Specific signaling pathways mediated programmed cell death in tumor microenvironment and target therapies

Abstract

Increasing evidence has shown that programmed cell death (PCD) plays a crucial role in tumorigenesis and cancer progression. The components of PCD are complex and include various mechanisms such as apoptosis, necroptosis, alkaliptosis, oxeiptosis, and anoikis, all of which are interrelated in their functions and regulatory pathways. Given the significance of these processes, it is essential to conduct a comprehensive study on PCD to elucidate its multifaceted nature. Key signaling pathways, particularly the caspase signaling pathway, the RIPK1/RIPK3/MLKL pathway, and the mTOR signaling pathway, are pivotal in regulating PCD and influencing tumor progression. In this review, we briefly describe the generation mechanisms of different PCD components and focus on the regulatory mechanisms of these three major signaling pathways within the context of global PCD. Furthermore, we discuss various tumor therapeutic compounds that target different signaling axes of these pathways, which may provide novel strategies for effective tumor therapy and help improve patient outcomes in cancer treatment.

Introduction

Cell death can be defined as the programmed cell death (PCD) and accidental cell death (ACD) [1]. The former is manipulated by signaling cascades and molecular effectors, occurring autonomously and in an orderly manner [1–3]. PCD includes apoptosis, necroptosis, ferroptosis, autophagy, pyroptosis, etc. It is characterized by cell contour shrinkage, nuclear condensation, abnormal mitochondrial cristae morphology, the formation of membrane bubbles, surface pores, apoptotic body, etc. [4, 5]. ACD, like necrosis, showed mainly the inflammatory response that is critically associated with ischemia–reperfusion damage. It’s triggered by cell swelling, loss of biofilm integrity, overflow of cell contents, and dissipation of ion gradients [6]. Cell death is closely associated with tumors and has been the research point of tumor research [7, 8]. Recently research indicated that PCD affected tumor progression and metastasis by regulating immunogenicity of the tumor microenvironment (TME). Cell death shows significant potential in cancer treatment. Different forms of cell death have an overlap in mechanism, thus it’s necessary to systematically analyze cell death as a whole. They will promote the conjunction utilization of PCD modulators in cancer treatment [9].

The signaling pathway has an essential role in the occurrence of PCD. Cysteinyl aspartate protease (caspase) mediates apoptosis, necroptosis and other PCD, thereby interfering with a variety of definite tumor progression [10]. PI3 K/AKT/mTOR signaling pathways mediated apoptosis in melanocyte tumors [11]. Receptor-Interacting Protein Kinase 3 (RIPK3) and Mixed Lineage Kinase Like (MLKL)-dependent necroptosis creates an immunosuppressive tumor microenvironment. It stimulates the immune system and induces cytotoxic CD8 + T lymphocytes to exert a strong adaptive immune response, and finally prevents tumor progression [12]. Previous studies summarized the mechanism of various signaling pathways which mediated the different cell death. However, it remained unknown how the signaling pathway works when considering the cell death as a whole. This will provide insight into the search for common signaling molecules in different cell death.

In this review, we first systematically characterized the components and depicted molecular mechanisms of PCD. Then we screened three signaling pathways that are significant in PCD, namely the caspase signaling pathway, RIPK1/RIPK3/MLKL signaling pathway and mTOR signaling pathway. We also reviewed the molecular mechanisms of four signaling pathways regulating PCD in the tumor microenvironment and elaborated on their essential roles in tumor progression. Finally, combined with the latest research progress, we summarize the targeted therapy of the three signaling pathways involved in PCD. This provides new ideas for the treatment of tumors and guide the study of combined drugs.

The components and mechanism of PCD in the TME

Apoptosis

Apoptosis can be categorized into exogenous and endogenous apoptosis, both of which involve intricate mechanisms encompassing multiple [13]. The extrinsic apoptotic pathway is initiated by the interaction of death receptors (belonging to the tumor necrosis factor receptor superfamily, TNFR) with their ligand, which activate pro-caspase 8, transforming it to caspase 8 [14]. Death receptor ligands encompass TRAILR2 (DR5)-TRAIL, TRAILR1 (DR4)-TRAIL, FAS (CD95, APO-1)-FasL, TNFR1-TNFα [15]. Adaptor proteins FADD and TRAD sequester caspase-8 and/or −10, resulting in the formation of the Death-Inducing Signaling Complex (DISC), which amplifies local pro-caspase concentrations, facilitating mutual autoactivation [16]. Activation of the initiator caspase leads to the processing of the downstream effector protein caspase-3, −6, and −7, resulting in the cleavage of vital substrates for cell survival, ultimately inducing cell death [17]. On the one hand, the endogenous apoptotsis (mitochondria-dependent) is triggered by intracellular signals such as irradiation and chemotherapeutic drug treatment [17]. Inner stimuli such as irreparable genetic damage, hypoxia, high intracellular calcium ion concentration, and severe oxidative stress initiate the endogenous mitochondrial pathways [18]. It has been established that pro-apoptotic proteins, such as Bak and Bax from the Bcl-2 family, are activated, disrupting the outer mitochondrial membrane permeability (MOMP), which allows the diffusion of apoptotic factors, like cytochrome c, normally confined to the mitochondrial membrane space, into the cytoplasm [19]. The pro-apoptotic proteins BH3-only members of the Bcl-2 family (Bak and Bax) and the anti-apoptotic proteins Bcl-2, Bcl-xL, and Mcl-1 are activated, leading to disruption of mitochondrial outer-membrane permeabilization (MOMP), thereby allowing the diffusion of apoptotic factors such as cytochrome c, which are normally confined to the membrane space, into the cytoplasm [19]. Cytochrome c binds to cytoplasmic Apaf-1(Apoptotic protease activating factor 1) and initiates the formation of a complex called apoptosome, which recruits the promoter pro-caspase-9 to its caspase recruitment domain (CARD) for autoactivation and proteolysis. Caspase 9 started the caspase cascade and activated the chain reaction of caspase-3/−6/−7/−9/−10 to lead to apoptosis [20–22].

Apoptosis is closely linked to genomic damage and tumorigenesis, influenced by interconnected signaling pathways and immune responses [23, 24]. During chemoradiotherapy, apoptosis can stimulate adaptive anti-tumor or antiviral immune responses by activating NF-κB signaling and cGAS/STING pathways [25–27]. Apoptosis triggered by drugs or cytotoxic immune cells also aids in eliminating cancer cells within the TME. However, although antineoplastic drugs exert potent in vitro anti-tumor effects, they often develop in vivo resistance, leading to weakened apoptosis [28].

Necroptosis

Necroptosis plays a role in lysing infected or damaged cells in inflammatory diseases. Various innate immune signaling pathways induce necroptosis, including RIGI-like receptor stimulation, Toll-like receptors (TLR), death receptor pathway, and ZDNA binding protein 1 (ZBP1). Current evidence suggests that RIPK1 kinase activity prompts RIPK3/MLKL-dependent necroptosis. Upon TNF-α binding to TNFR1 at the plasma membrane surface, RIPK1 is recruited intracellularly. When caspase-8 is absent or inhibited, RIPK3 assembles into RIPK1-RIPK3 necroplasts, leading to RIPK3 phosphorylation and MLKL recruitment, resulting in complex IIb formation and necroptosis induction. The phosphorylated RIPK1 and RIPK3 complex constitute the necrosome in this pathway. Auto-phosphorylated RIPK1 exposes the RIP Homotypic Interaction Motif (RHIM), prompting self-oligomerization and RIPK3 recruitment. RIPK3 phosphorylates MLKL, which further phosphorylates and oligomerizes to target the plasma membrane, rupturing it and causing necroptosis [29]. Moreover, ZBP1 participates in necroptosis by sensing intracellular nucleic acid changes. It has been shown that RIPK1 inhibits ZBP1's induction function, preventing RIPK3/MLKL-associated necroptosis. The conserved RHIM of RIPK1 hampers ZBP1 from combining and activating RIPK3 [30]. Interestingly, necroptosis holds both pro-tumor and anti-tumor effects in the TME [31]. On the one hand, low RIPK3 and MLKL expression in early tumor stages predict poor prognosis in various solid tumors, promotes dendritic cell maturation while suppressing CD8⁺ T cell anti-tumor immunity through RIPK1 and NF-κB signaling [31–34]. Notably, necrotic cells exhibit a limited ability to efficiently activate CD8⁺ T cells in vivo, distinguishing necroptosis from necrosis [34]. Additionally, RIPK3 depletion diminishes NKT cell cytotoxicity against tumors. Studies have shown that necroptotic cancer cell vaccines trigger dendritic cell maturation, CD8⁺ T cell cross-priming, and IFN-γ production, enhancing anti-tumor immunity. Hence, inducing tumor cell necroptosis while activating cytotoxic T cells effectively inhibits tumor progression [35]. On the other hand, it has been shown that the protein expression level of MLKL and RIPK3 is increased in the advanced stage of breast cancer (BRCA), promoting tumor metastasis and macrophage-induced T cell suppression [35, 36].

Ferroptosis

Ferroptosis is an iron-dependent programmed cell death sparked by uncontrollable lipid peroxidation and plasma membrane rupture, characterized by the accumulation of reactive oxygen species (ROS) [37, 38]. The regulatory mechanism of ferroptosis includes extrinsic and intrinsic pathways. The extrinsic pathway regulates effector ferroptosis inducers'interaction with different transporters. It has been shown that erastin inhibits the amino acid antiporter system (System xc⁻), hindering cysteine (GSH) metabolism and leading to lipid peroxide accumulation, damaging membrane protein and the membrane itself. Iron transporters, serotransferrin, and lactotransferrin, are activated to promote iron transportation, raising intracellular labile iron levels and triggering ferroptosis [39]. Intrinsic pathways are closely related to GSH and GPX4. GSH can transactivate GPX4 to perform its catalytic activity of reducing lipid peroxidation toxicity and maintaining membrane lipid bilayer homeostasis [40]. GPX4 inhibitors, Squalene synthetase, and HMG-CoA can directly counteract GPX4. Glutathione deficiency triggers GSH deficiency, deactivating GPX4 and causing ferroptosis [41]. Ferroptosis inflicts cellular damage and stimulates inflammation-linked immunosuppression in the tumor microenvironment, supporting tumor growth. Current evidence suggests that mutations in cancer-associated genes (RAS and TP53) and stress response pathways (NFE2L2 signaling) in ferroptosis are closely tied to tumor progression [42]. Ferroptosis also influences T cell anti-tumor effects in the TME. Glutathione peroxidase 4 (GPX4) deficiency in CD8⁺ and CD4⁺ T cells induces ferroptosis, impairing protection against infections and promoting cancer development [43].

Autophagy

It is now understood that autophagy initiation involves the ATG1/ULK1 (UNC-51-like kinase 1) complex and the VPS34/PI3 K C III (phosphatidylinositol 3-kinase III) complex. The ATG1/ULK1 complex, an upstream regulator of the PI3 K C III complex, stimulates the activated ULK1 complex to relocate the PI3 K C III complex from the dynein motor complex to the endoplasmic reticulum (ER) by phosphorylating autophagy/Beclin 1 regulator 1 (AMBRA1), a PI3 K C III complex component. The VPS34 complex in the ER, analogous to the PI3 K C III complex, phosphorylates PI to PI (3,4,5) P3, launching autophagy nucleation [44, 45]. Through acetylation, core elements of the ULK1 complex, the Beclin-1-PIK3 C3 complex, and the LC3 lipidation system foster expansion and closure, forming double-membrane autophagosomes [46–49]. These autophagosomes encase and subsequently ferry intracellular materials to lysosomes, where they undergo degradation [49]. ULK1 also activates the VPS34 complex, phosphorylating Beclin 1 in the VPS34 complex and inducing autophagy [50]. Interestingly, autophagy can be suppressed by mTORC1, which directly phosphorylates ULK1 and ATG13 within the ULK1 complex, as well as ATG14 within the PI3 K C III complex containing ATG14. In an inactive state, mTORC1 sequesters the PI3 K C III complexes containing ULK1 and ATG14. AMP-activated protein kinase (AMPK), a pivotal energy susceptor governing cellular metabolism for energy equilibrium, actively promotes autophagy. Both mTORC1 and AMPK exert control over autophagy by directly phosphorylating ULK1 [51]. In recent years, significant emphasis has been placed on the role of autophagy in cancer, spanning from the biology of tumor cells to clinical trials [52]. It is widely thought that autophagy holds a tumor-suppressive impact during tumorigenesis yet can exhibit a tumor-promoting effect in established tumors [53]. In addition, autophagy is widely believed to autonomously regulate CD8⁺ T cell metabolism, carrying implications for anti-tumor immunity [54].

Pyroptosis

Pyroptosis is a novel PCD process orchestrated by specific inflammatory caspases, including caspase-1/4/5/11 [55, 56]. The pyroptosis process is performed by gasdermin D (GSDMD) and manifests as continuous cell volume expansion until the cytomembrane ruptures, releasing cellular contents that activate a robust inflammatory response [57, 58]. Caspase-1/11/4/5 cleave and unleash the N-terminal domains, which interact with cell membrane lipids and perforate the membrane. This event disrupts cell osmotic pressure and triggers gradual swelling, resulting in cell membrane rupture [59–61]. Indeed, an inflammatory environment is associated with increased probability of tumor onset in cells and tissues subject to chronic exposure [62].

Pyroptosis shares similarities with apoptosis and ferroptosis in terms of critical signaling pathways, morphological transformations, and cell release [62]. In the context of regulating anti-tumor immunity within the tumor microenvironment, pyroptosis yields both pro-tumor and anti-tumor effects. In this regard, pyroptosis yields cytokines to promote inflammation such as IL-18 and IL-1β, which facilitate the infiltration of immune cells into the immunosuppressive tumor microenvironment, indicating its anti-tumor effect [63]. Conversely, pyroptotic cells release proinflammatory cytokines such as IL-1β, IL-6, and IL-18, promoting tumor progression and immune evasion [63]. Therefore, when employing pyroptosis-inducing chemotherapy agents, it is crucial to assess the impact of pyroptosis on tumor cells to select the optimal therapeutic strategy.

Other types of pan-cell death

Parthanatos is triggered by oxidative stress-induced DNA damage and chromatin lysis which is a poly (ADP-ribose) polymerase 1 (PARP1) -dependent form of non-apoptotic cell death [3, 64]. Under pathophysiological conditions, excessive PARP1 activation by DNA damage leads to the accumulation of PAR multimers and nuclear translocation of apoptosis-inducing factor (AIF). Eventually, this process leads to parthanatos [65]. Parthanatos has been found to play an increasingly important role in tumor progression [66, 67]. Therefore, the association between Parthanatos and tumors will become an essential field for PCD treatment of tumors, and the parthanatos-related pathways will also become a research hotspot.

NETosis is a cell death caused by neutrophils releasing their nuclear and mitochondrial DNA and being modified by cytoplasmic and granule proteins to form neutrophil extracellular traps (NETs) [68–71]. Increasing evidence suggests that NETs are associated with cancer metastasis [72–75]. NETosis affects the proliferation of tumor cells through proteases or activation signals, especially BRCA, lung cancer cells and chronic myeloid leukemia, by inducing neutrophils [76, 77].

Alkaliptosis, a PH-dependent form of regulatory cell death, was identified as a compelling novel anticancer strategy in pancreatic cancer (PCA) [78]. Oxeiptosis is a ROS-induced apoptosis-like cell death pathway that is non-caspase-dependent and non-inflammatory and does not depend on caspase [79]. Oxeiptosis was shown to be a novel tumor suppressor, but it is not well studied [80]. Anoikis is a kind of programmed cell death that occurs after detachment of cells from the original extracellular matrix, and disrupts integrin junctions sequentially. It is a critical mechanism that prevents dysplastic cells from growing or attaching to inappropriate substrates. Bcl-2 family proteins are critical factors involved in this process [81]. Disulfidptosis, a cell death triggered by metabolic therapy with glucose transporter (GLUT) inhibitors, has inhibited tumor growth [82]. Cuproptosis is a copper-dependent mode of death that can be distinguished from other cell death [83]. PANoptosis is a distinct pattern of inflammatory PCD regulated by the multifaceted PANoptosome complex [84].

Three signaling pathways and the target therapies

Caspase signaling pathway

The mechanism of caspase signaling pathway regulated PCD in tumor microenvironment

Caspases serve as central players in several critical signaling pathways. Within the context of apoptosis, caspases can be categorized into initiator caspases (− 2, − 8, − 9, − 10) and effector caspases (− 3, − 6, and − 7) [56, 85]. The extrinsic apoptosis pathway is initiated by initiator caspases 8 and 10, activated by the death-inducing signaling complex [86]. Within the intrinsic apoptosis pathway, initiator caspase-9 becomes part of the apoptosome with the assistance of CARD and undergoes proximity-induced autoactivation [87, 88]. RIPK1 stimulates the conversion of inactive pro-caspase 8 into activated caspase 8, triggering extrinsic apoptosis [89, 90]. In cases where caspase 8’s expression is blocked or inhibited in tumors, RIPK1 mediated the RIPK3/MLKL signaling in an active state, forming necroptotic bodies that ultimately lead to necroptosis [91].

The inflammatory caspase family includes caspases 1, 4, 5, 11, and 12, which are closely associated with pyroptosis [92]. In the realm of cancer, pyroptosis pathways linked to caspases can be categorized into canonical and non-canonical pathways. For the former, inactive pro-caspase 1 is activated by various inflammasomes (NLRP3, AIM2, NLRP1, PYRIN, NLRC4) [93–95]. Mechanistically, caspase 1 and caspase 11 cleave the linker region between the N- and C-terminal domains of GSDMD, releasing its N-terminal (p30) and C-terminal (p20) fragments, sustaining pyroptosis [59, 60, 95]. In the nonclassical pathway, lipopolysaccharide (LPS) activates Caspases 4/5/11 to cleave GSDMD to induce pyroptosis. Beyond these two pathways, caspase 3 cleaves GSDME, releasing its N-terminal fragment and triggering pore formation, which is crucial for pyroptosis [96, 97]. PD-L1 induces pyroptosis in BRCA through the caspase 8/GSDMC signaling pathway, shifting TNF-α or chemotherapeutic-induced apoptosis towards pyroptosis.

It is now understood that autophagy and apoptosis intricately regulate each other, largely through interactions between autophagy-related proteins (ATGs) and caspases. Caspase 1 fosters cytoprotective autophagy and eliminates damaged mitochondria following redox changes by activating LC3 and Beclin-1 [98]. Generally, caspase 2 exerts a negative regulatory effect on the autophagic process. This phenomenon was observed in model mouse embryonic fibroblasts (MEFs) and young adult mouse cortical neurons, where early-stage caspase 2 knockdown prompted mobilization of cytoprotective autophagy [99, 100]. In the absence of DISC, pro-caspase 8 can be activated in interaction with autophagosomes, which induces the occurrence of apoptosis [101]. Besides, Caspase 3/9 cleaves and partially inactivates ATG5 and Beclin-1 through interactions with autophagy-related proteins in various tumor cells.In murine bone marrow cells undergoing extended starvation, caspase 3 cleaves Beclin-1 to produce c-fragments that reduce autophagic fluxes enhancing apoptosis.

Blocking caspase activity does not stop parthanatos because caspase activation happens later in the process [102] (Fig. 1).

Fig. 1.

Caspase signaling can induce endogenous apoptosis through caspase 8,10,7,3 and exogenous apoptosis through the cascade of caspase3,6,7,9,10. Inhibition of Caspase 8 can induce necroptosis. Pyroptosis and autophagy can be induced through caspase 1. Caspase3,6,9 -mediated Beclin-1 degradation and Caspase 2 both inhibit autophagy

The targeted therapy of the caspase signaling pathway related to PCD

Targeting at caspases 3

Targeting genes with low expression in tumors, such as ULK1 in BRCA tissues, offers a viable strategy for cancer treatment. The ULK1 activator LYN1604 prompts autophagy by interacting with activator of RAD21 Recombinant Protein, caspase 3, and transcription 3 (ATF3) [103]. Besides, AZD5991 displays stable anti-tumor activity in hematologic malignancies such as multiple myeloma and AML. AZD5991 binds to Mcl-1, triggering the Bak-dependent intrinsic apoptotic pathway and activating caspase 3, ultimately inducing apoptosis [104]. Following AZD5991 monotherapy, about three-fifths of the patients experienced symptomatic relief. In addition, all patients were associated with varying degrees of elevated troponin levels [104]. Moreover, the 3',5'-degraded chalcone (C10) induces pyroptosis through the PKCδ/JNK pathway by activating caspase 3, leading to PARP and GSDME-dependent pyroptosis [105]. Interestingly, a therapeutic approach against metastatic triple-negative breast cancer (mTNBC) involving the conversion of a peptide-doxorubicin conjugate (PDC) mediated by tumor caspase 3 utilizes two separate caspase 3-cleaving PDCs: EMC-KGDEVD-DOX (MPD1) and RGDEVD-DOX (TPD1). These PDCs were conceived to target either tumor cells or vasculature associated with the aggressive advancement of mTNBC and exhibited notable effectiveness in inducing tumor cell death [106].

It is widely acknowledged that Ginsenosides, a traditional Chinese medicine, exhibits diverse pharmacological activities. The introduction of the piperazine group into ginsenoside upregulates Cl-Caspase 3, Cl-Caspase 9 and Cl-PARP expression was found to result in cellular apoptosis [107]. In patients with advanced hepatocellular carcinoma (HCC), Ginsenosides combined with catheter-based arterial chemoembolization (TACE) resulted in an overall survival of 13.2 months compared to 3.1 months with TACE alone [108]. Dihydroartemisinin (DHA), an artemisinin derivative found in artemisia annua, a traditional Chinese medicine, inhibits the proliferation and tumorigenicity of BRCA cells by activating caspase 3, upregulating the expression level of gasdermin E and melanoma 2, and inducing pyroptosis [109]. Adverse reactions such as anemia, nausea, and vomiting are 60% likely to occur during the treatment of solid tumors with Dihydroartemisinin. Notably, Dihydroartemisinin has a one in four success rate in controlling progression of solid tumors [110].

Besides, current evidence suggests that cisplatin and paclitaxel both induce significant apoptosis in lung cancer cells. However, cisplatin-induced caspase 3/GSDME-dependent pyroptosis yields a more sustained response than paclitaxel-induced pyroptosis, suggesting that cisplatin may offer extra benefits in treating lung cancer with GSDME overexpression [111–113]. In the phase 2 NeoSAC trial, the 36-month disease-free survival rate of apatinib and sindilizumab in combination with carboplatin and albumin-conjugated paclitaxel chemotherapy for early-stage triple-negative breast cancer was 94.1%, with a median follow-up time of 39.1 months. This demonstrates that this therapy shows promising prospects and a manageable safety profile for early TNBC [114]. Alantolactone (ATL), a terpenoid derived from the traditional Chinese herb Inula helenium, was documented to demonstrate remarkable anti-inflammatory, antibacterial, and anti-tumor properties. It was demonstrated that ATL triggers ROS-mediated activation of mitochondria-dependent caspases 3, 7, and 9, inducing apoptosis and GSDME-dependent pyroptosis [115]. BI 905711, a novel quadrivalent bispecific antibody targeting TRAILR2 and Cadherin-17 (CDH17), exhibits anti-tumor efficacy in CDH17-positive colorectal cancer (CRC) xenografts, activating caspases 3, 7, and 8, both in tumor tissue and plasma [116].

Green tea extract Polyphenon E (Poly E) halts the G0 and G1 checkpoint in PNT1a cells, activating caspases 3, 7 as well as 9. After autophagy, Polyphenon E^® prompts anoikis, a form of cell death [117]. In a phase II trial of Poly E for the treatment of patients at high risk for recurrent colorectal cancer, Poly E did not significantly reduce the number of abnormal crypt foci of the rectum, despite demonstrating good tolerability [118]. Checkpoint nano-proteolysis-targeting chimeras (nano-PROTACs) incorporate photosensitizer protoporphyrin IX (PpIX) and SHP2-targeting PROTAC peptide (aPRO), joined by caspase 3 cleavable fragments. Moreover, light-activated aPRO triggers SHP2 degradation via the ubiquitin–proteasome system due to increased caspase 3 expression in tumor cells post-light exposure. Persistent SHP2 deletion hinders immunosuppressive checkpoint signaling (CD47/SIRPα and PD-1/PD-L1), reactivating anti-tumor macrophages and T cells. This approach can work synergistically with immunogenic phototherapy, augmenting anti-tumor immune responses [119]. Dihydrotanshinone I (DHTS), a potent tanshinone, has been reported to exhibit strong anti-colon cancer activity. DHTS-induced cytotoxicity relies on ROS and induces activation of caspases 3, 2, and 9, promoting apoptosis and autophagy through the mitochondrial pathway in COAD cells. Accordingly, DHTS holds promise as a lead compound for anti-tumor drug development or as an adjuvant for COAD treatment [120].

Targeting caspase 3 holds significant potential in inducing the death of various tumor cells and effectively delaying tumor progression. This has positioned caspase 3 as a crucial target within the caspase pathway, with drugs directed toward it showing promising therapeutic effects against tumors.

Targeting at caspases 8, 9 and 10

It has been shown that the activation of caspase 8 by progesterone leads to apoptosis of cancer cells. By combining these two effects, the intrinsic and extrinsic apoptotic pathways can be simultaneously activated, effectively inhibiting tumorigenesis [121, 122]. A study revealed that ABBV-621, a TRAIL agonist, enhances caspase 8 aggregation and the formation of death signaling complexes, leading to the death of solid cancer cells at subnanomolar concentrations [123]. Besides, scutellarein (SCU), derived from Scutellaria baicalensis, activates caspases 8 and 3 by regulating Fas and FasL to overexpressed, inducing extrinsic apoptosis in Hep3B cells and exerting an anti-tumor effect [124]. Imipramine, an antidepressant, triggers extrinsic apoptosis in glioblastoma cells by upregulating FasL and activating caspases 8 and 3 [125]. CPT211, a novel camptothecin derivative, effectively suppresses BRCA cell proliferation by activating Fas/FADD/caspase 8 signaling and eventually inducing apoptosis [126]. Propofol triggers apoptosis in Leydig tumor cells by activating caspase 8, caspase 3, and caspase 9 through Akt pathway inhibition [127]. However, a multicenter randomized trial demonstrated that propofol management of elderly patients after major cancer surgery did not increase their overall or cancer-specific survival [128].

Targeting at caspases 1, 4, 5 and 11

Long non-coding RNA GAS5 enhance the assembly and expression of inflammasome by hinder the bioactivity of glucocorticoid receptors, thus increasing IL-1β and IL-18 inflammatory mediators through caspase 1, inducing pyroptosis [129]. It has been shown that aponin polyphyllin VI (PPVI) impedes non-small cell lung cancer progression by activating caspase 1 through the ROS/NF-κB/NLRP3/GSDMD signaling axis, prompting pyroptosis in lung cancer cells [130]. Simvastatin has been reported to mitigate NSCLC tumor growth by driving pyroptosis via Caspase 1, NLRP3, IL-18 and IL-1β activation [131]. However, a randomized phase II study showed no significant difference in survival between afatinib alone and afatinib combined with simvastatin in patients with non-small cell lung cancer [132].

Targeting at other caspases

Although additional exploration is needed for drugs that target different caspases, specific targeted drugs for certain caspases are already available. For instance, during the treatment of leukemia using the antibody conjugate gemtuzumab ozogamicin (GO; Mylotarg^®), Caspase 2 can facilitate apoptotic signal transmission mediated by the cleavage triggered by GO in human AML cells, subsequently initiating the apoptotic process. This finding could potentially lay the groundwork for future leukemia treatments [133]. In summary, the caspase signaling pathway plays a role in various forms of cell death, including apoptosis, pyroptosis, autophagy, and necroptosis. Nevertheless, the involvement of the caspase pathway in cell death processes such as Alkaliptosis and Oxeiptosis remains unclear. Notably, drugs targeting the caspase signaling pathway are well-developed, with a primary focus on caspases 3, 1, and 8. However, the exploration of targeted drugs for caspases 2, 12, and 14 is still in its preliminary stages. Caspase 2, known for cleaving specific substrates, acts as a tumor suppressor in adenocarcinoma, melanoma, and COAD [134]. The development of targeted drugs to enhance the caspase signaling pathway holds promise for introducing novel chemotherapy strategies for the prevention and treatment of tumors (Table 1).

Table 1.

Compounds targeting of the caspase signaling pathway related to PCD in cancer: ↓ represents decrease or inhibition. ↑ represents increase or activation

Compound name	Target	Current stage	Mechanism in PCD	Tumor type	Refs
LYN1604	ULKL, ATF3, RAD21 and caspase 3↑	Preclinical study	Induce autophagy	Breast cancer	[103]
AZD5991	Mcl-1↓ Bak and caspase 3↑	Phase 1/2 clinical trial	Induce apoptosis	Multiple myeloma, acute myeloid leukemia	[104]
3',5'-degraded chalcone (C10)	PKCδ/JNK, caspase 3↑	Preclinical study	Induce pyroptosis	Prostate cancer	[105]
RGDEVD-DOX (TPD1)	Caspase 3↑	Preclinical study	Induce apoptosis	Triple-negative breast cancer	[106]
EMC-KGDEVD-DOX (MPD1)	Caspase 3↑	Preclinical study	Induce apoptosis	Triple-negative breast cancer	[106]
Ginsenosides	Cl-Caspase 3, Cl-Caspase 9 and Cl-PARP↑	Phase 2/3 clinical trial	Induce apoptosis	Hepatocellular carcinoma	[107, 108]
Dihydroartemisinin	Caspase 3↑	Phase 1 clinical trial	Induce pyroptosis	Breast cancer	[110]
Paclitaxel	Caspase 3, caspase 8↑	Phase 2/3 clinical trial	Induce apoptosis	Lung cancer	[111–113]
Cisplatin	Caspase 3/GSDME↑	Phase 2/3 clinical trial	Induce apoptosis and pyroptosis	Lung cancer	clinicaltrials.gov
Alantolactone (ATL)	ROS, caspase 3, caspase 7 and caspase 9↑	Preclinical study	Induce apoptosis and pyroptosis	Breast cancer	[115]
BI 905711	Caspase 3, caspase 7, caspase 8↑	Phase 1 clinical trial	Induce apoptosis	Colorectal cancer	clinicaltrials.gov
Green tea extract Polyphenon E	Caspase 3, caspase 7 and caspase 9, cleaved poly (ADP-ribose) polymerase 1↑	Phase 1/2 clinical trial	Induce autophagy and anoikis	colorectal cancer	[117], clinicaltrials.gov
Nano-PROTACs	Caspase 3↑SHP2↓	Preclinical study	Induce apoptosis	Breast cancer	[119]
Dihydrotanshinone I (DHTS)	ROS, caspase 3, caspase 2 and caspase 9↑	Preclinical study	Induce apoptosis and autophagy	Colon cancer	[120]
Progesterone	Caspase 8↑	Phase 1/2 clinical trial	Induce apoptosis	Endometrial cancer	[121, 122]
ABBV-621	TRAIL, caspase 8↑	Preclinical study	Induce apoptosis	Colorectal cancer	[123]
Scutellarein (SCU)	Fas/FasL, caspase 8 and caspase 3↑	Preclinical study	Induce apoptosis	Hepatocellular carcinoma	[124]
Imipramine	FasL, caspase 8 and caspase 3↑	Preclinical study	Induce apoptosis	Glioblastoma	[125]
CPT211	Fas/FADD/caspase 8↑	Preclinical study	Induce apoptosis	Breast cancer	[126]
Propofol	Akt, caspase 8, caspase 3, and caspase 9↑	Phase 1 clinical trial	Induce apoptosis	Leydig tumor	clinicaltrials.gov, [127]
lncRNA GAS5	caspase 1, IL-1β and IL18↑	Preclinical study	Induce pyroptosis	Ovarian cancer	[129]
Aponin polyphyllin VI (PPVI)	ROS/NF-κB/NLRP3/GSDMD and caspase 1↑	Preclinical study	Induce pyroptosis	Lung cancer	[130]
Simvastatin	NLRP3, Caspase 1, IL-1β, and IL-18↑	Phase 2 clinical trial	Induce pyroptosis	Non-small cell lung cancer	[131], clinicaltrials.gov
Mylotarg®	Cleaved caspase 2↑	Post-Marketing Surveillance	Induce apoptosis	Leukemia	[133]

RIPK1/RIPK3/MLKL signaling pathway

The mechanism of RIPK1/RIPK3/MLKL signaling pathway regulated PCD in tumor microenvironment

RIPK1 is a TNF-induced pleiotropic adaptor protein, and phosphorylated RIPK1 contributes to RIPK1 self-oligomerization and RIPK3 recruitment. In the context of the cancer cell microenvironment, complex I could lead to necroptosis and apoptosis under the regulation of RIPK1. Consequently, it is feasible to prove the occurrence of complex I disruption during apoptosis by monitoring RIPK1 [135]. Following TNF-α binding to TNFR1 on the plasma membrane, RIPK1 begins to function in cytoplasm as a receptor, forming complex I [136]. Prolonged cancer cell survival is facilitated by the polyubiquitinated RIPK1 Lys63 domain, promoting the recruitment of the IĸB kinase (IKK) complex and the growth factor β-activated kinase growth factor β-activated kinase (TAK) complex, ultimately activating NF-κB through the IKKα/IKKβ comple [137]. Additionally, TANK-binding kinase 1 (TBK1), IKKε, and IKKα/IKKβ contribute to phosphorylated RIPK1’s dephosphorylation, impeding its translocation to complex II and consequently preventing RIPK1-dependent cell death which is non-NF-κB-dependent [138]. In the context of AML driver mutations, RIPK3 knockout significantly accelerates leukemia development in mice which is transplanted with bone marrow cells. This may be attributed due to reduced MLKL protein expression downstream of RIPK3, potentially resulting in the inhibition of necroptosis caused by the failure to form inflammasomes [139].

TGF-β-activated kinase 1 (TAK1) kinase functions as an inhibitory phosphatase. An increasing body of evidence suggests that RIPK1 can be suppressed by TAK1-mediated inhibitory phosphorylation and TAK1-activated kinases (MK2 and IKKs) [140–142]. Cells may directly trigger RIPK1-dependent apoptosis (RDA) in response to TNFα stimulation overcoming the inhibitory effect of TAK1 on RIPK1 kinase. [143–145]. Functioning as a crucial upstream regulator, RIPK1 governs various downstream signaling pathways of TNFR1 [136]. Upon TNF-α be joint to TNFR1 on the, protein molecules like RIPK1, LUBA, cIAPs, TRAF, and TRADD are recruited to form complex I. RIPK1 initiates apoptosis by associating with caspase 8-adaptor protein FADD, with the apoptotic process sustained through mitochondrial damage induction and cleavage of downstream caspase proteins like caspase 3 [141]. Current evidence suggests that TBK1, an endogenous inhibitor, mitigates RIPK1 expression in inflammatory apoptosis. Inhibition of TBK1 activates RIPK1, triggering apoptosis via direct phosphorylation [146]. Mutants of TGF-β-activated kinase 1 binding protein 2 (TAB2) impede the activation of RIPK1 kinase and the creation of the apoptotic complex (RIPK1, FADD, and caspase 8) through the TAK1-dependent pathway [147].

In the context of ferroptosis, Ferrostatin-1(Inhibitor of ferroptosis) inhibits the elevation of kidney RIPK3 and MLKL mRNA expression, suggesting that ferroptosis might augment susceptibility to necroptosis by amplifying RIPK3 and MLKL levels. In human fibroblasts, RIPK1 mutants D325 V or D325H mitigate RIPK1 expression, rendering them resistant to ROS generation and ferroptosis prevention [148]. A dual-active compound known as Nec-1f was reported to mitigate RIPK1 kinase activity and halt downstream MLKL phosphorylation, ultimately inhibiting ferroptosis progression [149].

During autophagy, RIPK1 is recruited to the early autophagosomes, leading to the activation of caspase 8 [150]. ULK1, an autophagy-initiating kinase, phosphorylates RIPK1 in a manner that hinders complex IIb and necrosome assembly, thus preventing cell death [151]. Under glucose starvation, AMPK induces autophagy by directly activating ULK1 through Ser 317 and Ser 777 phosphorylation [152]. Recent studies have identified RIPK3 as another member of the AMPK-activating kinase family, suggesting that RIPK3-dependent AMPK activation represents a reciprocal interaction between necroptosis and autophagy [152]. Moreover, RIPK3 undergoes selective autophagic degradation via tripartite motif containing 11 (TRIM11)-mediated ubiquitination, suppressing necroptosis [153]. RIPK1 upregulation can reportedly induce autophagy due to Beclin-1 activation upon dissociation from BCL2L11, itself phosphorylated by MAPK8/9 [154].

Neutrophil extracellular traps (NETs) induced by programmed necrosis-associated stimuli constitute a network of extracellular DNA and microbial-killing proteins that trap and destroy invading pathogens, which is dependent on the production of ROS, potentially linking the mechanisms of NETosis and RIPK1 at the molecular level [164]. Notably, activated MLKL may also translocate to various subcellular compartments, including mitochondria, exosomes, and lysosomes, where it contributes to diverse functions like aerobic respiration, ROS generation, extracellular vesicle generation, cytokine release, endocytosis, and autophagy [155–158]. Current evidence suggests the E3 subunit of the pyruvate dehydrogenase complex is directly phosphorylated and activated by RIPK3, promoting aerobic respiration and mitochondrial ROS production [155].

In the context of cancer, RIPK1-mediated mitophagy induction could prove effective for eliminating ECM-shed cancer cells. RIPK1 activation during ECM detachment stimulates mitophagy through a PGAM5-dependent mitochondrial phosphatase mechanism. Moreover, mitophagy-induced NADPH reduction in isolated-cell mitochondria leads to anoikis and nonapoptotic cell death due to increased ROS levels. Moreover, epigenetic changes repress RIPK1 to inhibit anoikis and enhance tumor cell metastatic potential, further contributing to tumorigenesis in head and neck squamous cell carcinoma (HNSCC) [159]. The antagonism of RIPK1/PGAM5 accentuates tumor formation in vivo [160]. However, it has been established that parthanatos does not lead to RIPK1 activation [1]. However, adenosine deaminase acting on RNA 1 (ADAR1) inhibits PANoptosis by interacting with the Zα2 domain of ZBP1, limiting the interaction between ZBP1 and RIPK3, promoting tumorigenesis [161] (Fig. 2).

Fig. 2.

The RIPK1/RIPK3/MLKL signaling pathway is involved in tumor cells through Complex I (TRADD, TRAF, cIAPs, LUBA and RIPK1) and Complex IIa(TRADD1, RIPK1,FADD, and pro-caspase 8) induces apoptosis, necroptosis and prevents PANoptosis through Complex IIb (RIPK1, RIPK3, MLKL). Phosphorylated MLKL can promote mitochondrial ROS generation, which induces NETosis and ferroptosis. Ubiquitinated RIPK3 can inhibit this process while inducing autophagy. Phosphorylated RIPK1 can also induce autophagy, which can promote the reduction of ROS production and NADPH production, thereby inducing anoikis

The target therapy of RIPK1/RIPK3/MLKL signaling related to PCD

Targeting at RIPK1

Ophiopogonin D’ (OPD’), derived from Ophiopon japonicas, triggers necroptosis in androgen-dependent PCA by enhancing FasL-dependent RIPK1 protein expression, offering therapeutic potential for tumors [162]. Moreover, Inula viscosa (L.) Aiton ethanolic extract hampers the growth of human AGS and human non-small-cell lung cancer (A549) cell lines. Studies have revealed that RIPK1 role in necroptosis pathway is essential in the toxicity induced by Enterobacteriaceae extracts [163]. NP-ALT, a therapeutic liposome peptide was shown to induce ROS and RIPK1-dependent necroptosis in xenograft models and endocrine therapy-resistant BRCA cells, which blocked tyrosine phosphorylation of p27kip1 (CDKN1B) as well as activation of CDK4 and CDK2 [164]. Resistance to apoptosis induction is closely linked to tumor drug resistance. RETRA, a small molecule that reactivates p53-regulated gene expression in mutant (MT) p53 cells, exerts its effect by affecting the phosphorylation level of the RIPK1/RIPK3/MLKL pathway in cervical cancer (CESC) cells. This leads to cell cycle arrest in the S phase, elevated p21 expression, reduced cyclin-d3 levels, mitochondrial hyperpolarization, ROS production, and ultimately selective necroptosis induction in the absence of p53. Co-administration of RETRA and Necrostatin-1 was found to reverse the impact of RETRA and prevent CESC cell death. RIPK1 inhibition with Nec-1 increases the survival of adrenocortical cancer (ACC) and anaplastic thyroid cancer (ATC) cells during radiotherapy, suggesting RIPK1's vital role in procedural necrosis induction [165]. The HSP90 inhibitor, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) treatment reduced the RIPK1 [166, 167]. In a phase I dose-escalation study, the combination of 17-DMAG and trastuzumab was shown to be effective in improving the efficacy of the treatment in ovarian cancer, and the appropriate weekly dose of 17-DMAG was found to be 80 mg/m [168]. As a robust third-generation platinum drug, lobaplatin has fewer side reactions and less cross-resistance to platinum. Lobaplatin promoted the proteasomal degradation of cell inhibitor of apoptosis protein-1/2 (cIAP1/2). This synergistic pyroptosis effect was inhibited by the cleavage of ROS and caspase 3 by inhibition of Ripoptosome (RIPK1/Caspase 8/FADD) with the cIAP1/2 antagonist birinapant [169]. The use of birinapant in the treatment of ovarian cancer revealed that although birinapant theoretically has a clear in vivo target inhibition, however, there was no significant efficacy in the ovarian cancer patients in the study [170]. In the context of cancer, as cancer cells resist anoikis, signaling via adhesion-related molecules is indispensable for cancer growth and metastasis. Focal adhesion kinase (FAK) is activated in focal adhesion, conveying survival signals dependent anchorage. In certain malignancies or malignant cell subsets, reduced FAK levels bolster cIAP2:RIPK1 complex formation, leading to decreased TPL2 expression levels and impeding tumor progression. When FAK inhibitors VS-4718, PF-573228, defactinib, GSK2256098 and IN10018 acted on cancer, cIAP2:RIPK1 complex formation was elevated, decreasing TPL2 expression level to induce anoikis and suppressed the tumor progression [171–174].

Targeting at RIPK3

It has been documented that resibufagenin induces programmed necrosis of CRC cells by activating glutamate dehydrogenase (GLUDL), glutamine synthetase (GLUL), and glycogen phosphorylase (PYGL), mediated by RIPK3 and consequently inhibiting tumor growth [175]. In recent years, curcumin has emerged as a potent RIPK3 inhibitor by upregulating necroptosis blockade, inhibiting the phosphorylation of MLKL and RIPK3 while sparing RIPK1. In PCA PC-3 AcT cells, proteins like p-RIP3 and p-MLKL contribute to necroptosis and apoptosis of PCA, eventually diminishing the viability of PC-3 AcT cells [176, 177]. There was no difference in the mean number or size of intestinal adenomas in the curcumin-treated group compared with the placebo group, but there were fewer adverse effects [178]. Neoalbuminol (NA), extracted from the fungus Albatrellus confluens, triggers autocrine TNFα production by modulating the RIPK/NF-κB signaling pathway and instigating ROS production dependent on RIPK3 in tumor cells, initiating necroptosis [215]. Studies have shown that the ubiquitin–proteasome system owns the possibility to regulate RIPK3-dependent necroptosis, and proteasome inhibitors acted as anticancer drugs to target the necroptosis pathway. It is widely thought that the ubiquitin–proteasome system might regulate RIPK3-dependent necroptosis, and proteasome inhibitors could serve as anticancer agents targeting the necroptosis pathway. The proteasome inhibitors MG132 and bortezomib can evoke RIPK3 and MLKL-dependent necroptosis human leukemia cells [179]. Polyinosinic: polycytidylic acid (PolyI: C), a viral dsRNA analog, has been convinced to trigger programmed necrosis in CESC cells, contingent upon RIPK3 expression [180]. Moreover, B-Raf(V600E) inhibitors like dabrafenib, essential anticancer agents for metastatic melanoma, may competitively bind with ATP of the RIPK3 enzyme to inhibit its activity in vitro [181, 182]. Polyphyllin D, a significant component of traditional herbal medicine, elicits RIPK3 expression and instigates various forms of programmed cell necrosis, exerting anti-tumor effects on neuroblastoma in vitro and in vivo [183]. Chloroquine (CQ) substantially enhances receptor-interacting RIPK3 expression involving CQ-related autophagy [184, 185].

Targeting at MLKL

MLKL, a pivotal regulatory gene in necrosis, can be significantly inhibited by gene deletion or low-dose chemical inhibitors, suppressing tumor recurrence and even reducing tumorigenicity both in vivo and in vitro. Staurosporine (STS), an alkaloid isolated from Streptomyces staurosporeus, induces RIPK1/MLKL-dependent necroptosis in leukemic cells when caspase activation is compromised [186, 187]. It has been established that IMB5036, a novel pyridazinone compound with potent cytotoxicity, exerts a lethal effect on pancreatic ductal adenocarcinoma (PDAC) cells. This compound triggers the translocation of MLKL and p-MLKL from the cytoplasm to the cell membrane, upregulating p-RIP1, p-RIP3, and p-MLKL, and partially inducing apoptosis and pyroptosis. Ultimately, IMB5036 impedes pancreatic transplantation tumor growth, augments the tumor necrosis area, and restrains human PDAC cell proliferation [188]. Sorafenib, a multikinase inhibitor and first-line treatment for advanced HCC, can be combined with the HSP90 inhibitor ganetespib. Notably, LAMP2 aids in MLKL degradation via the chaperone-mediated autophagy pathway. The synergistic use of ganetespib and sorafenib potentially engenders an anti-angiogenic impact by triggering necroptosis, suppressing macroautophagy, and offering a novel approach for HCC treatment [189–191]. In addition, ganetespib did not improve survival in patients with advanced lung adenocarcinoma treated with salvage therapy, along with adverse effects such as neutropenia time [192]. Clinical trials involving HSP90 inhibitors such as 17 AAG and Alvespimycin (IPI-504, MAG, and 17-D) have been conducted with cancer patients, potentially and indirectly inhibiting necroptosis [193–195].

Targeting at RIPK1 and RIPK3

Necrostatin-1 (NEC-1) functions as a traditional inducer of necroptosis, effectively blocking the interaction between RIPK1 and RIPK3. It inhibits programmed necrosis, providing a targeted approach for almost all common cancer types, particularly CRC and hematopoietic system malignancies. Notably, NEC-1 selectively suppresses programmed necrosis without affecting normal cellular functions or apoptosis [196]. Shikonin, a naturally occurring naphthoquinone present in traditional Chinese medicine, circumvents resistance regulated by drug transporters or anti-apoptotic Bcl-2 proteins by triggering necroptosis in human leukemia cell lines [197–199]. In PCA, shikonin induces necroptosis by modulating the expression of RIPK1 and RIPK3 [200]. Additionally, a study revealed that shikonin could stimulate mitochondrial ROS synthesis in TNBC cells, thereby initiating necroptosis or apoptotic elimination of BRCA cells [201]. Shikonin was also found to effectively inhibit primary tumor growth in an in vivo osteosarcoma model, primarily through RIPK1 and RIPK3-dependent necroptosis, and notably reduce lung metastasis [202]. Shikonin triggers RIPK1 and RIPK3-dependent necroptosis along with augmented autophagy, which, in turn, potentiates the upregulation of damage-associated molecular patterns (DAMPs). Importantly, the combined application of shikonin and chloroquine could potentially boost immunogenicity and vaccine efficacy, offering a novel strategy for cancer vaccine development [203]. Chelerythrine (CHE) orchestrates the formation of the RIPK1-RIPK3-Drp1 complex via mitochondrial ROS (mtROS), facilitating the mitochondrial translocation of Drp1 and promoting necroptosis [204].

Targeting at RIPK1, RIPK3 and MLKL

In the context of colorectal cancer, elaidic acid, a primary active component of the Active Fraction of Polyrhachis vicina Roger (AFPR), triggers necroptosis by activating ERK/RIPK1/RIPK3/MLKL. This compound offers a promising alternative for CRC [205]. Paeoniflorigenone (Paeo), an active substance extracted from the root bark of P. suffruticosa, significantly inhibits the migration and invasion of human YD-10B HNSCC cells. Paeo exerts this anti-metastatic effect by suppressing programmed cell apoptosis through the dephosphorylation of key programmed apoptosis proteins, including RIPK1, RIPK3, and MLKL [206]. Paeo's anti-metastatic action is attributed to the dephosphorylation of key programmed apoptosis proteins, namely RIPK1, RIPK3, and MLKL, leading to a substantial reduction in migration and invasion of human YD-10B HNSCC cells [206].

Targeting at producing ROS

The mitochondrial complex I inhibitor arctigenin induces necroptosis in PCA cells by inducing ROS-mediated mitochondrial damage and elevating CCN1 levels. This cascade eventually culminates in heightened levels of p-RIP3 and p-MLKL, contributing to the reduced viability of PCA cells [207]. Doramectin (DRM), an avermectin drug, exhibits anti-tumor effects by inducing ROS overproduction in glioma cells. This surge in ROS triggers necroptosis through the RIPK1/RIPK3/MLKL pathway, with mitochondria bridging these two pathways. Moreover, DRM holds huge promise as a potential therapeutic agent for inducing apoptosis and necroptosis in cancer treatment [208].

In conclusion, we summarized the mechanism of RIPK1/RIPK3/MLKL signaling in necroptosis, apoptosis, PANoptosis, autophagy, NETosis, and anoikis. In the occurrence and progression of tumors, there will be various PCD combined effects and unknown changes. Therefore, we provide a dynamic and holistic idea for studying the mechanism of tumor development and treatment. In addition, we summarized the chemotherapeutic agents targeting the RIPK1/RIPK3/MLKL signaling pathway, but we found that studies on drug combinations were imperfect. Therefore, we provide a reference for the combination of drugs targeting the RIPK1/RIPK3/MLKL signaling pathway to treat tumors (Table 2).

Table 2.

Compounds targeting of the RIPK1/RIPK/MLKL signaling pathway related to PCD in cancer: ↓ represents decrease or inhibition. ↑ represents increase or activation

Compound name	Target	Current stage	Mechanism in PCD	Tumor type	Refs
Ophiopogonin D’ (OPD’)	FasL and RIPK1↑	Preclinical study	Induce necroptosis	prostate cancer	[162]
Inula viscosa (L.) Aiton Ethanolic Extract	RIPK1 ↑	Preclinical study	Induce necroptosis	Lung cancer	[163]
NP-ALT	CDK4, CDK2, ROS and RIPK1↑	Preclinical study	Induce necroptosis	Breast cancer	[164]
RETRA	RIPK1/RIPK3/MLKL, p21, ROS↑cyclin-d3, ROS↓	Preclinical study	Induce necroptosis	Cervical cancer	[165]
17-DMAG	Hsp90, RIPK1↓	Phase 1 clinical trial	Inhibit necroptosis, induce apoptosis	ovarian cancer	[166, 167], clinicaltrials.gov
Birinapant	cIAP1/2, RIPK1/caspase 8/FADD, ROS, caspase 3↓	Phase 1/2 clinical trial	Inhibit pyroptosis	Solid tumor/ovarian cancer	[169, 170]
Defactinib	FAK↓ cIAP2:RIPK1↑	Phase 1/2 clinical trial	Induce anoikis	Pleural mesothelioma/Non-small cell lung cancer	clinicaltrials.gov
IN10018	FAK↓ cIAP2:RIPK1↑	Phase 1/2 clinical trial	Induce anoikis	Solid tumor	clinicaltrials.gov
Resibufagenin	RIPK3↑	Preclinical study	Induce necroptosis	Colorectal cancer	[175]
Curcumin	P-RIPK3 and p-MLKL↑	Phase 1/2 clinical trial	Inhibit necroptosis	Intestinal adenoma	[178]
Neoalbuminol (NA)	RIPK/NF-Κb, ROS↑	Preclinical study	Induce necroptosis and apoptosis	Cervical cancer, colorectal cancer, osteosarcoma	[215]
MG132	RIPK3 and MLKL↑	Preclinical study	Induce necroptosis	Leukemia	[179]
Bortezomib	RIPK3 and MLKL↑	Phase 1/2/3 clinical trial	Induce necroptosis	Leukemia/Refractory prostate cancer	clinicaltrials.gov, [179]
Polyinosinic: polycytidylic acid	RIPK3↑	Preclinical study	Induce necroptosis	Cervical cancer	[180]
Dabrafenib	RIPK3↓	Phase 1/2/3 clinical trial	Inhibit necroptosis	Metastatic melanoma	[181, 182]
Polyphyllin D	RIPK3↑	Preclinical study	Induce necroptosis	Neuroblastoma	[183]
Chloroquine	RIPK3↑	Phase ½ clinical trial	Induce autophagy	Colon cancer/Small cell lung cancer	[184, 185], clinicaltrials.gov
Staurosporine	MLKL↑	Preclinical study	Induce necroptosis	Leukemia	[186, 187]
IMB5036	p-RIPK1, p-RIPK3 and p-MLKL↑	Preclinical study	Induce apoptosis and pyroptosis	Pancreatic ductal adenocarcinoma	[188]
17 AAG	MLKL↓	Phase 1/2 clinical trial	Induce necroptosis	Colon cancer	[193–195]
IPI-504	MLKL↓	Phase 1/2 clinical trial	Induce necroptosis	Breast cancer	[193–195]
Ganetespib	MLKL↓	Phase 1/2/3 clinical trial	Induce necroptosis, inhibit autophagy	Hepatocellular carcinoma/	[193–195]
Necrostatin-1	RIPK1 and RIPK3↓	Preclinical study	Inhibit necroptosis	Colorectal cancer	[196]
Shikonin	RIPK1 and RIPK3↓	Preclinical study	Induce necroptosis and autophagy	Osteosarcoma, leukemia, pancreatic cancer and breast cancer	[197–199]
Chelerythrine	RIPK1-RIPK3-Drp1 and mtROS ↑	Preclinical study	Induce necroptosis	Glioma	[204]
Elaidic acid	ERK/RIPK1/RIPK3/MLKL↑	Preclinical study	Induce necroptosis	Colorectal cancer	[205]
Paeoniflorigenone (Paeo)	p-RIPK1, p-RIPK3 and p-MLKL↓	Preclinical study	Inhibit apoptosis	Head and neck squamous cell carcinomas	[206]
Arctigenin	ROS, CCN1, p-RIP3 and p-MLKL ↑	Phase 1 clinical trial	Induce necroptosis	Prostate cancer	[207]
Doramectin (DRM)	ROS and RIPK1/RIPK3/MLKL ↑	Preclinical study	Induce necroptosis and apoptosis	Glioma	[208]

mTOR signaling pathway

The mechanism of mTOR signaling pathway regulated PCD in tumor microenvironment

The atypical serine/threonine kinase mTOR acts as a significant role in mediating cell growth and metabolism. Indeed, mTOR signaling is activated in tumors generally, altering the expression levels or activities of critical metabolic factors in the pathway [209]. mTOR orchestrates tumor-related anabolic processes such as fatty acid, lipid synthesis, protein, and nucleotide while also governing PCD processes such as autophagy and apoptosis. mTOR is formed by two structurally and functionally distinct complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1, containing mTOR, is sensitive to rapamycin, whereas mTORC2 remains insensitive to rapamycin [210].

The PI3 K/AKT/mTOR signaling pathway is closely intertwined with angiogenesis, contributing to tumor growth, metastasis, and lethality. Both normal and tumor-related angiogenesis are influenced by the PI3 K/AKT/mTOR pathway [211]. Futhermore, The PI3 K/AKT/mTOR signaling pathway inhibits apoptosis. This pathway also exerts inhibition on apoptosis-related genes, including BAX, BAD, Forkhead Box Protein O1 (FOXO1), glycogen synthase kinase 3 (GSK-3), and caspase 9. Instead, it promotes the expression of anti-apoptotic proteins like NF-κB and cAMP response element-binding protein (CREB), thereby playing a crucial role in promoting cell survival [212, 213].

The PI3 K/AKT/mTOR signaling pathway inhibits cellular autophagic fluxes.During the initiation of autophagy, mTORC1 and AMPK work synergistically to inhibit the synthesis of ULK1 and VPS34 complexes. mTOR can counteract the inhibition of RIPK1/RIPK3 interaction through autophagy, thus enhancing 11-methoxytabersonine (11-MT)-mediated necroptosis [44, 214, 215]. In addition, inhibition of mtor-mediated mitochondrial autophagy resulted in limited ROS production, ultimately limiting ROS production and preventing necroptosis [216].

Interestingly, inflammasomes can serve as specialized executors of pyroptosis. The intricate interplay between pyroptosis and autophagy is linked to the characteristic activation of the pyrin domain-containing protein 3 (NLRP3) inflammasome [130]. Growing literature suggests that mTOR, by inhibiting autophagy, promotes ROS accumulation and exacerbates pyroptosis. Suppression of MTOR-induced mitophagy removes damaged mitochondria, thus decreasing ROS generation, alleviating oxidative stress-related damage, and inhibiting pyroptosis by suppressing the activation of NLRP3 inflammasomes [217–220].

The PI3 K/AKT/mTOR signaling pathway inhibits ferroptosis by reducing cellular iron loading and enhancing the intracellular oxidative system xc—glutathione (GSH)-GPX4.Current evidence suggests that mTORC1 promotes stearoyl-CoA via the PI3 K-AKT-mTORC1 axis through Sterol regulatory element-binding protein 1 (SERBP1) and stearoyl-CoA desaturase-1 (SCD-1), resulting in the production of monounsaturated fatty acids, which inhibits lipid peroxidation and, finally, ferroptosis [221, 222]. In addition, mTORC1 regulates the body's iron load, as its overactivation increases the levels of transferrin receptor 1 (TfR1) and ferroportin (FPN), resulting in reduced iron load and suppressed ferroptosis [223]. Ferroptosis can be restrained by targeting mTOR signaling pathways, especially mTORC1, which hinders the iron regulatory protein/iron response element (IRE/IRP) pathway, consequently reducing iron load and inhibiting ferroptosis [224]. In addition, mTORC1 mitigates ferroptosis by regulating the major antioxidant system Xc-glutathione (GSH)-GPX4. Additionally, mTORC1 inhibition-induced autophagy-mediated GPX4 degradation further suppresses ferroptosis [225].

Inhibition of anoikis in cancer cells after detachment from the extracellular matrix is a critical step in the metastasis process. In BRCA cells, the activation of AMPK-mediated mTORC1 inhibition and protein synthesis contributes to anti-anoikis effects [226]. F FDX1, a gene linked to cuproptosis, may obstruct PI3 K/AKT/mTOR signaling, playing a role in multiple chemoresistance scenarios [227]. The cuproptosis prognostic predictor PCA-associated transcript 6 (PCAT6) has been reported to trigger activation of the PI3 K/Akt/mTOR signaling pathway [228] (Fig. 3).

Fig. 3.

In the mTOR signaling pathway, PI3 K/AKT/mTOR axis can induce the occurrence of apoptosis. mTORC1 complex (mLST8, RAPTOR, DEPTOR, Tti1, Tel2, PRAS40) can induce anoikis. mTORC1 can promote autophagy through ULK1 complex and VPS34 complex, and can promote necroptosis through autophagy. mTORC1 can inhibit Fe2 + and thus ferroptosis. AMPK/mTOR axis inhibits ferroptosis and necroptosis by regulating the system Xc-glutathione (GSH)-GPX4 axis to inhibit ROS generation

Targeted therapies related to mTOR signaling pathways

Targeting at PI3 K/AKT/mTOR

ATP-competitive mTOR inhibitors represent an effective strategy to inhibit the PI3 K/AKT/mTOR axis. One such inhibitor is MLN0128, a pan-mTOR inhibitor that induces apoptosis and exhibits potent antitumor effects in vitro and in vivo, targeting tumors like bone and soft tissue sarcoma, BRCA, and primary effusion lymphoma [229–231]. The combination of sapanisertib and paclitaxel demonstrated significant clinical activity in the treatment of metastatic uroepithelial carcinoma (mUC) [232]. AZD8055, an ATP-competitive mTOR inhibitor, selectively inhibits class I PI3 K isoforms and other PI3 K-like kinase members. This leads to the induction of autophagy in conditions such as leukemia and BRCA [233–236]. Additionally, AZD6482, a specific inhibitor of pi3kβ, holds potential in inhibiting proliferation and inducing apoptosis in glioma cells [237]. SC66 is an AKT inhibitor that blocks the induction of apoptosis in CESC cell and glioblastoma cells by arresting cells in the G1 phase and inducing apoptosis by inhibiting mTOR activity and down-regulating Akt/β-catenin signaling pathway, respectively [238, 239]. The PI3 K/mTOR inhibitor VS-5584 can inhibit cell proliferation and promote apoptosis in acute lymphoblastic leukemia and glioblastoma [240, 241].

Melatonin combined with rapamycin treatment was found to block the negative feedback loop from the specific downstream effects of mTOR activation of S6 K1 to the Akt signaling pathway, thereby reducing cell viability, proliferation, and colony formation ability [242]. Apatinib is a vascular endothelial growth factor receptor-2 inhibitor that induces apoptosis and autophagy of ATC cells by downregulating p-mTOR and p-AKT signaling through the AKT/mTOR signaling pathway. In addition, apatinib promoted autophagy and elevated apoptosis in ATC cells, while the combined use of apatinib and autophagy inhibitor chloroquine significantly inhibited tumor growth both in vitro and in vivo. Therefore, molecular-targeted therapy combined with autophagy inhibitors is expected to advance the treatment of ATC patients [243]. Apatinib in combination with sindilizumab and carboplatin paclitaxel chemotherapy for treatment-phase early TNBC resulted in complete remission in 61.8% after imaging assessment, in addition to a disease-free survival rate of 94.1% at 36 months. This demonstrates the high level of clinical activity and manageable safety profile of apatinib [114]. Baohuoside-1, Mahanine, and Genipin inhibition can induce p-PI3 K, p-AKT, and p-mTOR, respectively, to induce apoptosis of oral cancer epithelial-mesenchymal stem cells, apoptosis of glioma cells, and autophagy of oral squamous cell carcinoma cells [244–246]. Isoglycyrrhizin (ISL) is a flavonoid extracted from licorice that inhibits mTOR by docking with its ATP-binding pocket (i.e., competing with ATP). It can effectively inhibit the proliferation and induce apoptosis of HCC and AGS cells by inducing autophagy in vivo and in vitro [247]. Pectin (PEC), geniposide (GJ), and baicalin are all flavonoids. The former induces autophagy in AGS cells, and the latter can enhance the induced apoptosis and autophagy in glioblastoma cells [246, 248, 249]. Tanshinone I (Tan—I) is isolated from the Chinese herbal medicine salvia miltiorrhiza, a critical fat-soluble monomer compound. Tan-I induces autophagy by inhibiting PI3 K/AKT/mTOR pathway in ovarian cancer (OV) [250].

Brusato, a natural quassinoid isolated from traditional Bruceae Fructus, can block PI3 K/Akt/mTOR to induce autophagy in HCC cells. Thus, it can effectively inhibit the proliferation and induce apoptosis of HCC cells and inhibit the invasion and migration of tumors in vitro and in vivo [251]. Gynostemma pentaphyllum (Thunb.) Makino is a traditional medicine commonly used in China, East Asia, and Southeast Asia. Importantly, gypenosides can induce apoptosis of renal cancer cells by regulating the PI3 K/Akt/mTOR signaling pathway by reducing the phosphorylation levels of Akt and mTOR [252]. Sinomenine (SIN), isolated from the traditional Chinese medicine Sinomenum, activates autophagy in renal cancer by inactivating the PI3 K/AKT/mTOR pathway [253]. Ricolinostat (ACY-1215) inhibits the proliferation and promotes apoptosis of esophageal squamous cell carcinoma through miR-30 d/PI3 K/AKT/mTOR and ERK pathways. ACY-1215 may be a promising antitumor drug for esophageal squamous cell carcinoma [254]. Novel quinazolinone MJ-33 induces AKT/mTOR-mediated autophagy-related apoptosis in 5 FU-resistant CRC cells [255]. Treatment of schwannoma cells with LiCl can lead to the generation of reactive oxygen species and the activation of the AKT/mTOR pathway, consequently inducing necroptosis. This effect was counteracted by the application of necrostatin-1, suggesting a potentially innovative mechanism of LiCl-triggered tumor cell [256]. Lorlatinib enhances the susceptibility of melanoma to ferroptosis by targeting the IGF1R-mediated PI3 K/AKT/mTOR signaling cascade. Consequently, the combination of lorlatinib significantly broadens the applicability of GPX4 inhibition in melanoma patients exhibiting elevated IGFR1 expression [257]. Loratinib was superior to crizotinib in the treatment of patients with ALK-positive non-small cell lung cancer, characterized by more durable benefits. However, the incidence of grade 3–4 adverse reactions with loratinib was 76%, leading to discontinuation of treatment in some patients [258]. In HCC cells, miR-21-5p functions to suppress ferroptosis by regulating MELK, which in turn modulates the AKT/mTOR signaling pathway [259].

Targeting at AMPK/mTOR

In non-small cell lung cancer A549 cell carcinoma, Resveratrol (RSV) enhances nerve growth factor receptor (NGFR) by increasing NGFR mRNA expression and prolonging the lifetime of NGFR mRNA and protein. It can induce the activation of AMPK/mTOR downstream and induce autophagy and apoptosis [260]. β-elemene is a potent anti-cancer compound extracted from Curcuma SPP. Moreover, β-elemene can induce apoptosis and autophagy of CRC cells by mediating ROS/AMPK/mTOR pathway, increasing ROS level, promoting AMPK protein phosphorylation, and inhibiting mTOR protein phosphorylation [261]. Ellagic acid induces apoptosis and autophagy in COAD cells through the AMPK/mTOR pathway. These results suggest that EA inhibits COAD growth and induces apoptosis and protective autophagy through the AMPK/mTOR pathway [262]. Besides, Periplocin inhibits the proliferation and induces apoptosis of PANC1 and CFPAC1 cells by activating the AMPK/mTOR pathway and inhibiting p70 S6 K [263]. It has been reported that Trifolirhizin induces autophagy-dependent apoptosis of COAD cells through AMPK/mTOR signaling pathway [264]. Chaga mushroom extract induces autophagy in BRCA cells by activating AMPK and inhibiting the mTOR signaling pathway, which involves activation of AMPK and inhibition of mTOR signaling pathway [265].

Targeting at MET/AKT/mTOR

The MET/AKT/mTOR signaling pathway regulates many biological processes, including cell apoptosis, autophagy, proliferation, tumorigenesis, and invasion. Cynaroside (Cy), a traditional Chinese medicine monomer, is a flavonoid glycoside compound which is widely present in plants. Cy can block the MET/AKT/mTOR axis by reducing the phosphorylation of AKT, mTOR, and P70S6 K. Therefore, the MET/AKT/mTOR axis can be a vital target of Cy, with anticancer efficacy and is expected to be a promising drug for treating AGS [266].

Targeting at EGFR/mTOR

Physakengose G (PG), a novel compound from the traditional Chinese medicine Physalis alkekengi var. franchetii, has shown promising anti-tumor effects in human osteosarcoma cells by inducing apoptosis through the EGFR/mTOR signaling axis. PG blocks the phosphorylation of EGFR and inhibits epidermal growth factor (EGF)-induced activation of downstream signaling molecules such as AKT and mTOR. In addition, PG induces apoptosis through the mitochondrial pathway [267]. GALNT14 regulates apoptosis and ferroptosis in OV through the EGFR/mTOR pathway. Importantly, GALNT14 inhibits the activity of the mTOR pathway by modifying the O-glycosylation of EGFR, providing a potantial method to overcome cisplatin resistance in OV patients [268]. The abnormal expression of GALNT14 can change the neogenesis, proliferation, migration, metastasis and drug resistance of tumor cells, and can be used as an important target for chemotherapy [269].

Targeting at other signaling pathway axis

Circ_0004585 plays a cancer-promoting role in the invasion and metastasis of PCA by targeting the miR-1248/TM9SF/mTOR axis, which provides a new idea for the development of therapeutic strategies for metastatic PCA [270]. The mTOR/S6 K1 signaling axis is known to regulate cell growth and exhibits a hyperactivated state in various cancers. miR-224 targets the 3'-UTR of mTOR, leading to the inactivation of apoptotic signaling and activation of cell proliferation [271]. Homoharringtonine (HHT) is a natural alkaloid from Taxus capitis with well-established anti-cancer effects on hematological malignancies. HHT inhibits the growth and promotes apoptosis of BRCA cells by regulating the miR-18a-3p/AKT/mTOR signaling pathway, and HHT may be a promising anti-tumor drug [272]. Sorafenib in combination with homoharringtonine in relapsed or refractory acute myeloid leukemia (R/R AML) demonstrated a highly active and manageable safety profile with an overall survival of 18.1 months, with fewer patients experiencing grade 3–4 adverse events [273]. Mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor U0126 induces BRCA cells (MDA-MB231 and S6 K) to undergo anoikis by simultaneously blocking extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR)/p70(S6 K) pathways, which may provide a therapeutic strategy for selectively targeting tumors that proliferate in the ectopic region [274]. GO induces autophagy and apoptosis in CRC cells through the ROS-dependent AMPK/mTOR/ULK-1 pathway [275]. The polyphyllin I ROS/AKT/mTOR pathway promotes autophagic death and apoptosis of COAD cells [276]. A recent study revealed that active metabolite of ginsenoside Compound K (CK) inhibited AKT/mTOR/c-Myc signaling pathway and its downstream hexokinase 2 (HK2) and pyruvate kinase isoenzyme M2 (PKM2) to promote the apoptosis of HepG2 and Huh7 human HCC cells [277].

Chrysin is a flavonoid that is abundant in honey and phytochemicals with an ERK/MTOR-mediated autophagy effect on oral cancer cells [278]. Chitooligosaccharide (COS) is used to trigger apoptosis and autophagy in osteosarcoma cells through the p53/mTOR signaling pathway [279]. Artematrolide A (AR—A) is a compound derived from Artemisia atrovirens that activates the ROS/ERK/mTOR signaling pathways. It orchestrates the transition of cell metabolism from aerobic glycolysis to mitochondrial respiration and prompts G2/M phase arrest and apoptosis in CESC cells. These findings suggest its potential as a therapeutic agent for CESC treatment [280]. Last but not least, in human oral squamous cell carcinoma, falcarinol inhibits cell proliferation, division, and metastasis by inducing apoptotic and autophagic cell death through the PI3 K/AKT/mTOR/p70S6 K pathway [281]. Novel conjugates of endoperoxide and 4-anilinoquinazoline inhibits the IGF1—R/AKT/mTOR signaling pathways and induces myeloma cell apoptosis [282]. Icariin and curcumol synergize to regulate miR-7/mTOR/SREBP1 pathway to induce autophagy and ferroptosis in PCA cells and affect lipid metabolism [283]. The natural product osthole exerts its anticancer effect in KRAS-mutated CRC cells by inducing ferroptosis by inhibiting the AMPK/Akt/mTOR signaling pathway [284].

We summarize our understanding of how mTOR signaling modulates the tumor microenvironment in PCD and summarize drugs targeting multiple mTOR signaling axes. mTOR signaling pathway has been used to study the relationship between apoptosis and autophagy. However, we found that autophagy, apoptosis, pyroptosis, anoikis, ferroptosis, and cuprotosis are closely related to the mTOR signaling pathway in the process of tumor development. Therefore, mTOR is closely related to regulating PCD and tumorigenesis. In addition, targeted drugs of the mTOR signaling pathway are abundant. Therefore, mTOR can be used as an essential target for the treatment of tumors (Table 3).

Table 3.

Compounds targeting of the mTOR signaling pathway related to PCD in cancer: ↓ represents decrease or inhibition. ↑ represents increase or activation

Compound name	Target	Current stage	Mechanism in PCD	Tumor type	Refs
MLN0128(Sapanisertib)	PI3 K/AKT/mTOR↓	Phase ½ clinical trial	Induce apoptosis	Soft tissue sarcoma, breast cancer, and primary effusion lymphoma	[229–231], clinicaltrials.gov
AZD8055	PI3 K↓	Phase 1 clinical trial	Induce autophagy	Leukemia and breast cancer	[233–236]
AZD6482	pi3kβ↓	Preclinical study	Induce apoptosis	Glioma	[237]
SC66	Akt/β-catenin and mTOR↓	Preclinical study	Induce apoptosis	Cervical cancer and glioblastoma	[238, 239]
VS-5584	PI3 K/mTOR↓	Phase 1 clinical trial	Induce apoptosis	Acute lymphoblastic leukemia and glioblastoma	clinicaltrials.gov, [240, 241]
Apatinib	p-AKT and p-mTOR ↓	Phase 2/3 clinical trial	Induce apoptosis and autophagy	Anaplastic thyroid cancer	[243], clinicaltrials.gov
Baohuoside-1	p-PI3 K, p-AKT and p-mTOR↓	Preclinical study	Induce apoptosis	Oral cancer	[244–246]
Mahanine	p-PI4 K, p-AKT and p-mTOR↓	Preclinical study	Induce apoptosis	Glioma	[244–246]
Genipin	p-PI5 K, p-AKT and p-mTOR↓	Preclinical study	Induce autophagy	Oral squamous cell carcinoma	[244–246]
Isoglycyrrhizin	ATP and mTOR↓	Preclinical study	Induce apoptosis and autophagy	Hepatoma and gastric cancer	[246, 248, 249]
Tanshinone I	PI3 K/AKT/mTOR↓	Preclinical study	Induce autophagy	Ovarian cancer	[250]
Brusato	PI3 K/AKT/mTOR↓	Preclinical study	Induce apoptosis and autophagy	Hepatocellular cancer	[251]
Gynostemma pentaphyllum (Thunb.)	PI3 K/AKT/mTOR↓	Preclinical study	Induce apoptosis	Renal cancer	[252]
Sinomenine	PI3 K/AKT/mTOR↓	Preclinical study	Induce autophagy	Renal cancer	[253]
Ricolinostat	miR-30 d/PI3 K/AKT/mTOR↓	Phase 1 clinical trial	Induce apoptosis	Esophageal squamous cell carcinoma	clinicaltrials.gov, [254]
Novel quinazolinone MJ-33	PI3 K/AKT/mTOR↓	Preclinical study	Induce apoptosis and autophagy	Colorectal cancer	[255]
LiCl	ROS and AKT/mTOR↑	Preclinical study	Induce necroptosis	Schwannoma	[256]
Lorlatinib	IGF1R-mediated PI3 K/AKT/mTOR and GPX4↓	Phase 2/3 clinical trial	Induce ferroptosis	Melanoma/Non-small cell lung cancer	[257], clinicaltrials.gov
miR-21-5p	GPX4 and AKT/mTOR↑	Preclinical study	Inhibit ferroptosis	Hepatocellular carcinoma	[259]
Resveratrol	AMPK↑, mTOR↓	Phase 1/2 clinical trial	Induce apoptosis and autophagy	Non-small cell lung cancer	[260]
β-elemene	ROS and p-AMPK↑, mTOR↓	Preclinical study	Induce apoptosis and autophagy	Colorectal cancer	[261]
Ellagic acid	AMPK↑, mTOR↓	Preclinical study	Induce apoptosis and autophagy	Colon cancer	[262]
Periplocin	AMPK/mTOR↑, p70S6 K↓	Preclinical study	Induce apoptosis	Pancreatic cancer	[263]
Trifolirhizin	AMPK↑, mTOR↓	Preclinical study	Induce apoptosis	Colon cancer	[264]
Cynaroside	MET/AKT/mTOR↓	Preclinical study	Induce apoptosis and autophagy	Gastric cancer	[266]
Physakengose G	EGFR/mTOR↓	Preclinical study	Induce apoptosis	Osteosarcoma	[267]
GALNT14	EGFR/mTOR↓	Phase 2 clinical trial	Induce apoptosis and ferroptosis	Hepatocellular carcinoma	[268], clinicaltrials.gov
circ_0004585	miR-1248/TM9SF/mTOR↓	Preclinical study	Induce autophagy and inhibit anoikis	Metastatic prostate cancer	[270]
Homoharringtonine	miR-18a-3p/AKT/mTOR↓	Phase 2 clinical trial	Induce apoptosis	Breast cancer/leukaemia	[272, 273]
U0126	mTOR/p70S6 K↓	Preclinical study	Induce anoikis	Breast cancer	[274]
Gemtuzumab ozogamicin	AMPK/mTOR/ULK-1↓	Phase 1/2/3 clinical trial	Induce apoptosis and autophagy	leukaemia	clinicaltrials.gov
Polyphyllin I	ROS/AKT/mTOR and CIP2 A/AKT/mTOR↓	Preclinical study	Induce apoptosis and autophagy	Colon cancer	[276]
Compound K	AKT/mTOR/c-Myc↓	Preclinical study	Induce apoptosis	Hepatocellular carcinoma	[277]
Chrysin	ERK/mTOR↓	Preclinical study	Induce autophagy	Oral cancer	[278]
Chitooligosaccharide	p53/mTOR↓	Preclinical study	Induce apoptosis and autophagy	Osteosarcoma	[279]
Artematrolide A	ROS/ERK/mTOR↑	Preclinical study	Induce apoptosis	Cervical cancer	[280]
Falcarino	PI3 K/AKT/mTOR/p70S6 K↓	Preclinical study	Induce apoptosis and autophagy	Oral squamous cell carcinoma	[281]
Novel conjugates of endoperoxide and 4-anilinoquinazoline	IGF1—R/AKT/mTOR↓	Preclinical study	Induce apoptosis	Myeloma	[282]
Icariin	miR-7/mTOR/SREBP1↓	Preclinical study	induce autophagy and ferroptosis	Prostate cancer	[283]
Curcumol	miR-7/mTOR/SREBP1↓	Preclinical study	induce autophagy and ferroptosis	Prostate cancer	[283]
Osthole	AMPK/Akt/mTOR ↓	Osthole	Induce ferroptosis	Colorectal cancer	[284]

Limitations of targeting three signaling pathways

These three signaling pathways have different pathway activities as well as differences in activation modes in different types of tumors. For example, in lung cancer, aberrant aggregation of FRMD6 activates mTOR expression and enhances the interaction between mTOR and S6 K to promote lung cancer progression [285]. However, in breast cancer, aberrant activation of mTOR is mainly associated with an increased flux of HIF-1α and a lack of the upstream gene, PTEN [286]. This is probably one of the reasons why Rapamycin has efficacy in specific types of cancer only. It is worth noting that the functions played by caspase 3 in different types of tumors are also highly variable. caspase-3 activation promotes oncogene-induced malignant transformation and accelerates breast cancer progression by triggering translocation of mitochondrial endonuclease G (EndoG), which activates the Src-STAT3 signaling pathway [287]. In colorectal cancer, the Caspase-3 generates the short form of IL-18 by cleaving interleukin 18 (IL-18), alters the phosphorylation state of STAT1, and promotes the secretion of ISG15, thereby strengthening the anti-tumor function of natural killer cells [288]. In addition, the upstream and downstream regulatory transduction of these three signaling pathways as well as the associated signaling pathways have not been fully revealed.

Targeting these three signaling pathways can lead to drug resistance, which will limit the use of targeted drugs. The apoptotic pathway is characterized by complexity and redundancy. Thus, when drugs targeting the caspase signaling pathway activate caspase 3 activity to induce apoptosis, aberrant activation of endogenous anti-apoptotic proteins prevents this task from proceeding [289]. It is worth noting that apoptosis-induced cell proliferation (AiP) remains a phenomenon that cannot be ignored by targeting the caspase signaling pathway, and a large number of tumor cells undergo apoptosis may lead to further tumor deterioration and progression [290]. In addition, apoptosis with compensatory activation of the necrotic pathway is an important mechanism for the development of drug resistance to drugs targeting RIPK1/RIPK3/MLKL. Specifically, inhibition of the RIPK1/RIPK3/MLKL pathway initiates the onset of other programmed cell deaths, including apoptosis and autophagy [291]. Interestingly, the O-GlcNAc glycosylation modification of RIPK1 was able to lead to resistance to sunitinib by inhibiting the RIPK1-dependent apoptosis (RDA) pathway [292]. When targeting the mTOR signaling pathway with mTOR inhibitors, cancer cells aberrantly activate the PI3 K/AKT pathway and the MAPK pathway to bypass the action of mTOR inhibitors [293]. Studies have shown that some cancer cells may upregulate the expression of the transporter proteins ABCB1 and ABCG2, which expel the mTOR inhibitors outside the cell, reducing intracellular concentrations and leading to resistance [294]. Therefore, how to solve the resistance of targeted drugs is an important direction for small molecule targeted therapy of PCD. Finally, targeting these three signaling pathways faces the problem of a large gap between preclinical studies and clinical applications. In our previous discussion, most of the drugs targeting these three signaling pathways are still in the preclinical research stage and have not entered the clinical research stage. Further, although some small molecule drugs have achieved good results in in vitro experiments, they still face significant problems when entering clinical studies. In conclusion, although targeting these three signaling pathways associated with PCD for the treatment of tumors is promising, there are still some limitations that restrict its application and development.

Novel targeted therapies associated with PCD

Novel therapeutic approaches such as the CRISPR/Cas gene editing system, immune checkpoint inhibitors and gene therapy have shown great potential in cancer treatment. These approaches can not only directly target cancer cells, but also regulate immune cells and signaling pathways in the tumor microenvironment. CRISPR/Cas can induce apoptosis and inhibit tumor growth by precisely editing key genes in cancer cells [295]. For example, editing of apoptosis-suppressing genes or activation of key proteins in apoptosis pathway can effectively induce apoptosis in cancer cells. Moreover, CRISPR/Cas can be used to repair or activate tumor suppressor genes, such as p53, thereby restoring the normal regulatory mechanisms of cells [296].

Immune checkpoint inhibitors restore or enhance the immune response of T cells against cancer cells by blocking immune-suppressing signals [297]. Studies have shown that immune checkpoint inhibitors not only enhance the cytotoxicity of T cells, but also exert anti-tumor effects by inducing PCD in cancer cells [298]. Gene therapy achieves targeted treatment of tumors by introducing exogenous genes into cancer cells or immune cells [299]. For example, the introduction of apoptosis-related genes into cancer cells via viral vectors can directly induce apoptosis. Although the CRISPR/Cas system, immune checkpoint inhibitors, and gene therapies have shown great potential in cancer treatment, single therapeutic approaches often struggle to overcome the complexity and heterogeneity of tumors. Therefore, an integrated therapeutic strategy combining PCD signaling pathways may be an important direction for future cancer treatment.

Conclusion

Different types of PCD play essential roles in tumor progression. The components of PCD can interact and transform each other, and the combined study of components in PCD can carry out more in-depth research on tumors and treatment. Signaling pathways play a massive role in inducing PCD. When signaling pathways induce multiple PCDS to occur, the nature and outcome of PCD may be altered. This review focuses on the mechanism and targeted drugs of the caspase signaling pathway, RIPK1/RIPK3/MKLK signaling pathway and mTOR signaling pathway in PCD. In this review, 32 drugs mainly target the caspase signaling pathway, 34 drugs primarily target the RIPK1/RIPK3/MKLK signaling pathway and 49 drugs principally target the mTOR signaling pathway. In addition, since the regulation of components in PCD in tumor immunity is a complex and holistic role, the role of PCD may even be reversed in different stages of tumor development, and the specific targets will be different due to the complexity of tumor components and types, so the use of targeted drugs needs more in-depth research. Combination of drugs, development of new drugs and avoidance of chemoresistance may provide new strategies for the treatment of cancer. In addition, the immune microenvironment plays a vital role in maintaining cell homeostasis and signaling pathways, and inflammation also has a causal relationship with PCD such as pyroptosis. The interaction between the immune microenvironment and PCD may be a new direction for tumor treatment in the future.

Drug development targeting the PCD pathway is driving a paradigm shift in tumor therapy. By constructing a dynamic model of the apoptosis signaling network, the imbalance in the regulation of Bcl-2 family proteins in the tumor microenvironment has been precisely addressed. Based on the structural analysis of caspase complexes, researchers successfully designed small molecules with specific binding sites, breaking through the bottleneck of death receptor signaling regulation. In addition, for the off-target risk of apoptosis inducers, the novel early warning system can identify the molecular features of Smac mimics that accidentally activate the NF-κB pathway, thus improving the efficiency of drug safety assessment. The current challenge lies in the heterogeneity of tumor cell death leading to biased pathway modeling, which needs to be combined with single-cell sequencing technology to address the signal crosstalk between necrosis and apoptosis in different subpopulations. Therefore, with the breakthrough of computational simulation technology, it will be a promising research direction to promote more drugs targeting PCD into clinical studies.

Abbreviations

11-MT

11-Methoxytabersonine

XB

2-Deprenyl-rheediaxanthone B

ACD

Accidental cell

AFPR

Active Fraction of Polyrhachis vicina Roger

ACC

Adrenocortical cancer

AGS

Advanced gastric cancer

ATL

Alantolactone

AMPK

AMP-activated protein kinase

ATC

Anaplastic thyroid cancer

AIF

Apoptosis-inducing factor

Apaf-1

Apoptotic protease activating factor 1

AR-A

Artematrolide A

AMBRA1

Autophagy/Beclin 1 regulator 1

ATGs

Autophagy-related proteins

CDH17

Cadherin-17

CREB

CAMP response element-binding protein

CARD

Caspase recruitment domain

cIAP1/2

Cell inhibitor of apoptosis protein-1/2

CESC

Cervical cancer

C10

Chalcone 3',5'-degraded

CHE

Chelerythrine

COS

Chitooligosaccharide

CQ

Chloroquine

COAD

Colon cancer

CRC

Colorectal cancer

CK

Compound K

Cy

Cynaroside

GSH

Cysteine

DAMPs

Damage-associated molecular patterns

DISC

Death-Inducing Signaling Complex

DHA

Dihydroartemisinin

DHTS

Dihydrotanshinone I

DRM

Doramectin

MPD1

EMC-KGDEVD-DOX

ER

Endoplasmic reticulum

EGF

Epidermal growth factor

ERK

Extracellular signal-regulated kinase

FADD

Fas-associated with death domain protein

FPN

Ferroportin

FAK

Focal adhesion kinase

FOXO1

Forkhead Box Protein O1

GSDMD

Gasdermin D

GO

Gemtuzumab ozogamicin

GJ

Geniposide

GLUT

Glucose transporter

GLUDL

Glutamate dehydrogenase

GLUL

Glutamine synthetase

GPX4

Glutathione peroxidase 4

PYGL

Glycogen phosphorylase

GSK-3

Glycogen synthase kinase 3

IMs

GSH/ROS dual response nanogel system

HNSCC

Head and neck squamous cell carcinoma

HCC

Hepatocellular carcinoma

HK2

Hexokinase 2

HHT

Highharringtonine

HDAC

Histone deacetylase

IKK

IĸB kinase

IRE/IRP

Iron regulatory protein/iron response element

ISL

Isoglycyrrhizin

MR

Mannose receptors

mTNBC

Metastatic triple-negative breast cancer

MOMP

Mitochondrial membrane permeability

mtROS

Mitochondrial ROS

MEK

Mitogen-activated protein/extracellular signal-regulated kinase

MLKL

Mixed Lineage Kinase Like

MEFs

Mouse embryonic fibroblasts

mTORC1

MTOR complex 1

mTORC2

MTOR complex 2

MT

Mutant

nano-PROTACs

Nano-proteolysis-targeting chimeras

NEC-1

Necrostatin-1

NA

Neoalbuminol

NGFR

Nerve growth factor receptor

NETs

Neutrophil extracellular traps

OPD’

Ophiopogonin D’

OV

Ovarian cancer

Paeo

Paeoniflorigenone

PCA

Pancreatic cancer

PDAC

Pancreatic ductal adenocarcinoma

PCAT6

PCA-associated transcript 6

PEC

Pectin

PDC

Peptide-doxorubicin conjugate:

PI3 K C III

Phosphatidylinositol 3-kinase III

PG

Physakengose G

PARP1

Poly(ADP-ribose)polymerase

PolyI: C

Polyinosinic: polycytidylic acid

PPVI

Polyphyllin VI

polyU

Poly-ubiquitinated

PCD

Programmed cell death

PCA

Proliferation of prostate cancer

PpIX

Protoporphyrin IX

NLRP3

Pyrin domain-containing protein 3

RICTOR

Rapamycin-insensitive mTOR chaperone

ROS

Reactive oxygen species

RIPK3

Receptor-Interacting Protein Kinase 3

RSV

Resveratrol

TPD1

RGDEVD-DOX

RHIM

RIP homotypic interaction motif

RDA

RIPK1-dependent apoptosis

SCU

Scutellarein

aPRO

SHP2-targeting PROTAC peptide

SIN

Sinomenine

SHP2

Src homology2 (SH2) domain-containg protein tyrosine phosphatase (PTPase)

STS

Staurosporine

SCD-1

Stearoyl-CoA desaturase-1

SERBP1

Sterol regulatory element-binding protein 1

TBK1

TANK-binding kinase 1

Tan–I

Tanshinone I

TAK1

TGF-β-activated kinase 1

TAB2

TGF-β-activated kinase 1 binding protein 2

TAK

The growth factor β-activated kinase growth factor β-activated kinase

TRAIL

TNFSF10

TLR

Toll-like receptors

ATF3

Transcription 3

TfR1

Transferrin receptor 1

TRIM11

Tripartite motif containing 11

TNBC

Triple-negative breast cancer

TME

Tumor microenvironment

TNFR

Tumor necrosis factor receptor

Ub

Ubiquitin

ULK1

UNC-51-like kinase 1

ZBP1

ZDNA binding protein 1

Author contributions

XGZ and KH conceived this study. CPS wrote the manuscript. YLS and JWG made the figures and tables. LH, JWG and YLS performed literature searching. All authors have read and approved the article.

Funding

The research project is supported by National Natural Science Foundation of China (Grant no. 82172989, 82273068 and 82260524); Key Research and Development projects in Jiangxi (Grant no. 20212BBG73021); Jiangxi Training Program for academic and technical leaders of major disciplines,Young talents program (Grant no. 20212BCJ23023); Key project of Science and Technology Innovation of Health Commission (Grant no. 2023ZD003); Jiangxi Provincial Natural Science Foundation(grant no. 20232BAB206101); Jiangxi Province Department of Education Science and technology research project, China (Grant no. GJJ210177); Project Fund of Jiangxi Provincial Health Commission (20204343, 202130873, 202210044).

Data availability 

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.