Isobavachalcone induces apoptosis of colorectal cancer cells mediated by interaction with survivin: Experimental and theoretical analyses

2.1 Materials and cell culture

Isobavachalcone [CAT Nr: SML1450, ≥98 % (HPCE]), 5-FU (F6627-1G, purity ≥ 99 %), and 1-anilino-8-naphthalenesulfonic acid hemi-magnesium salt hydrate (ANS) were purchased from Sigma and were dissolved in dimethyl sulfoxide (DMSO) for experimental assays. Recombinant human survivin protein (142 amino acid range, >90 % purity) was obtained from AbCam (ab87202). The solutions containing survivin (5 µM) were prepared in 20 mM phosphate buffer solution (PBS) containing 100 mM NaCl and 50 µM Zn²⁺, pH 8.0 (Gao et al., 2010). All of the other chemicals and reagents were from Sigma or Invitrogen.

2.2 Cell culture

Human colorectal carcinoma Lovo cells and WI-38 human fibroblast cells obtained from American Type Culture Collection (Manassas, VA, USA) were cultured in the F12 (Invitrogen, Carlsbad, CA, USA) and Dulbecco's modified Eagle's medium (DMEM/ Life Technologies), respectively containing 10 % FBS (fetal bovine serum, Invitrogen) and 1 % antibiotics (Sigma, USA) in a humidified atmosphere of 95 % air and 5 % CO₂ at 37 °C.

2.3 MTT assays

Cells (5 × 10³) per well seeded in 96-well tissue culture plates were incubated overnight and media were then replaced with fresh ones containing increasing concentrations of IBC (0, 1, 10, 20, 30, 50, and 100 µM). After 24 h, MTT assay was performed by replacing the media with 1 mg/ml MTT solution and incubation for 4 h in the dark. MTT solution was then gently removed and the wells were added by 100 μl of DMSO. The optical density of samples was read using a Dynex Opsys MR ELISA plate reader (Worthing, West Sussex, UK) at 570 nm.

2.4 Lactate dehydrogenase (LDH) assay measurements

After overnight incubation of cells, media were replaced with fresh media containing increasing concentrations of IBC (0, 1, 10, 20, 30, 50, and 100 µM). After 24 h of incubation, fixed amounts of media were collected and immediately used for the LDH assay. LDH assays were measured using a LDH Assay Kit (Colorimetric, ab102526) according to the manufacturer's protocol. The optical density of samples was read using a Dynex Opsys MR ELISA plate reader (Worthing, West Sussex, UK) at 450 nm.

2.5 Determination of reactive oxygen species (ROS)

ROS generation was assessed using a DCFH-DA fluorescence assay. The treated cells were incubated with DCFH-DA (10 μM, sigma, USA) for 30 min at 37 °C. Then the cells were washed twice with PBS and the fluorescence intensity was determined at 525 nm (λex = 488 nm).

2.6 Determination of lipid peroxidation

The presence of malondialdehyde (MDA) was assessed using the TBARS Assay Kit (Thiobarbituric Acid Reactive Substances, Cayman Chemical, Michigan 48,108 USA) as described previously (Ni et al., 2019). Briefly, after treatment, the cells were homogenized, incubated with thiobarbituric acid reactive substances and the absorbance of samples was read using a Dynex Opsys MR ELISA plate reader (Worthing, West Sussex, UK) at 530–540 nm.

2.7 Superoxide dismutase (SOD) and catalase (CAT) activity and glutathione (GSH) content assays

The biochemical assays for SOD and CAT were performed to assess the endogenous antioxidant activity in LoVo cells. The cell suspension after treatment was processed for SOD and CAT activity assays. The antioxidant activity in LoVo cells was analyzed by determining the SOD and CAT based on the recommended protocols provided with SOD assay kit (ab65354) and CAT assay kit (ab83464). Determination of GSH content was also done based on the method reported previously by Beutler et al (Beutler et al., 1963) and updated by Gu et al. (Gu et al., 2015).

2.8 Quantitative real-time PCR (qTR-PCR) analysis

Total RNA content of the LoVo cells was isolated using a RNeasy® Mini Kit (QIAGEN, Hilden, Limburg, Germany) according to the manufacturer's protocol. cDNA was synthesized using an RT-PCR kit (Takara, Kyoto, Japan). Real-time PCR was performed using SYBR Green Premix Ex Taq on an ABI ViiA 7DX RT-PCR machine. 2^-ΔΔct method was used for calculating the fold change and the primer sequences were used based on the previous papers reported by Afrin et al. and Zhang et al. (Afrin et al., 2018; Zhang et al., 2019).

2.9 Detection of mitochondrial membrane potential (MMP)

MMP assay was done with JC-1 staining as previously explained (Panada et al., 2023). Briefly, at 24 h after the IBC treatment (35 µM), LoVo cells were harvested and incubated with JC-1 at a final concentration of 2 µM at 37 °C in the dark for 15 min. The cells were then centrifuged, rinsed with HEPES buffer, resuspended, and MMP changes were assessed ratiometrically by determining the ratio of JC-1 fluorescence at 590 nm (red) and 530 nm (green) (λex = 490 nm).

2.10 Measurement of caspase-3 and −9 activity

Caspase-3 and caspase-9 activities were assessed using relevant kits according to the manufacturer's instructions (Beyotime Biotech, China). Briefly, LoVo cells (1 × 10⁶) were treated with IC₅₀ concentration of IBC (35 µM), harvested, washed twice with PBS, resuspended in lysis buffer, incubated on ice for 15 min, centrifuged at 18,000 × g for 10 min at 4 °C, and exposed to caspase activity assay. Reaction buffer with substrates acetyl-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNA, caspase-3) and acetyl-Leu-Glu-His-Asp-p-nitroanilide (Ac-LEHD-pNA, caspase-9), were used for detection of caspase activity. The pNA release was determined using a Dynex Opsys MR ELISA plate reader (Worthing, West Sussex, UK) at 405 nm.

2.11 Cytochrome c and survivin determination

ELISA kit (Abcam, Cambridge, UK) was used to analyze the release of apoptotic marker (Cyt C protein) and survivin protein expression according to the manufacturer’s protocols. Briefly LoVo cells (5 × 10⁵ cells) per well were treated with 35 µM IBC or 5-FU for 24 h. After harvesting, the cells were washed twice with ice-cold PBS, resuspended in cold lysis buffer for 30 min, and centrifuged at 12000 × g for 15 min at 4 °C. Then, supernatants were collected and used for protein assay. The color was detected and quantified at 450 nm with a microplate reader (Worthing, West Sussex, UK).

2.12 Spectroscopic analysis

2.13 Molecular docking study

A molecular docking study was done using AutoDock software. The structure of IBC (C₂₀H₂₀O₄) was downloaded from PubChem (PubChem CID: 5281255) in a 3D conformer (SFD). Low energy 3D conformer of the IBC was docked with survivin protein (PDB ID: 3UIH). During the docking process, charge optimization, hydrogen atom addition and removal of water molecules were performed. The docking procedure was done based on the default settings. The blind docking was done within the survivin protein as a rigid receptor. The AutoDock scoring set was then used to score the resulting interactions and the molecular docking results were visualized by relevant software.

2.14 Statistical analyses

Data are represented as the average of three experiments and error bars represent the standard deviation. Statistical analysis was performed via one-way analysis of variance (ANOVA). p < 0.05 was considered to be statistically significant.

3.1 Effects of IBC on the cell viability and membrane integrity

Fig. 1a displays that IBC reduced the proliferation of the human LoVo colorectal cancer cell based on a concentration-dependent manner, however, IBC did not significantly mitigate the growth of WI-38 normal cells up to 50 µM. Indeed, it was realized that upon incubation of the human LoVo colorectal cancer cell with 1, 10, 20, 30, 50, and 100 µM IBC, the cell viability was reduced from 100.00 % to 96.74 %, 83.19 %, 77.49 %, 48.92 %, 43.04 %, and 39.02 % respectively. While, the cell viability values were 100.00 %, 101.87 %, 94.37 %, 89.86 %, 88.39 %, and 80.44 % after incubation of WI-38 normal cells with 1, 10, 20, 30, 50, and 100 µM IBC at 24 h. Therefore, it was deduced that IBC at concentrations above 1 µM was able to significantly inhibit the proliferation of the human LoVo colorectal cancer cell, while it was only cytotoxic against WI-38 normal cells at a concentration of 100 µM.

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Fig. 1

(a) Effect of different concentrations of IBC on the viability of the LoVo cancer and WI-38 normal cells after 24 h assessed by MTT assay. (b) Effect of different concentrations of IBC on membrane damage of the LoVo cancer and WI-38 normal cells after 24 h assessed by LDH assay.

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Data are shown as mean ± SD (n = 3). * p < 0.05, * * p < 0.01, * ** p < 0.001.

Then the IC₅₀ concentration of IBC against human LoVo colorectal cancer cells was determined to be approximately 35 µM. Li et al. (Li et al., 2019) showed that IC₅₀ concentrations of IBC against HCT116 and SW480 colorectal cancer cells were 75.48 µM and 44.07 µM at 24 h, detected by CCK-8 assay. Furthermore, Table 1 shows the IC₅₀ concentrations of IBC against different cancer cell lines.

Destruction of membrane leakage induced by bioactive compounds can be one of the possible mechanisms of their anticancer activity (Zhu et al., 2016; Fang et al., 2023). For this reason, we aimed to investigate the effect of IBC incubation on the membrane integrity of the human LoVo colorectal cancer cells and WI-38 normal cells. We found that IBC incubation above concentrations of 10 µM could result in the membrane leakage in LoVo colorectal cancer cells (Fig. 1b). However, the relative LDH release values in WI-38 normal cells following incubation with increasing concentrations of IBC remained unchanged in comparison with the control group (Fig. 1b), revealing the minimum damage to membrane integrity of normal cells.

3.2 IBC increased intracellular oxidative stress

Oxidative stress is a crucial factor that mediates the bioactive compounds-induced anticancer effects. For example, Li et al. (Li et al., 2018) showed that IBC stimulated oxidative stress-mediated cell death by targeting antioxidant enzymes in human prostate cancer PC-3 cells. Also, He et al. reported that IBC prohibits acute myeloid leukemia, where oxidative stress through excessive ROS generation resulted in mitochondrial apoptosis (He et al., 2021). Furthermore, Zhao et al. (Zhao et al., 2022) displayed that IBC can disrupt mitochondrial respiration and stimulate cytotoxicity mediated by ROS generation. Excessive ROS generation and lipid peroxidation products have been shown to play a key role in the prevention of cancer progression and therapy (Barrera, 2012). Oxidative stress is a process by which a high intracellular ROS generation could destroy the lipids, proteins, and DNA structures (Chiang et al., 2022). Also, oxidative stress could be related to a variety of pathological factors (Chiang et al., 2022). This study showed that IBC could result in the production of excessive ROS in the human LoVo colorectal cancer cells (P < 0.01, Fig. 2a). In other words, when human LoVo colorectal cancer cells were exposed to 35 μM IBC for 24 h, the DCF fluorescence intensity level increased remarkably due to the formation of ROS.

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Fig. 2

(a) Effect of IC₅₀ concentration of IBC (35 µM) or 35 µM 5-FU on DCF intensity (ROS formation) of the LoVo cancer cells after 24 h. (b) Effect of IC₅₀ concentration of IBC (35 µM) or 35 µM 5-FU on MDA level of the LoVo cancer cells after 24 h. Data are shown as mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

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MDA is considered the main product of lipid peroxidation which prompts cytotoxicity in different cells (Niedernhofer et al., 2003). This study showed that IBC could give rise to elevation of MDA level in the human LoVo colorectal cancer cells (Fig. 2b). When the human LoVo colorectal cancer cells were incubated with 35 μM IBC for 24 h, the produced MDA level increased significantly. However, in both cases the effect of 5-FU as positive control was much higher in induction of oxidative stress in the human LoVo colorectal cancer cells than IBC-treated cells.

Both antioxidant enzymes, SOD and CAT, as well as non-enzyme antioxidant substance, GSH, are considered the key modulators in the regulation of oxidative stress (Gu et al., 2015; Jelic et al., 2021). Our data showed that SOD activity (Fig. 3a) and CAT activity (Fig. 3b) were significantly reduced by both IBC and 5-FU, further revealing that IBC may degrade or inactive SOD and CAT in the human LoVo colorectal cancer cells.

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Fig. 3

(a) Effect of IC₅₀ concentration of IBC (35 µM) or 35 µM 5-FU on SOD activity of the LoVo cancer cells after 24 h. (b) Effect of IC₅₀ concentration of IBC (35 µM) or 35 µM 5-FU on CAT activity of the LoVo cancer cells after 24 h. (c) Effect of IC₅₀ concentration of IBC (35 µM) or 35 µM 5-FU on GSH content of the LoVo cancer cells after 24 h. Data are shown as mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

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Additionally, intracellular GSH as the crucial non-enzymatic antioxidant system could serve as a free radical scavenger molecule to shield cells in response to oxidative stress (Estrela et al., 2006; Marini et al., 2023). We showed that both IBC and 5-FU reduced the content of GSH (Fig. 3c), indicating that IBC might be able to alter the redox status and consequently induce a high level of oxidative stress.

Together, these data suggested that IBC could elevate the level of oxidative stress in the human LoVo colorectal cancer cells, which may further cause increased cell death. More importantly, these data may suggest that incubation of the LoVo cancer cells with the IBC can be a potential strategy to deregulate oxidative/antioxidant balance in cancer cells as a main mechanism for the induction of anticancer effects.

3.3 IBC decreased expression of Nrf2 and HO-1 in LoVo cells

Nrf2 as a potential antioxidant pathway can protect cells against oxidative stress-mediated damage, indicating that this pathway could induce resistance to cancer therapy (Gallorini et al., 2023). Nrf2 could modulate several important molecules in antioxidant systems including CAT, SOD, and GSH (Liang et al., 2018). SOD, CAT and GSH could prevent cell apoptosis caused by free radicals (Liang et al., 2018). HO-1 is known as an antioxidative protein which is modulated by the Nrf2 as a transcription factor (Ghareghomi et al., 2023).

This study revealed that both Nrf2 (Fig. 4a) and HO-1 (Fig. 4b) expression at the mRNA level were downregulated significantly when the human LoVo colorectal cancer cells were treated with 35 μM IBC for 24 h.

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Fig. 4

(a) Effect of IC₅₀ concentration of IBC (35 µM) or 35 µM 5-FU on expression of Nrf2 mRNA of the LoVo cancer cells after 24 h. (b) Effect of IC₅₀ concentration of IBC (35 µM) or 35 µM 5-FU on expression of HO-1 mRNA of the LoVo cancer cells after 24 h. Data are shown as mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

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Kim et al. (Kim et al., 2020) reported that Nrf2 knockdown could result in re-sensitization of pancreatic cancer cells to 5-FU resistance through suppressing HO-1 expression. Also, Kweon et al. (Kweon et al., 2006) demonstrated that Nrf2-mediated overexpression of HO-1 plays a key role in resistance to apoptosis in human lung cancer cells induced by epigallocatechin 3-gallate. We then, demonstrated that IBC promotes oxidative stress in the human LoVo colorectal cancer cells via Nrf2/HO-1 signaling pathway, which is in good agreement with previous studies (Wei et al., 2021; Acquaviva et al., 2021).

3.4 IBC triggered mitochondrial dysfunction, release of cytochrome c, and activation of apoptosis in LoVo cells

The importance of mitochondrial dysfunction in cancer therapy has been widely reported in the literature (Luo et al., 2020; Księżakowska-Łakoma et al., 2014). Also, Cyt C is placed in the mitochondrial intermembrane/intercristal spaces and serves as an electron shuttle molecule in the respiratory chain. Different proapoptotic encouraging signals (Bcl-2-like proteins) promote the permeabilization of the outer membrane, mediate the interaction between intermembrane and intercristal spaces and assist the Cyt C release to the cytosol (Garrido et al., 2006; Hussar, 2022). Then, Cyt C promotes the activation of several mechanisms which are the main activator of caspase-9 and caspase-3. Activated caspases finally result in the induction of apoptosis. The caspase overactivation associated with the release of Cyt C to cytoplasm can be inhibited by Bcl-2 overexpression or Bax downregulation. Indeed, the Bcl-2 family (BCL-XL, MCL-1, BAK, BOK, BAD, BID, BIM) regulates MMP. These molecules could interact with each other to result in the formation of homo/hetero-complexes and play a key role in regulating MMP and prevention of apoptosis (Garrido et al., 2006; Hussar, 2022).

Therefore, in this study, the effects of IBC on the mitochondria destruction, release of Cyt C, Bax/Bcl-2 and caspase expression, and caspase activity in the human LoVo colorectal cancer cells were evaluated by relevant methods/techniques described in section 2.

Mitochondrial destruction is associated with alterations in MMP. Mitochondrial depolarization can be tracked by a reduction in the red/green fluorescence intensity ratio of JC-1 as a probe (Chiang et al., 2022). When the LoVo cells were subjected to IBC, the red/green fluorescence intensity ratio of JC-1 dye reduced significantly at 35 μM (Fig. 5a). As already discussed, when healthy cells are exposed to extracellular or intracellular anticancer stimuli, the MMP reduced, and Cyt C is released from mitochondria into the cytoplasm (Chiang et al., 2022). When the human LoVo colorectal cancer cells were incubated with 35 μM IBC for 24 h, the Cyt C release in the cytoplasm was enhanced significantly (Fig. 5b).

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Fig. 5

Effect of IC₅₀ concentration of IBC (35 µM) or 35 µM 5-FU on (a) MMP loss, (b) release of Cyt c, (c) expression of Bax mRNA, (d) expression of Bcl-2 mRNA, (e) expression of caspase-9 mRNA, (f) expression of caspase-3 mRNA, (g) caspase-9 activity, and (h) caspase-3 activity of the LoVo cancer cells after 24 h. Data are shown as mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

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As the Bcl-2 family plays a distinctive role in cell viability and apoptosis through the mitochondrial pathway, we aimed to devalue the effect of IBC on the expression level of Bcl-2 and Bax mRNA in the human LoVo colorectal cancer cells. This study showed that when the LoVo cells were subjected to 35 μM IBC for 24 h, the mRNA expression of Bax increased remarkably (Fig. 5c) but the mRNA expression of Bcl-2 decreased significantly (Fig. 5d). The same mRNA expression pattern was also detected for 5-FU (35 μM)-exposed LoVo cells for 24 h (Fig. 5c, d).

Apoptosis through the mitochondrial pathway is mainly modulated by the caspase-9 and −3. This study further explored whether IBC influenced the expression and activity of caspase-9 and −3 in the human LoVo colorectal cancer cells. When the LoVo cells were incubated with the IBC for 24 h, the caspase-9 mRNA expression (Fig. 5e), caspase-3 mRNA expression (Fig. 5f), caspase-9 activity (Fig. 5g), and caspase-3 activity (Fig. 5h) increased as the IBC concentration was 35 μM, which was also observed upon incubation of cells with 5-FU with the similar experimental procedure.

3.5 IBC downregulated the expression of survivin in LoVo cells

The antiapoptotic survivin is known as one of the main inhibitors of apoptosis, which is overexpressed particularly in cancer cells and increases the survival rate of cancer cells mediated by downregulation of caspase-linked apoptosis (Touloumis et al., 2020; Sabour et al., 2020). Upregulation of survivin is directly correlated with cancer cell proliferation, invasion and resistance and poor patient prognosis which necessitate the development of new and potential therapeutic approaches involving survivin targeting in cancer (Martínez-García et al., 2019). By searching the literature for IBC as an effective inhibitor of survivin, we found that IBC has been shown to have a significant effect on survivin expression (Li et al., 2019). We then aimed to determine whether IBC (35 µM) at 24 h leads to survivin downregulation and degradation in the human LoVo colorectal cancer cells as a metastatic cell model. As shown in Fig. 6, IBC similar to 5-FU significantly inhibited survivin expression at both mRNA (Fig. 6a) and protein (Fig. 6b) levels in LoVo colorectal cancer cells at a concentration of 35 µM.

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Fig. 6

Effect of IC₅₀ concentration of IBC (35 µM) or 35 µM 5-FU on (a) expression of survivin mRNA and (b) expression of survivin protein of the LoVo cancer cells after 24 h. Data are shown as mean ± SD (n = 3). ** p < 0.01.

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This data indicated that IBC might induce apoptosis in LoVo cells through downregulation of antiapoptotic (survivin, Bcl-2) and upregulation of apoptotic (Bax, caspase, Cyt C) factors, which these data are in line with findings reported by Li et al (Li et al., 2019).

3.6 Interaction properties of IBC and survivin

The intrinsic fluorescence of survivin arising from tryptophan residues can be used as a very important indicator of conformation alterations (dos Santos Rodrigues et al., 2023; Kyrychenko and Ladokhin, 2024). As shown in Fig. 7, when excited at 295 nm, the free survivin sample demonstrated the maximum emission intensity (λ_max) around 341 nm at all four studied temperatures (Fig. 7a-d). However, remarkable differences were detected in fluorescence intensity at λ_max following increasing the IBC in a concentration-dependent manner.

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Fig. 7

Effect of various concentrations of IBC on fluorescence quenching of survivin at four different temperatures of (a) 298 K, (b) 302 K, (c) 310 K, and (d) 315 K.

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These phenomena revealed that there were some structural changes and exposed hydrophobic moieties on the survivin-IBC complex (dos Santos Rodrigues et al., 2023). Similar phenomena in quenching of fluorescence intensity were also observed for bile salt hydrolases after interaction with IBC (Li et al., 2022). This might be associated with the partial unfolding of proteins occurring following interaction (Gooran and Kopra, 2024). These conformation changes could lead to displacement and masking of Trp residues or exposure to a polar environment (Gooran and Kopra, 2024).

3.6.1

3.6.1 Fluorescence quenching mechanism

Generally, static and dynamic quenching caused by complex formation (strong) and collision (weak), respectively can be distinguished by the Stern-Volmer Equation (Kenoth and Kamlekar, 2022):

(1)

$$F_0/F=1+K_{\mathit{SV}}\left[\mathit{IBC}\right]=1+kq{\tau }_0\left[\mathit{IBC}\right]$$ where F₀ and F, [IBC], K_SV, k_q, and τ₀ are the fluorescence intensities of survivin in the absence of the quencher (here, IBC), the fluorescence intensities of survivin in the presence of the IBC, concentration of IBC, Stern-Volmer quenching constant, the quenching rate constant, and the average lifetime of the free proteins (10⁻⁹ s), respectively.

Stern-Volmer plots in Fig. 8a show good linearity at the four different temperatures studied, indicating that almost one type of quenching mechanism is involved in the survivin-IBC interaction (Khalil and Kashif, 2023; Sun et al., 2024; Yi et al., 2024).

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Fig. 8

(a) Stern-Volmer plots, (b) modified stern-Volmer plots, and (c) Van’t Hoff plots of survivin-IBC complex.

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The quenching parameters are summarized in Table 2.

Table 2 Quenching parameters for the interaction of survivin with IBC at four different temperatures.

T (K)	KSV (×104 mol−1)	kq (×1013 mol−1s−1)	R2a
298	2.60	2.60	0.98
302	3.76	3.76	0.97
310	6.54	6.54	0.98
315	7.71	7.71	0.99

a R² is the correlation coefficient.

The data in Table 2 showed that quenching parameters increase with an elevation of temperature, while k_q values are much greater than 1 × 10¹⁰ M⁻¹ s⁻¹. In particular, k_q values play a key role in dictating the type of quenching mechanism occurring following the interaction of protein and ligands (Sun et al., 2024). For the dynamic quenching mechanism, the k_q values are close to the diffusion-controlled limit (1 × 10¹⁰ M⁻¹ s⁻¹), while higher k_q values reflect the contribution of static quenching (Sun et al., 2024). In the present study, k_q values are 3 orders of magnitude higher than the diffusion-controlled limit (1 × 10¹⁰ M⁻¹ s⁻¹), indicating that the survivin-IBC interaction is mostly static in nature. Similar findings have been reported in the interactions of IBC with bile salt hydrolases (Li et al., 2022).

3.7 Effect of IBC on survivin structure

The surface hydrophobicity of proteins can be determined using ANS fluorescence spectroscopy (Togashi and Ryder, 2008). ANS acts as an extrinsic fluorescent dye which is exceedingly sensitive to microenvironmental changes (Tang et al., 2020). ANS quantum yield extremely increases upon interaction with hydrophobic groups of proteins (Guliyeva and Gasymov, 2020). Therefore, ANS fluorescence spectroscopy may be used for detecting probable modifications in protein surface hydrophobicity triggered by drug/ligand binding. At a fixed concentration of survivin (3 µM) and ANS (30 μM), ANS fluorescence intensities were detected in the absence and presence of varying concentrations of IBC (Fig. 9a).

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Fig. 9

(a) ANS fluorescence spectroscopy, and (b) far-UV CD spectroscopy measurements of survivin-IBC complex.

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A significant increase in the ANS fluorescence intensity was observed upon increasing the IBC to the protein sample, which demonstrates the increase in the surface hydrophobicity of survivin. Indeed, the ANS fluorescence intensity of survivin in the absence of IBC is remarkably different in comparison to the presence of the IBC, which shows that the surface hydrophobicity of survivin is increased upon IBC binding.

CD spectroscopy measurement is used as a sensitive technique for recording the anticipated secondary structural changes of proteins upon ligand binding (Miles et al., 2021; Haque et al., 2022). Far-UV CD was then utilized to study the effect of IBC binding on survivin secondary structure. The far-UV CD spectra of survivin without and with varying concentrations of IBC are exhibited in Fig. 9b. The results showed that the ellipticity changes around 222 and 208 nm, as an indicator of α-helicity content (Haque et al., 2022), decreased for the survivin in the presence of IBC in a concentration-dependent manner. Thus, it can be concluded that IBC binding to survivin caused a reduction in the survivin α-helicity content. All these data indicated that IBC resulted in survivin structural changes accompanied by microenvironmental changes around Trp residues, an increase of surface hydrophobicity, and a decrease of α-helicity content.

3.8 IBC potential interaction with survivin evidenced by molecular docking study

Survivin with a homodimer structure plays a key role in the physiological function of cells (Sabour et al., 2020). Destabilization of the survivin following interaction with therapeutic small molecule into a domain that interferes with the dimerization hydrophobic interface, thereby further leading to cancer cell apoptosis.

To further provide insights on the obtained experimental results, IBC interaction with survivin was evaluated by theoretical study (Fig. 10). In this study, the IBC-survivin interactions were investigated by performing a molecular docking study using AutoDock software. The crystal structures of the IBC (Fig. 10a) and IBC-survivin complex (Fig. 10b) are shown in Fig. 10.

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Fig. 10

(a) IBC structure, (b) binding of IBC and survivin, (c) binding of 5-FU and survivin, (d), binding pocket of IBC-survivin, and (e) binding pocket of 5-FU-survivin.

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A docking study of IBC (Fig. 10b) showed that this compound was able to fit close to the binding pocket of survivin with a strong binding interaction with binding energy –9.80 kcal/mol (−41.00 kJ/mol). However, the binding energy of 5-FU with survivin was determined to be about −5.20 kcal/mol (−21.75 kJ/mol, Fig. 10c), revealing the higher affinity of IBC to survivin in comparison with 5-FU. It was then observed that hydrogen bonding between IBC and survivin was derived from the interaction of the Phe 13 and Lys91 with the IBC backbone (Fig. 10d, e). Also, Ile74, Phe86, Leu87, Val89, and Phe93 contributed to the formation of hydrophobic forces between IBC and survivin (Fig. 10 d, e), which is in good agreement with thermodynamic parameters determined by fluorescence quenching study.

Drawing from theoretical study, we can assert that IBC interacts with a survivin domain that plays a crucial role in the dimerization process. This compound can obstruct the dimerization hydrophobic interface, ultimately contributing to the apoptosis of cancer cells. This data is in good agreement with our experimental results and the finding reported by Sabour et al. which screened several cyanopyridine derivatives as survivin inhibitors by molecular docking study (Sabour et al., 2020).