Research ArticleExperimental Studies

The Dual Histone Deacetylase-Proteasome Inhibitor RTS-V5 Acts Synergistically With Ritonavir to Induce Endoplasmic Reticulum Stress in Bladder Cancer Cells

KAZUKI OKUBO, NINA REßING, WOLFGANG A. SCHULZ, FINN K. HANSEN, TAKAKO ASANO and AKINORI SATO
Anticancer Research December 2021, 41 (12) 5987-5996; DOI: https://doi.org/10.21873/anticanres.15417

Abstract

Background/Aim: Simultaneous inhibition of histone deacetylase and proteasomes induces endoplasmic reticulum (ER) stress efficiently. RTS-V5 is the first dual histone deacetylase–proteasome inhibitor, and we anticipated that combining it with the cytochrome P450 family 3 subfamily A member 4 inhibitor ritonavir would enhance its activity in bladder cancer cells. Materials and Methods: Using bladder cancer cells (human T-24, J-82, murine MBT-2), we evaluated the ability and mechanism by which the combination of RTS-V5 and ritonavir induced ER stress and killed cancer cells. Results: The combination of RTS-V5 and ritonavir triggered robust apoptosis and inhibited bladder cancer growth effectively in vitro and in vivo. It caused ubiquitinated protein accumulation and induced ER stress synergistically. The combination inhibited the mammalian target of rapamycin pathway by increasing the expression of AMP-activated protein kinase. We also found that the combination caused histone and tubulin hyperacetylation. Conclusion: Ritonavir enhances the ability of RTS-V5 to cause ER stress in bladder cancer cells.

Key Words:

Inducing endoplasmic reticulum (ER) stress is a novel anticancer strategy. Unfolded proteins in cells are repaired by molecular chaperones such as heat-shock protein 90 (1). If the repair fails, the unfolded proteins are ubiquitinated and degraded by proteasomes (1). This is the so-called ubiquitin–proteasome pathway and plays an essential role in maintaining protein homeostasis in cells (1). Disrupting this pathway causes unfolded proteins to accumulate, leading to ER stress induction and cell death (2). Histone deacetylase (HDAC) inhibitors are known to inhibit molecular chaperones (3) and we have shown that combining a proteasome inhibitor with an HDAC inhibitor caused ER stress in urological cancer cells and killed them effectively (4, 5).

RTS-V5 is the world’s first dual HDAC-proteasome inhibitor. It inhibits not only molecular chaperone function via HDAC inhibition but also proteasomes, increasing the amount of unfolded proteins in the cell and thereby inducing ER stress (6). A preclinical study on RTS-V5 revealed its efficacy against hematological cancer (6), however, it exhibits antitumor effects against bladder cancer cells at higher concentrations than used in that study (7).

The human immunodeficiency virus (HIV) protease inhibitor ritonavir is a potent inhibitor of the drug-degrading enzyme cytochrome P450 family 3 subfamily A member 4 (CYP3A4) and is clinically used to boost the activity of other drugs (8, 9). We investigated whether ritonavir would inhibit intracellular CYP3A4 and thereby enhance the antitumor effects of RTS-V5 in bladder cancer cells.

Materials and Methods

Cell lines and culture conditions. The human bladder cancer cell lines J-82 and T-24 were obtained from the American Type Culture Collection (Rockville, MD, USA). The murine bladder cancer cell line MBT-2 were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). The cells were maintained in recommended media supplemented with 10% fetal bovine serum and 1.0% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) at 37°C in a humidified atmosphere containing 5% CO2.

Reagents. RTS-V5 was synthesized as previously described (6) and dissolved in dimethyl sulfoxide (DMSO). Ritonavir and CYP3A-specific inhibitor cobicistat were obtained from Toronto Research Chemicals (North York, ON, Canada) and Selleck Chemicals (Houston, TX, USA), and dissolved in DMSO. Cycloheximide was obtained from Enzo Life Sciences (Farmingdale, NY, USA) and dissolved in distilled water. All reagents were stored in the dark at either –80°C or –20°C until use.

Cell viability assay. Cells were plated in 96-well culture plates at a density of 5×103 cells per well 24 h prior to treatment. They were then incubated for 48 h with 2.5-10 μM RTS-V5 with/without 20-40 μM ritonavir or with 2.5-10 μM RTS-V5 with/without 10-20 μM cobicistat. CCK-8 assay (Dojin, Kumamoto, Japan) was used to evaluate cell viability as previously described (7). The experiment was repeated two or three times with six wells per treatment.

Apoptosis assay. Cells (1×105) were incubated overnight in a 12-well culture plate and then cultured for 48 h with 10 μM RTS-V5 with/without 40 μM ritonavir or with 10 μM RTS-V5 with/without 20 μM cobicistat. Annexin-V assay was performed using flow cytometry as previously described (7). The experiment was performed three times.

Western blotting. After treatment for 48 h with 5-10 μM RTS-V5 with/without 40 μM ritonavir or with 5-10 μM RTS-V5 with/without 20 μM cobicistat or with 10 μM RTS-V5 and 40 μM ritonavir with/without 5 μg/ml cycloheximide, cells were lysed with radioimmunoprecipitation assay buffer and protein expression was evaluated by western blotting as previously described (4, 5, 7). The following primary and secondary antibodies were used: anti-CYP3A4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-cleaved poly(ADP-ribose) polymerase (PARP), anti-phospho-S6 ribosomal protein (p-S6), anti-S6, anti-phospho-eukaryotic translation initiation factor 4E-binding protein 1 (p-4EBP1), anti-4EBP1, and anti-endoplasmic reticulum protein 44 (ERp44) (Cell Signaling Technology, Danvers, MA, USA); anti-glucose-regulated protein 78 (GRP78), anti-sequestosome 1 (p62), anti-microtubule-associated protein 1 light chain 3 (LC3), and anti-AMP-activated protein kinase (AMPK) (Proteintech, Rosemont, IL, USA); anti-acetylated histone and anti-phorbol-12-myristate-13-acetate-induced protein 1 (NOXA) (Abcam, Cambridge, UK); anti-acetylated α-tubulin (Enzo Life Sciences); anti-actin (Millipore, Billerica, MA, USA); and horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit antibodies (GE Healthcare, Wauwatosa, WI, USA).

In vivo study. The procedures used in this in-vivo experiment were approved by the institutional Animal Care and Use Committee of National Defense Medical College (approval number 17039). C3H/He mice were obtained from CLEA Japan (Tokyo, Japan). MBT-2 cells (1×107) were subcutaneously injected into the posterior flank and treatment was started 5 days later (day 1), when all the tumors were palpable. The mice were randomly assigned to four groups each containing five mice. The control group was given intraperitoneal injections of DMSO, whereas the treatment groups received either 20 mg/kg RTS-V5 or 50 mg/kg ritonavir or both. The injections were given once a day for 2 weeks (5 days a week). Tumor volume and body weight were measured every 2 or 3 days. Tumor volumes were estimated as length × width2/2. On day 14, the animals were euthanized in accordance with the guidelines for the welfare and use of animals (10). The tumors were then harvested, lysed, and subjected to western blotting.

Statistics. Generation of isobolograms and calculation of combination indices were performed using CalcuSyn software (Biosoft, Cambridge, UK) by the Chou and Talalay method (11). Statistical comparisons between samples were performed using the Mann–Whitney U-test with JMP Pro14 software (SAS Institute, Cary, NC, USA). p-Values of less than 0.05 were considered to be statistically significant.

Results

The combination of RTS-V5 and ritonavir inhibited bladder cancer growth effectively. First, we evaluated the cytotoxicity of the combination of RTS-V5 and ritonavir against bladder cancer cells by CCK-8 assay. Each agent slightly reduced cell viability in a dose-dependent manner, and when the agents were combined, they exerted a synergistic cytotoxicity (Figure 1 and Table I). Thus, the combination was shown to inhibit bladder cancer cell growth more effectively than RTS-V5 alone.

Figure 1.
Figure 1.

The combination of RTS-V5 and ritonavir inhibited bladder cancer growth effectively. Cells were treated for 48 h with 2.5-10 μM RTS-V5 with/without 20-40 μM ritonavir, then cell viability was measured using CCK-8 assay. Data are the mean±SD, n=6.

View this table:
Table I.

Combination indices for the combination of 2.5-10 μM RTS-V5 and 20-40 μM ritonavir against bladder cancer cells. A combination index of <1, 1, and >1 indicate synergism, additive effect, and antagonism, respectively.

The combination of RTS-V5 and ritonavir induced apoptosis in bladder cancer cells. We then evaluated the induction of apoptosis by the combination. The combination increased the number of annexin-V-positive cells significantly compared to the treatment with the single compounds (Figure 2A). This apoptosis induction was also evidenced by the increased cleavage of PARP and expression of NOXA, both apoptosis-related proteins (Figure 2B).

Figure 2.
Figure 2.

The combination of RTS-V5 and ritonavir induced apoptosis of bladder cancer cells. A: Cells were treated for 48 h with 10 μM RTS-V5 with/without 40 μM ritonavir. Apoptotic cells were detected by annexin-V assay using flow cytometry, 10,000 cells were counted. Bar graphs show the percentages of apoptotic cells. Data are expressed as the mean±SD from three independent experiments. FITC: Fluorescein isothiocyanate; 7-AAD: 7-amino-actinomycin D. *Significantly different at p=0.0495. B: Cells were treated with 5-10 μM RTS-V5 and/or 40 μM ritonavir for 48 h then western blotting was carried out for cleaved poly(ADP-ribose) polymerase (PARP) and phorbol-12-myristate-13-acetate-induced protein 1 (NOXA). Actin was used for the loading control. Representative blots are shown.

The combination of RTS-V5 and ritonavir caused ubiquitinated protein accumulation and ER stress in bladder cancer cells. We postulated that ritonavir would boost the ability of RTS-V5 to cause the accumulation of ubiquitinated unfolded protein, thereby inducing ER stress effectively. Therefore, we next examined the changes in the expression of ubiquitinated proteins. RTS-V5 alone at 5-10 μM caused no or only slight ubiquitinated protein accumulation. On the other hand, in combination with ritonavir, 5 μM RTS-V5 caused marked accumulation of ubiquitinated proteins (Figure 3). Interestingly, increasing the RTS-V5 concentration to 10 μM did not result in further accumulation in MBT-2 cells, and in T-24 and J-82 cells, even reduced it (Figure 3). However, the increased expression of GRP78 and ERp44 confirmed that the combination of 5-10 μM RTS-V5 and ritonavir actually induced ER stress through ubiquitinated protein accumulation (Figure 3). Furthermore, the increased expression of the ER stress-associated pro-apoptotic protein NOXA (12) confirmed that ER stress itself induced apoptosis (Figure 2B). Thus, the drug combination was shown to induce ER stress cooperatively.

Figure 3.
Figure 3.

The combination of RTS-V5 and ritonavir caused ubiquitinated protein accumulation and endoplasmic reticulum stress in bladder cancer cells. A: Cells were treated for 48 h with 5-10 μM RTS-V5 with/without 40 μM ritonavir. Western blotting was then performed for ubiquitinated proteins, glucose-regulated protein 78 (GRP78), and endoplasmic reticulum protein 44 (ERp44). Actin was used for the loading control. Representative blots are shown.

The combination of RTS-V5 and ritonavir inhibited the mammalian target of rapamycin (mTOR) pathway. Because we previously showed that induction of ER stress increased the expression of AMPK and thereby inhibited the mTOR pathway (13-15), we thought that the combination of RTS-V5 and ritonavir might also induce AMPK expression and inhibit the mTOR pathway. As postulated, the combination increased the expression of AMPK and reduced the expression and phosphorylation of S6 and 4EBP1 (Figure 4A), both of which are key downstream proteins of the mTOR pathway (16), demonstrating that the combination indeed inhibited the mTOR pathway. Because inhibition of the mTOR pathway has been shown to induce autophagy (17), we next evaluated whether the combination also induced autophagy. The combination increased the expression of the autophagy marker LC3-II, and reduced the expression of p62 (Figure 4B). Thus, the combination was shown to inhibit the mTOR pathway.

Figure 4.
Figure 4.

The combination of RTS-V5 and ritonavir inhibited the mammalian target of rapamycin pathway. Cells were treated for 48 h with 5-10 μM RTS-V5 with/without 40 μM ritonavir then western blotting was carried out for AMP-activated protein kinase (AMPK), phospho-S6 ribosomal protein (p-S6), S6, phospho-eukaryotic translation initiation factor 4E-binding protein 1 (p-4EBP1) and 4EBP1 (A), in addition to sequestosome 1 (p62) and microtubule-associated protein 1 light chain 3 (LC3) (B). Actin was used for the loading control. Representative blots are shown.

The combination of RTS-V5 and ritonavir cooperatively induced histone acetylation. We have previously shown that extensive ER stress is closely associated with histone acetylation (13-15). So, we next evaluated whether the combination of RTS-V5 and ritonavir induces histone acetylation. As expected, the combination induced histone acetylation cooperatively (Figure 5). Furthermore, we also found that the combination induced α-tubulin acetylation (Figure 5), confirming that the combination inhibited HDAC6 and this would lead to the further suppression of molecular chaperone function.

Figure 5.
Figure 5.

The combination of RTS-V5 and ritonavir induced histone acetylation cooperatively. Cells were treated for 48 h with 5-10 μM RTS-V5 with/without 40 μM ritonavir. Western blotting for acetylated histone and acetylated α-tubulin was performed using actin for the loading control. Representative blots are shown.

CYP3A4 inhibition was a mechanism of enhanced RTS-V5 efficacy by ritonavir. To demonstrate that CYP3A4 inhibition is a mechanism through which ritonavir boosts the effects of RTS-V5, we first examined the expression of CYP3A4 in bladder cancer cells. Interestingly, T-24 and J-82 cells actually expressed CYP3A4 (Figure 6A). We then treated the cells with RTS-V5 in combination with cobicistat, a clinically approved CYP3A-specific inhibitor (18). The cobicistat–RTS-V5 combination likewise inhibited the growth of bladder cancer cells synergistically (Figure 6B and Table II), and significantly induced apoptosis (Figure 6C). Mechanistically, the combination of RTS-V5 and cobicistat, like the RTS-V5-ritonavir combination, caused marked ubiquitinated protein accumulation and induced ER stress (Figure 6D). Ritonavir thus seemed to enhance the effects of RTS-V5 by inhibiting CYP3A4.

Figure 6.
Figure 6.

Inhibition of cytochrome P450 family 3 subfamily A member 4 (CYP3A4) was a mechanism by which ritonavir enhanced RTS-V5 activity. A: Western blotting for expression of CYP3A4 in bladder cancer cells lines. Actin was used for the loading control. Representative blots are shown. B: Cells were treated for 48 h with 2.5-10 μM RTS-V5 with/without 10-20 μM cobicistat, and cell viability was measured using CCK-8 assay. Data are the mean±SD, n=6. C: Cells were treated for 48 h with 10 μM RTS-V5 with/without 20 μM cobicistat. Apoptotic cells were detected by annexin-V assay using flow cytometry; 10,000 cells were counted. Bar graphs show the percentages of apoptotic cells. Data are expressed as the mean±SD from three independent experiments. FITC: Fluorescein isothiocyanate; 7-AAD: 7-amino-actinomycin D. *Significantly different at p=0.0495. D: Cells were treated for 48 h with 5-10 μM RTS-V5 with/without 20 μM cobicistat then western blotting was carried out for ubiquitinated proteins, glucose-regulated protein 78 (GRP78), and endoplasmic reticulum protein 44 (ERp44). Actin was used for the loading control. Representative blots are shown.

View this table:
Table II.

Combination indices for the combination of 2.5-10 μM RTS-V5 and 10-20 μM cobicistat against bladder cancer cells. A combination index of <1, 1, and >1 indicate synergism, additive effect, and antagonism, respectively.

Induction of ER stress played a crucial role in the effects of the combination. To confirm that the induction of ER stress was essential for the increased efficacy of the combination, we then evaluated whether the inhibition of ER stress affects the efficacy of the RTS-5-ritonavir combination using cycloheximide, an inhibitor of protein synthesis and a suppressor of ER stress (19). Cycloheximide significantly attenuated the ability of the combination to induce apoptosis (Figure 7A), showing that the ER stress induction played a crucial role in the antineoplastic effect of the combination. In fact, cycloheximide inhibited combination-induced accumulation of ubiquitinated proteins, ER stress, and histone acetylation (Figure 7B). Thus, the induction of ER stress was shown to play a crucial role in the action of the RTS-V5–ritonavir combination.

Figure 7.
Figure 7.

Induction of endoplasmic reticulum stress played a crucial role in the action of the combination of ritonavir and RTS-V5. A: Cells were treated for 48 h with the combination of 10 μM RTS-V5 and 40 μM ritonavir with/without 5 μg/ml cycloheximide (CHX). Apoptotic cells were detected by annexin-V assay using flow cytometry; 10,000 cells were counted. Bar graphs show the increase in annexin-V-positive cells relative to cells without the RTS-V5-ritonavir treatment. Data are expressed as the mean±SD from three independent experiments. FITC: Fluorescein isothiocyanate; 7-AAD: 7-amino-actinomycin D. *Significantly different at p=0.0495. B: Cells were treated for 48 hours with the combination of 10 μM RTS-V5 and 40 μM ritonavir with/without 5 μg/ml CHX. Western blotting was then carried out for ubiquitinated proteins, glucose-regulated protein 78 (GRP78), and acetylated histone. Actin was used for the loading control. Representative blots are shown.

The RTS-V5–ritonavir combination was also effective in vivo. Finally, we examined the efficacy of the RTS-V5–ritonavir combination in vivo. In the murine subcutaneous tumor model using MBT-2 cells, a 14-day treatment with the combination suppressed tumor growth significantly (Figure 8A). The treatment was well tolerated and there was no significant difference in the body weight among the groups (Figure 8B). To explore the mechanisms of action of the combination in vivo, we then analyzed the tumor specimens by western blotting. The combination of RTS-V5 and ritonavir was shown to increase the expression of ubiquitinated proteins, NOXA, and acetylated histone (Figure 8C), confirming that the combination has the same mechanism of action in vivo that it does in vitro.

Figure 8.
Figure 8.

The RTS-V5–ritonavir combination was also effective in vivo. A murine allograft model was established using MBT-2 cells. The vehicle-treated group received intraperitoneal injections of dimethyl sulfoxide and the treatment groups received 20 mg/kg RTS-V5 or 50 mg/kg ritonavir alone or in combination. The injections were given once a day for 14 days (5 days on, 2 days off). A: Tumor volume. Data are the mean±SE, n=5. *Significantly different from all other groups at p=0.0079 at day 14. B: Body weight during the experiment. Data are the mean±SE, n=5. Note that there was no significant difference in the body weight among groups at day 14. C: After 14 days of the treatment, the animals were euthanized and the subcutaneous tumors were harvested, lysed, and subjected to western blotting for ubiquitinated proteins, phorbol-12-myristate-13-acetate-induced protein 1 (NOXA), and acetylated histone. Actin was used for the loading control. Representative blots are shown.

Discussion

Cisplatin-based chemotherapy has been the mainstay of treatment for advanced bladder cancer, but its efficacy is limited (20, 21). Although immune checkpoint inhibitors have been approved, there is no definitive treatment of advanced bladder cancer (22, 23). Inducing ER stress is a novel strategy for cancer treatment (24, 25), and we have shown its efficacy against urological cancer (7, 13-15). Furthermore, we have recently shown that ER stress-targeting drugs were effective in cisplatin-resistant bladder cancer cells (7). RTS-V5 is the world’s first dual HDAC-proteasome inhibitor and kills cancer cells by inducing ER stress (6). We tried in the present study to enhance the activity of RTS-V5 using a drug booster in combination with it.

Ritonavir is a classical HIV protease inhibitor and widely used for the treatment of HIV infection (8, 9, 26). It is also used as a drug booster to increase blood concentration of combined drugs by inhibiting the liver drug-degrading enzyme CYP3A4 (3, 4). In the present study we postulated that ritonavir would inhibit intracellular CYP3A4 in bladder cancer cells. We found that the bladder cancer cells themselves expressed CYP3A4 and postulated that ritonavir inhibited intracellular CYP3A4 and increased the intracellular concentration of RTS-V5. This was evidenced by the fact that the CYP3A-specific inhibitor cobicistat boosted the action of RTS-V5 activity and the changes in the expression of ubiquitinated proteins and the ER stress markers with the cobicistat-RTS-V5 combination were similar to those caused by the RTS-V5-ritonavir combination. According to the cell viability assay, MBT-2 is the least combination-sensitive cell line. However, in our animal study using MBT-2 cells, the combination inhibited the tumor growth almost completely. This suggests that ritonavir inhibited not only intracellular CYP3A4 but also that in the liver, thereby increasing both intracellular and blood concentration of RTS-V5.

Interestingly, in T-24 and J-82 cells, increasing the RTS-V5 concentration from 5 to 10 μM reduced the expression of ubiquitinated proteins (Figure 3). These effects are not actually due to decreases in the amount of ubiquitinated proteins: excessively accumulated ubiquitinated proteins aggregated and shifted to the detergent-insoluble fraction as previously described (7, 13, 14).

Ritonavir is also known to increase the amount of unfolded proteins by inhibiting molecular chaperone function (8, 9). We have furthermore shown that combinations of ritonavir with agents such as proteasome inhibitors and HDAC inhibitors increased the amount of unfolded proteins and thereby induced ER stress cooperatively (27-29). In the present study, ritonavir itself slightly increased the expression of ubiquitinated proteins and reduced viability of T-24 and J-82 cells, suggesting that the molecular chaperone activity was actually inhibited by ritonavir. This inhibition of molecular chaperone function might further enhance the effects of RTS-V5.

In the present study, the combination of RTS-V5 and ritonavir increased the expression of the mTOR inhibitor AMPK and inhibited the mTOR pathway, which is compatible with our previous finding that ER stress induction was closely related to increased expression of AMPK (13-15, 30). This inhibition of the mTOR pathway by the combination was also evidenced by the induction of autophagy because autophagy is generally induced by mTOR inhibition (17). The inhibition of the mTOR pathway might be another mechanism of the combination’s action.

The RTS-V5–ritonavir combination induced histone acetylation as well as ER stress, which is compatible with previous reports that there is a close relationship between ER stress induction and histone acetylation (7, 13-15, 31). Histone acetylation and deacetylation are associated with tumorigenesis and malignant tumor progression (32, 33), and triggering histone acetylation is attracting attention as a new option in cancer treatment (3). Furthermore, we also observed acetylation of α-tubulin by the combination. α-Tubulin acetylation is a well-known consequence of HDAC6 suppression (34), and the suppression of HDAC6 inhibits molecular chaperones and increases unfolded proteins, causing ER stress (34). Thus, the observed increase in α-tubulin acetylation is consistent with the induction of ER stress by the combination.

In summary, we have shown for the first time that the combination of RTS-V5 and ritonavir killed bladder cancer cells by inducing ER stress. mTOR inhibition and histone acetylation appear to be important mechanisms of action of this combination. The RTS-V5–ritonavir combination is a potentially promising next-generation treatment against bladder cancer.

Footnotes

  • Authors’ Contributions

    K.O. and A.S. designed the study. K.O. carried out all the experiments. N.R. and F.K.H. synthesized RTS-V5. K.O., T.A., N.R., W.A.S., F.K.H. and A.S. contributed to the interpretation of the results. K.O. wrote the article. N.R., W.A.S., F.K.H. and A.S. edited the article. A.S. supervised the study. All Authors read and approved the final article.

  • Conflicts of Interest

    None.

  • Received September 27, 2021.
  • Revision received November 4, 2021.
  • Accepted November 9, 2021.

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The Dual Histone Deacetylase-Proteasome Inhibitor RTS-V5 Acts Synergistically With Ritonavir to Induce Endoplasmic Reticulum Stress in Bladder Cancer Cells
KAZUKI OKUBO, NINA REßING, WOLFGANG A. SCHULZ, FINN K. HANSEN, TAKAKO ASANO, AKINORI SATO
Anticancer Research Dec 2021, 41 (12) 5987-5996; DOI: 10.21873/anticanres.15417
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