Comment on “Anti‐Cancer Potential of Copper‐NTB Complex via Damaging Cellular Organelles and Apoptosis in Triple‐Negative Breast Cancer Therapy”

Yuji Kamikubo has read the recent article by Balusamy et al. in Nano Select with great interest [1]. The authors report on a novel copper(Cu)-tris((1H-benzo[d]imidazol-2-yl)methyl)amine (NTB) complex with potent activity against triple-negative breast cancer (TNBC) cells, providing a systematic study of its cytotoxic mechanism. This work is a valuable contribution to the search for new TNBC therapies. While the study provides a solid foundation, Yuji Kamikubo believes several key points require further analysis to fully substantiate the conclusions and the compound's therapeutic potential. He offers comments on six aspects: statistical validation of superiority, quantitative analysis of apoptosis, the direct role of reactive oxygen species (ROS), classification of cell death, biochemical markers of apoptosis, and selective toxicity. First, the conclusion that the copper-NTB complex is “superior” to cisplatin is based on a comparison of their IC50​ point estimates (5.60 µg/mL vs. 6.95 µg/mL). A claim of superiority requires statistical validation. Since IC50​ values are derived from nonlinear regression and have associated confidence intervals, a formal statistical test comparing the curve-fit parameters is necessary to determine if the difference between the two compounds is significant. Without this, the claim of superiority remains an observation rather than a statistically supported conclusion. Second, the microscopic data for apoptosis in Figures 3–8 are presented qualitatively. A quantitative analysis would provide a more objective and robust comparison of the compound's effects. For instance, calculating the percentage of TUNEL-positive cells or quantifying the mean fluorescence intensity per cell from the Hoechst, LysoTracker, and MitoTracker assays would allow for rigorous statistical testing between dose groups and against the cisplatin control. This would shift the analysis from visual interpretation to data-driven conclusions. Third, the proposed mechanism involving ROS relies on indirect evidence. To confirm this central hypothesis, direct measurement of intracellular ROS is needed. An assay using a general probe like 2',7'-dichlorofluorescin diacetate (DCFDA) or CellROX (Thermo Fisher Scientific) would demonstrate whether ROS levels increase in a dose-dependent manner [2, 3]. To further pinpoint the mechanism, a mitochondrial-specific superoxide indicator like MitoSOX Red could be used to investigate the role of mitochondria in ROS production [4]. Crucially, a rescue experiment using an ROS scavenger, such as N-acetylcysteine (NAC), would be required to establish a causal link between ROS, cytotoxicity, and the observed downstream events [5]. Fourth, the description of the FACS analysis contains a minor inaccuracy. While the methods (Section 2.3.4) and Figure 2 axes (FITC-A) correctly imply a standard Annexin V/PI assay, the accompanying text in the results (Section 3.3) contains a point of confusion, stating that “PI staining (Red) detects the early stage of apoptosis.” Propidium iodide (PI) actually enters cells that have lost membrane integrity, a feature of late apoptosis or necrosis. The standard assay for distinguishing cell death stages is Annexin V/PI co-staining, which can differentiate viable, early apoptotic (Annexin V-positive, PI-negative), and late apoptotic/necrotic (Annexin V-positive, PI-positive) populations [6]. Using this standard terminology consistently would provide a more precise classification of the cell death pathways. Fifth, to provide definitive biochemical evidence of apoptosis, an assay for caspase activation is needed. The execution of apoptosis is dependent on a cascade of caspase enzymes. Measuring the activation of the key executioner Caspase-3, for example, via western blot for its cleaved form or a colorimetric activity assay, would confirm that the observed cell death occurs through the canonical apoptotic pathway [7]. This would strongly complement the morphological data. Finally, and most critically for translational potential, the study lacks an assessment of selective toxicity. A viable anticancer agent must preferentially kill cancer cells over normal cells. Therefore, determining the cytotoxicity of the copper-NTB complex in a noncancerous breast epithelial cell line (e.g., MCF-10A) is an essential experiment [8]. This would allow for the calculation of a selectivity index (IC50​ in normal cells/IC50​ in cancer cells), which is a key parameter for evaluating the therapeutic window and potential safety of a new drug candidate. This point reinforces the original authors' own conclusion, where they astutely state that “cytotoxicity towards normal cells must be elucidated for a better understanding.” In conclusion, the work by Balusamy et al. introduces a promising anticancer agent. The points raised here are intended as constructive suggestions to strengthen their findings. Addressing these issues of statistical rigor, quantitative analysis, direct mechanistic proof, and selective toxicity is critical for advancing this copper-NTB complex from a laboratory discovery toward potential clinical application. This work was supported by JSPS KAKENHI Grant Number 23K06170 and by the Nakatomi Foundation. The author declares no conflicts of interest. The author has nothing to report.