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By Zhanwei Zeng, Shiyi Liao, Huan Zhou, Hongyu Liu, Jiantao Lin, Yunsheng Huang, Chenhui Zhou, and Daohua Xu

Excerpt from the article published in Frontiers in Pharmacology, 31 August 2022, Sec. Pharmacology of Anti-Cancer Drugs, DOI: https://doi.org/10.3389/fphar.2022.952980

Editor’s Highlights

• Female breast cancer is one of the most common malignancies in the world, with an estimated 2.3 million new cases per year.
• Most anti-tumor drugs inevitably show reduced drug efficacy and tumors exhibit multidrug resistance (MDR) to chemotherapeutic drugs during long-term chemotherapy.
• ATP-binding cassette subfamily B member 1 (ABCB1) or P-glycoprotein (P-gp) and ATP-binding cassette subfamily G member 2 (ABCG2) play indispensable roles in cancer cell MDR.
•  Sigma-2 (σ₂) receptor ligands have been found to be potential agents for tumor therapy, with the capacity to inhibit proliferation and induce apoptosis in tumor cells.
• A σ₂ agonist compound demonstrated anti-MDR activity by inhibiting the transporting function of ABCB1 and ABCG2 transporters and thus was a potential therapeutic agent for the treatment of tumor resistance.

Abstract

Multidrug resistance (MDR) is thought to be one of the main reasons for the failure of chemotherapy in cancers. ATP-binding cassette subfamily B member 1 (ABCB1) or P-glycoprotein (P-gp) and ATP-binding cassette subfamily G member 2 (ABCG2) play indispensable roles in cancer cell MDR. Sigma-2 (σ₂) receptor is considered to be a cancer biomarker and a potential therapeutic target due to its high expression in various proliferative tumors. Recently, σ₂ receptor ligands have been shown to have promising cytotoxic effects against cancer cells and to modulate the activity of P-glycoprotein (ABCB1) in vitro experiments, but their specific effects and mechanisms remain to be elucidated. We found that A011, a σ₂ receptor ligand with the structure of 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline, showed promising cytotoxicity against breast cancer MCF-7 and adriamycin-resistant MCF-7 (MCF-7/ADR), induced apoptosis, and reversed adriamycin (ADR) and paclitaxel resistance in MCF-7/ADR cells. Furthermore, we demonstrated that A011 increased the accumulation of rhodamine 123 and mitoxantrone in MCF-7/ADR cells. A011 significantly decreased the ATPase activity of the ABCB1 and down-regulated ABCG2 protein expression. In addition, A011, administered alone or in combination with ADR, significantly inhibited tumor growth in the MCF-7/ADR tumor-bearing nude mouse model. A011 may be a potential therapeutic agent for the treatment of tumor resistance.

Introduction

Female breast cancer is one of the most common malignancies in the world. Breast cancer surpassed lung cancer to become the most prevalent cancer in 2020, with an estimated 2.3 million new cases (Sung et al., 2021). Over the past decades, tremendous progress has been made in the development of chemotherapeutic drugs. However, tumor recurrence and metastasis remain one of the major challenges in cancer treatment during the long-term course of chemotherapy (Warren, 2009). Most anti-tumor drugs inevitably show reduced drug efficacy and tumors exhibit multidrug resistance (MDR) to chemotherapeutic drugs during long-term chemotherapy, leading to tumor recurrence in patients. It has been reported that over 90% deaths in the chemotherapy population are associated with MDR (Bukowski et al., 2020). Various mechanisms have been reported to be involved in the development of MDR in tumor cells, including ATP-binding cassette (ABC) transporter family-mediated drug efflux, apoptosis down-regulation and epigenetic regulation, etc., of which the ABC transporter family is the most widely studied (Abraham et al., 2009; Rocha et al., 2018; Assaraf et al., 2019; Bukowski et al., 2020).

The ABC transporter superfamily is one of the largest families of transmembrane proteins, consisting of 49 ABC transporter members (Asif et al., 2020). Structurally, ABC transporters are characterized by a common structure: two nucleotide-binding domains (NBDs), which can bind and hydrolyze ATP, and two transmembrane domains (TMDs), which utilize the energy provided by ATP hydrolysis to transport substrates outside the cell. Functionally, most ABC transporters can transport the substrates produced during cellular metabolism (e.g., sugars, lipids, ions, peptides, amino acids and toxic components) from the cytoplasm to outside the cell membrane, and thus play an important role in maintaining normal physiological and pathological processes in the organism (Borst and Elferink, 2002; Theodoulou and Kerr, 2015). Dysregulated ABC transporters are associated with tumor resistance (Dean et al., 2001; Amawi et al., 2019). More importantly, several ABC transporters have been frequently found to be overexpressed in a variety of tumor resistant cells, such as P-glycoprotein (P-gp, ABCB1) and breast cancer resistance protein (BCRP, ABCG2). It has been found that the ABC transporters could bind and transport chemotherapeutic drugs outside the cell by utilizing the energy provided by ATP hydrolysis, resulting in lowering the concentration of intracellular drug accumulation, reducing the efficacy of chemotherapeutic drugs and the failure of tumor treatment (Amawi et al., 2019; Liu, 2019; Eckenstaler and Benndorf, 2020). Therefore, there is an urgent need to seek small molecule inhibitors targeting ABC transporters to restore the efficacy of chemotherapeutic drugs.

A lot of studies have shown that σ₂ receptor may be a potential therapeutic target for tumors (Zeng and Mach, 2017; Oyer et al., 2019; Schmidt and Kruse, 2019; Chen et al., 2021). The σ₂ receptor was found to be overexpressed in rapidly proliferating tumors such as lung, breast and pancreatic cancers and was identified as an important biomarker of tumor cell proliferation (Zeng et al., 2020). And σ₂ receptor ligands have been found to be potential agents for tumor therapy, with the capacity to inhibit proliferation and induce apoptosis in tumor cells (Nicholson et al., 2015; Cantonero et al., 2020). In addition, the σ₂receptor ligands exhibited promising anti-tumor proliferative activity against breast cancer MCF-7/ADR cells and could inhibit the activity of ABCB1, suggesting that σ₂ receptor ligands may be potential therapeutic agent in anti-tumor MDR (Azzariti et al., 2006; Abate et al., 2011).

It was reported that σ₂ receptor ligands with a 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline structure were capable of modulating the activity of ABCB1 (Pati et al., 2018). Recently, we synthesized a series of σ₂ ligands with 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline structural derivatives, and found many of them showed high affinity for the σ₂ receptor (Sun et al., 2018). Our previous study revealed that σ₂ ligand A011 with a 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline structure had high affinity for σ₂ receptor and showed good antitumor activity against a variety of tumor cells, including breast cancer MCF-7, MDA-MB-231 and lung cancer A549 cells (Li et al., 2022). However, its role and mechanism in tumor resistance remains to be further investigated.

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Results

Cytotoxicity, resistance fold and reversal effect of A011

We determined the cytotoxicity of A011 and positive drugs ADR, cisplatin and paclitaxel on three drug-resistant cells MCF-7/ADR, A549/DDP and HepG2/DDP and their parental cells by CCK-8 kits. ADR, cisplatin and paclitaxel showed good antitumor effects on MCF-7, A549, and HepG2 cells, with significantly decreased cytotoxic effects in MCF-7/ADR, A549/DDP, and HepG2/DDP cells, with RF value mostly >5, indicating that the 3 cell lines were multidrug resistant. In contrast, the IC₅₀ values of A011 were 3.79 ± 0.13 μM, 5.35 ± 0.42 μM, RF value 1.41, 4.59 ± 0.35 μM, 7.74 ± 0.32 μM, RF value 1.69, 4.07 ± 0.25 μM, 8.29 ± 0.04 μM, RF value 2.04 for MCF-7, MCF-7/ADR, A549, A549/DDP, HepG2 and HepG2/DDP cells, respectively (Table 1). A011 showed similar anti-proliferative effects on drug-resistant cells and their parental cells, indicating that A011 had excellent anti-tumor MDR effects (Figure 1A).

Based on the results of the CCK-8 assay, A011 had a stronger anti-proliferative effect in MCF-7/ADR cells relative to A549/DDP and HepG2/DDP cells, and therefore MCF-7/ADR cells were used as the subsequent experimental cell line. Moreover, A011 showed >90% survival of MCF-7/ADR cells at 1.25 and 2.5 μM concentrations and were therefore selected as the concentration for combination treatment. The results showed that A011 at 1.25 and 2.5 μM concentrations significantly increased the cytotoxicity of ADR, cisplatin and paclitaxel in MCF-7/ADR cells. No sensitization was observed in parental cells. And the inhibition effect of A011 combined with ADR was superior compared with that of ABCB1 inhibitor verapamil and ABCG2 inhibitor KO143 combined with ADR (p < 0.05) (Table 2). In addition, A011 significantly inhibited the clonogenic ability of MCF-7/ADR cells and their parental cells (Figure 1B). These results suggested that A011 has excellent anti-tumor MDR activity and could enhance the sensitivity of MCF-7/ADR cells to ADR, cisplatin and paclitaxel.

A011 induced apoptosis in MCF-7/ADR and its parental cells.

Hoechst 33,258 staining showed that MCF-7/ADR and MCF-7 cells exhibited increased cytoplasmic density, nuclear consolidation, nuclear membrane nucleolus fragmentation and increased apoptotic vesicles in a dose-dependent manner after A011 treatment compared to cells from control group with intact cell structure (Figure 2A). Furthermore, the flow cytometry results showed that A011 (5, 10, 20, and 40 μM) significantly induced apoptosis in MCF-7/ADR and MCF-7 cells, with apoptosis rate of 5.20% ± 0.55%, 25.15% ± 9.99%, 95.6% ± 6.35%, 99.47% ± 0.15% for MCF-7/ADR cells and 9.47% ± 1.25%, 11.87% ± 1.84%, 43.19% ± 8.81% and 87.51% ± 2.70% for MCF-7 cells (Figure 2B). The results indicated A011 could inhibit tumor cells growth by inducing cell apoptosis in MCF-7 and MCF-7/ADR.

A011 significantly increased the accumulation and decreased the efflux of Rh123 and mitoxantrone in MCF-7/ADR cells

To investigate the mechanism by which A011 reversed drug resistance in tumor cells, we determined the effect of A011 on the function of ABCB1 and ABCG2 transporters. Verapamil and KO143 were inhibitors of the ABCB1 and ABCG2 transporters respectively, and were able to inhibit the transport of substrates outside the cell membrane by the ABCB1 and ABCG2 transporters, so they were used as positive control. In addition, Rh123 and mitoxantrone were fluorescent substrates for the ABCB1 and ABCG2 transporters respectively, and were able to be quantified by flow cytometry. The results showed that the accumulation of Rh123 or mitoxantrone in MCF-7/ADR cells was significantly lower than their accumulation in parental cells. A011 significantly increased the accumulation of Rh123 and mitoxantrone in MCF-7/ADR cells. Moreover, the intracellular accumulation of Rh123 was higher in the A011 group compared to the verapamil group (Figures 3A,B). In addition, efflux experiments showed that the amount of Rh123 or mitoxantrone in MCF-7/ADR cells was significantly reduced during the time course, whereas the addition of A011 significantly inhibited the efflux of Rh123 or mitoxantrone, suggesting that the increased accumulation of Rh123 and mitoxantrone in MCF-7/ADR cells was due to inhibition of their efflux (Figures 4A,B). These results indicated that A011 could inhibit the transport function of ABCB1 and ABCG2 transporters.

A011 significantly decreased the ATPase activity of the ABCB1 transporter

To further investigate the role of A011 on the function of the ABCB1 transporter, we measured the effect of A011 on the ATPase activity of the ABCB1 transporter. The results showed that verapamil increased the activity of ABCB1 transporter ATPase, which was in agreement with the description of previous studies (Ledwitch et al., 2016). In contrast, A011 could significantly decrease the activity of ABCB1 transporter ATPase compared to the control group, indicating that A011 could inhibit the transport function of ABCB1 transporter by inhibiting the activity of ABCB1 transporter ATPase (Figure 4C).

A011 Decreased ABCG2 Protein Expression without Altering ABCB1 Protein Expression and Localization of ABCG2 and ABCB1 Proteins in MCF-7/ADR Cells

Given reducing the expression of transporter proteins and altering their localization in cells is one of the mechanisms to overcome MDR, we further investigated the effect of A011 on ABCB1 and ABCG2 proteins in MCF-7/ADR cells by immunofluorescence and western blot. Immunofluorescence assay showed that A011 did not alter the localization of ABCB1 and ABCG2 proteins in MCF-7/ADR cells (Figures 5A,B). And western blot showed that A011 down-regulated ABCG2 protein expression and did not alter ABCB1 protein expression (Figure 5C).

To evaluate the anti-tumor MDR activity of A011 in vivo, we established a xenograft model with MCF-7/ADR cells in nude mice. A011 (1 mg/kg) or ADR (5 mg/kg) alone showed low growth inhibitory activity against MCF-7/ADR tumors, with growth inhibition rates of 6.94% and 22.58%, respectively. However, when ADR (5 mg/kg) was co-administered with A011 (1 mg/kg), the anti-tumor activity of ADR was significantly enhanced with the growth inhibition rate of 58.43%. A011 (5 mg/kg) alone showed good anti-tumor activity against MCF-7/ADR tumors with the growth inhibition rate of 43.13% (Figures 6A–C). In addition, there was no significant change in body weight in the A011 group compared to the saline group (Figure 6D). These results suggested that A011 alone or in combination had promising anti-tumor MDR activity and was well tolerated in vivo.

To evaluate the effects of A011 in liver, kidney and tumor tissues in nude mice, we performed HE tissue staining, TUNEL staining and immunohistochemistry experiments respectively. HE staining of the liver and kidney showed that there was no difference between all treatment groups and control group, except for a small amount of inflammatory cell infiltration in the ADR (5 mg/kg) and A011 combined with ADR groups. In HE staining of tumor tissue, approximately 60% and 70% of tumor cells were necrotic in A011 (5 mg/kg) and A011 combined with ADR respectively, compared to 40% in the control group (Figure 7A). TUNEL staining showed that administration of A011 (5 mg/kg) alone or A011 (1 mg/kg) in combination with ADR (5 mg/kg) significantly induced an increase in MCF-7/ADR tumor apoptosis (Figure 7B). Immunohistochemistry of ABCG2 showed dark brown staining of ABCG2 protein in the saline and ADR (5 mg/kg) groups. As the concentration of A011 increased, the expression of ABCG2 was down-regulated (Figure 7C). A011 did not alter the expression of ABCB1 (Supplementary Figure S1). Above results suggest that A011 could induce apoptosis and down-regulate ABCG2 protein expression in MCF-/ADR tumor cells in vivo, without significant toxicity to liver and kidney tissues.

Discussion

Intrinsic MDR and acquired MDR are two of the major barriers to tumor treatment, seriously threatening the survival and affecting the lives of cancer patients (Zheng, 2017; Al-Akra et al., 2019). Due to the narrow therapeutic window of most chemotherapeutic agents, the emergence of tumor MDR has greatly limited the clinical use of chemotherapeutic agents, therefore it is particularly important for cancer treatment to overcome tumor MDR.

The σ₂ receptor has been found to be highly expressed in rapidly proliferating tumors such as breast cancer and is considered to be one of the potential targets for the treatment of tumors (Huang et al., 2014). Most of the σ₂ ligands not only show high affinity for the σ₂receptor but also exhibit excellent anti-tumor activity (Georgiadis et al., 2017; Sun et al., 2018; Liu et al., 2019), and those with a 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline structure could also interact with ABCB1 (Pati et al., 2015). Moreover, a few σ₂ receptor agonists were found to be collateral sensitive and their anti-proliferative activity was stronger in cells with high P-gp expression than in P-gp negative cells, suggesting that σ₂receptor ligands may have promising activity in drug-resistant tumors (Niso et al., 2013; Abatematteo et al., 2021). In this study, we found that σ₂ ligand A011 showed significant cytotoxic activity in three tumor cell lines MCF-7, A549 and HepG2 cells and generally showed no decrease in cytotoxic activity in drug-resistant cell lines MCF-7/ADR, A549/DDP and HepG2/DDP cells. In addition, A011 significantly increased the sensitivity of MCF-7/ADR cells to ADR at concentrations of 1.25 and 2.5 μM. And in vivo experiments, A011 (5 mg/kg) alone or A011 (1 mg/kg) co-administered with ADR showed promising anti-tumor activity, significantly inhibiting the growth of MCF-7/ADR tumors without significant toxicity, suggesting that A011 has the potential to overcome MDR.

Apoptosis is a form of programmed cell death that is co-regulated by multiple genes with important roles in maintaining the homeostasis of the internal environment and controlling cell proliferation (Goldar et al., 2015)_. Dysregulation of apoptosis is one of the hallmarks of cancer and is associated not only with tumorigenesis and progression, but also with tumor resistance to chemotherapeutic agents (Pistritto et al., 2016). We found that A011 could dose-dependently induce apoptosis in MCF-7/ADR and its parental cells, and the apoptosis induction of A011 was better in MCF-7/ADR than MCF-7 cells. In a MCF-7/ADR xenograft model, A011 (5 mg/kg) was able to induce an increased apoptosis relative to the control group, suggesting that A011 may also promote cell death by inducing MCF-7/ADR apoptosis.

ABC transporters have been found to be relatively highly expressed in drug-resistant tumors and able to transport intracellular chemotherapeutic agents to the extracellular compartment by relying on the energy provided by ATP hydrolysis, thereby mediating the resistance of tumor cells to chemotherapeutic agents (Choi and Yu, 2014; Beis, 2015). To elucidate the mechanism that A011 overcame MDR in MCF-7/ADR cells, we examined the activity and protein expression of the ABCB1 and ABCG2 transporters. Our results showed that A011 could inhibit ABCB1 transport function and ATPase activity, but had no effect on its protein expression. In addition, it was first discovered that A011 also inhibited the transport function and protein expression of ABCG2, which was further validated in ABCG2 protein immunohistochemical assay in vivo. Besides inhibiting ABC transporter activity and protein expression, ABC transporters as transmembrane proteins, altering their localization in cells is also part of the strategy to inhibit ABC transporter-mediated MDR (Zhao et al., 2019). However, A011 did not affect the localization of the ABCB1 and ABCG2 transporters in MCF-7/ADR cells. These results suggested that A011 could inhibit the function of ABCB1 and ABCG2 transporters and reduce the expression of ABCG2 protein, thereby overcoming MDR. However, the mechanisms of A011 inhibiting ATPase activity and expression of ABCG2 protein remain to be clarified.

A variety of σ₂ ligands are currently being developed for clinical diagnosis and cancer treatment with PET imaging of tumors. Phase I clinical trial results for the σ₂ radioligand [¹⁸F]ISO-1 showed that [¹⁸F]ISO-1 uptake values correlated with tumor Ki-67 (a gold standard proliferation biomarker) and are expected to be used for in vivo measurement of tumor proliferation status (Dehdashti et al., 2013). Studies have shown that a lot of selective σ₂ receptor ligands displayed cytotoxic effects on a variety of human cancer cells, and inhibited tumor growth (Asong et al., 2019; Liu et al., 2019). We previously found that A011 was able to increase intracellular ROS and Ca²⁺ levels in MCF-7 cells and induced endoplasmic reticulum stress and autophagy (Li et al., 2022). In addition, the study showed that A011 was well tolerated and had no significant toxicity to liver and kidney tissues. These results provided further insight into the pharmacological role of the σ₂ receptor and A011 may be a candidate for cancer treatment either alone or in combination with other anticancer agents.

Conclusion

In this study, we elucidated that the σ₂ ligand A011, containing a 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline structure, exhibited excellent anti-breast cancer MDR activity both in vivo and in vitro, either alone or in combination with ADR. A011 demonstrated anti-MDR activity by inhibiting the transporting function of ABCB1 and ABCG2 transporters and thus was a potential therapeutic agent for the treatment of tumor resistance.