ABSTRACT
Background
Docetaxel is an anti-microtubule chemotherapeutic agent classified in the taxane group and is widely used in the treatment of many types of cancer. However, systemic toxic effects can be observed in patients treated with docetaxel, and the development of de novo or acquired resistance limits its use and therapeutic efficacy. Thus, investigation of adjuvant treatment approaches has attracted interest. This study aimed to investigate the effects of toyocamycin-mediated inhibition of the Inositol-Requiring Enzyme 1 alpha (IRE1α)-X-box Binding Protein 1 (XBP-1) pathway, a physiological mechanism in mammalian cells, on the sensitivity of ovarian cancer cells to docetaxel.
Materials and Methods
Human ovarian adenocarcinoma cell lines SKOV3 and Caov-3 were used in the studies. The effects of toyocamycin and docetaxel on cell viability were investigated using the Water-Soluble Tetrazolium-1 assay. The inhibitory effect of toyocamycin on the IRE1α-XBP-1 pathway was confirmed by immunoblotting studies. The impact of combined treatment with toyocamycin and docetaxel on migration and invasion was evaluated using wound-healing and Matrigel-coated Boyden chamber invasion assays. The expression levels of some pro-apoptotic and anti-apoptotic genes, such as B-cell lymphoma 2 (BCL2)-associated X apoptosis regulator, BH3-interacting domain death agonist, BCL2 and BCL2-like 1, were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR).
Results
Our results showed that toyocamycin-mediated inhibition of IRE1α-XBP-1 signaling significantly suppressed cell viability, migration, and invasion in ovarian cancer cells. In combined treatment with toyocamycin and docetaxel, it was determined that the viability, migration, and invasion were more effectively suppressed in a dose-dependent manner with increasing concentrations of toyocamycin than with either agent alone. Similar results were obtained from qRT-PCR studies. Combination treatments upregulated pro-apoptotic genes and downregulated anti-apoptotic genes in both cell lines.
Conclusion
The present data suggest that pharmacological targeting of IRE1α/XBP-1 signaling could significantly enhance the sensitivity of ovarian cancer cells to docetaxel.
Introduction
Ovarian cancer is one of the leading causes of cancer-related deaths in women. According to the American Cancer Society, an estimated 20,890 women in the United States will be diagnosed with ovarian cancer in 2025, and approximately 12,730 of these women will die from ovarian cancer (1). Currently, many risk factors for ovarian cancer have been identified, including androgen levels, body weight, postmenopausal hormone therapy, and familial cancer syndromes (2). Local treatments, such as surgery and radiation therapy, and systemic treatment approaches, such as chemotherapy, hormone therapy, targeted drug therapy, and immunotherapy, are widely used in the treatment of ovarian cancer. However, the heterogeneity of cancer cells in tumor tissues, the development of resistance to systemic treatments, and recurrence after treatment may limit the effectiveness of treatment for ovarian cancer (3). Therefore, improving the utilization of existing agents with confirmed efficacy and enhancing the sensitivity of cancer cells to these agents continue to be important priorities. Adjuvant approaches remain important in this regard (4).
Docetaxel, an anti-microtubule agent belonging to the taxane family, is used as a first-line treatment for many cancers, including prostate and breast cancer. Docetaxel is a semi-synthetic, second-generation taxane with cytotoxic effects, derived from the needles of European yew trees (5). It blocks tubulin polymerization, leading to cell cycle arrest in ovarian cancer cells. However, patients develop de novo or acquired resistance to docetaxel following high-dose administration. This situation limits the use and therapeutic efficacy of docetaxel (6). We aimed to investigate the effects of combined application of docetaxel and toyocamycin, an adenosine analog obtained from Streptomyces diastatochromogenes, on the sensitivity of ovarian cancer cells to docetaxel.
Recent studies have reported that mechanisms involved in the control of endoplasmic reticulum (ER) stress, the unfolded protein response (UPR), and cellular proteostasis play a key role in tumor progression and the acquisition of drug resistance by cancer cells (7). UPR signaling is controlled by three main regulatory pathways: Inositol-Requiring Enzyme 1 alpha (IRE1α), PKR-like ER kinase, and activating transcription factor 6. Toyocamycin selectively inhibits the IRE1α–X-box Binding Protein 1 (XBP-1) signaling pathway (8). Under ER stress, IRE1α, localized in the ER membrane, oligomerizes, undergoes autophosphorylation of its cytosolic kinase domain, and mediates removal of a 26-bp intron from XBP-1 mRNA by alternative splicing through activation of its endoribonuclease domain. In this way, the expression of UPR target genes is selectively regulated by the formation of XBP-1s, the spliced form of XBP-1 (9, 10). Studies have reported that alterations in the activity of the IRE1α-XBP1 signaling pathway significantly affect tumorigenic properties of cancer cells, including survival, drug resistance, invasion, metastasis, and epithelial-mesenchymal transition (9–12). Although the UPR signal serves as an adaptive mechanism in mammalian cells, reprogramming cells in response to stress, it can also activate programmed cell death when cellular stress is insurmountable (13). Given these regulatory roles, the IRE1α-XBP-1 signaling pathway has been suggested as an important target for the development of cancer therapeutics.
Toyocamycin was originally isolated from Streptomyces species and is a nucleoside antibiotic analogue of adenosine (14). It blocks RNA synthesis and ribosomal function, and programmed cell death. Toyocamycin also negatively modulates the IRE1α-XBP1 pathway by affecting IRE1α autophosphorylation—a step required for IRE1α activation—thereby inhibiting the splicing of XBP-1 mRNA (14). Current in vitro and in vivo studies have revealed that it has potent anti-cancer activity against cancer cells (14–16). Considering this evidence, investigating the effect of toyocamycin as an adjuvant treatment is of particular interest.
Herein, we evaluated whether toyocamycin potentiates the anticancer activity of docetaxel in SKOV3 and Caov-3 human ovarian adenocarcinoma cell lines. For this purpose, their effects on cell viability and on the migratory and invasive abilities of ovarian cancer cells were assessed using several methods. Moreover, the effects on the expression levels of some pro-apoptotic and anti-apoptotic genes were examined by quantitative real-time polymerase chain reaction (qRT-PCR). Our results showed that combined treatment with docetaxel and toyocamycin has the potential to significantly enhance docetaxel’s anticancer effects in ovarian cancer cells. Taken together, these findings suggest that simultaneous pharmacological targeting of the IRE1α-XBP1 arm of the UPR signaling may significantly improve the sensitivity of ovarian cancer cells to docetaxel.
Materials and Methods
Materials
All cell culture-compatible plastic materials were supplied by Sarsdeth. The culture medium, fetal bovine serum (FBS), trypsin (0.25% and 0.05%), and other supplements were purchased from Lonza Bioscience. Dimethyl sulfoxide (#20385.01), of suitable quality for cell culture studies, was purchased from SERVA. Rabbit polyclonal antibody XBP-1s (#24868-1-AP) (1:2500), one of the primary antibodies used in immunoblotting studies, was supplied by Proteintech. Mouse monoclonal beta-actin antibody (#A5316) (1:10.000) was purchased from Sigma-Aldrich. Horseradish peroxidase (HRP)-conjugated anti-mouse (#31430) (1:500) or anti-rabbit (#31460) (1:500) immunoglobulin G (H+L) secondary antibodies were obtained from Thermo Fisher Scientific. Toyocamycin (#sc-362812) and docetaxel (#sc-201436) were obtained from Santa Cruz Biotechnology. Thapsigargin (#T9033) was provided by Sigma-Aldrich.
Methods
Cell Culture
SKOV3 (HTB-77TM) and Caov-3 (HTB-75TM) human epithelial ovarian adenocarcinoma cell lines were obtained from the American Type Culture Collection. SKOV3 cells were cultured in McCoy’s 5A Medium supplemented with 10% FBS and Caov-3 cells were cultured in Dulbecco’s Modified Eagle’s Medium containing 10% FBS and 2 mM L-glutamine under conventional cell culture conditions (37 °C and 5% CO2). Mycoplasma contamination was routinely monitored using the easy PCR™ Mycoplasma Detection Kit (#20-700-20) (Biological Industries). Cell lines used in the studies were passaged between 5 and 10 times.
Water-Soluble Tetrazolium-1 (WST-1)-Based Cell Viability Assay
Cells were seeded into 96-well cell culture dishes at a density of 10,000 cells/well; after 24 hours, toyocamycin, docetaxel or combinations of the two agents were applied to the cells for 48 hours. An equal volume of the solvent was applied as a control. The master stocks of all agents were prepared at a 2000-fold concentration. 48 hours later, cell viability was analyzed using WST-1 reagent (#MK400) (Takara, Japan), following the manufacturer’s recommended protocol. 20 µL of WST-1 was added to each well, and the culture dish was incubated for 2 hours at 37 °C. Absorbance was measured at 450 nm using a microplate reader (BioTek Epoch 2). Experimental studies were performed in 3 technical and 3 biological replicates. Results are presented graphically as % viability ± standard deviation (SD). Inhibitory concentration 50 (IC50) values for the agents were calculated using GraphPad Prism 8.0.
Quantitative Real-Time PCR
Total RNA was isolated using the Monoarch® Miniprep Total RNA Isolation Kit (#T2010S) (New England Biolabs, USA), following the manufacturer’s recommended protocol. The concentrations and purity of the obtained RNA samples were determined by A260/A280 absorbance measurements using a microspectrophotometer (Allsheng Nano 400A). 1 µg of RNA sample was used to synthesize complementary DNA (cDNA) using the iScript™ cDNA Synthesis kit (#1708890) (Bio-Rad). Real-time PCR analyses were performed on the CFX Connect Real-Time PCR system using iTaq Universal SYBR® Green Master Mix (#1725120; Bio-Rad). mRNA expression levels of BCL2-associated X apoptosis regulator (BAX), BCL2, BCL-xL, and BH3-interacting domain death agonist (BID) were analyzed. Expression levels of ribosomal protein lateral stalk subunit P0 were evaluated for use as a housekeeping gene. Relative gene expression changes were calculated using the Livak method. Results are presented as fold change ± SD in a bar graph. qRT-PCR studies were performed with three biological replicates; each cDNA sample was analyzed in three technical replicates. Melting curve analysis was performed at the end of each qRT-PCR run to evaluate the specificity of the PCR.
Western Blotting
Immunoblotting studies were conducted as previously described in Erzurumlu et al. (17). Cell pellets were lysed in radioimmunoprecipitation assay buffer containing 1% mammalian protease inhibitor (SERVA #39102.01) for 30 min. The samples were centrifuged at 14,000 r.p.m. for 20 minutes at 4 °C; the pellet was removed, and the supernatant was stored for use in subsequent steps. Total protein content was determined using bicinchoninic acid (#23225) (Thermo Scientific, MA). Protein samples were denatured in 4x Laemmli protein loading buffer at 70 °C for 15 minutes. Protein samples were loaded onto hand-cast sodium dodecyl sulfate–polyacrylamide gels at approximately 25 µg per lane and were subjected to electrophoresis for 2 h. Protein samples separated on the gel were transferred onto an Immun-Blot® polyvinylidene difluoride (PVDF) membrane (Bio-Rad; #1620177). The PVDF membrane was subjected to blocking, washing, primary antibody treatment, washing, HRP-conjugated secondary antibody treatment, and washing; chemiluminescence imaging was then performed using ClarityTM Western ECL substrate (#1705061) (Bio-Rad). Chemiluminescence imaging was performed on the Fusion Pulse (Vilber Lourmat) system.
Wound-Healing Assay
Cells were seeded into 12-well cell culture dishes at a concentration of 3.5 x 105 cells per well. 24 hours later, wound areas were created using a sterile 200-µl micropipette tip. After washing the cells with 1x DPBS, fresh complete medium was added to the cells, and the cells were treated with agents for 72 hours. Wound areas were photographed at 0 and 72 hours using a phase-contrast microscope equipped with a camera system (Sunny SopTop microscope ve kamera sistemi). Wound closure rates (%) were analyzed with ImageJ software (http://imagej.nih.gov/ij/). Each group was studied in three technical and three biological replicates, and the results were presented as % wound-closure area (mean ± SD) in a bar graph.
Matrigel-coated Boyden-Chamber Invasion Assay
Invasion assays were performed as previously described by Erzurumlu et al. (17). Matrigel (BD Biosciences) and serum-free medium were combined in a tube at a ratio of 1:8. 45 µl of the mixture was applied to the surface of a transwell (Sarstedt) with a pore size of 8 µm and incubated at 37 °C for 1 hour. The upper surface of the transwell was filled with 100 µl of serum-free medium and incubated for 30 minutes. 10,000 cells were seeded into each transwell. A culture medium containing 20% FBS was added to the culture dish holding the lower part of the transwell, and then the cells were treated with the agents. After 72 hours, cells that migrated to the lower surface of the membrane filter were fixed, stained with crystal violet, and counted. Each sample was analyzed in three biological and two technical replicates. Results are presented as percent invasion in a bar graph (mean ± SD).
Statistical Analysis
Statistical significance of differences between groups was determined by two-tailed Student’s t-test (assuming equal variances) or one-way analysis of variance, with a confidence level of at least 95%, using GraphPad Prism 8.0. Statistical significance was accepted at p < 0.05. Results are presented as mean ± SD.
Results
Evaluation of the Effects of Toyocamycin and Docetaxel on Viability in Ovarian Cancer Cells
The effects of toyocamycin and docetaxel on cell viability in SKOV3 and Caov-3 human ovarian adenocarcinoma cells were investigated using the WST-1 cell viability assay. For this purpose, cells were treated with 10, 12.5, 25, 32.5, 40, 50, 62.5, 75, and 100 nM toyocamycin, and with 0.1, 0.125, 0.25, 0.325, 0.45, 0.5, 0.625, 0.75, and 1 nM docetaxel for 48 hours, after which WST-1 viability analysis was performed. Our results show that docetaxel and toyocamycin significantly suppressed cell viability in SKOV3 and Caov-3 cells in a concentration-dependent manner (Figures 1A and 1B). The IC50 concentrations of toyocamycin were determined to be 55.08 and 51.33 nM for SKOV3 and Caov-3 cells, respectively, and for docetaxel, they were calculated to be 0.585 and 0.563 nM.
Investigation of the Effects of Co-Administration of Toyocamycin with Docetaxel on the Viability of Ovarian Cancer Cells
To evaluate whether toyocamycin enhances the suppressive effect of docetaxel on the viability of SKOV3 and Caov-3 ovarian cancer cells, toyocamycin at ¼x, ½x, and 1x IC50 and docetaxel at 1x IC50 were applied simultaneously to the cells, and cell viability was analyzed. We found that combined application of toyocamycin and docetaxel suppressed cell viability more strongly than either agent alone (Figure 2). Our findings revealed that co-administration of toyocamycin and docetaxel resulted in enhanced anticancer responses in ovarian cancer cells.
Confirmation of the Inhibitory Effect of Toyocamycin on XBP-1s Production by Immunoblotting
Toyocamycin, an adenosine analog obtained from S. diastatochromogenes, prevents the cleavage of XBP-1 mRNA and blocks the formation of XBP-1s, which function as active transcription factors in UPR signaling. (14). Immunoblotting studies were performed to confirm XBP-1 inhibition at the IC50 concentrations determined in cell viability assays. In these studies, thapsigargin, which can activate UPR signaling by inducing ER stress, was used as a positive control. SKOV3 and Caov-3 cells were treated with 5 nM thapsigargin for 1 hour to induce ER stress (18). As expected, thapsigargin administration resulted in increased XBP-1s levels in SKOV3 and Caov-3 cells compared to the control group. Thapsigargin-induced increases in XBP-1s levels were suppressed by toyocamycin in a dose-dependent manner (Figure 3). These results confirm that toyocamycin inhibits XBP-1s signaling at the applied concentrations in SKOV3 and Caov-3 ovarian cancer cells.
Investigation of the Effects of Combined Applications of Toyocamycin and Docetaxel on the mRNA Expression Levels of Apoptotic Genes in Ovarian Cancer Cells
To examine the effects of toyocamycin and docetaxel treatments on the expression of pro-apoptotic (BAX, BID) and anti-apoptotic (BCL2, BCL-xL) genes in SKOV3 and Caov-3 cells, mRNA levels were evaluated by qRT-PCR. For this purpose, cells were treated with docetaxel at 1x IC50 and with toyocamycin at ¼x, ½x, and 1x IC50 or with combinations of the two agents, for 24 h. Our data revealed that toyocamycin and docetaxel alone significantly up-regulated the expression levels of the pro-apoptotic proteins BAX and BID, while the expression levels of the anti-apoptotic proteins BCL2 and BCL-xL were down-regulated compared to the control group (Figures 4A and B). Combined treatment with increasing concentrations of toyocamycin and docetaxel strongly upregulated the expression of the pro-apoptotic proteins BAX and BID and downregulated the expression of the anti-apoptotic proteins BCL2 and BCL-xL, compared with treatment with toyocamycin or docetaxel alone (Figures 4A and B). These results indicate that co-treatment with toyocamycin and docetaxel induces apoptotic responses more effectively than either agent alone in SKOV3 and Caov-3 cells.
Evaluation of the Effects of Co-treatment of Toyocamycin and Docetaxel on the Migration and Invasion Ability of Ovarian Cancer Cells
To evaluate the effects of co-treatments with toyocamycin and docetaxel on the migratory and invasive abilities of ovarian cancer cells, SKOV3 and Caov-3 cells were treated with 1x IC50 docetaxel and ¼x, ½x, and 1x IC50 toyocamycin, or combinations of both agents, for 48 hours in migration assays and 72 hours in invasion assays. Our results showed that treatment with either toyocamycin or docetaxel alone significantly inhibited the migration and invasion of SKOV3 and Caov-3 cells compared with the control group (Figures 5A and 5B). In our trials in which combinations of increasing concentrations of toyocamycin and docetaxel were applied, co-treatment with toyocamycin and docetaxel more strongly inhibited the migratory and invasive abilities of SKOV3 and Caov-3 cells than either agent alone. Our results showed that the combination of toyocamycin and docetaxel produced more potent anti-migratory and anti-invasive effects.
Discussion
Gynecological cancers continue to be a significant health problem; they are commonly diagnosed and are a frequent cause of death (19). In many patients, resistance to conventional therapies or distinct genomic and proteomic profiles of tumor cells limit treatment success (20). Therefore, research on adjuvant approaches has gained importance.
Taxane-class agents are an important group of chemotherapeutic agents used in cancer therapy (21). Responses to treatments with taxane-class drugs may vary depending on whether patients have received previous therapy. Patients may develop resistance to these agents (22). In the present study, the combined effect of the taxane-class drug docetaxel and toyocamycin on improving its anticancer activity was evaluated in an in vitro ovarian cancer model using several methodologies.
Toyocamycin, also known as vengicide, is an adenosine analog. Isolated from S. diastatochromogenes, toyocamycin exhibits activity against multiple targets and functions as an antibiotic with antitumor activity (14, 23). Studies have shown that toyocamycin inhibits the IRE1α-XBP-1 signaling pathway, a physiological signaling mechanism in mammalian cells (14).
IRE1α signaling regulates numerous pathways in mammalian cells, including lipid biogenesis, chaperone synthesis, protein degradation, control of quality-control protein levels, regulation of autophagic responses, and increased expression of pro-survival genes (24, 25). Recent studies have revealed that changes in the activity of the IRE1α-XBP-1 signaling pathway promote carcinogenesis (9–12). Thus, it is suggested as one of the important mechanisms to be targeted in cancer therapy. Toyocamycin is a small-molecule agent that inhibits the IRE1α-XBP-1 signaling pathway by blocking the kinase activity of IRE1α (14). Its selective effect on IRE1α-XBP-1 signaling makes it an important agent for investigating its efficacy in anticancer therapies.
Our studies investigating the possible enhancing roles of toyocamycin on the anticancer activity of docetaxel determined that the combination of docetaxel and toyocamycin, across increasing concentrations, suppressed the viability of ovarian cancer cells to a significantly greater extent than docetaxel alone (Figure 1B). These findings suggest that targeting the IRE1α-XBP-1 pathway with toyocamycin, in combination with docetaxel as an adjuvant therapy, may represent an important approach to achieve more effective anticancer responses. Previous reports have shown that UPR signaling is an important mechanism of drug resistance in cancer cells (26). From this perspective, toyocamycin may offer an important approach to preventing resistance to docetaxel. However, our findings, which are limited to an in vitro experimental model of simultaneous combination therapy, should be validated in in vivo studies of long-term docetaxel treatment to confirm the effect of toyocamycin on docetaxel therapy.
Few studies have examined the combined effects of toyocamycin. Ri et al. (14) demonstrated that toyocamycin exerts therapeutic effects in multiple myeloma (MM) cells by inhibiting IRE1α-XBP-1. These studies also demonstrated that combinations of toyocamycin with bortezomib, a proteasome inhibitor, had greater antitumor activity in an in vivo MM model (14). Park et al. (15) reported that toyocamycin induces apoptosis through crosstalk between oxidative stress and the MAPK signaling pathway in human prostate cancer cells. Additionally, a Phase I study was conducted of toyocamycin in patients with advanced solid tumors. However, the study results were not carried forward to subsequent stages, in which patients did not demonstrate a significant clinical response to toyocamycin treatment. One important finding of this study was that no systemic side effects, such as organ dysfunction or cytopenia, were observed in association with toyocamycin treatment. Local foci of necrosis were observed only at the infusion sites in tissues treated with toyocamycin. This suggests that the side effects of toyocamycin infusion via central venous catheters would be manageable. This issue paves the way for its clinical use (27). Taken together, these findings suggest that toyocamycin is a promising agent and that further studies are required to examine its effects in different cancer groups and to determine its possible additive effects with different treatments.
Radiotherapy and systemic or targeted cytotoxic agents, commonly used in cancer therapy, stimulate the apoptotic cell-death pathway. Therefore, evasion of apoptosis by cancer cells is one of the main reasons for treatment failure (28). In our studies, we examined the expression levels of the pro-apoptotic genes BAX and BID and the anti-apoptotic genes BCL2 and BCL-xL. Our findings showed that toyocamycin and docetaxel reprogrammed the cells by upregulating pro-apoptotic gene expression and downregulating anti-apoptotic gene expression in SKOV3 and Caov-3 cells. Moreover, combination therapy induced the expression of apoptotic genes more effectively than either therapy alone (Figures 4A and B). These findings suggest that toyocamycin enhances docetaxel-mediated induction of apoptosis in ovarian cancer cells additively, thereby providing more effective anticancer responses.
One of the major limitations to successful cancer treatment is that cancer cells become more aggressive, exhibiting increased migration and invasion. This allows cancer cells to spread more rapidly. Furthermore, drug resistance contributes to the failure of currently applied therapies (29, 30). In this respect, the simultaneous use of combinatorial approaches alongside traditional treatments is important for improving therapeutic efficacy. Our findings showed that treatment of ovarian cancer cells with increasing concentrations of toyocamycin combined with docetaxel significantly reduced the migratory and invasive abilities of SKOV3 and Caov-3 cells compared with treatment with docetaxel or toyocamycin alone (Figures 5A and B). Previous studies have reported that pharmacological targeting of UPR signaling suppresses the motility-related properties of cancer cells (31). Consistent with this, present data suggest that toyocamycin treatment in combination with docetaxel, which has an inhibitory effect on cell division, results in more effective responses than either agent alone, due to the suppression of tumorigenic properties of ovarian cancer cells and to toyocamycin-mediated disruption of the UPR signal, which functions as an adaptive mechanism. Although our findings are limited to in vitro results, studies in the literature emphasize the importance of targeting the IRE1α-XBP-1 arm of the UPR signaling pathway for new therapeutic approaches. The importance of UPR-targeted approaches has been highlighted in numerous in vitro and in vivo studies, particularly in prostate, breast, pancreatic, ovarian, glioblastoma, and hematological cancers (11,12,32–38). Collectively, our research findings suggest that targeting IRE1α-XBP-1 signaling to improve cellular sensitivity to docetaxel in ovarian cancer may contribute to the development of new treatment protocols.
Conclusion
In conclusion, our in vitro findings suggest that the XBP-1 inhibitor toyocamycin, which targets the IRE1α arm of the UPR, has significant potential to sensitize ovarian cancer cells to docetaxel. Our data, limited to in vitro experimental findings, support further studies examining the potential use of toyocamycin in cancer cells. Although obtaining data from in vitro systems is a significant limitation of our study, the present findings suggest that the potential effects of toyocamycin may guide the design of further in vivo experimental models.
