**1. Introduction**

The search for better tumor treatments in glioblastoma (GBM) and other cancers is ongoing. Recently, a major step forward has been achieved on the basis of immune interventions. Specifically, humanized antibodies directed against immune checkpoint antigens are now available to reconstitute immune recognition of the malignant cell in vivo [1]. In addition to the ongoing search for novel checkpoint interventions, chimeric T cell antigen receptors recognizing clonal neoantigens of a given malignancy are promising candidates for an individualized approach [2]. For a broader range of tumors, however, the identification of novel chemotherapeutic drugs [2], potentially resulting in reduced tumor mass as well as improved stimulation of antigen presentation [3], and remain to be of high interest. In this context, we have been intrigued by the original observations of a small synthetic molecule, named Vacquinol-1, and its capacity to fulminant induce cell death in GBM. Even though the original description has been retracted due to non-reproducibility of the in-vivo data (http://retractionwatch. com/2017/07/20/not-want-create-false-hope-authors-retract-cell-paper-cant-replicate/), we here report novel findings on using Vacquinol-1 (here termed: Vac). The synthetic small molecule Vac appears to impair the hydrostatic balance of a tumor cell and induces vacuolization ending in cell rupture. The small therapeutical concentration window of action by Vac limits its application due to a cascade of side effects to occur in vivo [4, 5]. Interestingly, we recently reported that the discriminative effect by Vac against glioma, as opposed to non-transformed tissues could be increased in the presence of a plant derivative, carvacrol [6], likely acting by inhibiting TRPM7 [7]. We here provide evidence for a mechanism on Vac-induced cell death by deteriorating the mitochondrial membrane potential, increasing high intracellular calcium levels combined with endoplasmic reticulum (ER)-stress [8] and impaired calcium storage, followed by mitophagy and rupture of lysosomes and autophagolysosomes. Lysosomal rupture seems to constitute the final event leading to cell death by hydrostatic pressure-associated rupture of autophagolysosomes and the plasma membrane. The decisive effect of Vac against transformed vs. non-transformed tissues may be explicable by the functional sensitivity of TRPM7 to carvacrol in glioma. Essential aspects proving ER-stress and mitochondrial dysfunction have been unraveled by using 3D cryoelectron microscopy.

carvacrol, Vac-induced cell death was found to be increased in #12537-GB, as previously reported [6], an observation corresponding to the lower proliferative and less invasive phenotype of GBM when carvacrol was administered [7]. Carvacrol has been demonstrated to inhibit the transient receptor potential cation channel, subfamily M, member 7 (TRPM7 [7, 9], explaining the higher sensitivity of Vac-induced toxicity to the glioma cell line #12537-GB [6]. The gene encoding TRPM7 functions as both: An ion channel and a protein kinase [7]. In our experimental set-up, the addition of carvacrol at concentrations of 100 μM selectively increased the sensitivity to Vacinduced toxicity in our glioma cell lines, but not in non-transformed fibroblasts. To determine cell viability of pre-established cell layers, we applied the live cell imaging system IncuCyteZOOM® equipped with a 20× objective. Propidium-iodide staining was used to identify and count dead cells in a time-dependent manner. As shown in **Figure 1A**, high Vac concentrations >10 μM led to rapid cell death in both GBM cell lines as well as in non-malignant fibroblasts; 7 μM Vac resulted in significant cell death in #12537-GB cells (**Figure 1A**) after 12 h of incubation but not in #12794- GB cells (**Figure 1B**) and non-transformed fibroblasts (**Figure 1C**). The influence of carvacrol on Vac-induced cell death in glioma cell lines is shown in **Figure 1D** (#12537-GB), and **Figure 1E** (#12794-GB), and non-transformed fibroblasts (Hs68-Fi) in **Figure 1F**. After 56 h, carvacrol (100 μM) significantly (Sidak's Post hoc test after two-way ANOVA; p-values between 0.0198 and 0.0001) enhanced Vac-induced cell death in #12794-GB but not in non-transformed fibroblasts (**Figure 1D**–**F**). Accordingly, only glioma cell lines and not fibroblasts increased their sensitivity against Vac in the presence of carvacrol. To understand the action by carvacrol acting on TRPM7, we performed calcium measurements of glioma cells upon Vac stimulation with and without carvacrol pre-incubation. **Figure 2** demonstrates a rapid loss of cytoplasmic calcium in both GBM cell lines followed by subsequently elevated calcium levels. The addition of ionomycin was included at the end of the experiment to test the residual capacity of the target cell to mount a calcium response. This ionomycin-induced calcium peak was higher in #12537-GB than in #12794-GB. When carvacrol was present, Vac-treatment caused a rapid loss of cytoplasmic calcium again in both cell lines. However, the Vac-induced loss of cytoplasmic calcium was not followed by an increased cytoplasmic calcium level (**Figure 2C** and **D** cf. **Figure 2A** and **B**). The latter might have been due to the inhibition of TRPM7 and loss of control of store-operated calcium channels (SOCEs). Along with this line, Faouzi et al. recently, described the regulation of SOCEs by the kinase activity of TRPM7 [10]. As a consequence, blockade of the TRPM7 would

Mitophagy-Related Cell Death Mediated by Vacquinol-1 and TRPM7 Blockade in Glioblastoma IV

http://dx.doi.org/10.5772/intechopen.77076

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lead to an impaired reconstitution of calcium sequestration to cytoplasmic stores [10].

The molecular events by Vac affecting lipid bilayer integrity and its proton catching properties may contribute to dysfunction of calcium-storage organelles, such as ER and mitochondria. In the end, Vac appears to transiently affect the integrity of lipid bilayers in calcium storage organelles, resulting in transient leakage of bivalent cations such as calcium (**Figure 2**). The partial reconstitution of membrane integrity in the presence of Vac in the range of IC50 values appears to be significantly impaired in the presence of the TRPM7 blocker, carvacrol. As a consequence, calcium influx from external medium would be inhibited [10] and thereby lead to impaired calcium sequestration by ER and mitochondria. In the end, calcium measurements proved, that carvacrol appears to be essential to block endogenous membrane reconstitution. The observations of the combined effects by Vac and carvacrol remind of results obtained in a study performed with curcumin, which was tested on cell death in a melanoma cell line. Bakhshi and colleagues found a dramatic increase of ER-induced cell stress in curcumin-treated melanoma
