A Combination of SAHA and Quinacrine Is Effective in Inducing Cancer Cell Death in Upper Gastrointestinal Cancers Free

Gastric and esophageal cancers cause over 1,000,000 deaths each year worldwide (1). Although the incidence of most cancers is declining, esophageal adenocarcinoma is rising fast in the Western world (2). At the time of diagnosis, upper gastrointestinal cancers (UGCs) are often advanced or metastatic with an increased morbidity and mortality (3, 4). The majority of patients with UGCs have poor response to current therapeutic modalities with frequent tumor recurrence (5, 6). Despite significant improvements in surgical outcome, radiation techniques, and chemotherapy, the 5-year survival rarely exceeds 20% (7, 8). Drug resistance remains a challenging problem in UGC therapy as well as in the treatment of many other types of cancer. Developing novel therapeutic approaches to overcome drug resistance is desperately needed to improve the current therapeutic response and clinical outcome.

Quinacrine, an antimalarial drug, has been shown to possess anticancer effects both in vitro (9) and in vivo (10). In the cancer cells, quinacrine can simultaneously suppress NF-κB and activate p53 signaling with limited genotoxicity (9). Quinacrine can also suppress cellular inhibitors of apoptosis, independent of p53 (8). Suberoylanilide hydroxamic acid (SAHA), the first histone deacetylase inhibitor (HDACi) approved for cancer therapy (11), was found to inhibit class I HDACs and class II HDACs. SAHA inhibits HDACs' activity and has multiple cellular effects. These include induction of cell-cycle arrest, reactive oxygen species (ROS), apoptosis, and autophagy (12–15). However, its efficacy as a single agent in cancer treatment is considered moderate (16). Several preclinical studies and early clinical trials show evidence that SAHA can be combined with other anticancer drugs (17). Of note, SAHA is well tolerated in patients and has limited toxicity, which is rapidly reversible upon discontinuation of the drug (18).

Chemotherapy remains the bedrock for the fight against cancer. Several chemotherapeutics induce DNA damage that is sensed by p53, which, depending on the level, either arrests the cell cycle to allow DNA damage repair or induces apoptosis (19). The P53 mutation is one of the most prevalent genetic alterations in gastric cancer (20). Over 50% of human esophageal cancers harbor mutant p53 (mut-p53; ref. 21). Mutant p53 is considered a tumorigenic protein that acquires gain of oncogenic properties that promote transformation, tumor growth, and increased resistance to anticancer treatments (13, 22). The high mutation rate and hyperstability of mut-p53 protein in cancer can lead to failure of response to chemotherapeutics (23). Of note, there are data that indicate wt-p53, in some instances, can be a poor predictor of chemotherapy response (24, 25). Similar to mut-p53, patients with wt-p53 may be resistant to a variety of antitumor agents, including cisplatin, doxorubicin, and 5-FU (22, 26, 27). The presence of wt-p53 in advanced cancers, such as breast cancer, chronic lymphocytic leukemia, ovarian, and renal cell carcinoma, results in a significantly lower response and/or curative rates (24, 25, 28, 29). Wild-type p53 can mediate cell-cycle arrest and inhibit drug response, even in the context of heterozygous p53 point mutations or in the absence of p21 (25).

In this study, we investigated effects of combination of quinacrine and SAHA against wt- and mut-p53 UGC models. Our findings indicate that quinacrine and SAHA combination is more effective than a single-agent treatment in inducing cancer cell death. The results indicate that this combination augments therapeutic response by increasing DNA damage levels, promoting degradation of both wt- and mut-p53 protein levels, and suppressing oncogenic signaling pathways, such as AKT and NF-κB pathways. Additional investigations are needed to test quinacrine and SAHA combination in additional preclinical and/or clinical settings to determine the therapeutic efficacy of this combination.

Gastric (AGS, SNU1, MKN28, and MKN45) and esophageal (FLO1, OE33, and OE19) adenocarcinoma cells lines were used in the study. AGS, SNU1, and MKN45 are p53 wild-type cells, whereas MKN28, FLO1, OE19, and OE33 are mutant p53 cell models (http://p53.free.fr/Database/Cancer_cell_lines/p53_cell_lines.html; Supplementary Table 1). Immortalized nonneoplastic normal esophageal cell line EPC2, kindly provided by Dr. Anil Rustgi (University of Pennsylvania, Philadelphia, PA), was included as a control. AGS and SNU1 cells were purchased from ATCC. MKN28 and MKN45 cells were obtained from the Riken Cell Bank. The human esophageal adenocarcinoma cell lines (OE33 and FLO1) were kindly provided by Dr. David Beer (University of Michigan, Ann Arbor, MI). OE19 cells were obtained from Sigma-Aldrich. AGS and MKN28 cells were cultured in F12 media (GIBCO) supplemented with 5% FBS (Invitrogen Life Technologies) and 1% penicillin/streptomycin (GIBCO). SNU1, MKN45, and FLO1 were cultured in DMEM (GIBCO) supplemented with 10% FBS and 1% penicillin/streptomycin. OE33 and OE19 cells were maintained in RPMI medium (GIBCO) supplemented with 10% FBS and 1% penicillin/streptomycin. All cell lines were ascertained to conform to the original in vitro morphologic characteristics and were authenticated using short tandem repeat profiling (Genetica DNA Laboratories). All cell lines reported here have been tested and shown to be free of mycoplasma (R&D Systems). Horseradish peroxidase–conjugated anti-mouse (7074P2) and anti-rabbit (7062P2) secondary antibodies, p-STAT3 (Y705), STAT3, p-AKT (S473), AKT, p-NF-κB (S536), NF-κB, p-H2AX, H2AX, and Apoptosis Antibody Sampler Kit (9915) and β-actin (4970) antibodies were obtained from Cell Signaling Technology.

Cells were rinsed with PBS, trypsinized, and harvested in single-cell suspension. Cells (1,000 cells/well) were seeded in 6-well plates. The next day, cells were treated overnight with quinacrine (1 or 2 μmol/L) and SAHA (1 or 2 μmol/L) or vehicle, followed by replacement with a regular drug-free culture media. After incubation for 10 days, colonies were fixed with 4% paraformaldehyde and stained with 0.05% crystal violet. The plates were imaged and stained colonies were counted. In addition, we utilized the cell Titer ATP-Glo Cell Viability Assay (Promega) for quantitative estimation of cell growth and survival (IC₅₀). Cells were plated in 96-well microplates at 1,000 cells per well, and vehicle (DMSO) or various concentrations of quinacrine (0–20 μmol/L) or SAHA (0–20 μmol/L) were administered for 96 hours. Measurements using a Luminometer (BMG LABTECH) were conducted following the manufacturer's protocol.

The DNA content and cell-cycle distribution of gastric cancer cells treated with the quinacrine and SAHA were determined by flow cytometry. Cells were plated in 6-well plates at a seeding density of 1 × 10⁵ cells per well. Twenty-four hours later, cells were treated with quinacrine (2 μmol/L) and SAHA (2 μmol/L) overnight. The cells were then harvested and collected by centrifugation, washed twice in PBS (pH 7.2), and fixed with 70% cold ethanol. Cells were stained with 200 μL of propidium iodide (PI) solution [200 μg/mL RNase (Sigma), 20 μg/mL PI, and 0.1% Triton X-100 (Sigma) in PBS] and incubated at 37°C for 30 minutes. A total of 10⁴ events were acquired in a flow cytometer (BD Biosciences), and data were analyzed using CellQuest software (BD Biosciences).

Cells were treated with vehicle or quinacrine and SAHA (2 μmol/L) overnight. Cells were then collected and stained with Annexin V-FITC and PI (BioVision). The cells were washed with PBS and resuspended in a binding buffer for subsequent FACS analysis by a flow cytometer (Becton Dickinson). Apoptotic cell death was assessed by counting the numbers of cells that stained positive for Annexin V-FITC and negative for PI.

Cells were seeded on 6-well plates at a density of 2 × 10⁵ cells per well. Cells were treated with quinacrine (2 μmol/L) and SAHA (2 μmol/L) overnight, and then fixed by 3.7% formaldehyde/PBS for 10 minutes and washed once with 1× PBS. The cells were blocked for 30 minutes at room temperature with normal goat serum (10%; Thermo Fisher Scientific). Next, the cells were added to 1× anti–phospho-histone antibody solution and incubated overnight at 4°C. The cells were then washed five times with PBST and were incubated in 1× secondary antibody–FITC conjugate solution and washed five times with PBST. Coverslips were mounted with VECTASHIELD Mounting Medium containing DAPI to counterstain cellular nuclei. γ-H2AX foci were scored manually throughout the cell nuclei using an Olympus fluorescence microscope. The average number of foci per cell was calculated from a minimum of 250 cells per dose/time point. Experimental data represent the average of three independent experiments.

DNA single- and double-strand break (DSB) levels were evaluated using a Comet Assay Kit (Trevigen) under alkaline conditions following the manufacturer's instructions. Briefly, cells were nontreated or treated with quinacrine (2 μmol/L) and SAHA (2 μmol/L) overnight. Cells were suspended in PBS at 10⁵ cells/mL and mixed with Comet Agarose at 1:10 ratio (v/v). The cell mixture (75 μL) was immediately layered onto a comet slide. The slide was maintained at 4°C for 15 minutes for gelling and then immersed in lysis buffer at 4°C for 30 minutes. The slide was then placed in alkaline solution for 30 minutes in the dark, and electrophoresis was performed at 1 V/cm and 300 mA. After electrophoresis, the slide was rinsed with H₂O, immersed in 70% ethanol for 5 minutes, and allowed to air dry. The slide was then stained with Vista Green DNA Dye, and comet “tails” were visualized with an Olympus fluorescence microscope. Extent tail moment values of comet assay were quantitated using the OpenComet software (30).

Total RNA was isolated from cell lines by using the RNeasy Mini Kit (Qiagen). Total RNA (1 μg) was reverse transcribed by an iScript cDNA synthesis kit (Bio-Rad). qPCR was performed using a Bio-Rad CFX Connect Real-time PCR Detection System (Bio-Rad), with the threshold cycle number determined by Bio-Rad CFX manager software version 3.0. The primers for human HPRT1 were forward: 5′-TTGGAAAGGGTGTTTATTCCTCA-3′; reverse: 5′-TCCAGCAGGTCAGCAAAGAA-3′. The primers for human P53 were forward: 5′-AGAGACCGGCGCACAGAG-3′; reverse: 5′-GGTGAAATATTCTCCATCCAGTG-3′. Reactions were performed in duplicate, and results of three independent experiments were subjected to statistical analysis. Fold change was calculated using the ΔΔC_t method (31). The results of the target genes were normalized to HPRT1.

Cells were plated at a density of 2 × 10⁵ cells per well in 6-well plates and treated with vehicle or drugs. After the indicated time of treatment, cells were lysed by brief sonication in the RIPA Lysis Buffer (Santa Cruz Biotechnology), and cellular proteins were collected in the supernatant fraction after centrifugation at 13,000 rpm for 10 minutes. Proteins were separated on 12.5% SDS-PAGE and transferred to Immobilon PVDF membranes (Millipore). Membranes were probed with specific antibodies, and proteins were visualized by using horseradish peroxidase (HRP)–conjugated secondary antibodies and Immobilon Western Chemiluminescent HRP Substrate detection reagent (Millipore). Gel loading was normalized for equal β-actin. All immunoblots were imaged using the Bio-Rad ChemiDoc XRS+ System (Bio-Rad).

The PG13 luciferase reporter, which contains 13 copies of the wt-p53 binding-consensus sequence, was used as a measure of the transcription activity of p53 (Addgene plasmid #16442; ref. 32). Cells were seeded in 12-well plates and transfected with the PG13 luciferase reporter by using the DNAfectin transfection reagent (Applied Biological Materials). The cells were harvested for luciferase assays 48 hours later using a Luciferase Assay Kit (Promega) according to the manufacturer's protocol. β-Galactosidase expression plasmid was used as a control for normalization. Each transfection was performed in triplicate. Measurements using a Luminometer (BMG LABTECH) were conducted following the manufacturer's protocol.

Synergy was quantified using the Chou–Talalay method as described previously using the CalcuSyn software to calculate the values of the combination index (http://www.biosoft.com/w/calcusyn.htm, Biosoft; refs. 33, 34). The dose–effect curve for each drug alone is determined on the basis of experimental observations using the median-effect principle and is compared with the effect achieved with a combination of the two drugs to derive a combination index (CI) value. The CI indicates the level of synergism or antagonism: <0.9 indicates synergism (0.3–0.7 strong; 0.7–0.85 moderate; 0.85–0.9 slight), 0.9–1.1 nearly additive effect, and >1.1 antagonism (34).

Five-week-old female NIH-III nude mice were purchased from Charles River Laboratories, Inc. and maintained under specific pathogen-free conditions. UGC cells were injected subcutaneously (2 × 10⁶ cells/site) into the flanks. When the tumor volume reached approximately 150 mm³, the mice were randomly divided into four groups and treated with quinacrine (200 mg/kg, oral gavage), SAHA (50 mg/kg, i.p.), or a combination of quinacrine and SAHA three times per week for 3 weeks. Tumor growth was determined by measuring the width and length of the tumors with a caliper twice weekly, and body weight was measured to monitor drug toxicity. The tumor volume was calculated using the following formula: tumor volume (mm³) = 1/2 (W)² × (L). The Institutional Animal Care and Use Committee approved all animal work.

Values were expressed as mean ± SD of three independent experiments. With the GraphPad Prism 5 software, a one-way ANOVA Newman–Keuls multiple comparisons test was performed to compare the differences among three groups or more, and a two-tailed Student t test was used to compare the statistical difference between two groups. Differences with P values ≤0.05 are considered statistically significant.

Using the ATP-Glo cell viability assay, we found that the combination of quinacrine and SAHA significantly decreased cell viability in all tested UGC models (AGS, SNU1, MKN45, OE33, FLO1, OE19, and MKN28), as compared with single-agent treatments; these results were confirmed by determining the IC₅₀ in response to single agent or a combination of quinacrine and SAHA (Fig. 1A–G). The IC₅₀s of quinacrine, SAHA, and quinacrine and SAHA of all the tested UGC models were summarized in Fig. 1H. We did not detect significant changes in viability of immortalized normal esophageal cells (EPC2, Supplementary Fig. S1A and S1B). We tried to discern cell response to quinacrine and SAHA treatment. ATP-Glo assay was performed in AGS, SNU1, MKN45, MKN28, and OE33 cells following drug treatment. The cell survival ratio was decreased by 90% in AGS cells, 95% in SNU1 cells, 85% in MKN45 cells, and 90% in both MKN28 cells and OE33 cells following coadministration of 2 μmol/L quinacrine and 2 μmol/L SAHA compared with vehicle treatment cells (P < 0.01, Supplementary Fig. S2A–S2E). For increased stringency, we used quinacrine (1 μmol/L) and SAHA (1 μmol/L) for overnight treatment and long-term (10 days) clonogenic survival assay. The results indicated that quinacrine and SAHA cotreatment induced 70% cell death in both AGS and MKN28 cells and induced 60% cell death in FLO1 cells as compared with vehicle treatment cells (Supplementary Fig. S3A–S3C, P < 0.001). Furthermore, the combination index values were calculated using the Chou–Talalay isobologram equation (34). We combined the ratio of 1 with quinacrine and SAHA with twofold serial dilutions (start from 20 μmol/L). The results indicated synergistic activity between quinacrine and SAHA for almost all UGC cell lines tested, as indicated by combination index values of <0.7 (Fig. 1H). Taken together, these findings indicated that quinacrine and SAHA have a synergistic effect in induction of UGC cells death, which raised a question of how quinacrine sensitized UGC cells to SAHA treatment.

Disturbance of the cancer cell cycle is one of the therapeutic targets for development of new anticancer drugs (35). The cell-cycle distribution of each phase was examined by flow cytometry. As shown in Fig. 2, significant G₀–G₁ arrest was induced by combination treatment in AGS, SNU1, MKN28, and FLO1 cells in comparison with individual treatments and vehicle treatment cells (Fig. 2A–D, P < 0.01).

Consistent with these results, the Annexin V-FITC apoptosis assay showed that quinacrine and SAHA cotreatment induced apoptosis by approximately 10-fold in AGS cells, 20-fold in MKN45 cells, 16-fold in MKN28 cells, and 50-fold in FLO1 cells relative to control cells (Fig. 3A–D).

Furthermore, we analyzed the extent of DNA damage by examining the level of γ-H2AX. H2AX is considered a marker of DNA DSBs and activation of DNA damage response (36). Our results demonstrated that the combination of quinacrine and SAHA significantly increased the DSB levels as compared with individual treatments and vehicle treatment in AGS, SNU1, MKN28, and FLO1 cells (Fig. 4A–D, P < 0.01), as indicated by the increased number of γ-H2AX foci in these cells compared with vehicle treatment cells (Fig. 4A–D, P < 0.01). Next, we evaluated the DNA damage induced by quinacrine and SAHA treatment by performing comet assay in AGS and MKN28 cells; the results indicated that cotreatment of quinacrine could significantly enhance the DNA damages caused by SAHA in AGS and MKN28 cells compared with individual treatments and vehicle treatment cells (eightfold, P < 0.01; Supplementary Fig. S4A and S4B).

These results provide compelling evidence that DNA damage plays a causal role in the anti-UGC cytotoxic efficacy of combined quinacrine and SAHA.

Antimalarial drugs, such as quinacrine and chloroquine, have been previously shown to inhibit autophagy (37). Therefore, we tested the effects of quinacrine alone and in combination with SAHA. As expected, we observed induction of LC3B-II levels following treatment with quinacrine. These levels were further increased following combination treatment (Supplementary Fig. S5A and S5B).

We then investigated the levels of cell death following single-agent and combined treatments. We investigated the effects of treatment with quinacrine and SAHA alone or in combination on the endogenous levels of p53, γ-H2AX, and caspases-3 and 9; including wt-p53 (AGS, SNU1, and MKN45) and mut-p53 (MKN28, FLO1, and OE33) UGC cells. When treated with quinacrine alone, we observed induction of wt-p53 as shown in (AGS, SNU1, and MKN45; Fig. 5A; Supplementary Fig. S6A), with no effect on mut-p53 (MKN28, FLO1, and OE33; Fig. 5B; Supplementary Fig. S6B). However, SAHA alone and the combination of quinacrine and SAHA demonstrated a notable reduction in the levels of wt-p53 and mut-p53 (Fig. 5A and B; Supplementary Fig. S6). Western blot analysis also indicated that AGS, SNU1, MKN45, MKN28, OE33, and FLO1, following quinacrine and SAHA combination treatment, have increased activation of γ-H2AX and caspases-3 and 9 (Fig. 5A and B; Supplementary Fig. S6). Moreover, we found that there was no significant difference for p53 mRNA levels in mut-P53 cell lines after quinacrine cotreatment with SAHA as compared with control cells, but quinacrine treatment alone increased p53 mRNA expression in wt-P53 cell lines (Supplementary Fig. S7A–S7F). Luciferase assay showed that the quinacrine/SAHA treatment decreased P53 activity, as compared with controls (Supplementary Fig. S8, P < 0.01). It would be interesting to find out more about the mechanisms of P53 degradation induced by quinacrine and SAHA treatment in the future.

SAHA decreased the activation of AKT (38). Quinacrine inhibited AKT and the NF-κB pathway (8, 9). Here, we showed that when quinacrine was combined with SAHA, there was a remarkable decrease of AKT, STAT3, and NF-κB activities in AGS, FLO1, SNU1, and MKN28 cells (Fig. 5C and E; Supplementary Fig. S9). The luciferase reporter assay showed similar results; quinacrine/SAHA treatment caused significant decrease in NF-κB and STAT3 activities in AGS, FLO1, SNU1, and MKN28 cells (Fig. 5D and F, P < 0.01, Supplementary Fig. S10, P < 0.01). These findings suggest that concurrent interruption of cell survival signaling pathways warrants further investigation in cancer therapy.

Cisplatin (CDDP) is a first-line chemotherapeutic drug for gastric cancer, however, cancer cells often develop multiple mechanisms to overcome cisplatin-induced DNA damage and apoptosis, leading to cisplatin resistance (39). Targeted therapies have emerged as a new hope in cancer management during recent years. The vast majority of gastric cancer patients receiving gefitinib develop resistance (40). Chemotherapeutic resistance is a major challenge in our approaches to treat UGCs. There are two major forms of drug resistance: primary resistance and acquired resistance. To address this problem, we developed in vitro cell models of acquired resistance to CDDP (CDDP-R) using an AGS cancer cell line following continuous treatment with dose escalation for 6 months. We also tested our cell lines and found that OE19 cells have intrinsic resistance to CDDP (IC₅₀ > 10 μmol/L for OE19). Using AGS CDDP-R and OE19 cell models, the results indicated that these cells remain sensitive to the combined quinacrine and SAHA treatment (Supplementary Fig. S11A–S11D). Of note, AGS parental and AGS CDDP-R cell lines achieved similar IC₅₀ (around 0.80 μmol/L) for quinacrine and SAHA treatment (Supplementary Fig. S11B). The results also showed that OE19 cells are sensitive to the quinacrine and SAHA combination treatment (IC₅₀ = 0.55 μmol/L; Supplementary Fig. S11D). Next, we tested our cell lines and found that MKN45 cells have intrinsic resistance to gefitinib (IC₅₀ = 34.121 μmol/L), using MKN45 cell model. We found that these cells remain sensitive to the combined quinacrine and SAHA treatment (IC₅₀ = 0.56 μmol/L, Supplementary Fig. S11E and S11F). We included the IC₅₀ of gefitinib and CDDP for all the cell lines we used (Supplementary Fig. S11G). In the future, we will perform in vivo approaches to investigate the therapeutic efficacy of quinacrine and SAHA combination in preclinical models of resistance to CDDP and gefitinib.

We evaluated the effects of quinacrine and SAHA combination treatment in OE33 cells. Treatment with quinacrine or SAHA alone significantly inhibited tumor growth as compared with control tumors (P < 0.05, Fig. 6A), and treatment with quinacrine and SAHA combination significantly inhibited tumor growth as compared with individual treatments and control tumors (P < 0.01, Fig. 6A). The results demonstrated that tumors derived from OE33 cells combined quinacrine and SAHA treatment grew substantially slower than the individual treated tumor and control tumors (Fig. 6B and C). Immunostaining with the anti–cleaved caspase-3 and anti–Ki-67 antibodies indicated that the reduced tumor growth is likely due to a lower proliferation rate and higher apoptosis caused by quinacrine + SAHA treatment, as cleaved caspase-3 staining was much stronger in quinacrine + SAHA treatment tumors than individual treatments and vehicle treatment tumors (P < 0.01, Fig. 6D). Ki-67 staining was much weaker in quinacrine and SAHA treatment tumors than individual treatments and vehicle treatment tumors (P < 0.01, Fig. 6E).

Upper gastrointestinal cancers are characterized by resistance to chemotherapeutics and poor clinical outcome. Of note, these cancers are characterized by massive changes in DNA copy numbers showing chromosomal instability in the form of frequent gains and losses that affect large regions and subregions of the chromosomes (41–44). Although mutations and/or amplification of RTK genes have been reported, targeted therapy approaches have shown limited therapeutic efficacy (45–47). The recent next-generation sequencing and TCGA data reports indicate that mutations of p53 occur in more than half of these tumors, whereas mutations in other genes occur at low frequencies (43, 44). Given the complexity of UGCs, the use of combinatorial therapy could be an essential step to overcome the limited clinical efficacy of the current therapeutic strategies. In this study, we have investigated, for the first time, the effects of quinacrine in combination with SAHA on wt- and mut-p53 UGC cells. Our results demonstrate that a combination of quinacrine and SAHA is potent in inducing cancer cell death in preclinical models of UGCs. We further demonstrate that this combination targets both wt- and mut-p53 leading to their degradation, which could be an essential step in the observed significant response to this combination, as compared with single-agent treatments.

Quinacrine is an antimalarial drug that has been effectively used for several decades (48). The side effects and general toxicity of quinacrine are usually well tolerated (49). Over the past few years, there has been an interest in repurposing quinacrine for the treatment of cancer. In fact, earlier reports have shown that quinacrine treatment can suppress oncogenic pathways such as ERK, PI3K/AKT/mTOR, and NF-κB pathways (50, 51). However, quinacrine alone has not shown promising results in the treatment of cancer (52). SAHA is an HDAC inhibitor approved for cancer therapy (11). Unfortunately, its efficacy as a single agent in cancer treatment is only moderate (16). Some preclinical studies and early-phase clinical trials showed evidence that SAHA can be combined with other anticancer drugs (17). We have investigated the combination of quinacrine with SAHA. We found that quinacrine can induce LC3B-II protein expression, whereas combined quinacrine/SAHA treatment induced higher levels of LC3B-II than quinacrine treatment alone. Of note, quinacrine is a potent inhibitor of autophagy that has 60-fold higher potency of autophagy inhibition than chloroquine (53). Although, we have not specifically investigated the details of autophagy in our study, our findings suggest that the quinacrine and SAHA combination can promote cancer cell death.

P53 is a major pathway in regulating apoptosis and DNA damage repair. P53 is a double-sword protein that can induce apoptosis in response to high levels of DNA damage or DNA damage repair and cell survival in response to low and moderate levels of DNA damage (54). The DNA damage repair capacity of p53 can mediate resistance to chemotherapeutics. Indeed, a recent report has shown that wt-p53 can mediate therapeutic resistance by initiating and promoting the process of DNA damage repair (25). We found that treatment with quinacrine alone leads to induction of wt-p53, whereas the combination of quinacrine and SAHA demonstrated low levels of p53 protein, suggesting the presence of SAHA-mediated rapid degradation of quinacrine-induced p53 with abrogation of DNA damage repair. It has been shown that SAHA treatment can induce ROS and DNA damage (55, 56). In addition, quinacrine can also induce ROS and DNA damage (54). Our results demonstrate that with the suppression of wt-p53 levels, the combined treatment with quinacrine and SAHA led to accumulation of lethal levels of DNA damage. A recent study suggested that induction of p53 by quinacrine is mainly mediated by suppression of NF-κB activity (9). On the other hand, NF-κB activation by SAHA can lead to hyperacetylation, nuclear translocation, and activation of RelA/p65 (57). Therefore, it is possible that by using the quinacrine and SAHA combination, SAHA is antagonizing the quinacrine-mediated induction of p53. We conclude that the combination of quinacrine and SAHA augments the anticancer therapeutic efficacy not only by inhibition of autophagy, but also via suppression of p53 protein levels, inhibition of DNA damage repair, and accumulation of lethal levels of DNA damage.

Several studies have shown that mutant p53 acquires oncogenic functions and is an important factor in drug resistance in response to DNA damage and chemotherapeutics. In the current study, we show that the quinacrine treatment alone had no effect on mut-p53, whereas the quinacrine and SAHA combination promoted degradation of oncogenic mut-p53 and increased DNA damage levels higher than single-agent treatments. This was associated with a significant increase in cell death in vitro and reduction of tumor volume in in vivo preclinical models. Previous studies in other cancer types have shown that SAHA's inhibition on HDAC6 and HDAC8 decreases the mut-p53 protein level (58, 59). However, we acknowledge that the exact mechanism by which the combination of quinacrine and SAHA mediates degradation of mut-p53 in UGCs requires additional investigations.

Anticancer drugs upregulate the NF-κB pathway, which results in the development of drug resistance (60). Akt plays an important role in the signaling pathways in response to growth factors and other extracellular stimuli to regulate several cellular functions, including cell growth, apoptosis, and survival (61). Blocking signaling to STAT3 inhibits cancer cell growth, showing that STAT3 is crucial to the survival and growth of tumor cells and is an attractive therapeutic target for cancer (62). Here, we showed that when cells were treated with quinacrine and SAHA, there was a significant decrease of AKT, STAT3, and NF-κB activity in UGC cells. These findings suggest that concurrent interruption of cell survival signaling pathways warrants further investigation in cancer therapy.

Chemotherapeutic resistance is a major challenge in the treatment of upper gastrointestinal cancers. By testing gastric cancer cells with acquired resistance to cisplatin, as compared with their parental ones, we found that resistant and parental cells are highly sensitive to quinacrine and SAHA combination. A similar finding was noted in OE19 cells that has intrinsic resistance to cisplatin. Furthermore, the MKN45 cell model, resistant to gefitinib treatment, was also sensitive to this combination. Together, these results suggest that the quinacrine and SAHA combined treatment approach may be tested in clinical trials of UGCs.

In conclusion, our novel findings show that the quinacrine and SAHA combination is more effective than a single-agent treatment. Our results provide evidence that DNA damage may play an additional role in cytotoxic efficacy of combined quinacrine and SAHA. Our results demonstrate that this combination leads to abrogation of both wt and mut-p53 protein levels and augments the therapeutic response. Additional investigations are needed to fully explain the molecular and signaling effects of quinacrine and SAHA combination. Our findings may support clinical trials that test the activity of this combination in UGC patients.

No potential conflicts of interest were disclosed.

The contents of this work are solely the responsibility of the authors and do not necessarily represent the official views of the Department of Veterans Affairs, NIH, or University of Miami.

Conception and design: S. Zhu, W. El-Rifai

Development of methodology: S. Zhu, Z. Chen

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Zhu, Z. Chen, D. Peng

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Zhu, Z. Chen, L. Wang, D. Peng, A. Belkhiri, A.C. Lockhart

Writing, review, and/or revision of the manuscript: S. Zhu, A.C. Lockhart, W. El-Rifai

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): W. El-Rifai

Study supervision: W. El-Rifai

This study was supported by the NIH (R01CA93999 and R01CA131225), Research Career Scientist award (1IK6BX003787), and a merit award (I01BX001179) from the U.S. Department of Veterans affairs (to W. El-Rifai).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.