Comparison of ABQ-48 Multimodal Cytotoxicity Mechanism Against Lung, Colorectal, and Breast Cancer Cells

Simple Summary

Among main causes of death, cancer continues to be a significant cause worldwide. The diversity of cancer types and etiology creates the need for novel and active anticancer treatments. Our study evaluated the cytotoxic potential and mechanism of action of ABQ-48 (3-amino-7-benzylbenzimidazo[3,2-a]quinolinium chloride), a novel unnatural alkaloid of the BQ family, across three human cancer cell lines including: non-small cell lung carcinoma (NCI-H460), colorectal adenocarcinoma (COLO-205), and breast ductal carcinoma (T-47D). Our findings demonstrate that ABQ-48 has anticancer activity at low micromolar levels, especially on lung cancer cells, through the activation of multiple programmed cell death, mitochondrial damage and DNA fragmentation with consistent mechanistic profile across the three cancer types. Notably, ABQ-48 demonstrated superior cytotoxicity compared to cisplatin, a well-established and currently FDA approved and applied chemotherapy agent, supporting the potential and further investigation of ABQ-48 as a promising therapeutic candidate.

Abstract

Cancer continues to be a significant cause of death worldwide, particularly cancers with high incidence and mortality such as colorectal, breast, and lung, motivating the continued search for novel anticancer agents. Among potential new molecules with anticancer effects, members of the benzazolo[3,2-a]quinolinium salts (BQs) family, including ABQ-48, have shown promising cytotoxic activity in various cancer models. This study aimed to evaluate the cytotoxic potential and mechanism of action of ABQ-48 (3-amino-7-benzylbenzimidazo[3,2-a]quinolinium chloride) across non-small cell lung carcinoma (NCI-H460), colorectal adenocarcinoma (COLO-205), and breast ductal carcinoma (T-47D) cell lines. Cancer cells were treated for 48 h with ABQ-48, cisplatin, or vehicle, and cytotoxicity was assessed by determining IC₅₀ by fluorescence analysis. Mechanistic evaluation included Annexin V apoptosis detection, caspase-3/7/8 activation assays, mitochondrial membrane permeability analysis, and DNA fragmentation assessment. ABQ-48 exhibited dose-dependent cytotoxicity in all three cancer cell lines, with IC₅₀ values of 6.02 µM (NCI-H460), 14.33 µM (COLO-205), and 33.59 µM (T-47D), surpassing cisplatin’s overall efficacy. Annexin V assays confirmed apoptotic induction, while caspase activation demonstrated engagement of both intrinsic and extrinsic pathways. ABQ-48 demonstrates potent anticancer activity through activation of multiple programmed cell death mechanisms, supporting further investigation as promising therapeutic candidate.

1. Introduction

Cancer is the leading cause of death worldwide. In 2025, it is expected that 2,041,910 new cancer cases will be diagnosed in the United States, with 618,120 deaths attributed to this disease [1]. Although there have been advances in detection and treatment over the years, the mortality rate continues to rise, and resistance to conventional drugs drives the need for new therapeutic strategies. According to the National Cancer Institute (NCI), prostate, lung, and colorectal cancers are expected to account for approximately 48% of all new cancer cases in men this year. In women, breast, colorectal, and lung cancers are estimated to represent 51% of all newly diagnosed cases in the same year. This indicates that both men and women are susceptible to developing these types of cancer, highlighting the need for more effective treatments to improve patient prognosis [1].

Previous reported results [2] described the synthesis and screen, of two BQS, including ABQ-48 at the experimental concentration of 10 µM, utilizing the NCI-60 Human tumor cell line screening. Given the observed and promising biological activity of ABQ-48 we here expanded the analysis by determining the cytotoxic effect and mechanism of action comparison on three different cancer cell lines NCI-H460, COLO-205 and T-47D constitute models of lung, colon, and breast cancer, respectively. It is worth mentioning that these cell lines are widely used to investigate new anticancer drug candidates, what cell death mechanism is activated, and therapeutic resistance. On the other hand, COLO-205 cells, a colorectal adenocarcinoma, exhibit continuous signaling called WNT/β-Catenin, which regulates proliferation, apoptosis, and is responsible for drug resistance [3]. In addition, NCI-H460 cells, a non-small cell lung carcinoma, are characterized by alterations in the PI3K/AKT/mTOR, and MAPK/ERK pathways, and are frequently used to study apoptosis and chemotherapeutic resistance [4]. Finally, T-47D cells, a ductal breast carcinoma, are positive for estrogen and progesterone receptors, and it has been shown that they depend on ER/PR pathway signaling, which interferes with the PI3K/AKT and NF-κB pathways. Given these, among other characteristics, T-47D cells serve as a model of hormone-sensitive breast cancer with endocrine therapy resistance [5,6]. The continuous need for the development of novel anticancer agents with improved efficacy and alternative mechanisms of action has been emphasized by major research institutions, including the National Cancer Institute [7] driven us to aid in the search for active molecules such as the one reported.

The here studied experimental novel compound ABQ-48 (7-benzyl-3-aminobenzimidazo[3,2-a]quinolinium chloride) is a synthetic derivative of the benzazolo[3,2-a]quinolinium family of compounds, which are a family of cationic alkaloid-like compounds [2], that can be described as a cationic quaternary alkaloid with structural similarities to the anticancer agent ellipticine, with well documented anticancer properties [8,9,10]. ABQ-48 is protected under patent [11], which describes its chemical synthesis and potential biomedical applications. Some of these ellipticine analogs, for instance, can induce apoptosis in NCI-H460 lung cancer cells and compromise mitochondrial integrity, demonstrating potent dose-dependent cytotoxicity. Similarly, structurally modified anthracycline derivatives have shown increased toxicity in T-47D breast cancer and COLO-205 colorectal cancer cell lines [12]. Altogether, these results underscore the importance of continuing to evaluate analog compounds across multiple cancer models to optimize therapeutic outcomes.

Additionally, a structural modification was introduced in ABQ-48 through the incorporation of an amino group at position 3 of ring A (Figure 1;

Table 1. Molecular formula and R₁ substituents of the studied compound. ABQ-48 presents an amino group-substituted moiety at the R₁ position.

Name	R1	R2	R3	Molecular Formula	MW (g/mol)
ABQ-48: NSC D-763307	3-NH2	H	H	C22H18ClN3	359.8514

), which enhances its electron-donating activity, helping to improve its inhibitory activity [2]. Importantly, this modification strengthens its ability to interact with molecular targets involved in key processes essential for cancer cell survival, thereby ultimately promoting apoptosis. ABQ-48 was also evaluated in a preliminary NCI-H60 Human tumor cell lines screen, demonstrating significant antiproliferative activity across multiple cancer cell lines including lung (NCI-H460), colorectal (COLO-205) and breast (T-47D) reflecting a rational strategy for designing more potent and selective anticancer agents, especially for aggressive and treatment-resistant malignancies [2]. The mechanisms involved, for such effects, were not studied justifying the rationale of this study.

The objective of this study was to further describe the therapeutic potential of the compound ABQ-48 by determining the growth inhibitory concentration (IC₅₀) and mechanism of action in three cell lines: non-small cell lung carcinoma (NCI-H460), colorectal adenocarcinoma (COLO-205), and ductal breast carcinoma (T-47D). A key step in the study was to subsequently assess whether cell death occurred through apoptosis, programmed cell death including caspase activation, mitochondrial dysfunction, DNA fragmentation, and autophagy [13,14,15]. This approach allowed for a comparison of its cytotoxic effects with cisplatin, a drug currently used as an FDA approved chemotherapeutic treatment for a wide range of cancers, including breast, lung, and colorectal cancer. Finally, mechanistic features such as caspase activation, phosphatidylserine externalization, mitochondrial membrane permeability, and DNA fragmentation served as apoptotic indicators that function as biomarkers of therapeutic activity [16,17].

2. Materials and Methods

2.1. Compounds and Stock Solutions

ABQ-48 solutions were prepared at a concentration of 3 mM in nano pure water, and stored at 4 °C. The vehicle control (Negative control, nano pure water) was also stored at 4 °C under sterile conditions. The positive control, Cisplatin was prepared at a stock concentration of 3 mM using dimethyl sulfoxide (DMSO) as the solvent, and stored at –20 °C.

2.2. Chemicals

3-amino-7-benzylbenzimidazo[3,2-a]quinolinium chloride (ABQ-48: NSC D-763307) was synthetized as described by Cox et al. [2].

2.3. Cell Lines

Human cancer cell lines COLO-205 (Human colorectal adenocarcinoma, CCL-222), NCI-H460 (Human non-small cell lung carcinoma, HTB-177), and T-47D (Human breast ductal carcinoma, HTB-133) were obtained from the American Type Culture Collection (ATCC). COLO-205, NCI-H460, and T-47D cells were cultured in RPMI-1640 medium and supplemented with 10% fetal bovine serum (FBS). Cells were grown at 37 °C in a humidified incubator with 5% CO₂. Culture media were refreshed every 2–3 days, and cells were subcultured at 70–80% confluence using 0.25% trypsin-EDTA. All cell lines were handled under sterile conditions using a biological biosafety cabinet, class II.

2.4. IC₅₀ Determination Assay

Cells were harvested from a stock culture and seeded into a 96-well plate at a density of 50,000 cells per well in 200 µL of complete growth medium. Experimental wells included blanks (media only), Negative control (Nano-pure Water), Cisplatin (positive control) and ABQ-48 treatment with increasing concentrations from 10 to 100 µM. A 3mM stock solution of the compound was prepared, and serial dilutions were performed for the experimental concentrations in a total volume of 200 µL per well.

Cells were exposed to ABQ-48 as well as to negative and positive control treatments and incubated at 37 °C with 5% CO₂ for 48 h. After incubation, 10 µL of viability PrestoBlue dye (Thermo Fisher Scientific, cat A13262; Waltham, MA, USA) reagent was added to each well, followed by a 30 min incubation at 37 °C. Viability intensity was measured using the FLUOstar OMEGA microplate reader (BMG Labtech, Ortenberg, Germany) with an excitation wavelength of 560 nm and an emission wavelength of 590 nm, using a negative control for gain adjustment. Fluorescence readings were analyzed using MARS data analysis software (BMG LABTECH, version 3.32 R5; Ortenberg, Germany), with background fluorescence subtracted and viability percentages normalized to vehicle control. The dose–response curve was generated using nonlinear regression, and the IC₅₀ value, the concentration required to inhibit 50% of cell viability, was determined. Experiments were performed in triplicates.

2.5. Annexin V Assay

For the apoptosis, annexin V assay, Cells were cultured in RPMI-1640 media in 12.5 cm² flasks under standard conditions (37 °C, 5% CO₂), seeded at a density range of 2 × 10⁶ cells per flask. Cells were treated with negative control (Nano-pure water), positive control (Cisplatin) and ABQ-48 for 48 h before assessment of apoptosis. After the exposure to the IC₅₀ of ABQ-48, as well as to the positive and negative controls, approximately 1.0 × 10⁶ cells were resuspended in 100 μL Annexin V binding buffer, ensuring that all samples remained within the specified concentration range for accurate comparison. Subsequently, 2 μL of Annexin V-CF488A conjugate and 2 μL of Hoechst 33342 (final concentration: 10 μg/mL) were added, followed by gentle mixing via pipetting. The samples were incubated at 37 °C for 20 min, strictly adhering to the incubation time and temperature. Following incubation, cells were centrifuged at 400× g for 5 min at room temperature, and the supernatant was removed by pipetting. Each of the pellets was resuspended in 300 μL Annexin V binding buffer, centrifuged again at 400× g for 5 min, and the supernatant was removed. This washing step was repeated once more to ensure proper sample preparation. The final pellet was resuspended in 100 μL Annexin V binding buffer supplemented with 2 μL of Propidium Iodide (PI) to 100 μL of binding buffer, and the samples were immediately analyzed. The NucleoCounter NC-3000 was prepared, and 30 μL of each cell suspension was loaded into the chambers of a 2-chamber NC-Slide A2. The experiment was performed in triplicates.

2.6. Caspase Activity

2.6.1. Caspase 3/7

Cells were cultured in RPMI-1640 media in 12.5 cm² flasks under standard conditions (37 °C, 5% CO₂), seeded at a density range of 2 × 10⁶ cells per flask. Cells were treated with (Nano-pure water), Cisplatin and ABQ-48 for 48 h before assessment of apoptosis. After exposure to the IC₅₀ of ABQ-48, as well as to the positive and negative controls, the cells were harvested and their concentration determined using a Via1-Cassette, adjusting the final concentration to 1.0 × 10⁶ cells/mL. The reconstituted caspases FLICA reagent was diluted 1:5 in PBS (Phosphate Buffer Solution) by adding 200 μL PBS to 50 μL of reconstituted FLICA reagent. A volume of 5 μL of the diluted FLICA reagent was then added to 93 μL of the prepared cell suspension. Subsequently, 2 μL of Hoechst 33342 was added to achieve a final concentration of 10 μg/mL, and the samples were gently mixed by pipetting. Cells were incubated at 37 °C and 5% CO₂ for 60 min, with occasional swirling every 30 min to ensure uniform staining. Following incubation, cells were washed twice with 400 μL of 1X apoptosis wash buffer, centrifuged at <400× g between washes. After the final wash, the cell pellet was resuspended in 100 μL of apoptosis wash buffer supplemented with 10 μg/mL propidium iodide (PI) by adding 2 μL of PI to 100 μL of wash buffer, and samples were analyzed immediately. A total of 30 μL of each cell type and treatment suspension was loaded into the chambers of an NC-Slide A2, placed on the tray of the NucleoCounter NC-3000, and analyzed using the Caspase Assay protocol by selecting the appropriate software setting and initiating the RUN function. Experiment was performed in triplicates.

2.6.2. Caspase 8

Cells were cultured in RPMI-1640 media in 96-well plates under standard conditions (37 °C, 5% CO₂), seeded at a density range of 40,000 cells per well. Cells were treated with (Nano-pure water), Cisplatin and ABQ-48 for 48 h before assessment of apoptosis. Caspase-8 activity was measured using the Caspase-Glo 8 Assay (Promega, Madison, WI, USA). The Caspase-Glo 8 Reagent was prepared according to the manufacturer’s instructions, equilibrated to room temperature, and 100 µL of reagent was added to each well. To prevent cross-contamination, care was taken to avoid touching pipet tips to the well contents. The plate was gently mixed at 300–500 rpm for 0.5–2 min and incubated at room temperature for 30 min to 3 h, with luminescence measured at every 60 min using a FluoSTAR OMEGA (BMG Labtech, Ortenberg, Germany). Luminescence measurements were taken every hour, ensuring reading remained above 70% of peak luminescence. Data were analyzed using one-way ANOVA in Fluostar Omega (BMG LABTECH, version 5.50 R4). The experiment was performed in triplicates.

2.6.3. Mitochondrial Membrane Permeabilization

Cells were cultured in RPMI-1640 media in 12.5 cm² flasks under standard conditions (37 °C, 5% CO₂), seeded at a density range of 2 × 10⁶ cells per flask. Cells were treated with Nano-pure water, Cisplatin, and ABQ-48 for 48 h before assessment of apoptosis. JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) and DAPI (4′,6-diamidino-2-phenylindole) staining were performed to assess mitochondrial membrane potential and nuclear content, respectively. Approximately 1.0 × 10⁶ cells were suspended in 1 mL of medium or phosphate-buffered saline (PBS). JC-1, a cationic dye that accumulates in mitochondria in a potential-dependent manner, was added to a final concentration of 2.5 µg/mL by introducing 12.5 µL of Solution 7 (JC-1 stock solution at 200 µg/mL) followed by incubation at 37 °C and 5% CO₂ for 10 min in the dark. Staining time was adjusted as needed depending on cell type. After incubation, cells were centrifuged at 400× g for 5 min at room temperature, and the supernatant was carefully removed. The pellet was washed twice with 1–2 mL of PBS, centrifuging each time at 400× g for 5 min. After the final wash, the pellet was resuspended in 250 µL of Solution 8 (1 µg/mL DAPI), a fluorescent stain; and the samples were analyzed immediately. Approximately 30 µL of each cell suspension was loaded into an NC-Slide A2. The experiment was performed in triplicates.

2.6.4. DNA Fragmentation

Cells were cultured in RPMI-1640 media in 12.5 cm² flasks under standard conditions (37 °C, 5% CO₂), seeded at a density range of 2 × 10⁶ cells per flask. Cells were treated with Nano-pure water, Cisplatin and ABQ-48 for 48 h before assessment of apoptosis. DNA fragmentation analysis was performed using DAPI (Solution 3—DAPI Staining Reagent; ChemoMetec, Cat. No. 910-3003; Allerød, Denmark) staining to evaluate chromatin condensation and nuclear fragmentation. Cells were harvested by centrifugation at 500× g for 5 min at room temperature, washed once with PBS, counted, and resuspended at a density of 1 × 10⁶ cells in 0.5 mL PBS. For adherent cells, cells were detached by trypsinization, centrifuged at 500× g for 5 min, washed with PBS, and similarly resuspended in 0.5 mL PBS. Ice-cold 70% ethanol (4.5 mL) was added to each of the 10–15 mL centrifuge tube, and the cell suspensions were transferred into the ethanol-containing conical tubes, vortexed vigorously to ensure a single-cell suspension and fixed at 4 °C for a minimum of 12 h. Fixed cells were then centrifuged at 500× g for 5 min, ethanol was thoroughly removed, and the pellet was washed with 5 mL PBS for 50 s after centrifugation. The final cell pellet was resuspended in 500 µL mL of Solution 3, consisting of 1 µg/mL DAPI in PBS, and incubated for 5 min at 37 °C. Samples were then analyzed using the NucleoCounter NC-3000 system. The NC-Slide A2 (ChemoMetec, Cat. No. 943-0002; Allerød, Denmark) was used for loading approximately 30 µL of stained suspension per chamber. The experiment was performed in triplicates.

2.6.5. Statistical Analysis

Data are presented as means ± standard error (SE). Statistical comparisons were performed among vehicle, cisplatin, and ABQ-48–treated groups. One-way analysis of variance (ANOVA) was used to assess differences among groups, followed by post hoc multiple-comparison testing. Statistical analyses were performed using GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA). Differences between the negative control, positive control (cisplatin), and the experimental compound (ABQ-48) were assessed for statistical significance. at p < 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****). All experiments were performed with at least three independent biological replicates.

3. Results

Half-Maximal Inhibitory Concentration (IC₅₀)

As can be observed in

Table 2. Inhibitory Concentrations (IC₅₀) of ABQ-48 and Cisplatin Across Studied Human Cancer Cell Lines.

Inhibitory Concentrations (IC50)	Cell Lines
	T-47D	COLO-205	NCI-H460
ABQ-48	33.59 μM	14.33 μM	6.020 μM
Cisplatin	43.00 μM	20.40 μM	58.00 μM

, ABQ-48 proved to be consistently more toxic to cancer cells than the positive control, cisplatin. Across all three cell lines, ABQ-48 displayed lower IC₅₀ values, indicating higher potency. In T-47D cells, ABQ-48 reached 33.59 μM compared to 43.0 μM for cisplatin. The difference was stronger in COLO-205 (14.33 μM vs. 20.4 μM), and most notable in NCI-H460, where ABQ-48 showed an IC₅₀ of 6.02 μM while cisplatin required 58.0 μM to achieve similar inhibition. These results highlight that ABQ-48 is markedly more cytotoxic than cisplatin in all models tested.

4. Discussion

This study evaluated the cytotoxic activity and mechanisms of action of ABQ-48 (7-benzyl-3-aminobenzimidazo[3,2-a]quinolinium chloride) in three human cancer cell lines—NCI-H460 non-small cell lung carcinoma, COLO-205 colorectal adenocarcinoma, and T-47D breast ductal carcinoma, as summarized in

Table 3. Biological Effects of ABQ-48 Across Cancer Cell Lines.

Biological Endpoint	Cell Line
	T-47D	COLO-205	NCI-H460
Compound	ABQ-48
IC50	33.59 μM	14.33 μM	6.02 μM
Annexin V	+	+	+
Caspase (3/7)	+	+	+
Caspase (8)	+	+	+
Mitochondrial Membrane Permeabilization	+	+	+
DNA Fragmentation	+	+	+

The positive (+) symbol indicates activity determined experimentally.

. ABQ-48 exhibited clear cytotoxic activity with a cell line–dependent response profile (Figure 2). NCI-H460 cells were the most sensitive (IC₅₀ = 6.02 μM), followed by COLO-205 (IC₅₀ = 14.33 μM), whereas T-47D cells displayed the greatest resistance (IC₅₀ = 33.59 μM). These results are consistent with previous reports indicating that ABQ-48 induces cytotoxicity in cancer cells [2,18] but where mechanism was not determined. Importantly, differential sensitivity to anticancer agents has been widely associated with intrinsic genetic and signaling heterogeneity across cancer types, contributing to variable therapeutic responses and resistance mechanisms [19,20].

ABQ-48 belongs to the benzimidazo[3,2-a]quinolinium (BQ) family, a class of compounds with documented cytotoxic activity in multiple Human cancer cell models. Several benzazolo[3,2-a]quinolinium (NBQ) derivatives, including NBQ-38, NBQ-95, and NBQ-97, have previously been reported to exert cytotoxic effects in cancer cells, particularly in epidermoid carcinoma models [21]. These compounds were shown to interact with DNA and induce apoptosis primarily through activation of executioner caspases, including caspase-3/7, suggesting a caspase-dependent mechanism of cell death. In those studies, cytotoxicity was largely attributed to DNA damage–associated signaling and downstream apoptotic execution, with limited evaluation of mitochondrial involvement or pathway integration. Structurally, these NBQ derivatives contain a nitro group at the R₁ position, whereas ABQ-48 presents an amino-substituted moiety at the R₁ position. This substitution represents a key structural distinction within the BQ family and may influence the electronic properties and intermolecular interactions of ABQ-48, potentially contributing to its distinct biological behavior.

In contrast to previously reported NBQ derivatives, ABQ-48, while sharing the core BQ scaffold, demonstrated distinct biological activity in the present study. ABQ-48 exhibited differential cytotoxic potency across multiple human cancer cell lines, as reflected by cell line-specific IC₅₀ values, indicating variability in cellular sensitivity. Mechanistically, ABQ-48 induced robust apoptotic responses characterized not only by strong activation of caspases 3/7, but also by mitochondrial membrane depolarization (ΔΨm loss) and DNA fragmentation, suggesting engagement of both intrinsic and extrinsic apoptotic signaling pathways. The combined activation of mitochondrial dysfunction and caspase-dependent apoptosis distinguishes ABQ-48 from previously described NBQ derivatives, which were primarily associated with DNA interaction driven cytotoxicity.

Additional studies indicate that BQ compounds, including ABQ-48, promote apoptosis in lymphoma cells via activation of both intrinsic and extrinsic caspase pathways, as well as through the release of apoptosis-inducing factor (AIF) [22]. The results presented here support the role of ABQ-48 as a potent inducer of apoptosis, primarily mediated by mitochondrial permeabilization and caspase activation across all three cancer models, consistent with reports highlighting mitochondrial vulnerability as a critical determinant of drug sensitivity [19,23].

As reported here, Annexin V assays confirmed that ABQ-48 initiates apoptosis through early membrane phosphatidylserine externalization in all cell lines (Figure 3A, Figure 4A and Figure 5A) Consistently, caspase activation assays demonstrated increased activity of caspases-3/7 and caspase-8, indicating engagement of both intrinsic and extrinsic apoptotic pathways (Figure 3E, Figure 4E and Figure 5E). The activation of both pathways suggests that ABQ-48 overcomes single-pathway resistance mechanisms, a feature increasingly recognized as essential for effective anticancer agents, particularly in cancer models with complex resistance networks [20]. Notably, previous studies in NCI-H460 cells have demonstrated that cell death can also occur through caspase-independent mechanisms depending on the chemotherapeutic agent used [24], underscoring the relevance of the strong caspase activation observed here following ABQ-48 treatment.

Mitochondrial membrane permeabilization induced by ABQ-48 was consistently observed in all three cell lines and was greater than that produced by cisplatin, the positive control (Figure 3C, Figure 4C and Figure 5C). Given the central role of mitochondrial dysfunction in apoptotic signaling, these data reinforce the importance of mitochondrial pathways in ABQ-48–mediated cytotoxicity [2,25,26]. Dysregulation of mitochondrial integrity and redox homeostasis has also been linked to therapy resistance and survival advantages in lung and breast cancer models, particularly through metabolic and ferroptosis-related adaptations [19,23]. Thus, the pronounced mitochondrial effects induced by ABQ-48 may contribute to its enhanced cytotoxicity relative to cisplatin. DNA fragmentation assays provided further confirmation of apoptosis by demonstrating the occurrence of late-stage apoptotic events following ABQ-48 treatment (Figure 3F, Figure 4F and Figure 5F).

The observed differences in sensitivity among the cancer cell lines likely reflect distinct molecular and signaling contexts inherent to each model. NCI-H460 cells, which exhibited the lowest IC₅₀ value, appear particularly susceptible to ABQ-48. Based on prior studies, this heightened sensitivity may be associated with differential regulation of survival signaling pathways, such as the PI3K/AKT/mTOR axis, which is known to preserve mitochondrial integrity by phosphorylating and inactivating pro-apoptotic proteins, maintaining mitochondrial membrane potential, and preventing cytochrome c release [4]. Although PI3K/AKT activity was not directly evaluated in the present study, future investigations examining pathway activation status and mitochondrial dependence could clarify whether ABQ-48 sensitivity correlates with vulnerability to mitochondrial disruption in this cellular context. In COLO-205 cells, the intermediate sensitivity to ABQ-48 may reflect alternative survival or differentiation programs characteristic of colorectal cancer models. Notably, Wnt/β-catenin signaling has been implicated in modulating apoptotic thresholds and therapeutic resistance in colorectal cancer [3,27]. Future studies assessing β-catenin activity or downstream transcriptional targets following ABQ-48 treatment may help determine whether this pathway contributes to the observed response profile. By contrast, T-47D breast cancer cells exhibited relative resistance to ABQ-48, consistent with the cytoprotective roles of estrogen and progesterone receptor signaling and p21 associated survival mechanisms reported in hormone-responsive breast cancer models [5,6,20]. Further mechanistic studies evaluating hormone receptor status, cell-cycle regulation, and apoptotic checkpoint control in response to ABQ-48 would be valuable to define determinants of resistance in this model.

Importantly, the ability of ABQ-48 to engage in both intrinsic and extrinsic apoptotic pathways may represent a strategic advantage in overcoming cancer cell resistance mechanisms. Resistance to chemotherapy frequently arises from pathway redundancy, mitochondrial rewiring, or suppression of individual apoptotic routes, enabling tumor cells to evade cell death despite sustained drug exposure [19,20,23]. Agents capable of activating multiple apoptotic signaling axes, including mitochondrial permeabilization and caspase cascade amplification, have been shown to limit adaptive resistance and enhance therapeutic robustness across heterogeneous tumor populations. In this context, the multi-pathway apoptotic profile observed for ABQ-48 supports its potential efficacy in cancer models characterized by complex or compensatory survival signaling networks. While the present study demonstrates potent pro-apoptotic activity of ABQ-48 in malignant cells, evaluation of potential off-target effects and toxicity in non-malignant cells remains essential for translational advancement. Members of the BQ family have previously exhibited variable selectivity depending on substitution patterns and cellular context [21,22], underscoring the importance of defining therapeutic windows. Future studies will include comparative cytotoxicity analyses using at least one non-malignant human cell line (e.g., lung epithelial or fibroblast models) to assess cancer selectivity. Such analyses will be critical for distinguishing tumor-specific vulnerability from generalized cytotoxic stress and for guiding further preclinical development.

Together, these findings demonstrate that ABQ-48 induces apoptosis in lung, colorectal, and breast cancer cell models through activation of both intrinsic and extrinsic apoptotic mechanisms, with mitochondrial membrane permeabilization emerging as a central event. The pronounced activity observed in NCI-H460 cells highlights the potential utility of ABQ-48 as a promising therapeutic candidate for lung cancer models, particularly in contexts where resistance to conventional chemotherapeutics and pathway targeted agents limit clinical efficacy [19,23,24]. Although ABQ-48 demonstrated superior pro-apoptotic activity compared to cisplatin in vitro, further studies are required to fully evaluate its translational potential. Future work will focus on assessing the toxicity profile of ABQ-48 in non-malignant cells, as well as investigating its pharmacokinetic and pharmacodynamic properties. In addition, in vivo studies using appropriate tumor models will be necessary to evaluate therapeutic efficacy, biodistribution, and safety. These studies will be essential to determine whether the enhanced apoptotic activity observed in vitro can be translated into effective and tolerable anticancer responses in vivo.

5. Mechanism of Action of ABQ-48

Based on our experimental findings, the extrinsic and intrinsic apoptotic pathways are integrated in the mechanism of action of ABQ-48. This compound acts as an apoptotic stimulus leading to caspase-8 activation and subsequent activation of executioner caspases-3/7; additionally, phosphatidylserine externalization (Annexin V), mitochondrial membrane permeabilization (MMP), and DNA fragmentation contribute to this mechanism, as summarized in Figure 6.

In addition, based on previous findings with structural analogs within the chemical family capable of forming DNA adducts [28], we proposed a panel depicting a putative interaction of ABQ-48 with DNA. Although DNA adduct formation was not directly assessed in this study, such interactions may contribute to genotoxic stress and facilitate the activation of apoptotic signaling. The DNA fragmentation observed in this study might represent a late, caspase-dependent apoptotic event and does not constitute direct evidence of primary DNA damage.

Figure 6. Mechanism of action of ABQ-48. Schematic representation of the apoptotic mechanisms triggered by ABQ-48 through extrinsic and intrinsic pathways. As part of the extrinsic pathway, ABQ-48 acts as an apoptotic stimulus leading to caspase-8 activation and subsequent activation of executioner caspases-3/7.Experimentally validated events are indicated by star (*) symbols and include caspase-8 and caspase-3/7 activation, phosphatidylserine externalization (Annexin V), mitochondrial membrane permeabilization (MMP), and DNA fragmentation. As part of the intrinsic pathway, MMP a key apoptotic event can promote cytochrome c releaseand eventually downstream apoptosome formation, leading to caspase 9 activation and inducing the experimentally determined caspase-3/7 signaling. Cytochrome c release, apoptosome assembly, and caspase 9 activation are inferred based on established apoptotic pathways and were not directly assessed in this study. Components depicted in gray represent well-characterized apoptotic regulators included for mechanistic context. Based on previous findings with structural analogs within the chemical family capable of forming DNA adducts, we proposed a panel depicting a putative interaction of ABQ-48 with DNA.

Figure 6. Mechanism of action of ABQ-48. Schematic representation of the apoptotic mechanisms triggered by ABQ-48 through extrinsic and intrinsic pathways. As part of the extrinsic pathway, ABQ-48 acts as an apoptotic stimulus leading to caspase-8 activation and subsequent activation of executioner caspases-3/7.Experimentally validated events are indicated by star (*) symbols and include caspase-8 and caspase-3/7 activation, phosphatidylserine externalization (Annexin V), mitochondrial membrane permeabilization (MMP), and DNA fragmentation. As part of the intrinsic pathway, MMP a key apoptotic event can promote cytochrome c releaseand eventually downstream apoptosome formation, leading to caspase 9 activation and inducing the experimentally determined caspase-3/7 signaling. Cytochrome c release, apoptosome assembly, and caspase 9 activation are inferred based on established apoptotic pathways and were not directly assessed in this study. Components depicted in gray represent well-characterized apoptotic regulators included for mechanistic context. Based on previous findings with structural analogs within the chemical family capable of forming DNA adducts, we proposed a panel depicting a putative interaction of ABQ-48 with DNA.

6. Conclusions

In conclusion, this study demonstrates the cytotoxic activity of the novel compound ABQ-48 (3-amino-7-benzylbenzimidazo[3,2-a]quinolinium chloride) across lung (NCI-H460), colon (COLO-205), and breast (T-47D) cancer human cell lines, with differential effects depending on the genetic profile of each model. Biological assays showed that ABQ-48 induces apoptosis through both intrinsic and extrinsic pathways, involving activation of caspases 3, 7, and 8, mitochondrial membrane permeabilization, phosphatidylserine externalization, and DNA fragmentation. Notably, ABQ-48 exhibited greater efficacy than cisplatin in all three cell lines, highlighting its potential to overcome resistance mechanisms that limit the effectiveness of standard chemotherapeutic agents such as cisplatin. These findings support the notion that structural modifications of compounds can significantly enhance cytotoxic activity and selectivity toward cancer cells, as demonstrated here. Lastly, the multimodal cell death effects observed suggest that ABQ-48 may represent a promising therapeutic candidate for other aggressive malignancies with poor prognosis, including melanoma, pancreatic cancer, and hepatocellular carcinoma, warranting further investigation.

7. Patents

Members of the NBQ family of compounds including ABQ-48 are protected under patent [Patent No. US9,085,574B2], which describes its chemical synthesis and potential biomedical applications.

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Nov 2012|Patent 1—Zayas, B. and Cox, O. USPO granted Patent (US 8124, 770 B2). “Fluorescent Cellular Markers”. Also protected in China. China Patent (CN): CN201080021846.3. Granted April 29, 2015. “Fluorescent Cellular Markers”.

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March 2015|Patent 2—Zayas, B. and Cox, O. “Method for producing benzazoloquinolium (BQs) salts and using the biological activity of the composition”, US patent # US9,085,574B2, (21 July 2015).

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Feb 2018|Patent 3—Cox, O. and Zayas B. USPO US9889128B2 (13 February 2018). “Method for producing benzazoloquinolium (BQs) salts, using the composition as cellular markers, and using the biological activity of the composition.

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Dec 2020|Patent 4—US USPO 10,624,885B2 (21 April 2020). Title: Method for producing benzazoloquinolium (BQs) salts and using the biological activity of the composition.

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April 2022|Patent 5—USPO 11,311,531 (26 April 2022). Title: Method for producing benzazoloquinolium (BQs) salts and using the biological activity of the composition.