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. Author manuscript; available in PMC: 2024 Jul 24.
Published in final edited form as: Biomed Pharmacother. 2024 May 16;175:116743. doi: 10.1016/j.biopha.2024.116743

Nanocarrier mediated entinostat and oxaliplatin combination therapy displayed enhanced efficacy against pancreatic cancer

Paras Mani Giri a, Ashish Kumar a, Philip Salu b, Venkatachalem Sathish a, Katie Reindl b, Sanku Mallik a, Buddhadev Layek a,*
PMCID: PMC11268367  NIHMSID: NIHMS2000712  PMID: 38759290

Abstract

Pancreatic cancer is the third leading cause of cancer-related death in the United States, with a 5-year survival rate of only 12%. The poor prognosis of pancreatic cancer is primarily attributed to the lack of early detection, the aggressiveness of the disease, and its resistance to conventional chemotherapeutics. The use of combination chemotherapy targeting different key pathways has emerged as a potential strategy to minimize drug resistance while improving therapeutic outcomes. Here, we evaluated a novel approach to treating pancreatic cancer using entinostat (ENT), a selective class I and IV HDAC inhibitor, and oxaliplatin (OXP) administered at considerably lower dosages. Combination therapy exhibited strong synergistic interaction against human (PANC-1) and murine (KPC) pancreatic cancer cells. As expected, ENT treatment enhanced acetylated histone H3 and H4 expression in treated cells, which was even augmented in the presence of OXP. Similarly, cells treated with a combination therapy showed higher expression of cleaved caspase 3 and increased apoptosis compared to monotherapy. To further improve the efficacy of the combination treatment, we encapsulated OXP and ENT into bovine serum albumin and poly(lactic-co-glycolic) acid nanoparticles. Both nanocarriers showed suitable physicochemical properties with respect to size, charge, polydispersity index, and loading. Besides, the combination of OXP and ENT nanoparticles showed similar or even better synergistic effects compared to free drugs during in vitro cytotoxicity and colony formation assays towards pancreatic cancer cells.

Keywords: Pancreatic ductal adenocarcinoma, Synergistic cell killing, HDAC inhibitor, Platinum-based chemotherapeutics, BSA nanoparticles, PLGA nanoparticles

1. Introduction

Pancreatic cancer is one of the most lethal human malignancies, with a five-year survival rate of 12% [1]. Although pancreatic cancer accounts for only 3% of all new cancer cases, it is the 3rd leading cause of cancer-related death (7%) in the United States. In 2023, according to the American Cancer Society, an estimated 64,050 new pancreatic cancer cases will be diagnosed, and approximately 50,550 people will die of pancreatic cancer across the nation.

Given that it spreads quickly, pancreatic cancer is among the most aggressive types of cancer. The cancer itself, on the other hand, is frequently detected much later. The lack of early detection and the higher tendency of metastasis explain the poor survival rate of pancreatic cancer patients [2–4]. Like other cancers, surgery, radiation, and chemotherapy are the standard treatment approaches for pancreatic cancer [5,6]. The surgical removal of the tumor is not possible in more than 80% of patients since detection is often late, by which time the tumor has spread beyond the pancreas. The two current standards of care first-line treatment regimens for patients with inoperable pancreatic cancer are gemcitabine/nab-paclitaxel and FOLFIRINOX (FOL – folinic acid, F – 5-fluorouracil, Irin – irinotecan, and OX – Oxaliplatin) [7]. However, these treatments only extend life by less than five months compared to the best supportive care [8]. Therefore, novel therapeutic approaches for pancreatic cancer are a critical need. The poor treatment outcomes of conventional chemotherapeutic approaches are primarily associated with the inefficient delivery of cytotoxic agents to the tumor site and the tumor’s innate resistance to chemotherapy [9,10].

Due to enhanced permeability and retention (EPR) effects, nano-carriers have shown preferential tumor accumulation of their cargos while sparing normal tissues [11–13]. Therefore, nanoformulations not only improve the efficacy of the chemotherapeutic agents but also minimize their cytotoxicity by reducing the dose. Furthermore, combination therapies targeting different key pathways can reduce drug resistance while inhibiting tumor growth and metastatic potential [14]. Therefore, combination therapy is the cornerstone of numerous current chemotherapeutic regimens, if not all.

Histone deacetylase (HDAC) inhibitors are emerging therapeutic agents since HDAC plays an essential role in tumor cell proliferation, differentiation, and apoptosis, with minimal effects on normal tissue [15,16]. Several clinical trials are currently evaluating the HDAC inhibitor’s efficacy in pancreatic cancer [17]. HDAC inhibitors have significantly improved tumor remission rates and disease-free survival in hematological malignancies [18]. However, the efficacy of HDAC inhibitors as a single-agent therapy for solid tumors remains unsuccessful. Therefore, HDAC inhibitors were combined with other chemotherapeutic agents and radiation therapy. Nevertheless, combining HDAC inhibitors with gemcitabine, 5-fluorouracil, bortezomib, or radiation therapy has failed to improve clinical efficacy [19]. In addition, like conventional chemotherapeutic agents, their application in cancer therapy is also plagued by dose-limiting toxicities, including hematological and neurological toxicities [20]. Oxaliplatin (OXP) is a part of the FOLFIRINOX regimen to treat pancreatic cancer [21]. However, due to the severe toxicity of OXP and the combination, the approach is limited to a subset of patients, showing a median progression-free survival of 6 months only [22].

Here, for the first time, we investigate a new approach for treating pancreatic cancer with a combination of a selective class I and IV HDAC inhibitor, entinostat (ENT), and a leading chemotherapeutic agent, OXP, used at much lower concentrations. HDAC inhibitors reduce the affinity between histone protein and DNA, promoting chromatin opening. Such opening by histone hyperacetylation renders the DNA more accessible to chemotherapeutics like platinum drugs, leading to enhanced anticancer efficacy [23]. Initially, we determined the synergistic potential of OXP and ENT using both murine and human pancreatic ductal adenocarcinoma (PDAC) cells. After confirming synergistic combinations, we evaluated the underlying mechanism responsible for synergism by determining the expression of HDACs and cleaved caspase 3 in the treated cells. We also evaluated the cytotoxic effects of combination therapy via colony formation assays. Finally, both drugs were encapsulated in nanocarriers, and their cytotoxicity profiles were compared with the corresponding free solutions alone or in combination.

2. Materials and methods

2.1. Materials

OXP and ENT were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Cell culture grade dimethyl sulfoxide (DMSO) and thiazolyl blue (also known as MTT) were procured from Santa Cruz Biotechnology (Dallas, Texas, USA). Bovine serum albumin (BSA), Dulbecco’s Modified Eagle Medium (DMEM), phosphate-buffered saline (PBS), and Trypsin-EDTA solution 0.25% were obtained from Millipore Sigma (St. Louis, MO, USA). Poly (DL-lactide-co-glycolide) 50:50, ester terminated (PLGA) was purchased from Evonik Corporation (Birmingham, AL, USA). Antibodies such as acetyl-histone H3 (K9), acetyl-histone H4 (K8), histone H3 (D1H2), histone H4 (D2X4V), cleaved caspase-3 (D175), and caspase-3 (D3R6Y) and cell lysis buffer with protease inhibitors were procured from Cell Signaling (Beverly, MA, USA). β-actin antibody was purchased from Applied Biological Materials (Richmond, BC, Canada). Annexin/Necrosis kit was purchased from Abcam (Waltham, MA, USA).

2.2. Cell culture

The human pancreatic ductal adenocarcinoma cell line, PANC-1, was purchased from the American Type Culture Collection (Manassas, VA, USA). The murine pancreatic ductal adenocarcinoma cell line, KPC, was a kind gift from the Dr. Jiha Kim Laboratory, North Dakota State University (Fargo, ND, USA). Both PANC-1 and KPC cells were cultured in DMEM media supplemented with 10% FBS and 1% penicillin-streptomycin. These cells were maintained in a humidified atmosphere containing 5% CO2 at 37 °C.

2.3. Synergistic study

PANC-1 and KPC cell lines were seeded in 96-well plates at a density of 5×103 cells/well in a 200 μL culture medium. After 24 h of incubation, cells were treated with various concentrations of OXP and ENT (0.1 μM – 100 μM) as monotherapy and combination therapy. After 72 h of the treatment, the drug-containing medium was removed, 100 μL of MTT-containing medium (0.5 mg/mL) was added to each well and further incubated for 2 h. Then, the MTT-containing medium from each well was discarded, and formazan crystals generated by the live cells were dissolved in 150 μL of DMSO. The absorbance of the formazan solution was recorded using a microplate reader at 570 nm. Cells without drug treatment were used as a control to calculate relative cell viability.

To evaluate the additive or synergistic potential of OXP in combination with ENT, the cell viability data were analyzed using Combenefit software [24]. The software calculates a synergy or antagonism score via three established models: Highest single agent (HSA), Bliss, and Loewe [24]. A positive score indicates synergy, zero is an additive effect, and a negative score is an antagonist effect. The synergy score can also be represented graphically using a “Contour” or “Matrix” plot.

Viability data were also analyzed using CompuSyn software (ComboSyn, Inc., PD Science, LLC, Paramus, NJ, USA) that uses the Chou-Talalay method [25]. Combination index (CI) values were calculated over a wide range of drug concentration combinations using cell viability data (Fa) from 0.05 to 0.95 (5–95% cell death). The application analyzes the combination index of each drug combination where CIs value <1 represents synergy, CIs value =1 represents additive, and CIs>1 indicates antagonism. Furthermore, this software also determines the dose-reduction index (DRI) for the drug combinations. DRI demonstrates the fold decrease of a single agent when a combination of drugs is used, as compared to a single drug, to achieve the particular percentage of cell death.

2.4. Western blotting

PANC-1 and KPC cells were seeded in 6 well plates (0.3 ×106 cells/well). The cells were treated with either 5 μM of OXP solution, 10 μM of ENT solution, or their combination for 48 h. The cells were washed twice with PBS and treated with cell lysis buffer containing protease inhibitors for 15 min. The plate containing the lysate buffer was scrapped, and cell lysates were collected in low-protein-binding microcentrifuge tubes followed by centrifuging at 15,000 rpm (Hettich MIKRO 220 R microcentrifuge, Tuttlingen, Germany) for 15 min at 4°C. The supernatant was collected, and protein concentration was determined using a detergent-compatible protein assay kit (Bio-Rad). The extracted proteins were freshly used for western blot or stored at −80°C for future use.

From respective treatment groups, equivalent amounts of total protein were separated by electrophoresis on 4–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Criterion Gel System; Bio-Rad) gels. The proteins from the SDS-PAGE gel were transferred onto 0.22 μm polyvinylidene difluoride (PVDF) membranes using a Bio-Rad Trans-Blot Turbo rapid transfer system. The membranes were blocked with 5% BSA in Tris-buffered saline for 1 h at room temperature and incubated overnight at 4°C with specific primary antibodies. The membranes were then washed 3 times for 10 min/wash with Tris-buffered saline containing 0.1% (v/v) Tween 20, and blots were incubated with LiCOR near-red conjugated secondary (2°) antibodies at room temperature for 1 h, and the membranes were washed 3 times for 10 min/wash with Tris-buffered saline containing 0.1% (v/v) Tween 20. β-actin was used as a loading control. A Li-Cor Odyssey XL system imaged the protein expression on the membrane, and densitometry analysis was performed using ImageJ software. All experiments were repeated at least three times. The ratios obtained for western blot analysis were first normalized by dividing the raw values of proteins of interest with the raw values of β-actin (acetylated histones) or total protein (cleaved caspase 3). The obtained values were then normalized to control.

2.5. Apoptosis assay

PANC-1 and KPC cells were seeded in 6 well plates at a density of 3×105 cells/well and incubated overnight. The cells were treated with OXP (5 μM), ENT (10 μM), or their combination (5 μM OXP plus 10 μM ENT). After 24 h, cells were washed twice with DPBS, trypsinized, and centrifuged (SX47508A rotor, Allegra X-14R, Beckman Coulter, Brea, CA, USA) at 1000 rpm for 5 min at 4°C. The cell pellets were resuspended in 200 μL assay buffer and stained with Apoptosis/Necrosis Detection Kit (ab176749; Abcam) per the manufacturer’s protocol. Briefly, Apopxin Green indicator (2 μL) and 7 AAD (1 μL) were added to the cell suspension and incubated for 30 min. Finally, 300 μL of assay buffer was added to the cell suspension and analyzed immediately using a flow cytometer (BD FACSMelody, San Jose, CA, USA). The data were processed using FlowJo software.

2.6. Nanoparticle preparation

OXP-loaded BSA nanoparticles (OXP-BSA NPs) were prepared by the desolvation method [26]. Briefly, BSA (2% w/v) was dissolved in deionized water, and OXP (5 mg/mL) was added to the BSA solution. The solution was kept stirring for 30 min at room temperature. Ethanol was added to the solution (4:1 ratio of ethanol to BSA solution) at the rate of 1 mL/min. The formed BSA nanoparticles were stabilized via glutaraldehyde (10 μg of glutaraldehyde/ 1 mg of BSA) and incubated for 2 h. The resulting nanoparticles were ultracentrifuged (Beckman Coulter, Indianapolis, IN, USA) at 100,000 g for 35 min at 4°C, resuspended in deionized water, mixed with sucrose, and lyophilized using a Labconco freeze dryer (Kansas City, MO, USA) for 48 h. Similarly, blank BSA nanoparticles were fabricated without the use of OXP.

ENT-encapsulated PLGA nanoparticles (ENT-PLGA NPs) were fabricated via a single emulsion solvent evaporation method [27]. ENT (6 mg) and PLGA (30 mg) were dissolved in 1 mL chloroform. The organic solution was added to 7.5 mL 2% polyvinyl alcohol (PVA) aqueous solution, followed by sonication at 50 Hz using Qsonica 700 probe sonicator (Qsonica LLC, Newtown, CT, USA) for 5 min. The emulsion was stirred overnight in a chemical hood, followed by additional stirring under a vacuum desiccator for 1 h to remove organic solvent completely. The nanoparticle dispersion was centrifuged at 100, 000 g for 35 min at 4°C to collect nanoparticle pellet. The nanoparticle pellet was resuspended in deionized water using a plastic transfer pipette. The washing steps were performed twice more to remove excess PVA, unencapsulated drugs, and free polymer. After the final wash, nanoparticles were redispersed in deionized water, probe sonicated for one minute, and centrifuged at 1000 rpm for 5 min to remove precipitated drugs, if any, and formed microparticles. The supernatant was mixed with sucrose and lyophilized for 48 h. Blank PLGA nanoparticles were fabricated similarly without the drug.

2.7. Nanoparticle characterization

The lyophilized nanoparticles were dispersed in deionized water (0.1 mg/mL). Nanoparticles were characterized for particle size, polydispersity index (PDI), and zeta potential using Zeta Sizer Nano ZS (Malvern Instruments, Malvern, UK). For morphology characterization, nanoparticle dispersed in deionized water was dropped onto a copper grid with a thin carbon layer and dried. Images were captured using transmission electron microscopy (JEOL JEM-2100 high-resolution analytical TEM).

To determine OXP loading, 1 mg of drug-loaded nanoparticles were incubated with 1 mL trypsin (0.25% w/v) at 37°C for 6 h. Later, nanoparticle dispersion was centrifuged at 15,000 rpm for 10 min (Hettich MIKRO 220 R microcentrifuge), and the supernatant was analyzed for OXP using HPLC (Shimadzu HPLC system). The chromatographic condition was as follows: mobile phase: a mixture (95:5, v/v) of 0.01 M orthophosphoric acid and acetonitrile, flow rate: 1 mL/min, column: Roc C18 column (250 mm × 4.6 mm, particle size 5 μm), injection volume: 10 μL, UV–vis wavelength: 250 nM. The total run time was 10 min, and the OXP was eluted at 5.9 min.

ENT loading and encapsulation efficiency were determined by dispersing the nanoparticles in methanol. The dispersed nanoparticle was kept overnight at room temperature. PLGA polymer was separated via centrifugation (Hettich MIKRO 220 R microcentrifuge) at 15,000 rpm for 10 min. The amount of ENT in the supernatant was determined by HPLC. The chromatographic condition was as follows: mobile phase: a mixture (50:50, v/v) of ammonium acetate (10 mm, pH 4) and acetonitrile, flow rate: 0.3 mL/min, column: Accucore C18 (100 mm × 2.1 mm, particle size 2.6 μm), injection volume: 10 μL, UV–vis wavelength: 254 nM. The total run time was 10 min, and the ENT was eluted at 2.3 min.

An in vitro drug release study of OXP nanoparticles was conducted by adding 5 mg of OXP nanoparticles in 1 mL of PBS (n=6). The nanoparticle suspensions were placed into a dialysis tube (Spectra/Por 3 dialysis membrane MWCO: 3.5 kD). The dialysis tube was immersed into a beaker containing 25 mL of PBS. The release study was performed at 37 °C using an orbital shaker (MAXQ 4000, Thermo Scientific, USA) rotating at 100 rpm. At predetermined intervals, 1 mL of PBS media was removed and replenished with fresh PBS. The amount of ENT in the withdrawn media was determined via HPLC.

Similarly, in vitro drug release of ENT nanoparticles was conducted by adding 3 mg of ENT nanoparticles in 1 mL of PBS (n=6). However, the volume of total release media was 100 mL to maintain sink condition.

2.8. In vitro cytotoxicity

PANC-1 and KPC cell lines were seeded in 96-well plates (5×103 cells/well) and incubated for 24 h. Nanoparticle-encapsulated and free drugs were added to each well and incubated for 72 h. Following treatment, cell viability was determined by MTT assays [28,29]. Cells grown in DMEM media without treatment were used as controls.

2.9. Colony formation study

A colony formation assay was performed to determine the long-term ability of cells to survive and proliferate when treated with OXP, ENT, or a combination. Cells were seeded in 6-well plates (1×103 cells/well). The cells were treated with either OXP (1 μM and 5 μM solution or equivalent amount of nanoparticles), ENT (1 μM and 10 μM solution or equivalent amount of nanoparticles), or their combinations and incubated for 14 days. After 14 days, the media were removed, the cells were washed with DPBS, and stained with 0.5% w/v crystal violet in 10% ethanol. The cells were incubated for 30 min at room temperature, and then the crystal violet solution was discarded. The wells were washed with tap water to remove excess crystal violet and kept for drying. The number of colonies present in the wells was counted via ImageJ software.

2.10. Statistical analyses

Statistical analysis was performed using Prism version 10.1.1 (GraphPad, CA). One-way ANOVA (for multiple groups) followed by a Tukey post-hoc test was performed. A p-value less than 0.05 was considered statistically significant.

3. Results

3.1. Combination of OXP and ENT exhibited synergistic cell killing

Here, we investigated a new strategy to treat PDAC by combining OXP and ENT for possible synergistic cytotoxicity against KPC and PANC-1 cells. Cells were treated with variable concentrations of individual chemotherapeutic agents or their combination for 72 h, and cell viability was determined. The cell viability data were analyzed to evaluate the combinational effect (i.e., antagonistic, additive, or synergistic effect) between OXP and ENT using Combenefit and Compusyn software.

The Combenefit software utilizes three models (e.g., HSA, Bliss, Loewe) to determine combinational interaction and represent synergistic/antagonistic interaction as synergy scores and contour plots. The matrix and contour plots for the OXP and ENT combinations using the Lowe model are presented in Fig. 1. As expected, PANC-1 and KPC were less sensitive toward ENT than OXP. However, co-treatment of these drugs showed a strong synergistic effect rather than additive in both cell lines and significant cell killing was observed for multiple combinations of OXP and ENT (i.e., in a wide range of drug combinations) against both human and murine pancreatic cancer cell lines. For instance, the combination treatment showed a positive score in 58 and 44 combinations out of 70 tested for KPC and PANC-1 cells, respectively. The highest synergy scores obtained were 35 at a combination of 0.1 μM OXP and 10 μM ENT for KPC cells. Similarly, the highest synergy score for PANC-1 cells was 21, obtained with 10 μM OXP and ENT combination concentrations.

Fig. 1.

Fig. 1.

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Synergistic effects of entinostat (ENT) and oxaliplatin (OXP) combination against pancreatic cancer cells. The cells were treated with ENT and OXP in a 10 × 7 concentration grid for 72 h, and cell viability was determined using the MTT test. The viability data was analyzed by Combenefit software using Loewe model. Synergy and antagonism distribution matrix of drug combinations in (A) KPC and (B) PANC-1 cells. Synergy and antagonism surface of ENT and OXP combinations at indicated concentrations in (C) KPC and (D) PANC-1 cells. The darker the blue color, the greater the anticipated synergy between the drugs (n = 6).

We also determined the synergistic interaction using Compusyn software, which uses the Chou-Talalay Method for drug interaction. The software presents the possible interaction between drugs via CI. For any particular concentration of two drugs, CI = 1 indicates additive effects, CI < 1 presents synergistic effects, and CI>1 implies antagonistic effects. For instance, CI values were less than 1 for most drug combinations (i.e., 54/70), with fewer values greater than CI >1 (Tables S1 and S3). The software also calculates DRI, representing fold reduction in drug concentration when combined to achieve the same cytotoxicity using individual drug concentrations (Tables S2 and S4). For example, to achieve IC50, the OXP dose was 3.1 μM, and the ENT was 20.7 μM in KPC cells. However, in combination, DRI for OXP was 31.4 and ENT was 4.14, representing the dose of 0.01 μM (i.e., 3.1 μM/31.4) for OXP and 5 μM (i.e., 20.7 μM/4.14) for ENT. There was a significant dose reduction in a wide range of combination treatments for both cell lines.

3.2. Combination treatment suppressed HDAC activity in PDAC cells

ENT is an HDAC inhibitor associated with enhanced histone H3 and H4 acetylation in various cell types [30,31]. To understand the mechanism of synergistic interaction between OXP and ENT, we evaluated their effects on histone H3 and H4 acetylation in KPC and PANC-1 cells. Both cell lines were treated with either 5 μM OXP, 10 μM ENT, or their combination for 48 h. As expected, treatment with ENT significantly enhanced the acetylation of histone H3 (Ace-H3) and H4 (Ace-H4) in both cell lines (Fig. 2). There was no alteration in histone H3 or H4 acetylation in OXP-treated cells. Importantly, we observed that the Ace-H3 and Ace-H4 expression were significantly increased in cells treated with the ENT and OXP combination compared to individual treatment (Fig. 2). Although the exact mechanism of ENT’s increased histone acetylation capacity in the presence of OXP is unknown, similar results were also observed in other studies when HDAC inhibitors were combined with platinum-based drugs [32,33].

Fig. 2.

Fig. 2.

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ENT and OXP combination inhibit HDAC activity in KPC and PANC-1 cells. Cells were treated with ENT (10 μM), OXP (5 μM), or their combination for 48 h followed by protein extraction. Western blotting analysis of acetylated histone (Ace-H3 and Ace-H4) and total histone (H3 and H4) in (A) KPC and (D) PANC-1 cells. Quantitative analysis of Ace-H3 (B and E) and Ace-H4 (C and F) in KPC (B and C) and PANC-1 cells (E and F). Data represent mean ± SEM (n = 6). ‘*’ vs. CTRL, ‘#’ vs. ENT, ‘$’ vs. OXP,*/#p < 0.05, **/##/$$p < 0.01, ***/$$$p < 0.001.

3.3. Combination therapy enhanced cleaved caspase 3 expression in PDAC cells

Like other platinum-based substances, oxaliplatin primarily causes DNA damage to exert its cytotoxic effects. It forms intra-strand guanine-guanine and guanine-adenine DNA linkages in cancer cells, leading to apoptotic cell death [34]. On the other hand, ENT-mediated histone hyperacetylation promotes transcriptional activation of cancer-related genes, inhibiting tumor cell proliferation and differentiation while inducing apoptosis [35]. Therefore, to evaluate the synergistic attribute of the combination therapy, the KPC and PANC-1 cells were treated with either 5 μM OXP, 10 μM ENT, or their combination for 48 h, and apoptosis marker (cleaved caspase-3) expression was measured using western blotting. Compared to untreated control, cleaved caspase-3 expression was elevated in OXP or ENT-treated cells (Fig. 3). We observed a notable increase in the expression of cleaved caspase-3 when treated with the combination of drugs for both cell lines. However, we found no statistical significance for the respective treatment groups.

Fig. 3.

Fig. 3.

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Combined ENT and OXP treatment enhance cleaved caspase-3 expression in KPC and PANC-1 cells. Cells were treated with ENT (10 μM), OXP (5 μM), or their combination for 48 h followed by protein extraction. Western blot analysis of cleaved caspase-3 (Cleaved-C3) and total-C3 in (A) KPC and (C) PANC-1 cells. Quantitative analysis of cleaved-C3 activity in (B) KPC and (D) PANC-1 cells. Data represent mean ± SEM (n = 3). ‘ns’ indicates non-significant (p > 0.05).

3.4. OXP and ENT combination increases apoptosis

Oxaliplatin exerts its cytotoxic effect via DNA adduct formation, thereby inducing apoptosis and cell death. Here, we tried to determine the effect of the ENT on OXP-induced apoptosis of pancreatic cancer cells. Following various treatments, the apoptosis of KPC and PANC-1 cells was determined by flow cytometry using an Apoptosis/Necrosis Detection Kit. Mild apoptosis was observed in ENT (15.7%) and OXP (24.2%) treated KPC cells (Fig. 4). However, the ENT and OXP combination significantly increased apoptosis, with 47.9% of the cells being apoptotic. Similarly, for PANC-1 cells, the percentage of apoptosis with ENT, OXP, and their combination treatment was 18.1%, 25.4%, and 46.5%, respectively. These results demonstrate that a combination of ENT and OXP therapy induces a greater extent of apoptosis in PDAC cells and supports a synergistic cell killing by the drug combination.

Fig. 4.

Fig. 4.

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ENT and OXP combination enhance apoptosis in pancreatic cancer cells. Quantitative analysis of the percent of apoptosis for (A) KPC and (B) PANC-1 cells after ENT (10 μM), OXP (5 μM), or their combination treatment for 24 h. Data represent mean ± SEM (n = 5). Representative scatter plots of ApoGreen-A (y-axis) vs. 7-AAD (x-axis) for (C) KPC and (D) PANC-1 cells. ‘*’ vs. CTRL, ‘#’ vs. ENT, ‘$’ vs. OXP, *p < 0.05, ***/###/$$$p < 0.001.

3.5. Preparation and characterization of nanoparticles

OXP-BSA nanoparticles were prepared using the desolvation cross-linking method, while ENT-PLGA NPs were formulated using the emulsion solvent evaporation method. First, we assessed the morphological features of OXP-BSA and ENT-PLGA nanoparticles by TEM. Both nanoparticles appeared as spherical and discrete particles uniform in size (Fig. 5A and D). The mean hydrodynamic diameter of ENT-PLGA nanoparticles was 281.8 ± 17.8 nm, as determined using the DLS method (Fig. 5B). ENT-PLGA nanoparticles also demonstrated unimodal homogenous size distribution (PDI: 0.16). Nanoparticles showed a net negative surface charge with an average zeta potential of −17.0 ± 1.2 mV. The PLGA nanoparticles could encapsulate large amounts of ENT (11.3 ± 2.0% w/w) with higher entrapment efficiency (48.7 ± 6.6%).

Fig. 5.

Fig. 5.

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Characterization of drug-loaded nanoparticles. Representative TEM image of (A) ENT PLGA and (D) OXP BSA nanoparticles. Particle size distribution of (B) ENT PLGA and (E) OXP BSA nanoparticles was determined using the DLS method using Zetasizer Nano ZS. The drug release profile of (C) ENT PLGA and (F) OXP BSA nanoparticles in phosphate-buffered saline at pH 7.4. Data represents mean ± SD (n = 6).

The average hydrodynamic diameter of OXP-BSA NPs was 277.1 ± 5.2 nm, as determined by the dynamic light scattering (DLS) technique (Fig. 5E). The unimodal distribution with a small polydispersity index (PDI: 0.14) represents the homogeneous size distribution of nanoparticles. The surface charge of the OXP-BSA NPs was −18.1 ± 1.3 mV. OXP loading was 6.3 ± 0.3% (w/w) with an entrapment efficiency of 41.5 ± 2.4%.

In vitro drug release studies of ENT-PLGA and OXP-BSA nanoparticles were conducted using PBS media (pH 7.4) at 37 °C to mimic the physiological conditions. The ENT-PLGA nanoparticles showed an initial burst release, with approximately 16% of ENT released in the first hour, followed by a sustained release up to 7 days (i.e., ~84% of encapsulated ENT) (Fig. 5C). OXP-BSA nanoparticles also showed an initial burst release, with approximately 15% released in the first hour (Fig. 5F). The cumulative OXP release in 24 h was about 48%, followed by a sustained release of up to 7 days (i.e., ~82% of encapsulated OXP).

3.6. In vitro cytotoxicity study

A cell proliferation assay was conducted to evaluate the in vitro cytotoxicity of nanoparticles against KPC and PANC-1 cells compared to non-encapsulated drugs. Non-encapsulated ENT (10 μM) had minimal impact on cell viability, and about 75% of KPC cells remained viable after 3 days of treatment (Fig. 6A). On the other hand, OXP treatment induced significant cytotoxicity, and about 50% cell death was observed following treatment. However, it is noteworthy that the combination of ENT and OXP caused a dramatic increase in cytotoxicity, and ~33% of cells survived the treatment. Furthermore, significantly higher cytotoxicity was achieved with the corresponding nanoparticle formulations for all treatments.

Fig. 6.

Fig. 6.

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In vitro cytotoxicity of combination therapy using free and nanoparticle encapsulated drugs. Cells were treated with ENT (10 μM), OXP (5 μM), or their combination for 3 days and cell viability was determined by MTT assay. The percentage of relative cell viability for (A) KPC and (B) PANC-1 cells. Data represent mean ± SEM (n = 8) ‘*’ vs. respective solution, *p < 0.05, **p < 0.01, ***p < 0.001.

Similar results were observed with the PANC-1 cells (Fig. 6B). About 77%, 59%, and 30% of cells remained viable after 3 days of treatment with ENT solution, OXP solution, and their combination, respectively. We also observed a significantly greater extent of cell killing with the nanoformulations and their combination, which could be attributed to the higher cell uptake of drugs in nanoforms.

3.7. Colony formation assay

A colony formation assay was performed to evaluate the long-term therapeutic potential of combination treatments. PDAC cells were treated with ENT (1 μM), OXP (1 μM), or their combination for 14 days, and the number of viable colonies was counted using ImageJ software. ENT and OXP solutions treatment in the KPC cells reduced the total colonies to about 87% and 59% of the untreated group, respectively (Fig. 7A & B). However, when cells were treated with ENT and OXP combination, the colony number was reduced to 26% only. Similarly, in PANC-1, the number of colonies for ENT, OXP, and their combination was approximately 85%, 71%, and 47%, respectively (Fig. 7C & D). Most importantly, the nanoformulation-treated groups showed a significantly lower number of colonies than their solution counterpart in both the pancreatic cancer cell lines, which could be attributed to higher uptake of nanoencapsulated drugs by cancer cells, sustained release of drugs from nanoparticles, or a combination of these two factors.

Fig. 7.

Fig. 7.

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ENT and OXP combination treatment reduced colony formation in pancreatic cancer cell lines. Cells were treated with ENT (1 μM), OXP (1 μM), or their combination for 14 days, stained crystal violet, and colonies were counted using ImageJ software. Quantitative analysis of colonies formed by (A) KPC and (C) PANC-1 cells. Data represent mean ± SEM (n=6). Representative images of colonies of (B) KPC and (D) PANC-1 cells. ‘*’vs. respective solution. *p < 0.05, **p < 0.01, ***p < 0.001.

4. Discussion

Combination therapy has shown beneficial effects in many cancers because of the synergistic potential of two chemotherapeutic agents at lower drug concentrations than individual drugs, reducing dose-dependent toxicity [36]. The current standard of care for patients with metastatic pancreatic duct adenocarcinoma involves a platinum-based chemotherapy regimen called FOLFIRINOX [7,37]. However, compared to the best supportive care, these medicines only prolong life by fewer than five months, which advocates the importance of a new effective combination regimen.

The combination of HDAC inhibitors with various platinum-based compounds has emerged as a potential strategy against different solid tumors. It has been observed that DNA-damaging agents, such as platinum medications, have enhanced therapeutic efficiency when combined with HDAC inhibitors since the latter can expose nuclear DNA to cytotoxic DNA-damaging agents, hence increasing the efficacy of platinum therapies [38,39]. Zhou et al. [23] have shown that the combination of low doses of chidamide, a selective inhibitor of HDAC1, 2, 3, and 10, enhanced the cytotoxicity of carboplatin against non-small cell lung cancer. The synergistic potential of chidamide was observed when combined with cisplatin and OXP. A recent study showed that combining ENT with cisplatin enhanced cytotoxicity against small-cell lung cancer (SCLC) cells [40]. However, out of eighteen tested cell lines, only six showed strong synergistic cell killing, while the remainder displayed additive or slight synergism effect. Thus, combining HDAC inhibitors with platinum-based drugs does not guarantee synergistic interactions.

In this study, we investigated a new strategy to treat PDAC by combining OXP and ENT for possible synergistic cytotoxicity. The OXP and ENT combination treatments exerted strong synergistic cytotoxicity against KPC and PANC-1 cells over a wide range of combinations (Fig. 1). Bandolik et al. also reported a synergistic interaction between ENT and cisplatin against high-grade serous ovarian cancer [41]. Similarly, the combination of OXP with SBHA and ENT (MS275) exhibited synergistic cytotoxicity against colorectal cancer cell lines [42].

To understand the mechanism of synergistic interaction between OXP and ENT, we performed HDAC inhibition and apoptosis assays following monotherapy and combination therapy. The HDAC inhibition using western blotting analysis revealed that combining OXP with ENT significantly upregulated the acetylated histone expression in pancreatic cancer cell lines (Fig. 2). Similarly, the combination treatment notably enhanced the cleave caspase-3 expression in pancreatic cancer cells (Fig. 3). The synergistic interaction of combination therapy was also evaluated via apoptosis assay. A significantly higher number of apoptotic cells were observed in pancreatic cancer cell lines treated with the OXP and ENT combination than in monotherapy (Fig. 4). Therefore, an elevated level of acetylated histone and cleaved caspase 3 following combination therapy might be responsible for the synergistic cytotoxicity.

The selective killing of tumor tissues while minimizing harm to healthy tissues is an ideal approach for tumor therapy, yet making it a reality is difficult [43]. Over the years, nanoparticles have been extensively used for tumor-targeted delivery of chemotherapeutics [12, 44–47]. Therefore, nanocarrier-based OXP and ENT formulations were designed to achieve targeted delivery compared to their solution counterparts. OXP and ENT-loaded nanoparticles were prepared separately to ensure drug release at synergistic ratios. ENT was encapsulated in PLGA through solvent emulsification methods, while OXP was embedded in BSA via the desolvation cross-linking method. Initially, we tried to encapsulate OXP in the PLGA matrix. However, OXP, being a hydrophilic compound, resulted in very low entrapment (≤ 1%). On the other hand, OXP demonstrates a very high protein binding (> 90%), which motivates us to formulate albumin-based OXP nanoparticles.

Both the nanocarriers showed suitable parameters (Fig. 5) in terms of size, zeta potential, and release. In vitro drug release studies of ENT-PLGA and OXP-BSA nanoparticles showed an initial burst release (< 20%) in the first hour, followed by a sustained release up to 7 days (Fig. 5C). The initial burst release is a characteristic of matrix-type nanoparticles and is primarily due to the drug molecules that are loosely bound or present on the nanoparticle surface.

In vitro cytotoxicity was conducted to compare the cytotoxic potential of various formulations. The nanoformulations exhibited significantly reduced PDAC cell proliferation than the unencapsulated formulations, either as monotherapy or in combination (Fig. 6). Although in vitro cytotoxicity assays confirmed the synergistic interaction between OXP and ENT, a colony formation assay was performed to determine the long-term therapeutic potential of treatments on the ability of cells to survive and proliferate to form colonies [48]. Although we found a small number of colonies for the ENT (10 μM) solution, there were no visible colonies for the 5 μM OXP solution and the combination treatment groups. Therefore, colony formation assay was performed with ENT (1 μM), OXP (1 μM), and their combination. The OXP and ENT combination treatment resulted in fewer viable colonies than the individual drugs. Furthermore, the nanoformulation-treated groups showed a significantly lower number of colonies than their solution counterpart in both the pancreatic cancer cell lines, illustrating nanoparticle effectiveness for PDAC treatment (Fig. 7). Collectively, our data established the importance of combination therapy in PDAC, which can be further explored to develop novel therapeutics to alleviate resistance mechanisms in chemotherapy and examine the potential therapeutic edges in clinical settings in PDAC.

5. Conclusions

Pancreatic cancer is one of the deadliest human cancers, with a five-year survival rate of only 12%, indicating poor performance of the current treatment regimen. Here, we reported a novel therapeutic strategy against pancreatic cancer by combining a novel HDAC inhibitor ENT with a DNA-damaging agent, OXP. The combination therapy exhibited synergistic cell killing against murine and human PDAC cells over a wide range of drug concentrations. The synergistic cytotoxicity could be attributed to a more significant HDAC inhibition and a higher extent of apoptosis by the combination treatment. To enhance the therapeutic ability of this drug combination, both OXP and ENT were encapsulated in nanocarriers. In the future, we are planning to perform biodistribution and in vivo efficacy of the nanoencapsulated combination therapy in an orthotopic model of pancreatic cancer.

Supplementary Material

1
2

Acknowledgments

We greatly acknowledge the financial support provided by the National Institute of General Medical Sciences (NIGMS) COBRE award 2P20 GM109024 and DaCCoTA CTR pilot ready-to-go grant U54GM128729. V.S. and S.M. also acknowledge financial support from the NIH grant 2 R01GM114080.

Footnotes

Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors report financial support was provided by National Institute of General Medical Sciences

CRediT authorship contribution statement

Paras Mani Giri: Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. Ashish Kumar: Methodology, Investigation, Formal analysis. Philip Salu: Methodology, Investigation, Formal analysis. Venkatachalem Sathish: Writing – review & editing, Supervision, Resources. Katie Reindl: Writing – review & editing, Supervision. Sanku Mallik: Writing – review & editing, Supervision, Resources. Buddhadev Layek: Writing – review & editing, Writing – original draft, Resources, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.biopha.2024.116743.

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Supplementary Materials

1
NIHMS2000712-supplement-1.pdf (190.1KB, pdf)
2
NIHMS2000712-supplement-2.pdf (3.3MB, pdf)

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