When compared to monotherapy regimens, combination chemotherapy can prolong the management of many incurable cancers and is commonly utilized to cure specific cancer types. Because the likelihood of developing resistance to multiple drugs with distinct mechanisms is lower than that of developing resistance to a single drug, combining multiple, independently effective chemotherapeutic mechanisms could overcome tumor heterogeneity and lead to more patients experiencing longer-lasting remissions, if not cures [29, 30].

In addition to inducing the expression and activation of pro-survival molecules like GADD34 and GRP78, ER stress can also activate pro-apoptotic molecules such as CHOP (C/EBP homologous protein) and caspase-12. The equilibrium between these processes dictates the fate of the cell, which can either be adaptation or apoptosis. Therefore, the development of a resistant phenotype is determined by the tumor cells'tolerance to endoplasmic reticulum stress, which they achieve by preventing cell death by apoptosis [31]. There are many examples in the literature of anticancer drug combinations with chemicals that can exacerbate ER stress and upset the balance in favor of apoptosis [31,32,33].

As we previously demonstrated, BOLD-100 induces damage to cancer cells through a variety of methods, including apoptosis. BOLD-100 suppresses GRP78 and modifies the unfolded protein response (UPR), while also increasing reactive oxygen species (ROS), which causes DNA damage [22, 34]. This ROS production, comparable to that determined by standard chemotherapy drugs, is significantly magnified when BOLD-100 is used in combination with the individual drugs (Supplementary material 2). BOLD-100 can then increase cancer cell death by synergizing with cytotoxic chemotherapies and targeted medicines.

This study investigated three drugs that are currently used in the treatment of pleural mesothelioma: cisplatin, presently used as first-line systemic therapy in combination with pemetrexed [35, 36], gemcitabine and vinorelbine, used in second-line treatment [36], in combination with BOLD-100, that is currently tested in clinical trial in combination with oxaliplatin and fluorouracil [11, 12].

By binding to DNA and creating intra-strand DNA adducts, which stop DNA synthesis and cell division, cisplatin interacts with cellular macromolecules and causes cytotoxicity through a number of biochemical processes [37]. The antimetabolite gemcitabine, as nucleoside analog, inhibits tumor growth by converting into active triphosphorylated nucleotides that interfere with DNA synthesis and target ribonucleotide reductase [38]. Vinorelbine is an antimitotic anticancer drug whose primary mode of action is the suppression of microtubule dynamics, resulting in mitotic arrest and cell death [39].

The four mechanisms of action eventually lead to cell death by apoptosis, generating several signal cascades that, in three out of four situations, share DNA as a common target. Nevertheless, given that the main effect of BOLD-100 is on the activity of GRP78 and the UPR, we decided to analyze the synergy between the drugs and BOLD-100 using the combination index based on the Chou-Talalay model, which predicts an interaction between the two components of the mixture, and the Bliss independence model, in which the two components do not interact and act on two different targets [40]. After acquiring the dose–response curve for BOLD-100 and other drugs, a third experiment was specifically designed to assess if the interactions between BOLD-100 and the individual drugs were antagonistic, synergistic, or additive. A fixed molar ratio based on the respective potencies of drugs 1 and 2 was employed to determine the mixture’s therapeutic concentrations since in each pair, one of them is less potent than the other. As a result, a range of concentrations above and below the empirically determined EC50 will be evaluated, and the combination will be regarded as a novel drug [19].

Viability assessment following cytotoxic drug exposure fails to evaluate the cell death phenotype, hence drug treatment can induce any form of cell death phenotype, including passive necrosis [41]. Beginning with the idea that BOLD-100’s mode of action is able to disrupt the mitochondrial membrane potential (ΔΨm) [6], we also assessed whether the combination worked in concert to cause this event and the ensuing cell death.

To have a descriptive pool of cell lines representing the histological variability that characterizes PM, we used a line with a biphasic histotype (MSTO211H), one with a sarcomatoid histotype (570), three with an epithelioid histotype (MPP89, 718, 729) and one with an unavailable histotype (748). With particular reference to GRP78 expression, the variation in the protein's baseline between cell lines is visible.

When examining the combination indexes (CI) obtained from data gained with cell viability assay after treatment with the three combinations studied, it becomes clear that the combination with cisplatin is the most promising because a synergistic impact was produced for all of the cell lines that were considered, at least for the highest effect values.

On the other hand, gemcitabine is the least effective combination; in fact, only three out of the six lines showed a synergistic effect. Additionally, it is clear from comparing the cell lines that, when taking into account both the mitochondrial membrane potential assay and cell viability, MSTO211H were the most responsive to the treatments given utilizing the combinations. However, MPP89 cells showed the worst outcomes, which was in line with their reduced GRP78 expression. The sarcomatoid histotype, represented by line 570, offers more pertinent details.

Typically, cancers with this histotype have a more advanced start of resistance. A synergistic behavior was demonstrated by the two combinations, BOLD-100 + cisPt and BOLD-100 + Gem.

At the present, these data are not enough for a translational evidence regarding GRP78 as a biomarker in PM, but a greater knowledge on the role of this protein could be very useful in the evaluation of PM. However, multi-drug approach, with BOLD-100 as combination drug, can significantly increase the efficacy of methods currently employed for PM therapy.

This work establishes the groundwork for a more comprehensive exploration of GRP78 expression and tissue location in PM, its association with the emergence of resistance phenotype, and its potential utility as an extra therapeutic target. For the treatment of advanced gastrointestinal malignancies, BOLD-100 is presently undergoing phase 2 clinical development. We now showed that BOLD-100, when combined with anticancer drugs, showed synergistic behavior in some PM cell lines, especially in treatment-resistant cell lines. Our data clearly emphasizes the necessity for a clinical trial to assess the effectiveness of BOLD-100 in the PM population, given these findings and the dearth of viable therapies for PM.