Introduction

Around the world, hepatocellular carcinoma (HCC) is a leading cause of cancer-related fatalities1. Its incidence has increased during past several years. Liver cancer is anticipated to be the sixth most common illness diagnosed and the fourth leading cause of cancer mortality worldwide in 2018, with an estimated 841,000 new cases and 782,000 deaths2,3,4. Chemotherapy, radiation, and other adjuvant treatments cannot effectively cure HCC. Furthermore, due to the existence of cancer stem cells, HCC are more likely to develop drug resistance system5. Therefore, it is essential to identify chemotherapeutic medications that are toxic to HCC. Arsenic causes cancers by unidentified mechanisms and is a nonmutagenic human carcinogen. Despite being used as a medical medicine since the fifteenth century, ATO therapy was found to be effective in treating Acute Promyelocytic Leukemia (APL) in 1970s as it inhibits cell growth and promotes in vitro apoptosis in cancer cells6,7,8. It has since been tested on APL patients in clinical trials worldwide. ATO can kill a variety of malignant tumors, including lung and breast cancers9. During the year 2000, it was revealed that ATO reduced the growth of malignant glioma cell lines and promoted cell death10,11. Uncertainty exists regarding the exact mechanism by which ATO causes cell death. According to reports, it damages DNA and chromosomes structure, prevents DNA repair, and changes DNA methylation in mammalian cells12. According to Zhang et al.13, the primary mechanism of ATO-induced cell toxicity is telomere degradation and reduced telomerase activity. Telomeres are the repeating DNA oligomer sequences of (TTAGGG) present at the ends of eukaryotic chromosomes. The telomerase RNA protein complex and the shelterin proteins, whose regulation are controlled by the telomerase reverse transcriptase (hTERT) enzyme, are primarily responsible for maintaining telomere length. Normal somatic cells shorten their telomeres after every cell division14,15. Cellular apoptosis is caused by chromosomal end fusion once they reach a minimum length16. Telomerase may partially replace lost DNA and prevent chromosomal fusion by adding telomere DNA sequences (TTAGGG repeats)16. Malignancy and genomic instability may result from dysfunctional telomeres and telomerase activity16,17. Thus, preserving the ideal telomerase activity is essential for controlling the length of cellular life. Alterations in telomerase activity have been linked to aging and an increased risk of developing a number of chronic illnesses and cancers17. Although it is not always present, elevated telomerase activity is typically seen in advanced cancer cells and is essential for the continued proliferation of cancer cells18.The anti-proliferative effect of ATO on HCC cells, however, has not yet been proven to be caused by telomere or telomerase activity inhibition, with the exception of combinational setups19,20,21,22. The purpose of this study is to identify the mechanism by which ATO inhibit telomerase activity by in-silico analysis. This will be further verified by a number of in-vitro experiments, including the telomerase activity and the EMSA assay, which disrupts telomerase’s ability to bind DNA.

Telomere length maintenance is essential for controlling cellular life span. One of the key biomarkers that might be used to predict current or past illnesses is telomere lengthening or inhibition which is controlled by the telomerase enzyme (hTERT). In the meanwhile, research is being done on the effects of ATO exposure on this important enzyme, which has raised concerns. Although earlier research showed that arsenic has an impact on telomere length, but it was unclear from these findings whether arsenic exposure shortens or lengthens telomeres or how ATO causes induction or inhibition of telomerase. Therefore, this publication outlined and examined the allosteric inhibition of telomerase after exposure to ATO, as well as the potential dose–response impact of ATO on HCC and its influence on telomerase. Furthermore, arsenic speciation analysis may provide greater insight into how arsenic affects telomerase and HCC.

Materials and methods

Structure preparation of human telomerase and inhibitors

The RCSB Protein Data Bank (PDB) (Accession No. 7BGB) was used to retrieve the protein structure of human telomerase. There are four primary domains that make up human telomerase (TERT): the telomerase essential N-terminal (TEN) domain, the telomerase RNA-binding domain (TRBD), the reverse transcriptase domain, and the C-terminal extension (CTE), commonly known as the thumb domain. Hex software (http://hex.loria.fr/dist/index.php) was used to preprocess the protein structure using automated methods that were built-in. The Developmental Therapeutics Programme of the NCI (http://dtp.nci.nih.gov) database has the chemical make-up of ATO metalloid. Arsenic trioxide’s 3Datomic coordinates were retrieved in sdf format from the NCBI Pubchem database. On Linux version 16.0423, the open babel programme was used to further convert the sdf files into the pdb file format. All hydrogen and water molecules must be eliminated from the three-dimensional structure as part of the preparation procedures in order to minimize atomic collisions during the docking experiment that was carried out using the auto dock tool.

Molecular docking of ATO and DNA with telomerase

Active site prediction was done by assuming the binding of DNA to human telomerase. Reports suggested that telomerase structure has two binding sites (sites 1 and 2) for DNA binding that may be identified based on DNA-RNA duplexes. These interactions with conserved TERT motifs keep the DNA in the active site24. Arsenic trioxide’s docking was done using the Patchdock (https://bioinfo3d.cs.tau.ac.il/PatchDock/)&Hex programme (https://hex.loria.fr/dist/index.php), which automates the preparation processes for both receptors and ligands. The protein–ligand complexes that have been docked are stabilized by a variety of binding interactions, including polar, hydrophobic, hydrophilic, pi-pi stacking, salt bridges, and others. Using the structure visualization tools PyMOL version 2.325, Chimera10.126, and LigPlot, the molecular interactions were examined and assessed based on their polar contact.

Molecular dynamic simulation

The binding interaction stability between protein–ligand was validated through MD simulations using using GROMACS 5.1.2 tool ((https://manual.gromacs.org/5.1.5/download.html). Protein, Protein-DNA and Protein-DNA-ATO complex systems were built using AMBERGS force field in dodecahedron periodic boundary conditions (PBC)27,28. The systems were solvated using the suggested SPC water model and positioned at the center from 1 nm’s margin from the PBC box’s inner surface. By adding NA + and Cl- ions to the solution, the ions were balanced in order to neutralize the systems after that energy minimization were performed using the steepest descent approach. Further, NVT and NPT ensembling were carried out, where in NPT step the systems were gradually heated up to 300 K for 10,000 ps, and the systems were equilibrated at 300 K by providing 1 atm pressure for 10,000 ps with a leap-frog integrator28,29. The final production run of all complex systems was carried out for 100 ns time steps. The simulation lasted 2 fs, with coordinates saved every 20 ps. Analysis of resulting simulated trajectories was carried out in terms of root mean square deviation (RMSD), root mean square fluctuation (RMSF), Radius of gyration (Rg), solvent accessibility surface area (SASA), hydrogen bond using GROMACS tools and the results were visualized with XMGRACE tool in Linux platform and hydrophobic interactions29,30.

Culture and treatment

Cell culture

Wrl-68 treated cells as a control liver cells and HepG2 liver cancer cells were obtained from the NCCS (Pune, India). Cellgene beta actin (cat # 14C10). Afters were grown in T75 culture flasks with MEM media containing 10% FBS. When the cells reached confluence, they were detached with a Trypsin EDTA solution. HiMedia supplied the cell culture reagents. The stock solution of 5 mM ATO (Sigma) was produced in phosphate buffered saline, filtered, and the working concentrations were prepared fresh each day by diluting with serum-free MEM medium.

Treatment plans

In culture media, ~ 5 × 105cells/ml plated in 12 well plates in triplicate at 37 °C for 24, 48 & 72 h. For preliminary experiments, cells were treated with varying concentrations of arsenic trioxide (0.2–20 µM), Following optimization, the best results were obtained at i.e. (ATO 4 µM). The dose of ATO was prepared and administered to separate cell groups both control & HCC cell lines for 24, 48 & 72 h. Only sterile PBS was given to the drug control group. Sigma Aldrich (USA) supplied all chemicals.

Cell proliferation assays

MTT tests were used to determine ATO’s cytotoxicity against liver cancer cells. Prior to treatment, cells in the log growth phase were seeded at a density of 5 × 103 cells per well in 200 μl of media in 96-well microplates. After then, the final ATO concentration was adjusted to (0, 2, 4, 6 or 10 μM). The vehicle control was dimethyl sulphoxide (DMSO). 20 μl of MTT solution (5 mg/ml) were introduced four hours before incubation. After dissolving cellular formazan crystals in DMSO, the absorbance at 570 nm was measured using a microplate reader.

Wound healing assay

A monolayer of cells with a confluence of about 100% was obtained by cultivating the HepG2 & Wrl-68 treated cells to be examined in a cell culture plate. Using a sterile 200 µl pipette tip, a straight scratch was formed on the cell monolayer, guaranteeing uniformity and consistency. To lessen interference with non-specific migration, the cells were gently washed with PBS to get rid of cell debris and cells that had come off during scratching. The cells underwent further culture at 37 °C with 5% CO2 in an incubator. An inverted microscope (ECLIPSE Ti2, Nikon) was used to acquire pictures of the scratch area at predetermined intervals (e.g., 0 h, 24 h, and 48 h).

Transwell invasion and migration assay

5 × 104 cells were moved from serum-free medium into the upper chamber of the 8-μm pore size membrane for the migration test. 6 × 105 cells in serum-free medium were placed in the top chamber of an insert covered with Matrigel (BD Biosciences) for the invasion tests. The bottom chamber was filled with medium containing 10% FBS. Cotton applicators were used to remove the cells that remained in the top membrane after a 24-h incubation period. Under an inverted microscope (Olympus), the cells that moved or infiltrated through the membrane were counted, photographed, and stained with methanol and 0.1% crystal violet.

ROS detection using 2’,7'-dichlorofluorescein diacetate (DCFDA) staining

DCFDA fluorescent dye was used to identify the generation of ROS in both untreated and cells treated with different doses of (0, 2, 4, 6 & 10 μM) of ATO after 48 h. Trypsinization and resuspension of the cells was done in fresh media, containing 10 µg/ml of DCFDA. After that, the mixture was incubated in the dark for 30 min at 37 °C. Cells were observed under a fluorescent microscope after two PBS washes.

Flow cytometric assays

For the flow cytometry apoptosis experiment, 1 × 105 cells were seeded per ml in six-well plates overnight before being treated with above mentioned doses of ATO for 48 h. Apoptotic cells were identified by fluorescent labeling, as described by Looi et al.,31. Cells were treated for 15 min in binding buffer with propidium iodide (PI) & FITC-annexin V (BD Biosciences). Flow cytometry studies were performed immediately on stained cells using a BD Biosciences FACS Canto II flow cytometer.

Real-time polymerase chain reaction

Total RNA was extracted from both cells using an RNA isolation reagent (Sigma) as directed by the manufacturer, and the recovered RNA was purified using the Qiagen RNeasy Mini Kit. Reverse Transcription Kit (Qiagen) was used to prepare cDNA as indicated by the manufacturer. The relative gene expression of target gene was normalized to GAPDH expression (reference gene) using the 2−ΔΔCqapproach was performed with primer sequences: hTERT forward, 5′-TTTCTGGAGCTGCTTGGGAA-3′ and reverse, 5′-GAAGAGCCTGAGCAGCTCGA-3′ and GAPDH forward, 5′-TCCTCTGACTTCAACAGCGACACC-3′ and reverse, 5′-TCTCTCTCTTCCTCTTGTCGTCTTGG-3′0.5.0 µl of 10X PCR buffer, 4.0 µl of MgO, 2 µl of dNTP, 2 µl of cDNA, 1 µl of each primer (1 mmol/l), and 2 µl of Taq DNA polymerase were employed as part of the amplification system. Double distilled water was added to get the volume down to 20 µl. hTERT underwent 30 cycles of denaturation at 94 °C for 50 s, annealing at 58 °C for 45 s, and extension at 72 °C as part of the amplification conditions.

Western blotting

Total protein was isolated from cell samples using RIPA buffer (Cell Signalling Technology, Inc.). The quantification of the protein was done through BCA Protein assay kit (Thermo Fisher Scientific, Inc.). Equal amount protein (30 μg) was resolved on 10% SDS gel and was transferred to nitrocellulose membrane. One hour blocking in fat free milk was done followed by overnight primary antibody incubation for anti-hTERT antibody (cat # 2085972) and control gene beta actin (cat # 14C10). After 3 times washing in TBST, membrane was incubated for 1 h in suitable HRP conjugated secondary antibodies. Clarity western ECL substrate (cat: 170–5060) was used for protein detection by chemi-luminescence (Bio Rad).

Telomeric repeat amplification protocol assay

Telomerase enzyme activity of HepG2 cells was assessed using a TRAP assay after 48 h exposure of ATO (4 μM). As reported by32, the TRAP test was carried out. The effects of ATO on HepG2 and Wrl-68 cell lysates were assessed using a TRAPeze kit (Roche Diagnostics). Each PCR employed (2 μg) of total cellular protein. A PAGE gel was used to separate the PCR products. TRAP assay primer sequences are (R4, 5′-AATCCGTCGAGCAGAGTTAG[GGTTAG] 3 -3′) to eight (R8, 5′-AATCCGTCGAGCAGAGTTAG[GGTTAG] 7 -3′) telomeric repeats were used in the TRAP assay. Anchored return primers were developed (ACT, 5′-GCGCGG[CTAACC]3-3′; ACX, 5′-GCGCGG[CTTACC]3CTAACC-3′) that have a 6 bp ‘anchor’ at the 5′-end which is neither telomeric nor complementary to telomeric sequences, followed by sequences that hybridize to telomeric repeats32.

EMSA

An electrophoretic mobility shift experiment was performed 48 h after incubation to determine the effect of ATO on the telomeric DNA and telomerase binding relationship. With 1 μg protein concentrations and a constant concentration of (100 ng) 243 bp DNA, the Gel Retardation Assay was utilized to evaluate DNA–protein interaction. A binding solution comprising 50 mM Tris–HCl (pH 7.4), 50 mM NaCl, 1 mM DTT, 5 mM MgCl2, and 6% glycerol was used to attach protein to DNA. The reaction mixture was incubated at 4 °C for 30 min. The DNA–protein complex was resolved on a 1% agarose gel at 3 Volt/cm for approximately 1–2 h at 4 °C in TBE buffer at 0.5X in TBE buffer on a 1% agarose gel at 3 Volt/cm. Following EtBr (ethidium bromide) staining (0.5 g/mL), the gel was visualized using the Chemi Doc TM MP imaging system.

Supershift assay

The Supershift assay was also used to examine how the hTERT antibody interacted with DNA. The binding solution, which contains 1 mM DTT, 5 mM MgCl2, 50 mM Tris–HCl (pH 7.4), 50 mM NaCl and 6% glycerol, was used to test the DNA binding activity of the protein and antibody with the telomeric DNA (Fig. 9). The reaction mixture was incubated with hTERT antibody (0.5 µg/ml) at 4 °C for 30 min.

Statistical analysis

Graph Pad Prism 5 software was used to plot and analyze graphs (Graph Pad Software, Inc.). The mean ± SEM is used to represent data in graphs. Experiments were conducted at least three times, with three replicates per condition. Three different trials with similar findings were used to create morphological pictures. Unpaired Student’s t test or one-way ANOVA were used for significant comparisons between treatment and control groups, and Tukey’s test was used for comparisons among multiple groups.

Result

Molecular docking, binding affinity & evaluation of in-silico anti-cancer activity of ATO

In order to comprehend the structural complexity of this protein’s target specificity, the structural complexes of hTERT (the target) and ATO (the ligand) were examined using a computational protein–ligand docking technique. Docking was carried out by Patch Dock (Protein-DNA docking), Autodock Vina (protein–ligand docking) and Hex 8.0 (protein-ATO docking) software and the results were analyzed in term of energy of interaction of ATO with hTERT and assigned “Etotal” score-92.0. A complete structure of Human Telomerase Reverse Transcriptase with DNAas shown in supplementary Fig. S.1. The target enzyme (hTERT), effectively docked with ligands along with their known inhibitor silibinin, according to the minimal binding energy (Table 1). The possible binding modes of ATO at telomerase active sites as shown in Fig. 1 along with amino acid residues of hTERT protein Glu341, Glu372 and Met371 form interaction with ligand molecule.

Table.1: Energy obtained during docking analysis of ATO as ligand molecules and hTERT as target protein.
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Fig. 1
figure 1

Visuals of docking results, (a) The hTERT in complex with ATO (b). Binding interaction of ATO with hTERT.

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Moreover, MD simulation was conducted to evaluate the stability of the interaction between inter and intra molecules. RMSD analysis indicates more stable binding of DNA to hTERT compared to hTERT alone during the course of MD simulations (100 ns). Moreover, the structural movement occur more with slight fluctuations in backbone (Cα) of hTERT-ATO complex after the DNA binding, means RMSD analysis suggested that the binding of ATO create the hindrance in the binding of DNA to telomerase (Fig. 2a). RMSF also indicates that the less residual fluctuations after the ATO binding to hTERT, suggested (Fig. 2b). While the MD simulations, several time the structural decompactness occurs in hTERT-DNA complex compared to hTERT-ATO-DNA complex. The Rg plot was also validated through the SASA analysis indicating more or less similar patterns of accessible interaction from surrounding solvent but in case of hTERT-ATO-DNA complex light unstable intra-molecular interaction were depicted (Fig. 2c and d). The molecular dynamics analysis is also focus to calculate the H-bond between hTERT–ATO complex to check the molecular stability of ATO with hTERT, MD analysis depicted the fluctuating H-bond behavior of ATO to hTERT and it stable with one H-bond at end of MD simulation (Fig. 2e). The calculation of H-bond confirmed that ATO shown the potential stability with hTERT, means the potential binding of ATO play key role to allosterically inhibit the binding of DNA.

Fig. 2
figure 2figure 2

Stability of the interaction between inter and intra molecules by Molecular Dynamics study. (a) RMSD of hTERT, DNA-hTERT and DNA-in complex with ATO-hTERT, (b) RMSF of hTERT, DNA-hTERT and DNA-in complex with ATO-hTERT, (c) Rg of hTERT, DNA-hTERT and DNA-in complex with ATO-hTERT, (d) SASA of hTERT, DNA-hTERT and DNA-in complex with ATO-hTERT, (e) H-bond interaction of ATO with hTERT.

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ATO is cytotoxic in HepG2 cells and inhibits cell migration and invasion

We examined effect of ATO on the proliferation of Wrl-68 treated cells & HepG2 cells by MTT assays at concentrations ranges from (0-10micromolar). Obvious dose-and time-dependent inhibition of growth was observed in HepG2 cell types as shown in Fig. 3a, the cells in the untreated Wrl-68 group are neatly organized and strongly adherent. Treatments with 4 μM doses of ATO cause noticeable changes in cellular morphology, leading to the observation of fewer, less adherent, and rounder cells. These modifications became more noticeable in cancer cells after a longer incubation period (48 h) (Fig. 3). Additionally, MTT experiment on both cells supported our findings and demonstrated that 4 μM of ATO is lethal to HCC cells (Fig. 3b). The IC50 results that were obtained from the cytotoxicity assays are presented in Fig. 3b.

Fig. 3
figure 3

4 μM of ATO inhibits the development of cancer cells. Different concentration of ATO was applied to control (Wrl 68) and cancer (HepG2) cells for various time intervals of 24, 48, and 72 h. The microscopic images demonstrate changes in cellular morphology: (a), phase contrast microscopic images after 48 h of 4 μM of ATO; (b) Methylthiazolyltetrazolium (MTT) assay graphs demonstrating ATO-induced cytotoxicity in HepG2 cells after (b.a). 24, (b.b) 48 & (b.c) 72 h. Overall, the data indicate that the ATO has a significant (p < 0.05), dose (4 μM) and time-dependent (48 h) cytotoxic effect on cancer cells. Although control cells’ viability is reduced, it does so over a longer period of time than cancer cells and to a lesser extent. Each observation is supported by at least three distinct experiments. Observations revealed that 4 μM of ATO therapy had a strong inhibitory effect on cancer cells while maintaining the viability rate of normal cells. Statistical significance 3b. was determined using a two-tailed unpaired t-test (n = 3) or one-way ANOVA followed by Tukey’s post-test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Data are mean ± SEM.

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ATO inhibit the migration and invasion potential of HepG2 cells

A wound healing test and transwell migration experiment were used to confirm if 4 µM of ATO had the anti-migration effect. In comparison to the untreated control cells (Wrl-68 treated cells), the relative migratory distance of HepG2 cells was significantly decreased in a time-dependent manner following treatment with 4 µM for 48 h, according to the findings of the wound healing experiment shown in (Fig. 4a and b). 4 µM of ATO considerably reduced the number of cells passing through the filter over 48 h, according to the transwell migration experiment data in (Fig. 4c and d). Together, the findings of the transwell migration experiment and wound healing test indicated that ATO had a considerable ability to prevent HepG2 cell migration and, consequently, tumor formation.

Fig. 4
figure 4

ATO inhibits the migration and invasion potential of HepG2 cells. Both cells were treated with ATO (4 μM) for 48 h. During a 48 h wound closure assay pictures were taken using an inverted microscope. (a) Representative pictures at 0, 24 and 48 h are showing migration potential of ATO on cancer cells and scale bars (100 μm) are added for wound width measurement. The experiments were repeated three times independently. Scale bars indicate 100 μm. (b) Shows a comparative graphical histogram of wound area covered by HepG2 cells in absence or presence of ATO treatment after 48 h with 4 μM of recommended dose.4c.The Transwell assay confirms that ATO (4 μM) inhibits the metastatic potential of HepG2 tumor cell line after 48 h, and significantly reduced the invasive capabilities compared to the untreated tumor cells 4d. Quantitive analysis of number of migrated cells following ATO treatment after 48 h. The wound healing rate was calculated using the following formula: wound healing rate (%) = [1 − (wound area at 48-h time point/wound area at the 0-time point)] × 100. Statistical significance was determined using a two-tailed unpaired t-test (n = 3) or one-way ANOVA followed by Tukey’s post-test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Data are mean ± SEM.

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ATO is cytotoxic and induced ROS in HepG2 cells

HepG2 cells treated with 4 μM of ATO had higher fluorescence intensities than untreated cells. After 48 h of treatment, however, Wrl-68 cells showed little to no fluorescence in either the treated or untreated cells (Fig. 5).

Fig. 5
figure 5

ROS induction after 48 h of ATO (4 μM) treatment in normal verses cancer. (a) shows the fluorescence detection of ROS production using DCFDA dye. (b) demonstrate the fold enhancement of fluorescenceintensity in HepG2 cells with and without ATO. Scale bars indicate 100 μm. Statistical significance was determined using a two-tailed unpaired t-test (c, n = 3), (** p < 0.01, *** p < 0.001). Data are mean ± SEM.

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ATO evokes cell apoptosis

We examined whether ATO caused ROS-mediated apoptosis by measuring the incidence of apoptosis in the cells stained with Annexin V and PI using flow cytometry with 4 μM of ATO which is lethal to HCC cells as demonstrated in dose optimization of our previous paper Chaudhary et al.,22. ATO has the ability to cause apoptotic cell death as seen by the dose-dependent rise in the number of apoptotic cells in the upper and lower right quadrants (Fig. 6a and b). All of these findings point to telomerase suppression and ROS-based apoptosis in liver cancer cells when exposed to ATO.

Fig. 6
figure 6

ATO induces apoptosis in HepG2 cells as shown by Facs after 48 h. Following treatment with 4 µM ATO, representative FACS analysis demonstrates that ATO promoted apoptosis in HepG2 cells. Cells were double-stained with annexin V and PI before being analyzed using flow cytometry. After being exposed to the appropriate concentrations of ATO, apoptotic cells are shown in (a) and (b) shows a representative cellular histogram of 4 μM ATO response on cancer cells after 48 h of incubation. Scale bars indicate 100 μm. Statistical significance was determined using a two-tailed unpaired t-test (C, n = 3) (** p < 0.01, *** p < 0.001). Data are mean ± SEM.

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ATO modulates telomerase expression

To explore the potential mechanism of action, PCR and Western blotting were used to examine the expression of telomerase in both Wrl-68 and HepG2 cells. Following a 48-h incubation with ATO in both Wrl-68 and HepG2 cells, hTERT gene and protein expression was decreased in the HepG2 cell line (Fig. 7a and b). On the other hand, ATO partially restored these expressions in the Wrl-68 cell line. According to the data, ATO dramatically suppress hTERT expression after 48 h.

Fig. 7
figure 7

Effect of ATO on expression analysis of telomerase on both HepG2 and Wrl-68 at different time interval. (Fig. 7a) In comparison to normal cells, the fold differences in mRNA expression are shown. SYBR green-based qRT-PCR was used to assess the mRNA expression (A) after 24 h; (B) after 48 h; and (C) after 72 h, as specified in the protocols. To determine the degree of hTERT expression at protein concentration, immunoprecipitations was performed. The figures indicate relative protein band density on densitometry normalized to the equivalent GAPDH loading control (Fig. 7b). After 48hof time interval, hTERT expression clearly demonstrates that ATO have potential telomerase inhibitory properties in cancer cells (HepG2). Statistical significance was determined using a two-tailed unpaired t-test (7a, n = 3) or one-way ANOVA followed by Tukey’s post-test (7b, n = 5) (** p < 0.01, *** p < 0.001). Data are mean ± SEM. The original blots/gels are presented in Supplementary Fig .S.2.

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Inhibitory effect of ATO on telomerase activity

We used telomerase protein from both cancer and control cells in telomeric repeat amplification protocol (TRAP) tests to determine the impact of ATO on the enzyme’s activity. We did not notice any activity or band pattern in control cells since there was no telomerase activity in those cells. Then, using grey scale analysis, activity levels in cancer cells were measured. We discovered that HepG2 cell types’ telomerase activity was significantly dose- and time-dependently inhibited by ATO (Fig. 8a and b).

Fig. 8
figure 8

Inhibition of telomerase activity by ATO in HepG2 cells. Untreated HepG2 cells were represented by lane 1. HepG2 cells subjected to 4 μM ATO treatment are shown in lane 2 (Fig. 8a) after 48 h of incubation. According to densitometry, the graphs display relative protein activity (Fig. 8b). Statistical significance was determined using a two-tailed unpaired t-test (8b, n = 3). The original blots/gels are presented in Supplementary Fig. S.3.

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ATO block telomere telomerase interaction in HCC cell line

The ability of ATO to inhibit telomere and telomerase interaction was investigated via electrophoresis. Cell lines were treated and the interaction was measured by EMSA and supershift assay (Fig. 9). Supershift and EMSA assays were performed using nuclear extracts (5 μg), pre incubation with anti-hTERT antibody. Protein DNA interaction from HepG2 cells after 48 h at 4 °C. The observed inhibition in the shifting pattern of the band in response of ATO clearly depicts that ATO inhibits this telomere protein binding.

Fig. 9
figure 9

EMSA was used to examine telomerase’s interaction with telomeric DNA. In untreated HepG2 cells, the HTR domain of telomerase binds to (TTAGGG)3 in the presence of competitive DNA as indicated in the first lane. Lane 2nd indicates ATO treatment, which disrupts the corresponding binding. Lane 3rd depicts the impact of ATO on the super sift assay’s measurement of telomerase binding to TAGGGTTAGGGTTAG in presence of anti-hTERT antibody. It represents that ATO treatments prevent the corresponding binding, whereas without ATO a visual interaction and shifting was observed in lane 4. The original blots/gels are presented in Supplementary Fig. S.4.

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Discussion

To a wide variety of anti-cancer medications, tumors typically develop resistance against majority of the drugs33. Drug resistance is thus a significant barrier in treating cancer and particularly liver cancer34. We have previously shown that ATO, when combined with other herbal substances, remarkably reduced telomerase expression and caused apoptosis22. Here, we demonstrate how ATO disrupts the binding of protein-DNA interactions for telomerase activity, and how it alters the activity of telomerase enzymes, and causes apoptosis in a liver cancer cell line. These findings demonstrate that ATO has potent telomerase inhibitory effect, preventing DNA protein interaction at the active site. We then carried out an in-silico docking experiment to learn more about the type of enzyme inhibition brought on by ATO. According to the in-silico docking result the binding energy of telomere and telomerase in control or native environment without any inhibitor is -36.0 kcal/mol which we could say a good interaction. In the presence of ATO, the binding energy of telomere and enzyme remarkably drops to -11.95 kcal/mol, but still the interaction persist between them with least stability which fulfill the criteria of allosteric inhibition in which the inhibitor does not interact to the enzyme at its active site but it alters the active site or make it unfit for its ligands (Figs. 1, 2 and Table 1). Moreover, MD simulation analysis indicates more stable binding of DNA to hTERT after ATO binding during the course of MD simulations (100 ns) compared to the binding of DNA to hTERT without the ATO. MD analysis depicted the fluctuating H-bond behavior of ATO to hTERT and it stable with one H-bond at end of MD simulation as shown in Fig. 3. According to the study by Sherin et al.35, hTERT’s oscillations after engaging with ligands at its binding pocket also reached stable conformations during the course of simulations. So, the molecular docking and MD simulation study suggest that ATO may act as a potential inhibitor for the enzyme telomerase. Such inhibition was also shown in A549, HepG2, MGC-803, and SGC-7901 cell lines due to tetrahydropyran macrolide antibiotic produced from Streptomyces sp. 569N-3 and 11 flavonoid derivatives36,37. Our current study thus reveals the anticancer potential of ATO for the first time with least toxic effects on normal cells. To further confirm this in-silico-enzyme inhibitory role of ATO, we performed several in-vitro experiments. The MTT assay was used to confirm and evaluate the cell viability following exposure to ATO at final concentrations of 4 µM and at different time periods (24, 48, and 72 after drug treatment) (Fig. 3b). As various doses of ATO were independently performed and demonstrated in the supplementary data (Fig. S.2), there was a discernible growth inhibition of malignant cells in comparison to normal cells following 48 h of incubation with 4 µM of ATO. We chose this optimized time and concentration for the rest of the experiments. According to the morphological findings, ATO treatment reduced the number of living cells in HepG2 cancer cell lines in a dose- and time-dependent manner, which is consistent with the cell viability findings discovered by MTT assay (Fig. 3). Similar effect was shown by Propolis, also known as bee wax or propolis extract, is a sophisticated resinous compound that bees gather from vegetable plants, embellish, and utilize as a sort of cementing ingredient to insulate and preserve the beehives, exhibits the similar cytotoxicity towards MG63 osteosarcoma cells37,38.

According to wound healing test & trans well invasion assay, 4 µM ATO may be more effective in preventing HCC cells from proliferating and migrating than Wrl-68 treated liver cells after 48 h incubation. It inhibits cancer cell migration and invasion in a time- and dose-dependent manner. Interestingly, following ATO therapy, there were no discernible changes in migration or proliferation rate of Wrl-68 treated liver cells (Fig. 4). These results suggested that ATO had substantial inhibitory effects against HCC in in vitro. DCFDA a cellular esterase enzyme de-acetylate, a membrane-permeable dye, making it non-fluorescent. The latter is oxidized by ROS to create 2′,7′-dichlorofuorescein (DCF), which exhibits strong fluorescence when activated at 485 nm. By increasing fluorescent intensity, treated HepG2 cells were shown to produce more ROS than control cells, which may have led to cell death. Figure 5 illustrates that even after prolonged incubation with the same dose of ATO, the fluorescence intensity was the same as control, indicating that Wrl-68 treated cells’ production of reactive oxygen species (ROS) was unaffected. The DCFDA intensity rises when HepG2 cells are treated to 4 µM of ATO for 48 h because more ROS are created, which results in increased cell death. Our investigation optimized 4 μM of ATO, which kills HepG2 cells mainly by inducing apoptosis and inhibiting telomerase via increasing intracellular ROS. Curcumin derivatives also showed the importance of ROS up-regulation in the suppression of carcinogenesis, and these compounds may be useful in developing an anti-cancer medication with low side effects39.

Apoptosis is the most common and well-organized form of intentional cell death and it is essential for several biological processes40. Deregulation of apoptosis can result in a wide range of clinical disorders, including different types of cancer41. Therefore, stimulating apoptosis was one of the driving concepts for developing novel chemotherapeutic medications. In the present study, we found that ATO induced apoptosis in the HCC cell lines in a way that was dependent on both time and dose. These results confirm that ATO has strong anti-cancer effects against hepatocellular carcinoma by inducing cellular apoptosis and inhibiting cell growth in HepG2 cells without significantly interfering with the apoptotic pathway of control cells, as seen by the flow data (Fig. 6). However, as Fig. 6 illustrates, prolonged exposure of higher doses of ATO, around and above 10 μM, may be linked to noticeably greater chances of Wrl-68 treated liver cell damage. Reports also suggest that higher and prolong ATO treatment may include certain toxicity to healthy liver cells, especially with regard to liver damage, despite the impressive therapeutic results to cancer cells42. Liver may be a target for arsenic toxicity in addition to being the main location for arsenic methylation. Arsenic metabolites also bioaccumulate and can cause liver damage42. According to research, patients with APL who receive higher concentration of ATO have a greater incidence of hepatotoxicity than those who receive lower concentration43. Hence, the dosage, length of therapy, and general health of the patient all affect the likelihood of hepatotoxicity from ATO. For patients with liver insufficiency, it is crucial to assess liver function at the start of ATO use and to continuously monitor it. One to four weeks following therapy is the most crucial time for monitoring. While ALT, AST, and γ-GT levels momentarily rose with ATO therapy, total protein, albumin, the albumin/globulin ratio, total bilirubin, pre-albumin, and cholinesterase levels were constant. These results suggest that acute injury is the main cause of ATO-induced hepatotoxicity and that dynamic monitoring of hepatic function indicators is required during ATO therapy44.

Arsenic (As) is a naturally occurring metalloid that serves two different purposes: as a medication at lower concentrations and as a health hazard at greater doses above 10 μM. Even though ATO is poisonous and acts as a carcinogenic agent with prolonged exposure, its medicinal benefits have been valued for millennia at doses below 5 μM. For acute promyelocytic leukemia (APL), arsenic trioxide (ATO), a well-known arsenic-based medication, is now utilized as a first-line treatment when combined with all-trans retinoic acid45.Though the precise mechanism of action has not been fully confirmed, there is growing evidence that drugs containing arsenic have therapeutic potential for treating solid and hematologic malignancies other than APL. In the present study, we highlight their diverse mechanisms of action and recent advancements in their application against a variety of cancers by effectively inducing apoptosis in HCC cells at the appropriate dosage and timing, therefore blocking the essential enzyme telomerase. Polyphyllin VII (PVII) and formosanin C (FC) together cause lung cancer cells to undergo apoptosis, according to a similar finding46.

The current study found that a variety of signals and pathways, such as the activation of ROS, apoptotic pathways, and telomerase enzyme inhibition, were responsible for ATO’s anticancer effects on cancer cells. Regarding regulating telomerase expression in both mRNA and protein, we found that ATO inhibits telomerase enzyme activity in HepG2 cells after 48 h at the indicated level of 4 µM. However, Wrl-68 cells were unaffected by ATO at the same dose (Figs. 7 and 8). The telomerase enzyme plays a critical role in the battle against human cancer by regulating cell growth or triggering apoptosis47. Telomeres are specialized DNA structures located at the ends of chromosomes that progressively shorten with each cell division48. By maintaining stable telomeres, telomerase enables cells to overcome this growth barrier49. Since hTERT, the catalytic subunit of telomerase, has been demonstrated to be hyperactive in 85–90% of cancers, including HCC, it has gained recognition as a tumor marker and a popular target for anticancer treatments50. Thus, malignant gliomas can be selectively targeted with a therapy strategy that uses telomerase inhibition. Telomerase is usually blocked by nucleus assembly and export, mRNA interference, expression regulation, or hTERT phosphorylation51. Our research first revealed that the primary factor causing real cytotoxicity in HCC cells is telomerase inhibition. Dose- and time-dependent ROS generation seems to be the main source of hTERT deregulation and displacement52. Elevated ROS levels can damage telomeres because they oxidize guanine and disrupt their regulation. However, telomerase activation has been shown to reverse the decline in mitochondrial function brought on by telomere degradation. One important link between telomere shortening and mitochondrial dysfunction is the DNA damage response, which activates the tumor suppressor protein p53 and reduces mitochondrial biogenesis and metabolic disruptions. This illustrates how ROS induced telomere preservation and mitochondrial activity are correlated. This study looks at the complex interactions between ROS and telomerase activity in both Wrl-68 treated and cancer cell lines offering insight into the interconnected mechanisms underlying aging and cellular function.

HepG2 cells experienced telomere dysfunction and death as a result of the displacement of hTERT interfering with the subunit’s capacity to catalyze telomere repair, as demonstrated by in-silco studies and the TRAP test (Fig. 8). Cisplatin treatment at the right timing and concentration also demonstrated such telomerase inhibition or telomere shortening in BEL-7404 human hepatoma cells53. After 48 h, ATO causes hTERT suppression in HepG2 cells, based on evaluations of hTERT expression using western blot and real-time PCR. The fact that ATO inhibits the telomerase enzyme, which restricts the development of malignancies, may be the reason for this decrease in telomerase gene expression (Fig. 8).

An essential technique for studying the interactions between proteins and DNA is the electrophoretic mobility shift assay, or EMSA54. The growth of cancer and the survival of tumor cells depend on telomeric DNA lengths and telomerase activity, which may be a target for anti-cancer treatment55. Electrophoresis mobility sift assay (EMSA) was used to examine the interactional pattern of telomere and telomerase in response to ATO inhibitor in tumor cell lines. ATO has the ability to block telomere-telomerase interaction, which means it can inhibit the telomerase enzyme (Fig. 9). This is demonstrated by the obvious suppression of band shifting in response to ATO in HepG2 cells. Similarly, it has been shown that when topo-I is sumoylated, the breast cancer cell lines ZR-75-1 and BT-474 interact56. Following 15 min of CPT injection, Topo-I-SUMO conjugates started to build in both cell lines. However, when cells were pretreated with 2-D08 for many hours, this effect was prevented. Crucially, the meta- and para-analogs 2 and 3 were completely inactive at the same dosage in cells, highlighting the effect’s specificity55,56. We obtained super imposable results from EMSA super shift experiments using the hTERT antibody in a manner similar to these findings. Even when hTERT antibody is present, ATO prevents the band from gradually migrating higher; after 48 h, the band is merely reversed compared to those with the untreated control (Fig. 9). These findings demonstrate that our suggested substance (ATO) effectively inhibits telomerase, preventing DNA–protein interaction.

Telomeres and the enzyme telomerase are essential for maintaining the integrity of the genome and chromosomes. Arsenic is recognized to be both a human carcinogen and an anticancer agent due to its clastogenicity57,58. To better understand the molecular mechanisms underlying arsenic’s actions, we investigated the effects of ATO on telomere and telomerase, evaluated cell proliferation and telomerase activity, expression on the mRNA and protein level, and telomere and telomerase interaction in wrl-68 and HepG2 cells in vitro. With least toxicity to the Wrl-68 treated cells, ATO at doses of 4 micromolar significantly inhibits cellular proliferation, conserved or extended telomere length, and decreased telomerase activity in HepG2 cell line. In the HCC cell line, the recommended dose of ATO dramatically downregulated telomerase expression, activity, and its association with the telomere. This suggests that telomerase allosteric inhibition may play a major role in arsenic-induced apoptosis and telomerase inhibition. These findings suggest that the anticancer application of ATO may be partially accounted for an increase in telomerase inhibition and activity, which encourages HCC cell death and apoptosis.

Conclusion

This study offers the first proof that ATO may have anticancer effects on HCC cell lines by preventing cell growth, causing ROS generation, cell death, halting telomerase function, translation activity, and interfering with the telomere-telomerase interaction. The anticancer effects of ATO may be related to molecular changes that affect many cellular signaling pathways. The current study offers mechanistic evidence for the therapeutic potential of ATO in the treatment of HCC. However, the goal of the current study is to clarify how ATO prevents liver cancer in vitro. More research will be done on this substance’s potential as a treatment in animal models of HCC.