Combinatory anti-tumor activities of AQ4 and temsirolimus against colorectal cancer cells

doi:10.21203/rs.3.rs-1984936/v1

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Combinatory anti-tumor activities of AQ4 and temsirolimus against colorectal cancer cells

https://doi.org/10.21203/rs.3.rs-1984936/v1

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Purpose: Banoxantrone is a topoisomerase II inhibitor that is selectively activated in hypoxia. Although it has exhibited anti-tumor activity against several types of cancers in preclinical models, its efficacy against colorectal cancer (CRC) remains unclear.

Methods: We examined the antitumor effects of AQ4, an activated metabolite of banoxantrone, in CRC cell lines (HT-29, CaR-1) using in vitro experiments under normoxic and hypoxic conditions. The inhibition of cell growth was assessed using a proliferation assay. The induction of apoptosis and changes in the cell cycle were measured using flow cytometry. Signaling pathways involved in apoptosis and hypoxia were analyzed. The anti-tumor activity of temsirolimus, an inhibitor of mammalian target of rapamycin, and the combined effects of temsirolimus and AQ4 were also evaluated.

Results: Regardless of the oxygen condition, a single drug treatment with AQ4 or temsirolimus inhibited proliferation and induced apoptosis in both cell lines, accompanied by a reduction in the phosphorylation of S6. AQ4 induced G2/M cell cycle arrest, whereas temsirolimus induced G0/G1 arrest. Moreover, the combined treatment markedly reduced the proportion of cells in the S phase and enhanced apoptosis, as evidenced by an increased Bax/Bcl-2 ratio. The hypoxia-induced activation of the HIF-1α pathway was suppressed by AQ4 and temsirolimus.

Conclusion: Based on the cooperative anti-tumor activity of AQ4 and temsirolimus in vitro, the combination of banoxantrone plus temsirolimus has potential as a treatment option for CRC in preclinical and clinical settings.

temsirolimus

AQ4

colorectal cancer

hypoxia

anti-tumor activity

Colorectal cancer (CRC) is the fourth most commonly diagnosed and third most deadly cancer worldwide (Bray et al. 2018; Sawicki et al. 2021). The mainstay of treatment for patients with metastatic CRC is systemic chemotherapy based on cytotoxic drugs, such as 5-fluorouracil, oxaliplatin, and irinotecan, or their combination with or without biologics (Hashiguchi et al. 2020; National Comprehensive Cancer Network. 2022a, b; Van Cutsem et al. 2016). Although these regimens have achieved tolerable response rates and prolonged survival (Hashiguchi et al. 2020; National Comprehensive Cancer Network. 2022a, b; Van Cutsem et al. 2016), many metastatic CRC patients do not respond to these treatments. Therefore, there is an imminent need for new drugs and a broader range of therapeutic options against CRC.

Temsirolimus (TEM), a water-soluble inhibitor of mammalian target of rapamycin (mTOR), has been shown to exhibit anti-tumor activity against a wide range of cancers, including CRC (Johnson et al. 2018; Kaneko et al. 2014; Nguyen et al. 2012; Shiratori et al. 2019), and functioned as a chemosensitizer in preclinical models (Dancey et al. 2009). Regarding the mechanisms underlying the anti-tumor effects of TEM, we previously demonstrated that TEM exhibited not only direct anti-tumor activities, but also inhibited angiogenesis in CRC tumors in mouse models (Kaneko et al. 2014). Therefore, we hypothesized that hypoxia-activated drugs may further increase the efficacy of TEM.

Banoxantrone dihydrochloride (AQ4N) is an aliphatic N-oxide that is metabolized by cytochrome P (CYP450) isozymes and inducible nitric oxide synthase to AQ4, and is categorized as a potent inhibitor of topoisomerase II (TOP2) (McKeown et al. 1995; Patterson and McKeown 2000; Phillips 2016; Smith et al. 1997). TOP2 inhibitors block the ligation step of DNA during its replication of transcription, which, in turn, activates the apoptosis or necrosis cascade (McKeown et al. 1995; Patterson and McKeown 2000; Phillips 2016; Smith et al. 1997). Notably, AQ4N is selectively reduced to its active form, AQ4 by hypoxia, and, thus, displays cytotoxicity to hypoxic tumor cells that may manifest a more aggressive phenotype or acquire resistance to chemotherapy (Patterson and McKeown 2000; Phillips 2016). AQ4N was shown to exert anti-tumor effects on human fibrosarcoma as a single agent or in combination with radiation in vitro (Mehibel et al. 2016). Previous studies reported that AQ4N inhibited the growth of murine mammary carcinoma, squamous cell carcinoma, and fibrosarcoma in combination with cyclophosphamide and cisplatin (Friery et al. 2000; Gallagher et al. 2001). The cytotoxic mechanisms of AQ4N are distinct from those of the aforementioned drugs currently used to treat CRC patients. These findings prompted us to investigate the potential anti-tumor effects of AQ4N on CRC cells.

A previous study demonstrated that mTOR induced HIF-1α in hypoxic conditions, leading to the suppression of CYP450 (Legendre et al. 2009). Therefore, we speculated that AQ4N may be effectively activated by CYP450 into AQ4 under hypoxia induced by an mTOR inhibitor. Before moving forward to future treatments with a combination of AQ4N and TEM in vivo, we herein investigated the synergic effects of both drugs on CRC cell lines in vitro.

Cells and reagents

The human CRC cell lines, HT-29 and CaR-1, were purchased from the Japanese Cancer Research Resource Bank (Osaka, Japan). Cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum and 1% antibiotics/antimycotics, and were then incubated in a 5% CO₂ and 21% O₂ incubator at 37°C. Under hypoxic conditions, cells were cultured in a modular incubator chamber filled with 5% CO₂ and 1% O₂ at 37°C. AQ4 (Sigma-Aldrich, St. Louis, MO) and TEM (Sigma-Aldrich) were dissolved in dimethyl sulfoxide at a concentration of 1 mM. Stock solutions were diluted to the desired final concentrations with growth medium before use.

Incorporation of AQ4 into cells

CRC cells were treated with AQ4 (4 µM) for 4 h and then observed under a fluorescence microscope (BZ-X810, KEYENCE, Osaka, Japan).

Cell proliferation

To separately evaluate the effects of AQ4 and TEM on cell proliferation, cells were treated with AQ4 (0, 1, 2, or 4 µM) for 48 h or TEM (100 nM) for 48 h. To assess the combined effects of AQ4 and TEM on proliferation, cells were treated with AQ4 (2 µM) and TEM (100 nM) for 48 h. As a control, cells were incubated for 48 h in the presence of the vehicle alone. All assays were examined under normoxic and hypoxic conditions. Cell proliferation was evaluated by the MTS assay using the Celltiter 96 AQueous One solution Cell Proliferation assay kit (Promega, Madison, WI) according to the manufacturer’s instructions. The absorbance of formazan at 490 nm was directly measured using a microtiter plate reader (VARIOSKAN FLASH, Thermo Fisher Scientific, Waltham, MA).

Analysis of the cell cycle by flow cytometry

Cells were treated with AQ4 (2 µM) and/or TEM (100 nM) for 48 h under normoxic or hypoxic conditions. As a control, cells were incubated for 48 h in the presence of the vehicle alone. Treated cells were analyzed by flow cytometry using the fluorescein isothiocyanate (FITC) BrdU Flow Kit (BD Pharmingen) according to the manufacturer’s instructions. Cell cycles were analyzed with fluorescence-activated cell sorting (FACS) and Accuri C6 Plus software.

Detection of apoptosis by flow cytometry

Cells were treated with AQ4 (2 µM) and/or TEM (100 nM) for 72 h under normoxic or hypoxic conditions. As a control, cells were incubated for 72 h in the presence of the vehicle alone. Cells were then stained with Annexin V-FITC and propidium iodide using the Annexin V-FITC Apoptosis Detection kit (BD Pharmingen, San Jose, CA) according to the manufacturer’s instructions. Cells were analyzed using a FACS flow cytometer (Accuri C6 Plus, Becton-Dickinson, Mountain View, CA) and apoptotic cells with Accuri C6 Plus software (Becton-Dickinson).

Western blotting of cultured cell extracts

Cells were treated with AQ4 (2 µM) and/or TEM (100 nM) for 48 or 72 h under normoxic or hypoxic conditions. As a control, cells were incubated for 48 or 72 h in the presence of the vehicle alone. Cultured cells were lysed with 0.2 ml 25% Bolt LDS Sample Buffer (Thermo Fisher Scientific) containing 10% Sample Reducing Agent (Thermo Fisher Scientific) and cOmplete ULTRA tablet (Roche Diagnostics, Mannheim, Germany). Total protein concentrations were measured by Qubit 3.0 Fluorometer (Invitrogen, Carlsbad, CA). Cell lysates were separated on a Bolt 4–12% Bis-Tris Plus Gel (Thermo Fisher Scientific) and transferred to polyvinylidene difluoride membranes (pore size, 0.2 µm) using the iBlot 2 Dry Blotting System (Thermo Fisher Scientific). After blocking with 1× Tris-buffered saline plus 2% w/v nonfat dry milk at room temperature for 10 min, membranes were incubated with the following primary antibodies at 4°C overnight: Bax (Santa Cruz Biotechnology, Santa Cruz, CA; dilution 1:500), Bcl-2 (Santa Cruz Biotechnology; dilution 1:500), cyclin B1 (Santa Cruz Biotechnology; dilution 1:100), cyclin D (Santa Cruz Biotechnology; dilution 1:100), HIF-1α (Abcam; dilution 1:500), REDD1 (ProteinTech, Rosemont, IL; dilution 1:1000), phospho-S6 ribosomal protein (p-S6, Cell Signaling Technology; dilution 1:1000), and ß-actin (Sigma-Aldrich; dilution 1:1000). After washing with 1× Tris-buffered saline plus 0.05% tween 20 for 30 min, membranes were incubated with horseradish peroxidase-linked anti-rabbit IgG or anti-mouse IgG (Cytiva, Marlborough, MA) at room temperature for 60 min. Immunoreactive bands were visualized by enhanced chemiluminescence using the ECL detection system (Amersham Pharmacia Biotech, Buckinghamshire, UK). The density of each band was measured semi-quantitatively using ImageJ software (http://rsb.info.nih.gov/ij/) and standardized by the density of β-actin.

Statistical analysis

Statistical analyses were performed using JMP Pro 15.0.0 (SAS Institute, Cary, NC). The significance of differences was evaluated by an unpaired Student’s t-test. All reported p-values were two-sided and considered to be significant if less than 0.05.

Inhibition of CRC cell proliferation by AQ4 and TEM

AQ4 was taken up into the cytoplasm and nuclei of all cultured HT-29 and CaR-1 cells within four hours of its administration (Fig. 1).

We investigated the effects of AQ4 alone on the proliferative activity of CRC cells using the MTS assay. As shown in Fig. 2, the proliferation of HT-29 and CaR-1 cells was inhibited by AQ4 in a dose-dependent manner under normoxia and hypoxia. In comparisons with untreated controls, the relative rates of the proliferation of HT-29 and CaR-1 cells treated with 4 µM AQ4 were 0.73 ± 0.04 and 0.38 ± 0.05, respectively, under normoxia (Fig. 2A). Similar rates were observed when cells were treated with 4 µM AQ4 under hypoxia (0.63 ± 0.12 for HT-29 cells and 0.35 ± 0.12 for CaR-1 cells, Fig. 2B).

We then evaluated the combined effects of AQ4 and TEM on the proliferation of the CRC cell lines. In comparisons with untreated controls, the dual treatment with 2 µM AQ4 and 100 nM TEM further reduced the proliferation rates of HT-29 cells to 58% and 69% under normoxia and hypoxia, respectively. The proliferation rates of CaR-1 cells decreased to 33% under normoxia and 39% under hypoxia with the dual treatment. Proliferation rates in both cell lines were lower with the dual treatment than with AQ4 or TEM alone (Fig. 3).

Cell cycle and apoptosis of AQ4 in combination with TEM

To assess whether the inhibitory effects of AQ4 and TEM on the proliferation of CRC cells was dependent on the induction of apoptosis or cell cycle arrest, we performed a cell cycle analysis and apoptosis detection assay on cells treated with AQ4 and TEM.

In the cell cycle analysis, AQ4 induced arrest at the G2/M phase in HT-29 and CaR-1 cells. The G2/M fraction of HT-29 or CaR-1 cells increased by 52.2% and 2.6%, respectively, under normoxia (Fig. 4A). On the other hand, the TEM treatment slightly increased the cell fraction in the G0 phase in both cell lines. The combination of 2 µM AQ4 and 100 nM TEM reduced S phase cells by 35.2% in HT-29 cells and by 18.3% in CaR-1 cells. Under hypoxia, the AQ4 treatment increased the G2/M fraction of HT-29 or CaR-1 cells by 63.8% and 14.3%, respectively. The dual treatment reduced S phase cells by 40.4% in HT-29 cells and 11.9% in CaR-1 cells (Fig. 4B).

Apoptotic cells increased after a 72-h treatment with AQ4 in both HT-29 and CaR-1 cells under normoxia; the percentage of Annexin V (+) HT-29 cells increased from 10.0–23.3% and that of CaR-1 cells from 11.4–15.6% (Fig. 5A). Although the TEM treatment for 72 h did not change the percentage of apoptotic cells in either cell line, further increases in the number of apoptotic cells were observed when AQ4 was added in combination with TEM to HT-29 and CaR-1 cells; the percentage of Annexin V (+) increased up to 34.0% in HT-29 cells and 22.4% in CaR-1 cells (Fig. 5A). Similarly, under hypoxia, the percentage of Annexin V (+) HT-29 cells increased from 12.5–20.5% and that of CaR-1 cells from 11.0–16.2% by the AQ4 treatment alone (Fig. 5B). The combination of AQ4 and TEM increased the percentage of Annexin V (+) HT-29 cells to 30.0% and CaR-1 cells to 20.6% (Fig. 5B).

Changes in key mediators in cell cycle arrest and apoptosis by the AQ4 and TEM treatment

To elucidate the mechanisms underlying cell cycle arrest and apoptosis induced by AQ4 and TEM, we assessed cyclin B1, cyclin D, Bax, and Bcl-2 expression levels in CRC cells under normoxia by western blotting. As shown in Fig. 6, AQ4 up-regulated cyclin B1 expression, and down-regulated cyclin D in both cell lines. The combination of AQ4 and TEM further pronounced the changes of the cyclin expression levels; compared to the untreated controls, the expression level of cyclin B1 increased 14.9-fold in HT-29 cells and 2.67-fold in CaR-1 cells, whereas that of cyclin D decreased by 95% in HT-29 cells and 41% in CaR-1 cells.

AQ4 increased the expression levels of Bax and decreased those of Bcl-2 in both cell lines. TEM also increased Bax and decreased Bcl-2. The Bax/Bcl-2 ratio was increased by AQ4 or TEM, while the highest ratio was reached with the co-treatment; the combination of AQ4 and TEM increased the Bax/Bcl-2 ratio by 29.9-fold in HT-29 cells and by 17.4-fold in CaR-1 cells (Fig. 6).

Changes in HIF-1α, REDD1, and phospho-S6 expression by the AQ4 and TEM treatment

We examined changes in the HIF-1α signaling pathway by the AQ4 and TEM treatment and hypoxia. As shown in Fig. 7, the relative levels of HIF-1α in HT-29 cells treated with AQ4 or TEM alone for 48 h decreased to 47% and 58%, respectively. The co-treatment of AQ4 and TEM further reduced the HIF-1α expression level to 30% in HT-29 cells. Similar changes in HIF-1α expression were observed in CaR-1 cells by the AQ4 and/or TEM treatment (Fig. 7).

We then investigated the mechanisms by which the AQ4 and/or TEM treatment and hypoxia affect the expression of REDD1, a downstream mediator of HIF-1α. The expression of REDD1 was up-regulated under hypoxia in both cell lines. REDD1 expression levels were decreased by AQ4 or TEM alone, and the magnitude of the reduction by the combination of AQ4 and TEM was prominent; the co-treatment decreased REDD1 expression by approximately 90% under normoxia and by 50% under hypoxia regardless of the cell lines (Fig. 8).

We also examined the expression of phospho-S6 (p-S6) in AQ4 and TEM-treated CRC cell lines under normoxia and hypoxia (Fig. 9). In normoxic HT-29 cells, p-S6 levels were decreased by AQ4 and/or TEM. Hypoxia slightly decreased p-S6 expression in HT-29 cells, while the single treatment with AQ4 or TEM further reduced its expression. With the dual treatment of AQ4 and TEM, only a trace amount of the band for p-S6 was detected in hypoxic HT-29 cells. In CaR-1 cells, AQ4 slightly reduced p-S6 expression, whereas TEM completely suppressed the phosphorylation of S6 under normoxia. Hypoxia halved the expression of p-S6 in CaR-1 cells without the drug treatment. An additional treatment with AQ4 further decreased p-S6 expression, whereas p-S6 was not detected in TEM-treated hypoxic CaR-1 cells.

AQ4N is effective against various types of tumors as a single agent or in combination with other chemotherapeutic drugs. However, the anti-tumor effects and mechanisms of action of AQ4N on CRC have not yet been elucidated in detail. Lalani et al. reported that AQ4 exhibited anti-tumor activity against CRC cell lines including HT-29 cells in vitro (Lalani et al. 2007). The proliferation of CaR-1 cells as well as HT-29 cells was suppressed by AQ4 in the present study. Regarding the anti-tumor mechanisms of AQ4, previous studies showed that AQ4 inhibited TOP2 activity and induced cell cycle arrest at the G2 phase in human and rodent fibroblast cell lines (Patterson 1993; Smith et al. 1997). We also demonstrated that AQ4 induced G2/M cell cycle arrest and apoptosis in CRC cell lines.

Although previous studies evaluated AQ4 combined with other cytotoxic drugs or radiation (Friery et al. 2000; Gallagher et al. 2001; McKeown et al. 1995; Phillips 2016), it currently remains unclear whether the combined inhibition of TOP2 and mTOR exhibited anti-tumor activity against CRC. In the present study, when AQ4 was administered together with TEM, CRC cells in the S phase decreased further, while the number of apoptotic cells markedly increased. Our group previously demonstrated that TEM induced G0/G1 cell cycle arrest in CRC cell lines by down-regulating the PI3K/AKT/mTOR signaling pathway (Kaneko et al. 2014). Moreover, Lyu et al. recently reported that the silencing of TOP2 alpha resulted in the inactivation of the mTOR pathway in gallbladder cancer cells (Lyu et al. 2020). Based on these findings, the marked decrease in S phase induced cells by AQ4 and TEM may partly result from their synergistic inhibitory effects on the mTOR pathway. We speculate that the induction of apoptosis through increases in the Bax/Bcl-2 ratio, together with cell cycle arrest at multiple phases, strongly contributed to the inhibition of CRC cell proliferation by AQ4 and/or TEM.

HIF-1α is a key regulator for energy metabolism and angiogenesis under hypoxic conditions. In response to the hypoxic tumor environment, HIF-1α is frequently overexpressed, and this is considered to be associated with resistance to chemotherapy and poor outcomes in a wide range of tumors (Aebersold et al. 2001; Birner et al. 2000). Therefore, the inhibition of the HIF-1α pathway needs to be considered in the treatment of CRC. A previous study using human breast cancer cells showed that the activation of the PI3K/AKT/mTOR pathway up-regulated the expression of HIF-1α, while TEM inhibited HIF-1α protein synthesis in vitro and in vivo (Del Bufalo et al. 2006). We also found that TEM decreased the expression of HIF-1α and REDD1. On the other hand, regarding the inhibitory effects of TOP2 inhibitors on HIF-1α, Toh et al. indicated that independently of the mTOR pathway, mitoxantrone may interfere with the translation of HIF-1α by suppressing ribosome biogenesis or intercalating into HIF-1α mRNA (Toh and Li 2011). The structure of AQ4 is based on mitoxantrone with modifications to alkylamino side chains (Smith et al. 1997), and, thus, AQ4 may exhibit similar activities to mitoxantrone in terms of the down-regulation of the HIF-1α pathway. In the present study, AQ4 markedly reduced the expression of HIF-1α and REDD1 in HT-29 and CaR-1 cells, nevertheless p53, a negative regulator of HIF-1α, was mutated in both cell lines. Moreover, reductions were prominent when AQ4 was combined with TEM. Therefore, the dual treatment may inhibit HIF-1α expression through at least two distinct mechanisms, namely, 1) the suppression of the HIF-1α protein via positive feedback from the mTOR pathway by TEM, and 2) the p53-independent direct inhibition of HIF-1α by AQ4 (Fig. 10).

We and several groups showed that AQ4 and other topoisomerase inhibitors suppressed the phosphorylation of S6 (Cam et al. 2014; Gaur et al. 2011; Ki et al. 2019). However, the underlying mechanisms for the inactivation of S6 induced by TOP2 inhibitors remain unclear. Cam et al. suggested that TOP2 inhibitors suppressed the activation of the MAPK/ERK pathway, and down-regulated the phosphorylation of S6 in mouse embryonic fibroblasts in vitro (Cam et al. 2014). On the other hand, a review article reported the phosphorylation of ERK by DNA damaging agents, including TOP2 inhibitors, in various human cancer cells in vitro (Cagnol and Chambard 2010). Further studies are needed to elucidate the status of signal transduction in the PI3K/AKT/mTOR pathway and MAPK/ERK pathway by TOP2 inhibitors.

In conclusion, AQ4 in combination with TEM inhibited CRC cell growth through the induction of cell cycle arrest and apoptosis and the suppression of HIF-1α expression under hypoxia in vitro. To establish whether AQ4N and TEM are an effective treatment for CRC clinically, further in vivo studies are needed.

Funding

This research is supported by Grants-in-Aid for Scientific Research (C: grant number; 18K07194, C: grant number; 19K09114, C: grant number; 19K09115, C: grant number; 20K0905, Challenging Research (Exploratory): grant number; 20K21626, B: grant number; 21H02778) from Japan Society for the promotion of Science. This research is supported by the Project for Cancer Research and Therapeutic Evolution (P-CREATE), grant number: JP 19cm0106502 from the Japan Agency for Medical Research and Development (AMED).

Competing Interests

The authors have no conflict of interest.

Author contributions

KO, HN, HS, MK and SI developed the study design and concept, retrieved the data of experiments and carried out the analysis. All authors participated in writing and revising the manuscript critically. All authors read and approved the final manuscript.

Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval

This study did not require ethics approval.

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No competing interests reported.

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Combinatory anti-tumor activities of AQ4 and temsirolimus against colorectal cancer cells

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Abstract

Figures

Introduction

Materials And Methods

Cells and reagents

Incorporation of AQ4 into cells

Cell proliferation

Analysis of the cell cycle by flow cytometry

Detection of apoptosis by flow cytometry

Western blotting of cultured cell extracts

Statistical analysis

Results

Inhibition of CRC cell proliferation by AQ4 and TEM

Cell cycle and apoptosis of AQ4 in combination with TEM

Changes in key mediators in cell cycle arrest and apoptosis by the AQ4 and TEM treatment

Changes in HIF-1α, REDD1, and phospho-S6 expression by the AQ4 and TEM treatment

Discussion

Declarations

References

Additional Declarations

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