Cytoplasmic Vacuolization: A Fascinating Morphological Alteration From Cellular Stress to Cell Death

2.1. Pathophysiological Factors

Physiological vacuolization plays a pivotal role in cellular function [14, 15]. For instance, autophagosomes engulf damaged organelles and proteins, merging with lysosomes for degradation [16, 17]. Lipid droplets regulate lipid metabolism and shield cells from stress [18, 19]. In this context, vacuolization is not directly associated with cell death.

Moreover, polysaccharides from bacteria have been utilized in treatment of ascites tumors and have been linked to CVACD [20]. Viruses such as dengue virus and zika virus exhibit cytotoxic effects in infected cells, prompting cytoplasmic vacuolization [21, 22]. Cytoplasmic vacuolization has also been documented in intraductal papillary neoplasm of the bile duct [23]. Additionally, conspicuous vacuolization around the cell membrane has been noted in the leukemic phase of peripheral T Cell lymphoma [24]. Here, mild vacuolization is typically viewed as a self‐preserving cellular strategy, assisting in the purgation of harmful bacteria and viruses. Nevertheless, when cytoplasmic vacuolization instigated by bacterial and viral onslaughts converges with intense inflammatory responses, it may precipitate cellular pyroptosis or necrosis.

2.2. Drugs or Synthetic Compounds Treatment

Cytoplasmic vacuolization is frequently observed following exposure to natural products, biosynthesis compounds, clinical medications, and weakly basic substances (Figure 1A). The administration of ammonium acetate, autonomic drugs, and weakly alkaline drugs has been shown to induce a robust vacuolization response [25, 26, 27]. Furthermore, natural products, such as curcumin, celastrol, taxol, etc., have been found to provoke cytoplasmic vacuolization [5, 28, 29, 30, 31]. These compounds hold significant promise in anticancer therapy.

Additionally, innovative benzo[a]quinolizidine derivatives have been created and demonstrated to initiate vacuolization [32]. Targeting mitotic cells with Ru65 (Ru(II) polypyridyl complexes) allowed for photodamage to cancer cells through cytoplasmic vacuolization [33, 34]. The copper‐containing nanoparticles and compound 4‐PQBH induce cytoplasmic vacuolization [35]. Novel anthraquinone compounds have been identified to induce cancer cell death through vacuolization [36]. PDT also operates by impairing specific organelles, resulting in CVACD [37].

2.3. Activation or Overexpression of Certain Proteins

As indicated in Table 1, HRasV12 induce methuosis accompanied by cytoplasmic vacuolization in human glioma and gastric cancer cells [4, 40, 44]. Introducing IGF‐1R in different cancer cell lines has been shown to trigger paraptosis, the earliest recognized form of CVACD [3, 41]. Previous studies have verified that the TAJ/TROY protein induces PLCD in HEK293T cells [38]. Moreover, when recombinant mouse macrophage colony stimulating factor (mM‐CSF)‐expressing glioma cells are implanted into immunodeficient mice, they are rejected through paraptosis [39]. Overactivation of steroid receptor coactivators (SRCs) selectively induces CVACD in cancer cells [8]. Our work has illustrated that activation of MscL can induce cytoplasmic vacuolization originating from the ER, mitochondria, and peroxisomes [12, 13]. The morphological alterations of cytoplasmic vacuolization elicited by MscL are depicted in Figure 1B–F, as captured by different imaging modalities. In general, the emergence of CVACD induced by these proteins holds great promise in the realm of oncological therapeutics.

3.1. Paraptosis and MAPK Signaling Pathways

Paraptosis is initiated by the overexpression of IGF‐1R, leading to an alternative caspase‐9 activity independent of Apaf‐1 [3]. Subsequent investigations have unveiled the participation of the MAPK family members (c‐JNK1, MEK‐2, and ERK) in paraptosis [7]. Further connections between paraptosis and the MAPK signaling pathways have been depicted in Figure 2A. The enantiopure titanocene complex CpR2TiCl2 activates EGFR, MAPK, and AKT signal transduction, leading to paraptosis in MDA‐MB‐468 and HCT‐116 cells [42]. Curcumin, PDT, and swainsonine, induce paraptosis through ERK and JNK activation [28, 45, 46]. Interestingly, ferroptosis induced by curcumin treatment may signify a shift from paraptosis to ferroptosis, indicated by the reduction in p62 expression, as paraptosis is intricately linked with ER stress and p62 overexpression [47]. Additionally, elaiophylin triggers paraptosis by inducing ER stress through hyperactivation of the SHP2/SOS1/MAPK pathway [9].

ER stress and the MAPK signaling pathway are crucial in paraptosis. Inhibition of paraptosis can be achieved with substances like actinomycin D, cycloheximide, or AIP‐1/Alix. The components of the MAPK signaling cascade (p38, JNK, ERK), along with ER stress and cell death, are closely linked. Further research is needed to identify the key molecules triggering paraptosis and coordinating cell death.

3.2. Paraptosis/PLCD and ER Vacuolization

ER vacuolization is involved in PLCD (Figure 2B). For instance, SRC3 activation induces PLCD through ER stress and ROS generation [8]. Intriguingly, PDT combined with Ru65 initiates distinct mechanisms of cell death in cells during interphase (PLCD) or mitosis (apoptosis) [34]. The autophagy‐associated FYVE (Alfy) protein has been demonstrated to impede PLCD caused by wheat germ agglutinin, and depletion of Alfy can result in cytoplasmic vacuolization linked to ER stress and UPR [29]. Zika virus induces PLCD through the PI3K/Akt signaling pathway involving UPR and Sec61 ER translocation [22]. Honokiol diminishes the viability of human APL cell lines by triggering PLCD, accompanied by ROS production, mitochondrial impairment, ER stress, heightened LC3 processing, and participation of the mTOR and MAPK signaling pathways (p‐P38, p‐ERK, and p‐JNK) [48]. Furthermore, there are investigations proposing that paraptosis/PLCD can be induced by concurrent expansion of the ER and mitochondria. For instance, 4‐PQBH, targeting Nur77 (an orphan nuclear receptor), induces paraptosis via ER stress [35]. Notably, there appears to be no discernible distinction in mechanisms between paraptosis and PLCD. Anticancer therapies, such as sorafenib, PDT, and the combined administration of geldanamycin and bortezomib, have been linked with various forms of cell death characterized by unspecified mechanisms of ER‐derived cytoplasmic vacuolation [2, 49, 50]. These therapies have been directly associated with the UPR and the molecular chaperone GRP78 [51]. In all of these instances of CVACD, there is apparent ER vacuolization.

In summary, research on ER involvement in CVCAD includes ER stress‐related ubiquitination detection, proteasome‐related protein detection, and ion channels on the ER and mitochondria. Identifying downstream signaling pathways and common regulatory molecules is crucial. Understanding commonalities among triggers is essential for developing effective anticancer medications and enhancing CVCAD mechanisms.

3.3. Paraptosis/PLCD and Mitochondrial Dysfunction

As demonstrated in Table 2, the ROS generation, ATP depletion, and ∆Ψm loss, all indicative of mitochondrial dysfunction, are common events in the context of paraptosis/PLCD. Additionally, ROS generation is a prevalent feature in cell death instigated by ER‐derived vacuolization prompted by selenium, celastrol, honokiol, and SRC overstimulation [8, 48, 53, 57]. Interestingly, PLCD induced by SRC hyperactivation and HRC can be hindered by antioxidants, which also act as inhibitors of ferroptosis, suggesting that there may be overlapping signaling pathways between PLCD and ferroptosis [52, 58].

Furthermore, reducing oxidative stress can protect against vacuolization formation and mitigate CVACD [11]. PDT generates ROS for the fenton reaction, potentially enhancing ferroptosis, cytoplasmic vacuolization, and the efficacy of cancer therapy [54]. A combination of diethyldithiocarbamate and B_12b induces paraptosis through ER stress, mitochondrial impairment, proteasome suppression, and actin depolymerization [59]. Targeted expression of MscL in mitochondria increases cytoplasmic vacuolization, emphasizing the role of mitochondrial permeability [13]. ER‐mediated calcium overload in mitochondria causes vacuolization from both ER and mitochondria, inducing paraptosis with ΔΨm loss [55, 60]. These findings underscore the complex interplay between cell death, stress severity, and cytoplasmic vacuolization as a response to cellular stress.

3.4. CVACD and Ion Imbalance

As depicted in Figure 3, cyclometalated iridium (Ir) complex‐peptide (IPH) 4, CGP37157 (a mitochondrial Na⁺/Ca²⁺ exchanger inhibitor), and Triptycene−Peptide Hybrids (TPHs) possess the ability to induce paraptosis [55, 60]. The underlying mechanism entails the fusion of mitochondrial and ER membranes, the transfer of calcium from the ER to mitochondria, and the loss of ΔΨm. Furthermore, BKCa channels activation may function as a downstream pathway resulting from the interaction between mM‐CSF transduced tumor cells and anti‐tumor immunity, causing mitochondrial calcium overload and potassium efflux [56, 61]. Additionally, the cytosolic calcium overload was observed in vacuolization cells induced by MscL activation [12]. Interestingly, both MscL and BKCa channels are characterized by their large conductance and ability to modulate calcium levels. These channels can induce cytoplasmic vacuolization originating from the ER, mitochondria, or lysosome. Overall, calcium overload in the cytosol or mitochondria, along with potassium efflux, may play a role in the formation of cytoplasmic vacuolization. Furthermore, vacuolization has also been linked to cuproptosis and ferroptosis, though its precise role remains insufficiently explored [43, 62, 63].

3.5. CVACD and Dysfunctional Endosomal‐Lysosomal System

4.1. Overcoming Apoptosis/Drug Resistance of Cancer Cells by CVACD

Multiple studies have illustrated that natural products can induce CVACD in apoptosis/drug‐resistant cancer cell strains. For instance, elaiophylin has been shown to enhance MAPK levels and selectively eliminate drug‐resistant cells through paraptosis [9]. An additional potential therapeutic approach for apoptosis‐resistant cervical cancer cells involves inducing PLCD by administering wheat germ agglutinin [29]. Furthermore, a high concentration of taxol displays significant anti‐cancer properties by inducing CVACD in vitro and in vivo, without significant toxicity [31]. PDT and GRP78‐targeting cytotoxin treatment can effectively eliminate cancer cells regardless of their apoptotic competence and provoke CVACD [49]. Briefly, CVACD may leverage the caspase‐independent signaling pathway to achieve an anticancer effect.

4.2. Selective Lethality of CVACD Towards Cancer Cells

CVACD inducers are employed for targeted cancer treatment, with the goal of enhancing treatment efficacy while minimizing harm to healthy cells. Oncogenic Ras mutations are prevalent in tumor cells, with rare Ras oncogenic mutations observed in glioma and gastric cancer, leading to self‐destruction of these cells through methuosis [4, 40, 44]. Curcumin has been found to induce paraptosis in breast cancer cells but not in normal breast cells [28]. Overstimulating the SRC oncogenic program can effectively trigger cancer cell death through paraptosis‐like mechanisms by pushing cancer cells to their proliferation limits [8]. Methuosis‐inducing compound effectively inhibits tumor growth in a HeLa xenograft model without toxicity [75]. Additionally, the iron chelator deferriferrichrysin induced paraptosis in MCF‐7 and H1299 cells, while having minimal impact on normal human keratinocyte HaCaT cells [76]. CVACD is commonly observed in photodynamic therapy, where cancer‐specific sensitizers can selectively target cancer cells [34, 49, 54]. Agents inducing CVACD can reduce protein degradation, increase ROS production, and heighten ER and oxidative stress in cancer cells, making them more vulnerable to these treatments. Integrating CVACD with the study of cellular stress and metabolism correlations may enhance the precise eradication of cancer cells.

4.3. Immunogenicity of CVACD

The immunogenicity of CVACD in cancer therapy plays a crucial role in the intricate interplay between cancer progression and immune responses. CVACD is often associated with regulated necrosis characterized by membrane rupture, releasing various intracellular antigens that can enhance tumor cell antigen presentation and potentially boost immune responses. However, there is limited research on the immune response elicited by CVACD in cancer therapy. Studies suggest that glioma cells expressing mM‐CSF can act as tumor vaccines, stimulating immune responses in mice and inhibiting tumor growth [39, 56, 61]. By enhancing the natural immunogenicity of tumors, CVACD treatment may pave the way for adjunctive therapies that could lead to improved long‐term survival. Activation of BK channels through paraptosis may harness the tumor cell's natural immunogenicity [1]. The elimination of the endoplasmic reticulum stress‐related kinase, PERK, in endoplasmic reticulum‐stressed melanoma cells initiates SEC61b‐mediated paraptosis, thereby promoting immunogenic cell death and systemic anti‐neoplastic responses [77].

In addition, combining CVACD inducers with current medications can enhance the anti‐cancer effect. Celastrol, when used in conjunction with erbB2‐targeted drugs like Trastuzumab and Lapatinib, shows a strong synergistic effect, increasing cytotoxicity at lower doses [53]. The combination of honokiol and paclitaxel may offer a novel approach to overcoming paclitaxel‐resistant non‐small cell lung cancer cells [78]. Moreover, the concurrent administration of curcumin and apigenin has demonstrated a synergistic anti‐tumor response by regulating mechanisms leading to cellular death, such as apoptosis, autophagy, and ER stress‐induced paraptosis in cervical carcinomas [79]. In summary, CVACD presents a promising alternative for managing drug‐resistant malignancies by precisely targeting malignant cells, inducing selective cytotoxicity, stimulating immune responses, and potentially enhancing the efficacy of current treatments through synergistic approaches. Further research into the molecular mechanisms of CVACD is crucial to fully harness its therapeutic potential.