Gliomas consist of a heterogeneous group of tumors graded by the World Health Organization based on their microscopic and molecular profiles. Glioblastoma (GBM) is the most aggressive form of high-grade glioma, characterized by a poor prognosis and high mortality rates, with a median patient survival time of less than 15 months. Despite extensive efforts to combat this tumor, GBM remains one of the most lethal neurological malignancies. Its invasive and infiltrative features hinder complete surgical tumor resection, leading to a lack of complete remission (Li et al., 2024; Pichol-Thievend et al., 2024). Furthermore, the tumor microenvironment and the blood–brain barrier, coupled with cellular heterogeneity, the presence of tumor stem cells, and alterations in signaling pathways, significantly contribute to the resistance of GBM to radiotherapy and chemotherapy. The current standard approach involves tumor resection, followed by radiotherapy and adjuvant chemotherapy. However, the inherent features often result in inadequate recovery and disease recurrences (Ferguson et al., 2022; Giordano et al., 2024).

Cisplatin ((SP-4-2)-diamminedichloridoplatinum (II), CDDP) has demonstrated significant clinical efficacy. Once inside the cells and after aquation, it leads to the formation of CDDP–DNA adducts that interfere with DNA replication and transcription. Nonetheless, similar to many other anticancer drugs, CDDP is associated with side effects and drug resistance (Wen et al., 2023), impacting its overall efficacy (Jeon et al., 2021). Consequently, several studies are currently exploring the therapeutic opportunities with platinum-based drugs to leverage their efficacy and overcome the associated limitations. In this field, the analysis of combination therapy using platinum(IV) complexes has shown promising results (Ponte et al., 2023).

Combination therapy exploits the synergistic effects of two or more compounds to surpass the potential of single-drug treatments. It has been demonstrated that by targeting multiple key pathways, drug combinations may achieve superior efficacy, reduced toxicity, or dosage reduction compared to single drugs (Duarte and Vale, 2022). Octahedral Pt(IV) complexes offer an efficient way to combine a “traditional” Pt(II) moiety (e.g., CDDP) with a second drug in the axial position, having a synergistic or at least additive effect with CDDP. Octahedral Pt(IV) complexes designed in such a way are specifically reduced in hypoxic tumor tissues, resulting in the release of the cytotoxic Pt(II) metabolite alongside the concomitant loss of the second drug in the axial position, a mechanism known as “activation by reduction” (Ravera et al., 2022). The final result is the combination of the selective and stimuli-responsive targeting of cancer cells with the synergistic effect of the platinum moiety and the adjuvant drug (Gabano et al., 2022). Among the octahedral Pt(IV) complexes, the prodrug (OC-6-44)-acetatodiamminedichlorido(2-(2-propynyl)octanoato)platinum(IV), also named Pt(IV)Ac-POA (Figure 1), has been reported to exhibit efficient antitumor activity both in vitro and in vivo (Gabano et al., 2017). Pt(IV)Ac-POA includes, as an axial ligand, the free acid POA—a medium-chain fatty acid and histone deacetylase inhibitor—along with an inert acetate (Ac) group. POA, as a medium-chain fatty acid, enhances the lipophilicity of the drug compared to cisplatin alone, thereby increasing cellular drug uptake (“synergistic cellular accumulation”) (Raveendran et al., 2016; Novohradsky et al., 2015; Ravera et al., 2019). Moreover, the histone deacetylase inhibitor appears to facilitate the formation of Pt(II)–DNA adducts, possibly by reducing the chromatin compaction levels, thus increasing DNA accessibility (Ferrari et al., 2020).

In a similar approach, the platinum moiety has been combined with the naturally occurring rhein (4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-carboxylic acid or cassic acid), a molecule known for its anti-inflammatory, anti-oxidant (Yin et al., 2021), and anticancer effects by regulating apoptosis, cell proliferation, migration, and invasion (Henamayee et al., 2020; Zhang H. et al., 2023). This combination constitutes the prodrug (OC-6-44)-acetatodiamminedichlorido(4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-carboxylato)platinum(IV), also referred to as DB178 (Figure 1). The complexes demonstrated anti-proliferative activity not only higher than that of the parent compounds CDDP and rhein but also higher than the reference drug temozolomide in the same and other human glioblastoma cell lines (Poon et al., 2021; Gabano et al., 2022). Moreover DB178 retained its activity under hypoxia and caused a significant reduction in the motility of both cell lines, which can be related to its ability to inhibit MMP2 and MMP9 matrix metalloproteinases (Gabano et al., 2022).

Cathepsins constitute a class of lysosomal proteases that play a central role in protein degradation; numerous studies have investigated their role in the progression of various tumors; hence, they are being evaluated as potential target candidates for both diagnostic and treatment purposes. In detail, a specific member of the cathepsin family, cathepsin B (CatB), is involved in several physiological and pathological processes, particularly in gliomas, where it is commonly overexpressed, promoting angiogenesis, regulating cell death, and influencing the onset of pharmacological resistance (Ma et al., 2022; Huang et al., 2024). However, the role of CatB in cell survival and its basis of execution exhibit deep variance (Mijanović et al., 2019; Chevriaux et al., 2020); thus, a comprehensive analysis of CatB’s role in GBM is still unavailable, and in particular, no data are available regarding therapies with Pt(IV)-based drugs. Importantly, Pt(IV)-based prodrugs are activated via reduction under hypoxic conditions typical of tumor microenvironments and do not rely on CatB for their activation. Nonetheless, due to its ability to modulate the apoptotic and autophagic pathways, CatB represents a biologically relevant marker when evaluating programmed cell death (PCD) mechanisms triggered by Pt(IV) complexes. This is supported by a substantial body of literature focusing on CatB targeting in GBM. Rempel et al. (1994) first demonstrated increased CatB mRNA expression in glioblastoma cells, correlating it with malignancy and paving the way for emphasizing the contribution of CatB to multiple aspects of GBM progression and patient survival (Norton et al., 2024). Several studies have since incorporated CatB in the characterization of novel therapies, either by developing CatB-responsive programmed brain-targeted drug delivery systems (Jiang et al., 2024), assessing CatB levels following therapeutic interventions, or modulating its expression to observe functional outcomes. For instance, Zhang et al. (2018) reported that irradiation significantly increases CatB expression, and that silencing CatB enhances radiosensitivity in GBM cells by promoting apoptosis through the downregulation of RAD51 and homologous recombination efficiency, hence revealing a possible role for CatB in radioresistance. Despite these findings, data regarding the involvement of CatB in Pt(IV)-based treatments are still lacking, prompting us to include CatB analysis in this study.

For these reasons, this study aims to investigate the effects of the two prodrugs Pt(IV)Ac-POA and DB178 by comparing them with CDDP to evaluate their impact on different molecular pathways. Particular attention will be paid to CatB, considering its central role in tumor progression and its association with other markers indicative of cell proliferation, therapeutic resistance, cellular damage, and cell death. The study will deepen the existing research conducted on the glioblastoma cell line U251 and, concurrently, assess the effects of the drugs on human glioblastoma T98G cells. Particular attention will be paid to the analysis of CatB localization and mitochondria, given the growing number of studies linking these two elements in the context of PCD. Several reports have shown that CatB, especially when released from lysosomes into the cytosol, can trigger a cascade of cell signaling events that ultimately induce the mitochondrial apoptotic pathway through a mechanism involving cytochrome c release and the subsequent promotion of apoptosome assembly (Kavčič et al., 2020; Xie et al., 2023; Zhang Z. et al., 2023).

GBM is the most common malignant primary brain tumor, and current therapies only extend overall patient survival and are unsuccessful in providing a definitive cure preventing recurrence phenomena. The development and characterization of novel drugs are essential for the supply of new strategies and therapeutic targets. In this context, octahedral Pt(IV)-based prodrugs are currently being investigated for their potential superior anticancer efficacy. This study aims to characterize the impact of two Pt(IV)-based chemotherapeutic complexes, namely, Pt(IV)Ac-POA and DB178, with specific attention to their effects on markers of proliferation and cell death pathways. These drugs are of particular interest in the context of glioblastoma due to their lipophilic properties, which may facilitate their passage across the blood–brain barrier. Based on previous in vitro and in silico data, Pt(IV)Ac-POA showed an increased accumulation rate through biological membranes; similarly, promising results were obtained when investigating the lipophilicity of DB178, suggesting that both compounds could the blood–brain barrier more effectively than cisplatin and temozolomide (Gabano et al., 2017; 2022).

PCD is a fundamental physiological mechanism for maintaining cellular homeostasis, and the dysregulation of this process is commonly associated with a variety of human diseases, including cancer. PCD is commonly categorized according to the cellular and molecular findings, and a detailed comprehension of the triggered forms of PCD is vital to characterize the effectiveness of a drug and its putative limitations, particularly regarding the development of resistance (Chen et al., 2024).

In the first part of the work, we focused our attention on DB178. The toxicity profile of this prodrug was assessed through a viability MTT test. This allowed the identification of an IC₅₀ value of 25 µM and 10 µM for the U251 and T98G cell lines, respectively. According to data from Cellosaurus ¹ (Bairoch, 2018), the differing sensitivity of T98G and U251 cells to DB178 may be attributed to their distinct TP53 and PTEN mutations, which influence apoptotic regulation and survival pathways. ClinVar ² (Landrum et al., 2014) reports that the mutations found in U251 cells are associated with higher oncogenic potential and increased resistance to cell death. Notably, the observed dose-dependent effect was significantly more pronounced when cells were treated with the prodrug, highlighting its superior efficacy compared to the individual components (Pt(IV) or rhein) administered separately. This suggests an adjuvant effect, where the prodrug enhances the activity of its constituents, underscoring the importance of further characterizing the compound, in parallel with the other novel chemotherapeutic prodrug, Pt(IV)Ac-POA, whose synergistic activity has already been proved (Gabano et al., 2017). Unlike what was observed with DB178, Pt(IV)Ac-POA exhibited comparable toxicity in both T98G and U251 cells. Encouragingly, both compounds proved to be more effective at lower doses than CDDP. Although further studies are needed to clarify their specific mechanisms of action and potential mechanisms of resistance, the current findings offer valuable insights into the induction of PCD mechanisms.

Growing evidence supports the idea that cathepsin B is involved in PCD pathways at multiple levels (Liu H. et al., 2023). CatB is the most characterized member of the C1 family of papain-like lysosomal cysteine peptidases. It is synthesized as an inactive pre-proenzyme, glycosylated in the Golgi apparatus, and then transferred to lysosomes (Chou et al., 2023). Although extensive research has been carried out, an unambiguous explanation of the role of CatB under pathological conditions is still not available. For instance, the upregulation of CatB has been associated with increased angiogenic potential, as well as with anti-necroptotic activity and pharmaco- and radioresistance (Mijanović et al., 2019). On the contrary, other studies emphasized the pro-apoptotic activity of CatB due to its role in triggering cytochrome C from mitochondria and subsequent apoptosis (Wang et al., 2023), particularly when released from the lysosomes (Ni et al., 2022). Our data indicate the maintenance of basal CatB levels after treatments in both U251 and T98G cells, suggesting the therapeutic potential by neither stimulating angiogenesis nor promoting resistance onset in surviving cells. The only exception is represented by T98G cells treated with Pt(IV)Ac-POA. To further characterize this aspect, immunogold labeling was employed to localize cathepsin B within specific cellular compartments. This technique has previously been applied to investigate lysosomal proteins with high spatial resolution (Haraguchi et al., 2005; Mahanty et al., 2024). In this condition, CatB immunogold labeling showed elevated labeling of free cytosolic and nuclear CatB compared to the lysosomal counterpart. The release of CatB from lysosomes is known for its important role in determining apoptosis (Yadati et al., 2020; Yoon et al., 2021), which may confirm the positive effect in inducing PCD, as also supported by ultrastructural evidence. The release of CatB into the cytoplasm has been associated with the phenomenon of lysosomal membrane permeabilization, leading to regulated cell death (Reinheckel and Tholen, 2022). Several studies have reported that this release is involved in the activation of intrinsic apoptotic pathways in addition to the extrinsic apoptosis cascade (Pratt et al., 2009). In this context, both genetic manipulation and the use of pharmacological inhibitors have demonstrated that cytosolic CatB contributes to apoptosis through a multilevel mechanism. Notably, CatB can promote the activation of Bid, leading to its translocation to mitochondria and the subsequent release of cytochrome c, which initiates caspase activation and apoptotic cell death. Simultaneously, apoptosis is further favored by the degradation of anti-apoptotic proteins such as Bcl-2, Bcl-xL, Mcl-1, and XIAP (Yadati et al., 2020; Anes et al., 2022). Moreover, CatB has been identified as a key mediator in TNF-α-induced apoptosis (Xie et al., 2023). The presence of these findings in the literature reinforces the relevance of investigating Pt(IV)-based compounds, where the cytosolic relocalization of CatB may indicate effective activation of programmed cell death pathways, alongside the formation of Pt(II)–DNA adducts. Nonetheless, given the dual role of CatB in cancer, further analyses may be necessary to better define the relationship between Pt(IV)-based prodrugs, glioblastoma cell proliferation, and PCD.

For these reasons, we focused our attention on Parkin, a 465 amino acid-long protein encoded by the parkin RBR E3 ubiquitin-protein ligase (PRKN). Parkin, with its E3-ligase activity, has been shown to reduce tumor cell proliferation by blocking access to the G1/S phase of the cell cycle. Parkin expression is reduced in gliomas compared to that in healthy brain tissues, and lower parkin levels are associated with a poor prognosis (Clausen et al., 2024). Pt(IV)Ac-POA has previously been associated with an increase in the Parkin level in U251 cells (Gaiaschi et al., 2023b), while no data were available regarding neither its effect on T98G cells nor DB178 outcome on U251 or T98G cell lines. In this study, we showed that DB178 induced an even greater increase in Parkin levels in U251 cells, demonstrating its potential role in regulating cancer cell progression. Additionally, T98G cells exposed to DB178 exhibit higher Parkin immunolabeling, further supporting the efficacy of this drug compared to standard CDDP treatments.

PINK1, a serine/threonine kinase, acts as a negative regulator of multiple cellular pathways exploited by cancer cells. PINK1 reduces glioblastoma proliferation by controlling aerobic glycolysis, reducing ROS production, and regulating the Warburg effect; furthermore, PINK1 plays a pivotal role in mitophagy (Raimi et al., 2024). Previous studies highlighted an increase in PINK1 levels in U251 cells following Pt(IV)Ac-POA treatment, indicating effective anti-proliferation activity (Gaiaschi et al., 2023b). Our data showed that DB178 treatment also led to a statistically significant increase in PINK1 immunolabeling in U251 cells, supporting the positive effect of this drug. Analogously, we reported a statistically significant increment following DB178 treatment in T98G cells, confirming the drug’s effectiveness on an additional cell line. Moreover, ultrastructural investigations revealed the presence of mitophagy events, consistent with the role of PINK1 in mitochondria quality and abundance control.

To further characterize the effect of drugs on mitochondrial status, we assessed the levels of two different mitochondrial enzymes, COX4 and ACO2. COX4 is the largest regulatory subunit of cytochrome c oxidase, whose overexpression is correlated with the repression of reactive oxygen species (ROS) production, higher respiration rates, mitophagy, and the induction of regulated cell death pathways (Oliva et al., 2022). Pt(IV)Ac-POA has previously been demonstrated not to impact COX4 expression in U251 cells (Gaiaschi et al., 2023b). Interestingly, we found that DB178 correlates with higher levels of COX4. Mitochondrial ACO2 is the Krebs cycle enzyme designated for the conversion of citrate to isocitrate (Kim et al., 2023). Its overexpression has been associated with increased mitochondrial mass and the activation of mitophagy, suggesting an active role in mitochondrial turnover during the quality control process (Jeong et al., 2024). Pt(IV)Ac-POA has already been linked to alterations in ACO2 levels in U251 cells (Gaiaschi et al., 2022). In this study, we demonstrated a similar trend in T98G cells and deregulations of ACO2 following DB178 treatment. The altered levels of ACO2 are associated with cell death induction and mitophagy (Zhu et al., 2023), supporting the activity of Pt(IV) complexes against GBM.

LC3B, an RNA-binding protein and an mRNA decay factor, plays a central role in autophagosome formation during autophagy (Hwang et al., 2022), a process contributing to cell survival and adaptation (Feng et al., 2020; Molina et al., 2022). Recent findings highlighted a worse prognosis in GBM patients with high levels of autophagy-related genes due to increased tumor aggressiveness and therapy resistance (Bashiri and Tabatabaeian, 2023; Danish et al., 2024). Our data indicated that while LC3B levels remained at baseline in U251 cells following treatment with either Pt(IV)Ac-POA (Gaiaschi et al., 2023b) or DB178, a reduction is observed in T98G cells after exposure to Pt(IV)Ac-POA. This decrease may reflect an early block in the autophagic process, which could interfere with the ability of tumor cells to activate survival strategies. Consistently, SQSTM/p62 levels appear globally similar across treatments (Supplementary Material). As a multifunctional adapter protein that links ubiquitinated proteins to the autophagic machinery for degradation—and itself degraded during autophagy (Bjørkøy et al., 2009)—these results support the hypothesis of an incomplete autophagic flux.

Taken together with the other findings, these results suggest that the treatments induce significant mitochondrial damage, triggering mitophagy and the formation of vesicles containing damaged mitochondria, as confirmed by ultrastructural analysis. However, at the time point analyzed, it appears that the autophagic flux was not fully completed (Liu S. et al., 2023). Further investigations, for instance, assessing the colocalization of LC3B with p62 while using reference compounds such as rapamycin and bafilomycin or performing activity and apoptosis assays (e.g. caspase), could represent a valuable approach to clarify this aspect.

Nrf2, a transcription factor with cytoprotective activity, stimulates the expression of genes containing antioxidant response-like sequences in their promoters. Nrf2’s role as a cellular protector applies to both healthy and cancer cells: in normal cells, it protects against oxidative damage and inhibits malignant transformation, whereas in cancer cells, its protective effect may result in resistance to radiotherapy or chemotherapy by preventing apoptosis and cellular death and stimulating drug metabolism and/or drug efflux (Zimta et al., 2019; Ulasov et al., 2022). Cancer cells are known to tolerate moderate levels of oxidative stress, and increased ROS levels contribute to proliferation and evasion from senescence, while excessive exposure to ROS may lead to apoptosis. In this context, the involvement of Nrf2 in NADPH production may prevent excess oxidative damage as a protective mechanism (Pölönen et al., 2019; Cano et al., 2021; Micalizzi et al., 2021). The Nrf2 pathway is overactive in gliomas, contributing to tumorigenesis, cell invasion, and stemness properties (Gumeni et al., 2021; Shahcheraghi et al., 2022; Crisman et al., 2023; Lin et al., 2023). Interestingly, analysis of Nrf2 on T98G cells clearly indicates that Pt(IV) complexes under investigation effectively reduce the levels of this enzymatic cellular protector. This suggests an improved efficiency in limiting resistance acquisition in glioblastoma cells. Although not statistically significant, a similar trend is observed in U251 cells, confirming the potential beneficial effect of these treatments. The combination of these markers supports the hypothesis of reduced development of pharmacological resistance to the assessed Pt(IV) drugs compared to standard cisplatin.

In conclusion, taken together, these findings underscore the pivotal role of the two investigated octahedral Pt(IV)-based prodrugs in the regulation of GBM proliferation, favoring PCD mechanisms. Pt(IV) complexes are specifically designed to combine a Pt(II) moiety with a second compound in the axial position, allowing for a synergistic or additive effect that is selectively activated in the hypoxic tumor tissue via the established “activation by reduction” mechanism (Ravera et al., 2022). While Pt(IV)Ac-POA incorporates a medium-chain fatty acid to enhance lipophilicity and cellular uptake, along with a histone deacetylase inhibitor to promote the formation of Pt(II)–DNA adduct formation (Ferrari et al., 2020), DB178 is characterized by the presence of rhein, aiming to couple efficient DNA adduct formation with the known anti-proliferative properties of this natural molecule, including its ability to reduce cell motility (Gabano et al., 2022). Although some data on the efficacy of Pt(IV)Ac-POA on U251 glioblastoma cells were already available, this study expanded the current knowledge by confirming its capacity to induce apoptosis in T98G cells. Furthermore, for DB178, no prior data were available on the investigated molecular pathways. Our analysis demonstrated the effectiveness of DB178 against both investigated cell lines. In addition to their efficacy in eliciting PCD mechanisms, both compounds did not significantly induce increases in the detected levels of cathepsin B, further confirming their potential to reduce the possible onset of pharmacological resistance. Furthermore, in the only case in which we found an increase in the cathepsin B signal, immunogold labeling revealed cytosolic relocalization, which aligns with the activation of the apoptotic pathway. Further studies are needed to unravel the complex molecular mechanisms underlying the efficacy of drugs targeting GBM. For instance, the maintenance of basal levels of cathepsin B supports the investigation of Pt(IV)-based prodrugs in combination with cathepsin modulators, which may positively impact the efficacy of the treatments by additional reduction of the proliferative potential of GBM cells. Given the dual role of cathepsin B in either promoting cell proliferation or inducing PCD pathways such as apoptosis, it would be of interest to investigate the effect of the tested prodrugs in combination with cathepsin B inhibitors or, even more intriguingly, functional modulators. This could be pursued in future studies by incorporating commercially available molecules (Siddiqui et al., 2024) and exploring newly synthesized compounds specifically designed for this purpose (Yadav et al., 2024) or through drug repurposing strategies (Alrouji et al., 2024), with the latter two approaches representing rapidly growing areas of research. Such an approach would allow for a deeper understanding of the role of cathepsin B in either sustaining cell proliferation or promoting cell death, depending on its functional modulation. Since tumor recurrence is common with standard therapies, identifying novel and specific molecular targets could support the development of improved drugs that act on multiple signaling pathways, thus improving the efficacy of available treatments and, ideally, taking steps forward in this complex field.