells were later quantified under a
   microscope.

4.9. Apoptosis Assay

   For apoptosis assays, Muse™ Annexin V and Dead Cell kits (Luminex Corp,
   Austin, TX, USA) were utilized following the manufacturer’s guidelines.
   In these experiments, 5 × 10^5 cells were seeded per well in 6-well
   flat plates. Following treatment with Gem, ABE, and their combination
   for 48 h, the cells were harvested. Subsequently, 100 μL of cell
   suspension was mixed with 100 μL of Muse Annexin V & Dead Cell Reagent.
   The percentage of apoptotic cells was assessed using a Muse™ Cell
   Analyzer (Millipore Corp, Billerica, MA, USA) in accordance with the
   manufacturer’s instructions.

4.10. Gene Enrichment and Pathway Analysis

   To identify differentially expressed genes in Gem-R PDAC cells, gene
   expression profiles from two publicly available datasets
   ([164]GSE148200 and [165]GSE140077) sourced from the Gene Expression
   Omnibus (GEO) database ([166]https://www.ncbi.nlm.nih.gov/geo/,
   accessed on 28 August 2022) were analyzed. Differential gene expression
   across various groups was examined using the “DEseq2” package in R,
   with a significance threshold of p < 0.05 and a Log[2]FC (Logarithm of
   Fold Change) exceeding ±0.5.

   The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was
   performed using the DAVID bioinformatics database
   ([167]https://david.ncifcrf.gov/, accessed on 28 August 2022). The KEGG
   pathway enrichment analysis was visualized using the “ggplot2” package
   in R.

4.11. Isolation of Cytosolic and Nuclear Extracts

   After treatment with Gem, ABE, and their combination for 48 h,
   cytosolic and nuclear extracts were isolated from cells using a nuclear
   extraction kit (Active Motif, Carlsbad, CA, USA) according to the
   manufacturer’s instructions. These extracts were collected and stored
   in aliquots at −80 °C.

4.12. Protein Isolation and Western Blot

   Total protein was extracted from PDAC cell lines treated for 48 h with
   Gem, ABE, or their combination. The cells were harvested using a
   plastic scraper. Subsequently, the cells were lysed with ice-cold
   protein extraction solution RIPA containing a protease inhibitor
   cocktail (Thermo Fisher Scientific). Protein concentration was
   determined using the BCA procedure (Thermo Fisher Scientific). Equal
   amounts of protein samples were separated via SDS-PAGE using 6% or 10%
   Mini-PROTEAN TGXTM Precast Gels (BIO-RAD, Hercules, CA, USA) and then
   transferred onto nitrocellulose membranes, followed by an additional
   transfer onto a 0.45 μm PVDF membrane (Cytiva, Marlborough, MA, USA).
   The membranes were blocked with 5% bovine serum albumin in
   Tris-buffered saline with 0.1% Tween-20 for 1 h at room temperature.
   Subsequently, the membranes were incubated with primary antibodies,
   including anti-Bax (1:1000, 5023S; Cell Signaling Technology [CST],
   Danvers, MA, USA), anti-Cleaved Caspase 9 (1:1000, 9505; CST),
   anti-PARP (1:1000, 9532S; CST), anti-TLR3(1:1000, PA5-20183; Thermo
   Fisher Scientific), anti-MYD88 (1:1000, 23230-1-AP; Proteintech,
   Rosemont, IL, USA), anti-P65 (1:1000, 3034; CST), anti-Lamin A/C
   (1:2000, 10298-1-AP, Proteintech), anti-Vimentin (1:2000, 10366-1-AP,
   Proteintech), anti-MMP-9 (1:1000, 10375-2-AP, Proteintech),
   anti-E-cadherin (1:2000, 10375-2-AP, Proteintech), and anti-GAPDH
   (1:2000, 5174T; CST) at 4 °C overnight. After three washes with TBST,
   the membranes were incubated with corresponding anti-rabbit (1:2000,
   7074; CST) or anti-mouse (1:2000, 7076; CST) secondary antibodies for 1
   h at room temperature. The blot was visualized using an HRP-based
   chemiluminescence kit (Thermo Fisher Scientific) with Gel Imaging
   Systems (BIO-RAD). GAPDH protein served as an internal control, and the
   intensities of the protein bands were quantified using ImageJ software.

4.13. Quantitative Reverse Transcription PCR (qRT-PCR)

   Total RNA extraction was performed using the Qiagen miRNeasy Kit
   (Qiagen, Hilden, Germany). Subsequently, cDNA was synthesized by
   reverse transcription of total RNA using the High-Capacity cDNA Reverse
   Transcription Kit from Thermo Fisher Scientific. RT-PCR assays were
   conducted with the QuantStudio 6 Flex RT-PCR System (Applied
   Biosystems, Foster City, CA, USA), following the manufacturer’s
   instructions and utilizing the SensiFAST SYBR Lo-ROX Kit from Bioline
   (London, UK). The relative mRNA expression levels of MYD88 were
   determined using the 2^−ΔΔCt method and normalized to the expression of
   β-actin, acting as the internal control. The primer sequences can be
   found in [168]Supplementary Table S1.

4.14. Immunofluorescence Assay

   For the immunofluorescence assay, 5 × 10^5 cells per well were seeded
   in 6 cm flat plates with slides. Following an 18-h incubation period,
   the cells were treated with Gem, ABE, or their combination for 48 h.
   The slides were subsequently fixed with 4% paraformaldehyde for 10 min
   at room temperature, then permeabilized with 0.5% Triton-X-100 (Thermo
   Fisher Scientific) for 10 min at room temperature. To remove endogenous
   peroxides, the slides were treated and subsequently blocked in TBST
   (Tris-buffered saline with Tween-20) containing 3% BSA for 1 h at room
   temperature. The slides were then incubated with an anti-P-glycoprotein
   Polyclonal antibody (1:500, 22336-1-AP, Proteintech) overnight at 4 °C,
   followed by incubation with a FITC-labeled secondary antibody (1:1000,
   A-21202; Thermo Fischer Scientific) for 1 h at 37 °C. Following
   incubation, the slides were subjected to three additional washes with
   phosphate-buffered saline, each lasting five minutes. Finally, the cell
   nucleus was stained with DAPI (Thermo Fischer Scientific), and all
   images were captured using a Carl Zeiss fluorescent microscope (Zeiss,
   Oberkochen, Germany).

4.15. Patient-Derived 3-Dimensional Tumor Organoids (PDOs)

   PDOs from PDAC patients were generated as described in a previous study
   [[169]77]. With approval from the institution’s ethics committees,
   written informed consent was obtained from all patients. To ensure
   confidentiality and anonymity, patients were coded following ethical
   guidelines outlined in the Declaration of Helsinki. For the human
   organoids, the Human Complete Feeding Medium (hCPLT) consisted of
   PancreaCultTM Organoid Growth Medium (STEMCELL Technologies, Cambridge,
   MA, USA), supplemented with EGF (STEMCELL Technologies) and
   prostaglandin E2 (STEMCELL Technologies), following the manufacturer’s
   instructions. PDAC organoids were seeded in a 24-well plate to form a
   dome in 40 μL Matrigel (Corning, Tehama County, CA, USA) with 500 μL of
   hCPLT. The domes were then divided into 4 groups and appropriate
   concentrations of Gem (800 nM), ABE (90 μg/mL), and their combination.
   After 7 days of treatment, the number and size of the organoids were
   analyzed through microscopy (magnification ×100) and measured using
   ImageJ software.

4.16. Statistical Analysis

   Statistical analysis was conducted utilizing SPSS software version 21.0
   and GraphPad Prism version 6.0. Student’s t-test was employed to assess
   the significance of observed differences between the two groups, while
   one-way analysis of variance (ANOVA) was utilized to evaluate
   differences among multiple comparisons. All experiments were performed
   in triplicate on independent biological replicates, and the data were
   presented as mean ± standard deviation. A significance level of p <
   0.05 was considered statistically significant.

5. Conclusions

   We firstly provided a novel insight into the potential of ABE to
   overcome Gem-R in PDAC cells using a systematic series of Gem-resistant
   cell culture and patient-derived tumor organoids experiments.
   Additionally, we observed that the MYD88/NF-κB/P-gp axis plays a
   crucial role in Gem resistance in PDAC, and ABE could overcome this
   resistance by downregulating MYD88 and its downstream signaling
   pathways in PDAC cells ([170]Figure 6C), suggesting ABE as a promising
   and cost-effective approach to enhance therapeutic outcomes in PDAC.
   These results warrant further exploration of ABE in clinical settings
   to validate its potential as an adjunct therapy for improving treatment
   efficacy in pancreatic cancer patients. In the next phase of our
   research, we should further investigate the optimal dose of ABE to
   effectively counter Gem resistance at the minimum required dosage.

Acknowledgments