This article is mentioned in:

Introduction

Gastric cancer is the fifth most common tumor and the fourth leading cause of cancer related death worldwide. Although the incidence and mortality rates of gastric cancer have been declining in previous decades, 1,000,00 newly diagnosed cases are still reported each year, and close to 800,000 deaths from gastric cancer were estimated in 2020 (1). Moreover, ~10% of all cancer-related deaths are caused by gastric cancer (1,2). Gastric cancer may be classified into two main subtypes, the intestinal and diffuse type, using the Laurén classification, based on histological appearance (3). Intestinal gastric cancer represents 54% of all gastric cancers, and in general, has an higher prognostic value compared with diffuse gastric cancer (4). By contrast, the diffuse type presents in 32% of all gastric cancer, has a worse prognosis and is highly invasive and prone to metastasis (5).

Approximately one-third of all gastric cancer cases advance into metastatic sites, usually into the liver, peritoneum, lungs and bones (6,7). The clinical outcome for patients with advanced gastric cancer is poor, with a 5-year survival rate of 5–20% and a 10-month median overall survival time (8). The low survival time may be attributed to a lack of early diagnosis, as well as the high rate of recurrence and metastasis (9,10). Nonetheless, the majority of therapies such as surgical or endoscopical resection and peri-operative or adjuvant chemotherapies only target primary tumors and there is no standard regimen for metastatic or advanced gastric cancer. The drugs frequently used as first-line therapy are cisplatin or oxaliplatin, pyrimidine analogs such as 5-fluorouracil or capecitabine and anthracyclines, such as doxorubicin or epirubicin (11). However, when the cancer has metastasized, chemotherapy has only a palliative effect, and elevated concentrations of these drugs may produce toxic adverse effects in patients (12,13).

Due to the poor response of advanced gastric cancer to conventional chemotherapy, alternative therapeutics such as monoclonal antibodies, which include trastuzumab, ramucirumab, pembrolizumab and nivolumab, have been evaluated and approved by the United States Food and Drug Administration (8,14,15). Although positive results with the use of these drugs have been reported (16–19), there are a number of associated limitations. For instance, patients with HER2-positive tumors may potentially benefit from monoclonal antibody therapy using trastuzumab, hence there is a need to identify clinically useful predictive biomarkers (20,21). In addition, further studies are necessary to advance the current understanding of the adverse effects caused by monoclonal antibody therapies (22,23). Furthermore, clinical outcomes were most improved when these drugs were administered in combination with chemotherapy, despite an increased risk of adverse reactions (14,24–26).

Retinoids, which are natural and synthetic vitamin A derivatives, are ligands of retinoic acid receptors (RARs) and retinoic X receptors. In the nucleus, these receptors serve key roles in the regulation of cellular processes, including differentiation, cell growth and cell death. Due to their effects promoting cell differentiation, the naturally-occurring retinoids, all-trans retinoic acid (ATRA) and 13-cis-retinoic acid, are frequently used in pediatric oncology, primarily in acute myeloid leukemia and for the treatment of skin diseases such as psoriasis, acne vulgaris and keratinization disorder (27–29). However, treatments using these compounds on solid tumors have been less effective, mainly attributed to difficulties in effective delivery due to their limited solubility in aqueous solutions and reduced half-life in plasma (30,31). Fenretinide [N-(4-hydroxyphenyl) retinamide], also termed 4-HPR, is a synthetic amide of retinoic acid. Fenretinide has been used as a chemopreventive agent against breast cancer (32). In vitro, fenretinide has shown cytotoxic effects in neuroblastoma, melanoma and breast cancer cell lines (33–35), and it is well established that the mechanisms that mediate fenretinide cytotoxicity include p53-and caspase-independent apoptosis, activation of the cellular stress response and the induction of autophagy (36–38). In the previous 20 years, there have been several clinical trials involving fenretinide in patients with different solid tumors such as neuroblastoma, and breast, prostate and bladder cancer, however, the majority of the results from these trials have been less promising compared with those from in vitro studies (32,39–44). A predominant limitation is the low oral bioavailability of fenretinide, thus only a low concentration reaches the plasma. However, in the last five years, novel research has focused on developing improved oral formulations to achieve higher plasma drug concentrations (45,46).

The aim of the present study was to evaluate the effect of the retinoid fenretinide on the viability and migration of two cell lines that serve as models of primary and metastatic intestinal gastric cancer. To achieve this, the effect of several chemotherapeutic agents and the natural retinoid ATRA on the viability of these cell lines were compared, and the possible beneficial effect of combining fenretinide and cisplatin was evaluated.

Materials and methods

Cell culture

Human gastric cancer AGS (CRL-1739) and NCI-N87 (CRL-5822) cell lines were purchased from the American Type Culture Collection. The AGS cell line was derived from a gastric adenocarcinoma that was located in the stomach (47), whereas the NCI-N87 cell line was derived from a liver metastasis of a gastric carcinoma (48). Both cell lines were cultured in RPMI-1640 medium (cat. no. R8758; Sigma-Aldrich; Merck KGaA) supplemented with 10% fetal bovine serum (FBS; cat. no. F2442; Sigma-Aldrich; Merck KGaA) and 100 U/ml penicillin-streptomycin (cat. no. 15140122; Gibco; Thermo Fischer Scientific, Inc.) at 37°C in a humidified incubator containing 5% CO2.

Evaluation of cytotoxicity using the MTT assay

AGS (1.5×104 cells/well) and NCI-N87 (3×104 cells/well) cells were seeded in 96-well plates and incubated at 37°C overnight. After which, cells were exposed to different concentrations of cisplatin (Pfizer, Inc.), doxorubicin (cat. no. 44583; Sigma-Aldrich; Merck KGaA), ATRA (cat. no. R2625; Sigma-Aldrich; Merck KGaA) or fenretinide (cat. no. H7779; Sigma-Aldrich; Merck KGaA) for 48 h at 37°C. As all the drugs evaluated were light-sensitive, drugs and treated cells were protected from light. Doxorubicin and the retinoids, ATRA and fenretinide, were dissolved in dimethyl sulfoxide (DMSO) according to the manufacturer's instructions. Cisplatin solution was directly dissolved in cell culture media.

After the treatments, the media was removed and the cells were incubated with MTT (final concentration, 0.5 mg/ml) solution dissolved in RPMI-1640 medium for 2 h at 37°C. The media was then removed and 95% ethanol was added to the wells to dissolve the formazan crystals. Cells incubated in media with only the vehicle (DMSO<0.05%) were used as a control and were used to define 100% viability. Absorbance values were determined at a wavelength of 570 nm using a microplate reader (BioTek Cytation 3 Imaging Multi-Mode Reader; Biotek; Agilent Technologies, Inc.).

Evaluation of the combined effect of fenretinide and cisplatin

AGS (1.5×104 cells/well) and NCI-N87 (3×104 cells/well) cells were seeded in 96-well plates and incubated at 37°C overnight. Fenretinide and cisplatin were added simultaneously for 48 h and the experiment was conducted with non-fixed ratio combinations. Two doses of cisplatin were selected, corresponding to values below the calculated IC50. By contrast, three doses of fenretinide were chosen, one dose at the IC50 value, one below it and one above it. This strategy aimed to evaluate whether the combination with fenretinide could reduce the concentration of cisplatin required, thereby mitigating the adverse effects associated with cisplatin.

Combined drug action was studied through median effect analysis according to the Chou-Talalay method (49). The combination index (CI) for non-constant combination and drug reduction index (DRI) were calculated using the CompuSyn software (version 1.0; ComboSyn, Inc.). The DRI provided an estimation of the extent to which a dose of an agent in combination can be decreased to achieve effect levels similar to a single drug. The CI was defined as follows: CI<1, synergistic effect; CI>1, antagonistic effect; and CI=1, additive effect.

Determination of reactive oxygen species (ROS) production

Intracellular generation of ROS was measured using 2′,7′-dichlorofluorescein diacetate (DCFDA; cat. no. D6883; Sigma-Aldrich; Merck KGaA). DCFDA is a non-fluorescent probe that is internalized by the cell and when oxidized by ROS, is converted into the highly fluorescent 2′,7′-dichlorofluorescein. AGS (1.5×104 cells/well) cells were seeded in black 96-well plates and left to adhere at 37°C for 5 h, before treatment with fenretinide (3 µM) for 20 h at 37°C. As a positive control of ROS production, cells were also treated with the oxidizing reagent tert-butyl hydroperoxide solution (TBHP; 1 mM; cat. no. B2633; Sigma-Aldrich; Merck KGaA) at 37°C for 2 h. Before the conclusion of TBHP incubation, the cells were incubated with DCFDA (60 µM) at 37°C for 30 min. Finally, the wells were rinsed twice with phosphate-buffered saline (PBS) and the intensity of the fluorescence was assessed using a BioTek Cytation 3 Imaging Multi-Mode Reader (Biotek; Agilent Technologies, Inc.), using 485 nm as the excitation wavelength and 528 nm as the emission wavelength. The basal production of ROS was determined in cells treated with medium alone; this value was subtracted from the fluorescence detected when cells were incubated with fenretinide.

Role of ROS inhibition on cell viability

AGS (8×103 cells/well) and NCI-N87 (1.6×104 cells/well) cells were seeded in 96-well plates and incubated at 37°C overnight. Cells were then treated with the antioxidant quercetin (80 µM) for 1 h before the addition of fenretinide (20–40 µM) or TBHP (0.6 µM, AGS; 4.8 µM, NCI-N87) as a positive control of ROS production for 4 h at 37°C. For quercetin, a concentration of 80 µM was selected as it was the highest dose that did not show toxicity in either cell lines. In the case of fenretinide, concentrations >IC50 value that were reported from the MTT assay were used as the experiments were performed at 4 h instead of 48 h.

As various antioxidants interfere with the MTT assay, the sulforhodamine B assay was instead used to determine cell viability. Briefly, cells were fixed in 10% trichloroacetic acid for 1 h at 4°C and stained with 0.04% sulforhodamine B solution (cat. no. S1402; Sigma-Aldrich; Merck KGaA) for 30 min at room temperature. After which, the wells were rinsed three times, eachfor 30 sec, with 1% acetic acid at room temperature to remove the excess dye. Protein-bound dye was dissolved in 10 mM Tris base solution and the absorbance was determined at 530 nm using a microplate reader (BioTek Cytation 3 Imaging Multi-Mode Reader; Biotek; Agilent Technologies, Inc.).

Intracellular lipid droplet (LD) staining and fluorescence quantification

AGS (8×103 cells/well) and NCI-N87 (1.6×104 cells/well) cells were seeded in black 96-well plates and incubated at 37°C overnight. After which, cells were treated with a moderately toxic dose of fenretinide (10 µM), which corresponded to the IC25 value or avasimibe (20–40 µM) for 48 h. A fenretinide concentration <IC50 value was selected as it allowed detection of a toxic effect while ensuring that most of the visual fields contained sufficient cells for meaningful quantification. This approach was particularly important as visual fields were randomly selected. Avasimibe (cat. no. PZ0190; Sigma-Aldrich; Merck KGaA), an inhibitor of acyl-CoA:cholesterol O-acyltransferases (ACATs), reduces LD synthesis and was therefore used as a control.

Cells were fixed with 4% paraformaldehyde for 10 min at room temperature and incubated in a 0.5 µg/ml solution of Nile red (cat. no. 19123; Sigma-Aldrich; Merck KGaA) in PBS for 10 min at 37°C. Images were captured using a fluorescence microscope with a red fluorescence protein filter (RFP; 531 and 593 nm) with a 20X objective under the same conditions of LED exposure, including integration time and gain, using a BioTek Cytation 3 Imaging Multi-Mode Reader (Biotek; Agilent Technologies, Inc.). For each treatment, images were captured from five independent wells. The Gen5 Image software (version 3.09; Agilent Technologies, Inc.) was used to quantify the fluorescence intensity. All images from each cell line were included in the experiment and an automatic setting was applied for preprocessing for RFP and bright-field channels. The cellular analysis tool was used to select the areas containing cells in the bright-field images, with threshold values for AGS and NCI-N87 of 500 and 4,000, respectively. The object size selection was set between 15–500 µm for both cell lines. The total signal fluorescence object sum intensity [Tsf (RFP 531,593)], total signal fluorescence object sum area [Tsf (bright field)] and the relative object sum intensity/object sum area were calculated for all images. The mean value and standard deviation were determined and statistical analyses were performed.

Wound healing assay

AGS (1×106 cells/well) cells were seeded on plastic 24-well plates and incubated at 37°C overnight. Wounds were scraped in the middle of the well with a sterile pipette tip. To remove floating cells, wells were carefully rinsed twice with PBS. After which, cells were incubated with medium or fenretinide at a nontoxic concentration (6 µM) for 24 h. Each condition was performed in duplicate in media containing 5% FBS. AGS cell viability decreases considerably when FBS is completely depleted (50). It has been established that, when necessary, the medium can be supplemented with 2–5% FBS to maintain cell health during the assay (51). Since a low concentration of FBS was used in both the control and treated groups, cell proliferation was monitored through the entire wound-healing assay using the sulforhodamine B assay (data not shown). Light microscopy pictures were captured using a microplate reader (BioTek Cytation 3 Imaging Multi-Mode Reader; Biotek Instruments, Inc.) at 0 and 24 h time points. The percentage migration was calculated as the mean of three random measurements in each picture and expressed as a percentage of the control (time 0 h for each treatment) using the ImageJ software (version 1.49; National Institutes of Health).

Statistical analysis

For all the viability assays, dose-response curves were generated and the IC50 values were calculated using the Slide Write Plus software (version 6.10; Advanced Graphics Software, Inc.). All quantitative experiments were independently repeated three times and data were expressed as the mean ± standard deviation. To evaluate the significance of the results between two groups, the Mann-Whitney test or independent t-test was applied. For comparisons of ≥3 groups, one-way ANOVA followed by Bonferroni's post-hoc test was applied. All tests were performed using GraphPad Prism (version 5; Dotmatics) or SPSS (version 22; IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.

Results

Effect of cisplatin and doxorubicin on cell viability

The present study evaluated whether the AGS and NCI-N87 cell lines had a different response to cisplatin and doxorubicin, which are both frequently used to treat gastric cancer. The primary gastric cancer cell line AGS was sensitive to both cytotoxic agents, however, doxorubicin was more efficient, as the IC50 value was 0.39 µM, compared with cisplatin that had an IC50 value of ~20 µM (Table I). The metastatic cell line NCI-N87 demonstrated the opposite effect; cells exhibited a notably low response to doxorubicin, with doses as high as 90 µM being poorly toxic, since cell viability remained close to 80%. By contrast, both cell lines were affected in a similar manner by cisplatin treatment at concentrations <20 µM (Fig. S1). However, at concentrations >20 µM, AGS cells presented lower viability compared with the NCI-N87 metastatic cell line.

Table I. IC50 values for retinoids and chemotherapy agents for AGS and NCI-N87 cells after 48 h.

Table I.

IC50 values for retinoids and chemotherapy agents for AGS and NCI-N87 cells after 48 h.

Retinoids/chemotherapy agents	AGS (IC50, µM)	NCI-N87 (IC50, µM)
Cisplatin	18.49±4.54	27.25±2.74
Doxorubicin	0.39±0.02	>90.00a
All-trans retinoic acid	46.47±3.73	122.07±4.86a
Fenretinide	13.91±1.17	19.65±0.96

{ label (or @symbol) needed for fn[@id='tfn1-ol-28-6-14694'] } A comparison of IC₅₀ values between cell lines, for each treatment at the same time point. Values are presented as the mean ± standard error of three independent experiments.

a P<0.001.

Effect of fenretinide and ATRA on cell viability

The impact of the synthetic retinoid, fenretinide, was assessed using an MTT assay and its effects compared with those of its natural analog ATRA were recorded. ATRA was used to compare whether fenretinide-treated cells showed the same response observed with doxorubicin or cisplatin. Since ATRA has been shown to be ineffectively delivered in solid tumors (52), it was hypothesized in the present study that for fenretinide to be promising as a therapeutic drug in gastric cancer, the IC50 values should be significantly lower than those for ATRA.

The results of the present study showed not only that NCI-N87 cells were less sensitive to ATRA compared with AGS cells, but also that both cell lines had lower IC50 values when treated with fenretinide compared with ATRA. In fact, both cell lines had a similar response when treated with fenretinide, and the IC50 values were in a similar range as those obtained when using cisplatin (Table I; Fig. S1).

As MTT assays are primarily based on the activity of mitochondrial enzymes and cell metabolic health (53,54), a sulforhodamine assay was also performed to allow the determination of cell density based on the measurement of cellular protein content (55). The results obtained using the MTT and sulforhodamine assays were consistent with each other (Fig. S1C-F).

Potential synergism of fenretinide and cisplatin on cell viability

It has been suggested that retinoids could be used as co-adjuvants in combination with chemotherapy (56), therefore it was evaluated whether fenretinide increased the toxic effect of cisplatin on gastric cancer cells. Cisplatin was selected as it was the only chemotherapy drug that showed a toxic effect on both cell lines in the present study. Additionally, the current first-line treatment for advanced or metastatic unresectable gastric cancer is a dual chemotherapy regimen based on a platinum compound and fluoropyrimidine, which suggests that anthracyclines may not be needed for optimal results (57).

Following combined treatment with fenretinide and cisplatin for 48 h, CompuSyn software was used to calculate the CI and DRI for cisplatin. The combination of three non-fixed toxic doses of fenretinide (similar to IC50) with two different doses of cisplatin was evaluated (Table II; Fig. 1). Based on the CI value, it was suggested that in both cell lines, there may be an additive or partial synergistic interaction between both drugs when a high fraction of cells is affected (fraction affected >0.7). Notably, the additive effect was stronger in the NCI-N87 metastatic cell line; combination index values were closer to 0.9 and the drug reduction index values for cisplatin were at least twice those observed in AGS cells. However, when a low fraction of cells were affected, it was possible to observe CI values that suggested antagonism. However, for anticancer agents, synergy is more relevant to therapy when a high fraction of cells are affected (49).

Figure 1. Effect of non-constant combinations of Fen with CP. Dose response curves, CI plots and normalized isobolograms were obtained through computer simulation using the Compusyn software. Representative graphs of the (A) dose-effect curve, (B) CI plot and (C) normalized isobologram for AGS cells are presented. Representative graphs of the (D) dose-effect curve, (E) CI plot and (F) normalized isobologram for NCI-N87 cells are presented. Each graph corresponds to a representative experiment of each cell line. For each cell line, three independent experiments were performed. In the dose-effect curves the solid and dashed lines correspond to the dose-effect curves for Fen and CP, respectively, each administered as a single therapy. Fen, fenretinide; CP, cisplatin; CI, combination index.

Table II. CI and DRI values for non-constant combinations of fenretinide and cisplatin after 48 h.

Table II.

CI and DRI values for non-constant combinations of fenretinide and cisplatin after 48 h.

Cell line	Cisplatin, µM	Fenretinide, µM	Fraction of affected cells (range, 0–1)	CI	DRI for cisplatin
AGS	5	10	0.46±0.01	1.37±0.02	2.15–2.70
		15	0.74±0.01	0.96±0.02	5.63–8.78
		20	0.82±0.01	0.99±0.01	5.63–8.78
	10	10	0.55±0.02	1.40±0.06	8.79–11.93
		15	0.78±0.01	0.95±0.02	3.54–5.34
		20	0.84±0.02	1.01±0.05	4.82–7.68
NCI-N87	5	15	0.45±0.02	1.21±0.05	2.66–3.83
		25	0.67±0.01	0.92±0.07	11.25–18.80
		30	0.73±0.02	0.88±0.07	14.84–30.83
	20	15	0.53±0.02	1.45±0.10	1.17–1.74
		25	0.67±0.01	1.14±0.12	2.63–4.70
		30	0.72±0.02	1.07±0.02	3.93–5.66

[i] Values are presented as the mean ± standard error of three independent experiments performed. CI>1 indicates antagonism; CI=1 indicates an additive effect; and CI<1 indicates synergism. DRI is the dose reduction required for cisplatin when used in combination with fenretinide. CI, combination index; DRI, drug reduction index.

Effect of fenretinide on ROS levels

Cancer cells exhibit increased ROS stress compared with normal cells, therefore targeting ROS levels may be useful as a therapeutic strategy (58). It was assessed whether fenretinide could induce an alteration in redox homeostasis. Following preincubation of both cell lines with the antioxidant quercetin and fenretinide, protection from cell death was only demonstrated in AGS cells (Fig. 2A and B). Nonetheless, the results of the present study suggested that other mechanisms besides ROS induction may also be implicated, as cell viability did not recover to levels similar to those in the control conditions. By contrast, for NCI-N87 metastatic cells, ROS production did not appear to be relevant for this effect as fenretinide did not enhance cell viability following TBHP exposure. The increase in ROS stress induced by TBHP was confirmed by the fact that the antioxidant quercetin was able to restore cell viability.

Figure 2. Effect of Fen on ROS levels and the role of ROS on the viability of gastric cancer cell lines treated with Fen. (A) Viability of AGS cells treated for 48 h with Fen after preincubating with Qrc. (B) Viability of NCI-N87 cells treated for 48 h with Fen after preincubating with Qrc. The oxidizing agent TBHP was used as a control for ROS induction. Values are expressed as the mean ± standard error of three independent experiments. (C) Fluorescence of the DCFDA probe after AGS cells were treated with Fen for 20 h. Values are presented as the mean ± standard error of three independent wells *P<0.05, **P<0.01 and ***P<0.001.UA, units of absorbance; ROS, reactive oxygen species; TBHP, tert-butyl hydroperoxide solution; Qrc, quercetin; DCFDA, 2′,7′-dichlorofluorescein diacetate; Fen, fenretinide.

Notably, the TBHP concentration used to produce minimal toxic effect was increased ~8-fold compared with AGS cells.

To confirm the results observed in AGS cells, a DCFDA probe was used to detect intracellular ROS levels after treatment with fenretinide (Fig. 2C). After 20 h, a significant elevation in ROS was detected. However, this effect was smaller than the effect of TBHP, which was used as a positive control as it is a well-established ROS inducer (59).

Effect of fenretinide on LD content

Autophagy is implicated in fenretinide induced cell death (38). Xu and Fan (60) reported that LD content can be modified by autophagy. To assess this in the present study, AGS and NCI-N87 cells were stained with Nile red, a lipophilic agent that stains intracellular LDs. As a control, cells were incubated with avasimibe, an ACAT inhibitor that reduces LD formation (61).

In AGS cells, fenretinide significantly reduced Nile red fluorescence, which suggested a decrease in the number of LDs. By contrast, NCI-N87 cells treated with fenretinide showed no change in Nile red fluorescence in comparison with cells without treatment. Of note, when the NCI-N87 metastatic cell line was treated with avasimibe, LD content was significantly increased compared with the control cells (Fig. 3). A previous study reported that NCI-N87 cells regulate intracellular cholesterol differently from AGS cells. Furthermore, it was reported that NCI-N87 cells were more resistant to the effect of avasimibe when cell viability was assessed, suggesting the presence of a compensatory mechanism for cholesterol esterification in addition to ACAT-1 (50). This may partially explain why when treated with an inhibitor of LD formation, the opposite effect was observed. Since avasimibe changed LD content in these cells and fenretinide did not, these results suggest that fenretinide is not affecting the autophagy mechanisms that may break down LDs.

Figure 3. Effect of Fen on lipid droplet content. (A) Nile red staining of intracellular lipid droplets in gastric cancer cells. (B) Fluorescence quantification adjusted by area. Ava acts by inhibiting ACAT-1 and therefore reduces lipid droplet synthesis. Values are expressed as the mean ± standard error of at least five independent images. *P<0.05, **P<0.01 and ***P<0.001. Fen, fenretinide; Ava, avasimibe; ACAT1, acyl-CoA: cholesterol O-acyltransferase; RFU, relative fluorescence units.

Effect of fenretinide on cell migration

Previous studies have demonstrated that, under basal conditions, NCI-N87 cells require a prolonged period, usually 48 h, to exhibit a migration effect in the wound healing assay (62–64). In comparison with other gastric cancer cell lines, including AGS cells, NCI-N87 cell line demonstrate a more differentiated epithelial phenotype characterized by high expression levels of E-cadherin and zonula occludens proteins (65). This phenotype facilitates the formation of highly cohesive monolayers, thereby hindering their migration. As NCI-N87 cells have a limited capacity to migrate in the wound healing assay, only the effect of fenretinide in the migration of AGS cells was evaluated. After 24 h incubation with a nontoxic concentration of fenretinide, a significant reduction in the capability of AGS cells to migrate and close the wound was observed (Fig. 4).

Figure 4. Migration of AGS cells treated fenretinide for 24 h. (A) Representative images from a wound healing assay of control cells and cells treated with fenretinide. (B) Degree of migration is presented as the percentage of the wound measurement from 0 h. Values are expressed as the mean ± standard error of at least three independent experiments performed in duplicate. *P<0.05. Fen, fenretinide.

Discussion

A major limitation in gastric cancer treatment is the lack of early diagnosis. For advanced gastric cancer, surgery is not curative and conventional treatments such as radiotherapy and chemotherapy achieve very modest outcomes (57). Despite the use of several combinations of chemotherapeutics in advanced gastric cancer, the median overall survival time remains <1 year (66). There is consequently a need to investigate alternative options that overcome resistance to chemotherapy.

In the present preclinical study, AGS and NCI-N87 cell lines were used as they are well- characterized models of intestinal gastric cancer (48,67,68). AGS is a primary gastric cancer cell line that is considered to be highly invasive and moderately differentiated (69). The metastatic cell line NCI-N87 demonstrates upregulated levels of HER2, and thus has been extensively used as a model system for studying trastuzumab, and its mechanisms of acquired resistance over time in gastric cancer (70). Trastuzumab, a humanized monoclonal antibody that targets the extracellular domain of HER2, has shown significant therapeutic benefit in the treatment of patients with HER2-overexpressing gastric cancer (16). Simeone et al have demonstrated that HER2 overexpression reduced the sensitivity of breast cancer cells to fenretinide (71).

In the present study it was determined that the AGS and NCI-N87 cell lines respond differently to the chemotherapeutic agents, cisplatin and doxorubicin, demonstrating that the NCI-N87 cell line was markedly insensitive to doxorubicin, while AGS cells were affected at concentrations less than 1 µM. By contrast, it was demonstrated that both cell lines had similar sensitivity to cisplatin, especially at doses corresponding to the IC50 values. It has been hypothesized that the use of ATRA could be beneficial for the treatment of gastric cancer; however, its main limitations include its low availability and high resistance (30). Hence, the present study aimed to assess the cellular response of both cell lines to fenretinide, with the response to ATRA serving as a comparative benchmark. It was observed that NCI-N87 cells showed less sensitivity compared with AGS cells. However, in both cell lines, the IC50 values were more than twice as high as those observed for fenretinide. These results were consistent with those observed by Guarrera et al where, from a panel of gastric cancer cell lines, they classified AGS and NCI-N87 cells as low ATRA-sensitive (72).

The presence of a benzene ring instead of a cyclohexane ring allows fenretinide to be more hydrophilic, resulting in a higher bioavailability compared with ATRA (73). To the best of our knowledge, there is only one previous study evaluating the effect of fenretinide on gastric cancer cells. Liu et al reported that fenretinide inhibited the growth of the gastric cancer BGC 823 cell line and suggested that the effect was partly mediated by RARβ2 (74). In the present study, it was demonstrated that fenretinide not only had a similar toxic effect on both AGS and NCI-N87 cell lines, but according to the IC50 values, this effect was comparable with those observed for cisplatin.

The idea combining naturally occurring agents with conventional chemotherapeutic agents has been gaining interest (75). Although cisplatin has been extensively used to treat gastric cancer, drug resistance and adverse side effects are its two main limitations. It has been previously suggested that combining cisplatin with certain natural products such as flavonoids, saponins and alkaloids may enable treatments to overcome the cisplatin resistance of cells and the adverse side effects of cisplatin use (76). The present study did not investigate the doxorubicin-fenretinide combination because a primary aim was to evaluate fenretinide as a potential co-adjuvant therapy for the treatment of metastatic tumors, as they present a poor response to current chemotherapy strategies. There are several chemotherapy regimens used for gastric cancer, and some of which involve simultaneous administration of three chemotherapy drugs that include doxorubicin or epirubicin in addition to cisplatin and 5-fluorouracil; however, according to the European Society of Medical Oncology, for metastatic gastric cancer the response rate is higher with dual therapy, combining only cisplatin and 5-fluorouracil (57).

The results of the present study indicated that combination of cisplatin with fenretinide had an additive effect in primary and metastatic gastric cancer cells, and according to the dose reduction index, this combination could enable cisplatin dose reduction up to 20-fold, which potentially benefits the survival of patients with gastric cancer by reducing the adverse effects of cisplatin. Fenretinide as an oral capsule has been reported to be well tolerated in clinical studies (35). However, achieving an optimal plasma drug concentration has been an obstacle due to the low bioavailability of fenretinide (35,73). Nonetheless, novel attempts to improve fenretinide solubility have been under development, including oral formulations with improved bioavailability and intravenous formulations that result in significantly higher plasma levels, alongside acceptable toxicity responses (73,77–80).

Multiple studies have suggested that fenretinide-induced cell death occurs through apoptosis, either through the production of ROS or the involvement of lipid secondary messengers such as dihydroceramide and dimethylsphingosine (38,81–86). In the present study it was observed that fenretinide induced ROS production in AGS cells. Furthermore, it was determined that ROS were relevant in the fenretinide-associated mechanism of cell death only in the primary gastric cancer cells. Lai and Wong (84) reported that in leukemia and cervical cancer cells, fenretinide caused a rapid increase in ROS levels, and this triggered the activation of the unfolded protein response. In addition, Darwiche et al (82) described in leukemia cells that fenretinide induced growth inhibition associated with ROS accumulation at early time points (<12 h), therefore it is possible that for the NCI-N87 cell line, the time evaluated (20 h) was not sufficient for this effect to be observed in the present study. However, it appears that NCI-N87 cells have different compensatory mechanisms to protect against oxidative stress as the response to the ROS-inducer TBHP was also low when compared with AGS cells.

Autophagy has a dual role in cancer by inhibiting tumor initiation but also supporting a pro-tumorigenic role in established disease and mediating treatment resistance (87). It has been previously reported that fenretinide is able to trigger an autophagic cell death pathway in breast cancer cells (38). As autophagy regulates the mobilization of fat from cellular deposits, such as LDs, Nile red staining was used as a means to indirectly study autophagy in fenretinide-treated cells. Notably, fenretinide treatment significantly reduced the number of LDs in the AGS cell line, but in NCI-N87 cells there was no significant effect when compared with the control. The effect of autophagy on the levels of LD formation has not been elucidated at present. However, Nguyen et al (88) reported that during autophagy, under starvation conditions, the amount of LDs increased (88). Although, this association between autophagy and LD content is complex, as in liver cells under starvation or induction by an acute stimulus, a selective autophagy process termed lipophagy degrades LDs in lysosomes (89). The crosstalk between LDs and autophagy still requires further study, but the results of the present study suggest that in AGS cells, fenretinide may affect autophagy pathways.

Retinoids have been reported to prevent the conversion of primary tumors to invasive malignancies by inhibiting invasion as well as angiogenesis (90,91). In the present study a significant reduction in AGS cell migration was observed when treated with fenretinide. In the liver cancer cell line HepG2, fenretinide inhibited its migration properties by regulating the p38-MAPK signaling pathway (92). There is also evidence of fenretinide acting on proteases involved in the degradation of the extracellular matrix. In vivo experiments have shown that fenretinide prevents the development of different metastases in mouse models of prostate and liver cancer (93). Due to fenretinide capacity to disrupt pro-invasive signaling pathways, its combination with chemotherapy may enhance treatment outcomes in patients with advanced gastric cancer.

Although fenretinide had a similar cytotoxic effect in the primary and metastatic cell lines in the present study, it is clear that there are different mechanisms involved in the cytotoxic effects that should be further investigated. The results of the present study suggest that in the metastatic cell line NCI-N87, neither ROS nor autophagy mediate the fenretinide mechanism of action. Apraiz et al (85) reported that in human leukemia cells, sphingolipid accumulation and ROS induction were not necessarily essential in fenretinide-mediated cytotoxicity.

Overall, the present study provided preclinical evidence that fenretinide exhibits anti-tumoral activity on primary gastric cancer cells through the induction of ROS, possibly due to cell death by autophagy. Moreover, fenretinide may be a potential therapeutic option for metastatic gastric cancer as it may inhibit cell migration and act together with cisplatin. Currently, there is an imperative need to improve the poor prognosis resulting from conventional chemotherapy in patients with advanced gastric cancer, and further preclinical research evaluating the potential benefits of fenretinide alone or as a co-adjuvant for the treatment of gastric cancer, especially for metastatic tumors, should be performed.

The main limitation of the present study was that only two cancer cell lines were evaluated. However, these cell lines are well-characterized and have been widely used in previous literature (15,48,65,94–96). Future studies should examine the effect of fenretinide in other gastric cancer cell lines derived from metastatic sites. Additionally, given the complex role of autophagy in cancer, further studies are required to determine the causative mechanisms of the effect of fenretinide on primary cell lines, and whether the targeting of autophagy could be a promising therapeutic strategy for gastric cancer.

Although improving fenretinide low drug solubility remains a challenge, as aforementioned, in recent years there has been a growing interest in developing improved oral formulations and using alternative routes of administration that may overcome the low plasma concentration seen in earlier clinical trials. The results of the present study provide a basis for further research that could aid the understanding of the molecular mechanisms responsible for the cytotoxic effect and inhibition of migration caused by fenretinide, either alone or in combination with cisplatin, particularly those observed with the metastatic cell line. It is important to continue the investigation of novel therapeutic alternatives for primary and metastatic gastric cancer that may improve the survival of patients with this disease and reduce the adverse effects of the use of chemotherapy at high doses.

Supplementary Material

Supporting Data

Acknowledgements

Not applicable.

Funding

The present study was supported by Vicerrectoría de Investigación, Universidad de Costa Rica (grant no. 741-B5-104).

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

NO and CD conceived and designed the study, analyzed the results and wrote and reviewed the manuscript. NO and CD confirm the authenticity of all the raw data. Both authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References