Anti-tumor activity of butorphanol in colorectal cancer via targeting SIGMAR1

Abstract

Colorectal cancer (CRC) stands for a prevailing gastrointestinal neoplasm, concomitant with considerable occurrence and lethality rate. Butorphanol, a synthetic opioid analgesic medication targeting the opiate receptor, has been recently reckoned to harbor anti-oncogenic properties. This study proposes to delineate the impacts of butorphanol on CRC and the interrelated response mechanism. In sigma non-opioid intracellular receptor 1 (SIGMAR1)-overexpressing CRC cells treated by varying concentrations of butorphanol, the functional experiments including CCK-8 method, EDU staining, wound healing and transwell assays severally appraised the capabilities for CRC cells to proliferate, migrate as well as invade. TUNEL staining assayed the cellular apoptotic level. The expressions of proteins implicated in proliferation, metastasis as well as apoptosis were ascertained by Western blot. CB-Dock2 server predicated butorphanol-SIGMAR1 interaction and Western blot also examined SIGMAR1 expression. Noticeably, butorphanol profoundly eliminated the capabilities of CRC cells to proliferate, migrate and invade whilst intensified the cellular apoptotic level with the ascending doses. Butorphanol was identified to possess an interrelation with SIGMAR1 and concentration-dependently lowered SIGMAR1 expression. Elevation of SIGMAR1 partially blunted the affection of butorphanol on the biological events of CRC cells. To sum up, butorphanol may extenuate the aggressive cellular behaviors to produce tumor-suppressing activity on CRC via binding with SIGMAR1.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12672-024-01581-1.

Introduction

Colorectal cancer (CRC) stands for a prevailing primary intestinal neoplasm involving colon and rectum [1]. In the United States, CRC was estimated to result in 153,020 new cases, taking a death toll of over 52 thousand deaths in 2023 [2]. Specifically, CRC is reckoned to be attributed to diversified pathogenic factors including lifestyles, genetic and environmental factors [3]. At present, curative resection integrated with chemoradiotherapy remain the mainstay curative approach for CRC patients, apart from which, immunotherapy and targeted therapy are also well established as novel treatment alternatives [4, 5]. Pressingly, tumor relapse and distant metastasis are frequent events which greatly restrain the therapeutic outcomes, however. Accordingly, there is an imperative need to identify the latent mechanism of CRC metastasis and develop novel treatment regimens.

Butorphanol is a lipid-soluble anesthetic drug that possesses a potent affinity for κ-opioid receptor (KOR) and a weak antagonist activity for μ-opioid receptor (MOR) [6]. In clinical practice, butorphanol is extensively applied to pain relief [7]. In recent years, substantial evidence has supported that butorphanol can function on important organs such as heart, brain, lung, and so on to elicit protective effect in diversified human diseases [8–10]. More importantly, emerging literatures have unmasked that butorphanol can retard the aggressive transformation of cancer cells to produce anti-tumor activity in osteosarcoma [11], ovarian cancer [12] as well as hepatocellular carcinoma [13]. Despite these findings, the participation of butorphanol in CRC remains explicit.

Sigma non-opioid intracellular receptor 1 (SIGMAR1) is a ubiquitously expressed ligand-operated chaperone or scaffolding protein that predominantly localizes at the interface of mitochondria-endoplasmic reticulum and is implicated in a wide array of cellular functions [14, 15]. Previous review has summarized the potential regulatory role of SIGMAR1 in human malignancies [16]. In particular, SIGMAR1 displays aberrantly boosted expression in CRC samples and cells and is concerned with advanced tumor grade, lower overall survival and cancer cell migration [17, 18].

Intriguingly, Sea (https://sea.bkslab.org/; access data 2023.10.23) and targetnet databases (http://targetnet.scbdd.com/; access data 2023.10.23) predicted the possible binding of butorphanol with SIGMAR1. Taking all these findings into consideration, the current study is committed to figuring out the possible functions of butorphanol on the process of CRC and the underlying crosstalk between butorphanol and SIGMAR1.

Materials and methods

Cell culture and treatment

CRC cell lines SW620 and HCT116 accessed from BeNa Culture Collection (BNCC, Beijing, China) were severally nourished by Dulbecco's modified Eagle medium (DMEM; Sangon Biotech, Shanghai, China) and Roswell Park Memorial Institute (RPMI)-1640 medium (Sangon Biotech, Shanghai, China) enhanced with 10% fetal bovine serum (FBS; PAA Clone, Coelbe, Germany) in a water-saturated atmosphere equipped with 5% CO₂ at 37 °C. Additionally, both cells were treated by butorphanol (Cayman Chemical, Ann Arbor, MI, USA) at varying doses (0, 2.5, 5, 10, 20, 40 and 80 μg/ml).

Gene overexpression

With reference to Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), cells were treated with the SIGMAR1 adenovirus (Ov-SIGMAR1) and control adenovirus (Ov-NC) gained from Vigenebio (Shandong, China), abiding by the kit recommendations.

Cell counting kit-8 (CCK-8)

Cells sowed into the 96-well flat-bottomed plate (5 × 10³ cells/well) were administrated with ascending doses of butorphanol (0, 2.5, 5, 10, 20, 40 and 80 μg/ml), each well of which was subsequently given 10 μl CCK8 working liquid (Abmole Bioscience, USA) and left for 2 h. Under a microplate reader, the absorbance reading was implemented at 450 nm.

5-Ethynyl-2′-deoxyuridine (EDU) staining

Cell proliferation was assayed sticking to the protocol of the EdU Labeling Kit (Sangon Biotech, Shanghai, China). After the treatment of cells distributed into 96-well plates (1.5 × 10⁴ cells/well) with ascending doses of butorphanol (0, 20, 40 and 80 μg/ml), they were processed with pre-warmed complete medium nourished with 10 µM EDU working solution for 1 h. Afterwards, cells were exposed to 4% paraformaldehyde for half an hour and 0.5% Triton X-100 for 10 min for separate immobilization and perforation. Following being covered with reaction buffer and dyed by DAPI, the viable cells were observed by employing a fluorescence microscope.

Wound healing assay

Following cells dispensed into 6-well plates (5 × 10⁵ cells/well) were treated with ascending doses of butorphanol (0, 20, 40 and 80 μg/ml), the longitudinal cell-free regions were formed in the confluent monolayer by means of a micropipette tip. The wound closure extent was recorded at predetermined time intervals under a light microscope.

Transwell assay

Transwell compartments were paved with Matrigel for invasion assay. After being treated with ascending doses of butorphanol (0, 20, 40 and 80 μg/ml), a total of 5 × 10⁴/well cells suspended in FBS-deprived media and 500 μl medium were severally drawn into the upper and bottom compartments. The membrane of the upper level was exposed to methanol and dyed by 0.5% crystal purple. The transmigrated cells were photographed under a light microscope.

Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL)

After the treatment of cells distributed into 96-well plates (4 × 10⁴ cells/well) with ascending doses of butorphanol (0, 20, 40 and 80 μg/ml), they were exposed to 4% paraformaldehyde for immobilization. Following being mixed with 50 μl TUNEL working buffer (Yeasen, Shanghai, China) for 1 h shielded from light, cells were processed with 100 μl reaction buffer. After DAPI nuclear staining, the stained apoptotic cells were visible under a fluorescence microscope.

Western blot

Through RIPA buffer (Real Times, Beijing, China), the protein samples were prepared for quantification based on the BCA method (Real Times, Beijing, China). Then, PVDF membranes that were to transfer the proteins resolved by 10% SDS-PAGE were closed with 5% BSA and treated by primary antibodies overnight at 4 °C and goat anti-rabbit HRP antibody for 1 h. The interaction was monitored with the RealECL reagent (Real Times, Beijing, China).

Statistical analyses

The statistical data plotted utilizing GraphPad Prism 8 software (GraphPad Software, Inc.) were denoted as the mean ± standard deviation. The statistical significance of the difference among means were determined by analysis of variance (one-way) with Tukey’s test. The normality of the data was tested using the Shapiro–Wilk test. The mean values were acknowledged to possess significance in statistics when p ≤ 0.05.

Results

Butorphanol eliminated the capability of CRC cells to proliferate in a concentration-dependent manner

With the goal of establishing the detailed role of butorphanol in CRC, cell viability was assayed upon treatment with varying concentrations of butorphanol (0, 2.5, 5, 10, 20, 40 and 80 μg/ml). As portrayed by CCK-8 method, SW620 and HCT116 cell viability were both on a downward trend by butorphanol with the gradually ascending doses (Fig. 1A). Considering that, butorphanol at 20, 40 and 80 μg/ml doses harboring the most profound effect were applied to the follow-up assays. The results from EDU staining also rendered that the green fluorescence in SW620 and HCT116 cells was robustly diminished when exposed to butorphanol, exhibiting a dose-dependent manner (Fig. 1B). Through Western blot analysis, the protein expressions of proliferation-associated Ki67 and PCNA appeared to be both depleted by increasing doses of butorphanol (Fig. 1C).

Fig. 1.

Butorphanol eliminated the capability of CRC cells to proliferate in a concentration-dependent manner. A CCK-8 method assayed cell activity. B EDU staining estimated the capacity of CRC cells to proliferate. C Western blot ascertained Ki67 and PCNA expressions

Butorphanol weakened the ability of CRC cells to metastasize in vitro in a concentration-dependent manner

Moreover, it was observable from Fig. 2A that the wound closure level was reduced in SW620 and HCT116 cells administrated with ascending concentrations of butorphanol, underpinning that butorphanol strongly eliminated the migratory potential of CRC cells. In addition, the data from transwell assays delineated that butorphanol treatment also brought about the noticeable decline on the number of invaded cells (Fig. 2B). As expected, in SW620 and HCT116 cells, metastasis-associated MMP2 and MMP9 protein expressions were both lowered upon exposure to butorphanol (Fig. 2C).

Fig. 2.

Butorphanol weakened the ability of CRC cells to metastasize in vitro in a concentration-dependent manner. A Wound healing and B transwell assays estimated the capacities of CRC cells to migrate and invade. C Western blot ascertained MMP2 and MMP9 expressions

Butorphanol intensified the apoptotic level of CRC cells in a concentration-dependent manner

Simultaneously, TUNEL staining evidenced that following treatment with incremental concentrations of butorphanol, the apoptotic ratio of SW620 and HCT116 cells was remarkably raised (Fig. 3A). Coincidently, BCL2 expression was reduced whereas Bax expression was elevated in butorphanol-administrated SW620 and HCT116 cells dose-dependently (Fig. 3B).

Fig. 3.

Butorphanol intensified the apoptotic level of CRC cells in a concentration-dependent manner. A TUNEL staining assayed the cellular apoptotic level. B Western blot ascertained BCL2 and Bax expressions

Butorphanol possessed an interrelation with SIGMAR1 and lowered SIGMAR1 expression

Based on the prediction from Sea and targetnet databases that butorphanol might bind with SIGMAR1, it was consequently speculated that butorphanol might function in CRC cells via mediating SIGMAR1. According to the docking tool CBDOCK2 server, the interaction between butorphanol and SIGMAR1 was affirmed (Fig. 4A). Furthermore, it turned out that butorphanol concentration-dependently cut down SIGMAR1 protein expression (Fig. 4B), highlighting that butorphanol might be negatively correlated with SIGMAR1. Besides, 80 μg/ml of butorphanol was applied to the ensuing experiments for its most noticeable efficacy.

Fig. 4.

Butorphanol possessed an interrelation with SIGMAR1 and lowered SIGMAR1 expression. A CB-Dock2 server predicated butorphanol-SIGMAR1 interaction. B Western blot ascertained SIGMAR1 expression

Butorphanol down-regulated SIGMAR1 to produce anti-proliferation activity in CRC cells

To unveil the significance of the interactive mechanism of butorphanol with SIGMAR1 in the process of CRC, SIGMAR1 expression was raised by transfection of OV- SIGMAR1 (Fig. 5A). As speculated, butorphanol notably repressed SW620 and HCT116 cell viability, which were both improved again after SIGMAR1 was overexpressed (Fig. 5B). As Fig. 5C depicted, EDU staining substantiated that the attenuated green fluorescence in butorphanol-treated SW620 and HCT116 cells was intensified again by up-regulation of SIGMAR1. Consistently, the descending Ki67 and PCNA expressions in SW620 and HCT116 cells exposed to butorphanol rose again when SIGMAR1 expression was elevated (Fig. 5D).

Fig. 5.

Butorphanol down-regulated SIGMAR1 to produce anti-proliferative activity in CRC cells. A RT-qPCR and Western blot ascertained SIGMAR1 expressions after transfection of OV-SIGMAR1. B CCK-8 method assayed cell activity. C EDU staining estimated the capacity of CRC cells to proliferate. D Western blot ascertained Ki67 and PCNA expressions

Butorphanol down-regulated SIGMAR1 to produce anti-metastatic activity in CRC cells in vitro

Expectedly, it was noticed from wound healing assay that elevation of SIGMAR1 boosted the number of migrated cells which receded by butorphanol treatment (Fig. 6A). Additionally, after SIGMAR1 was overexpressed, the deteriorative invasive potential of SW620 and HCT116 cells on account of butorphanol administration was invigorated again (Fig. 6B). Also, butorphanol exposure resulted in the reduction on MMP2 and MMP9 expressions which were partially raised when SIGMAR1 was up-regulated (Fig. 6C).

Fig. 6.

Butorphanol down-regulated SIGMAR1 to produce anti-metastatic activity in CRC cells in vitro. A Wound healing and B transwell assays estimated the capacities of CRC cells to migrate and invade. C Western blot ascertained MMP2 and MMP9 expressions

Butorphanol down-regulated SIGMAR1 to produce pro-apoptotic activity in CRC cells

At the same time, as presented by the data from TUNEL staining, the enhanced apoptotic ratio of SW620 and HCT116 cells imposed by butorphanol was impaired again by SIGMAR1 elevation (Fig. 7A). The depleted BCL2 expression and the augmented Bax expression in butorphanol-administrated SW620 and HCT116 cells dose-dependently were both partially reverted by SIGMAR1 overexpression (Fig. 7B).

Fig. 7.

Butorphanol down-regulated SIGMAR1 to produce pro-apoptotic activity in CRC cells. A TUNEL staining assayed the cellular apoptotic level. B Western blot ascertained BCL2 and Bax expressions

Discussion

The molecular genesis of CRC is a multifactorial and intricate process encompassing multi-gene alternation [19]. Herein, we illustrated the promising tumor-suppressing function of butorphanol in CRC in vitro, as evidenced by the dose-dependent protection against the abilities to proliferate, migrate and invade as well as the inducible influence on apoptosis. With respect to the possible response mechanism, SIGMAR1 was targeted and inactivated by butorphanol.

Butorphanol is an agonist of opioid receptors serving dual pharmacological functions, in view of which, the existing researches have underlined the potential protective role of butorphanol in human malignancies. For instance, butorphanol has been disclosed to hamper cell proliferation, metastasis and angiogenesis in hepatocellular carcinoma [13]. piRNA hsa_piR_006613 expression can be up-regulated by butorphanol to hinder osteosarcoma cell proliferation and migration [11]. All these findings have supported that butorphanol can act as an anti-cancer agent through mediating cellular aggressive events. Cancer cell survival relies on the balance between proliferation and apoptosis. Ki67 that primarily resides in the nucleus is known as a cellular proliferation marker. PCNA is a scaffold protein essential for proliferation via acting as an indispensable maestro of DNA replication and repair. Elevated Ki67 and PCNA expression may be an early event in CRC carcinogenesis and development [20, 21]. Our experimental results illuminated that ascending doses of butorphanol dramatically suppressed CRC cell viability and proliferation, coupled with the depleted Ki67 and PCNA expression. BCL2 and Bax are deemed as hub determinants of apoptosis, the enhanced ratio of which may represent the reduction on apoptosis in CRC [22]. On the contrary, butorphanol concentration-dependently exaggerated the apoptosis of CRC cells, manifested by the raised apoptotic level, the eliminated BCL2 expression and the boosted Bax expression.

Metastasis, a complex and multi-step process, is recognized as a dominant cause of CRC-associated mortality, during which migration and invasion are extensively perceived as central events. Matrix metalloproteinases (MMPs) constitute proteolytic enzymes that degrade multiple components of the extracellular matrix, the destruction of which is believed as a basic step of tumor invasion and metastasis [23]. MMPs family members MMP2 and MMP9 have been developed to be accountable for the metastatic behavior of CRC cells [24, 25]. Through investigation, it was noticed that the migrated and invaded potentials of both SW620 and HCT116 cells were both abated, MMP2 and MMP9 expression were both lowered upon treatment with varying doses of butorphanol. Accordingly, it was concluded that butorphanol retarded cellular aggressive transformation to elicit tumor-suppressing influences in CRC, which was in compliance with the study conducted by Wang et al. which have introduced that butorphanol represses the abilities of ovarian cancer cells to proliferate, migrate, invade and aggravates cell apoptosis [12].

Based on Sea (https://sea.bkslab.org/) and targetnet databases (http://targetnet.scbdd.com/), butorphanol was forecasted to interact with SIGMAR1. More importantly, SIGMAR1 has been deemed as a selectively multifunctional drug target in the context of cancer [16]. Thereafter, the relationship of butorphanol with SIGMAR1 in the context of CRC was surveyed. As a result, butorphanol had a tight interaction with SIGMAR1. It is noteworthy that higher SIGMAR1 expression was observed in CRC samples and cells [17, 18]. Consistently, in the current paper, SIGMAR1 expression was boosted in CRC cells and was negatively modulated by butorphanol. As is well documented, SIGMAR1 has been proposed as a tumor promoter in breast cancer and prostate cancer by potentiating tumor cell aggresiveness [16–18, 26]. Particularly, SIGMAR1 contributes to cell migration in vitro and cell invasion in vivo in CRC [17, 18]. As expected, our study proved that when SIGMAR1 was up-regulated, the retarded proliferation, migration, invasion and the strengthened apoptosis of CRC cells imposed by butorphanol administration were partially compensated again, further validating the oncogenic role of SIGMAR1 in CRC.

Conclusion

Taken together, for the first time, we herein for the first time demonstrated the anti-oncogenic function of butorphanol in CRC through inhibiting cell proliferation, migration, invasion and promoting cell apoptosis. The interaction between butorphanol and SIGMAR1 represent a novel mechanism to regulate the occurrence and development of CRC. This finding suggested that butorphanol might serve as an effective therapeutic target for CRC.

Abbreviations

CRC

Colorectal cancer

SIGMAR1

Sigma non-opioid intracellular receptor 1

KOR

κ-Opioid receptor

BNCC

BeNa Culture Collection

DMEM

Dulbecco's modified Eagle medium

RPMI

Roswell Park Memorial Institute

FBS

Fetal bovine serum

Ov

Overexpression

NC

Negative control

CCK-8

Cell Counting Kit-8

EDU

5-Ethynyl-2′-deoxyuridine

TUNEL

Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling

RT-qPCR

Reverse transcription-quantitative PCR

RIPA

Radioimmunoprecipitation assay

BCA

Bicinchoninic acid

PVDF

Polyvinylidene fluoride

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

BSA

Bovine serum albumin

HRP

Horseradish peroxidase

ECL

Enhanced chemiluminescence

PCNA

Proliferating cell nuclear antigen

MMP2

Matrix metallopeptidase 2

MMP9

Matrix metallopeptidase 9

BCL2

B-cell lymphoma 2

Bax

BCL-2-associated X

MMPs

Matrix metalloproteinases

Author contributions

JG conceived the study. XH and LQ performed the experiments, analyzed the data, wrote the original manuscript and revised the manuscript. YX and JL put forward some constructive suggestions and revised the manuscript. All authors reviewed the manuscript.

Funding

None.

Data availability

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

Declarations

Competing interests

The authors declare no competing interests.