Alkaliptosis

1.1 Mechanism Overview

Alkaliptosis is a novel form of regulated cell death driven by intracellular alkalinization, first proposed by Song et al. in pancreatic cancer research in 2018. Studies have revealed that the small molecule JTC801 induces a cell death modality distinct from apoptosis, necroptosis and ferroptosis, characterized by sustained elevation of intracellular pH and disrupted acid‑base homeostasis. Mechanistic investigations demonstrate that JTC801 activates the IKK/NF‑κB signaling pathway to promote nuclear translocation of the transcription factor NF‑κB p65 (RELA), which subsequently suppresses the expression of carbonic anhydrase 9 (CA9). As a critical regulator maintaining acid‑base balance in tumor cells, CA9 catalyzes the conversion of CO₂ and H₂O into HCO₃⁻ and H⁺, participating in intra‑ and extracellular pH modulation. Inhibition of CA9 impairs cellular pH buffering capacity, hinders H⁺ extrusion and triggers intracellular HCO₃⁻ accumulation, leading to persistent intracellular alkalinization. Elevated pH further disrupts metabolic homeostasis, ionic equilibrium and protein function, initiating irreversible cellular damage and alkaliptosis.

Beyond the NF‑κB/CA9 axis, emerging evidence links alkaliptosis to plasma membrane transport systems, metabolic reprogramming and the tumor microenvironment. Na⁺/H⁺ exchanger 1 (NHE1/SLC9A1), vacuolar H⁺‑ATPase (V‑ATPase) and bicarbonate transporters collectively sustain cellular pH homeostasis, and their dysregulation may determine cellular sensitivity to alkalinizing stimuli. Furthermore, tumor cells generate an acidic microenvironment via the Warburg effect and rely heavily on pH regulators such as CA9 for survival. Therefore, targeting pH homeostasis represents a promising strategy to trigger alkaliptosis and enhance anti‑tumor efficacy. Current data indicate that alkaliptosis can function as an independent death modality and engage in crosstalk with other regulated cell death pathways including ferroptosis, pyroptosis and cuproptosis, jointly governing tumor cell fate.

Alkaliptosis Model Diagram. JTC801 inhibits CA9 expression by activating the IKK/NF‑κB signaling pathway, disrupting the CO₂/HCO₃⁻/H⁺ buffering system and inducing persistent intracellular alkalinization. This subsequently triggers metabolic disturbance, mitochondrial damage, ROS accumulation and ionic imbalance, ultimately initiating a novel regulated cell death distinct from classical cell death modalities, namely alkaliptosis.

1.2 Target List

Mechanism Module	Target	Full Name	Biological Function	Mechanism in Alkaliptosis	Research Value / Detection Index
Core NF‑κB Signaling Pathway	NF‑κB p65 (RELA)	Nuclear factor kappa B subunit p65 (RELA)	Transcription factor regulating inflammatory and stress‑response genes	Key upstream regulator; NF‑κB activation suppresses CA9 expression and promotes intracellular alkalinization	Core signaling activation marker
	p50 (NFKB1)	Nuclear factor kappa B subunit 1	Forms canonical NF‑κB heterodimer with p65	Participates in NF‑κB transcriptional regulation during alkaliptosis	Indicator of canonical NF‑κB activation
	IKKα (CHUK)	Inhibitor of nuclear factor kappa‑B kinase subunit alpha	Serine/threonine kinase initiating NF‑κB activation	Phosphorylates IκB proteins and promotes NF‑κB pathway activation	Upstream NF‑κB activation marker
	IKKβ (IKBKB)	Inhibitor of nuclear factor kappa‑B kinase subunit beta	Kinase responsible for IκBα phosphorylation and degradation	Promotes release and nuclear translocation of NF‑κB	NF‑κB pathway activation indicator
	IκBα (NFKBIA)	NF‑κB inhibitor alpha	Inhibitory protein retaining NF‑κB in cytoplasm	Reduced expression indicates NF‑κB activation	Negative regulator of NF‑κB signaling
Core Execution Axis	CA9	Carbonic anhydrase IX	Carbonic anhydrase regulating intracellular pH balance	NF‑κB‑mediated CA9 suppression decreases CO₂/HCO₃⁻ buffering capacity, resulting in intracellular alkalinization	Most critical marker of alkaliptosis
pH Homeostasis Regulation	NHE1 / SLC9A1	Sodium/hydrogen exchanger 1	Exchanges intracellular H⁺ with extracellular Na⁺ to regulate pH	Modulates intracellular alkalization through H⁺ extrusion	pH regulation marker
	V‑ATPase	Vacuolar‑type H⁺‑ATPase	ATP‑dependent proton pump maintaining proton gradients	Regulates proton transport and intracellular pH adaptation	H⁺ transport regulation marker
	SLC4 family (HCO₃⁻ transporters)	Solute carrier family 4 bicarbonate transporters	Maintains bicarbonate buffering system	Regulates intracellular bicarbonate balance during alkalinization stress	pH buffering capacity indicator
	MCT1/MCT4 (SLC16A1/SLC16A3)	Monocarboxylate transporter 1/4	Transports lactate and H⁺ across membranes	Participates in tumor cell pH adaptation	Tumor pH regulation marker
pH Detection Assays	Intracellular pH (pHi)	Intracellular hydrogen ion concentration / intracellular pH	Direct measurement of intracellular acid‑base status	Increased pHi represents intracellular alkalinization, the defining feature of alkaliptosis	Direct confirmation of alkaliptosis occurrence
	HCO₃⁻ level	Bicarbonate concentration	Major intracellular buffering component	Altered bicarbonate levels reflect disruption of pH buffering	Evaluation of buffering system changes
Mitochondrial Injury	COX IV	Cytochrome c oxidase subunit IV	Component of mitochondrial respiratory Complex IV	Alkaliptosis‑associated stress impairs mitochondrial respiratory function	Mitochondrial quality control marker
	ATP5A	ATP synthase F1 subunit alpha	Catalyzes ATP synthesis during oxidative phosphorylation	Mitochondrial dysfunction reduces ATP production capacity	Energy metabolism indicator
	TOM20	Translocase of outer mitochondrial membrane 20	Maintains mitochondrial protein import and structure	Reflects mitochondrial structural damage	Mitochondrial integrity marker
	JC‑1	Mitochondrial membrane potential fluorescent probe	Detects mitochondrial membrane potential (ΔΨm)	Loss of ΔΨm indicates mitochondrial dysfunction	Mitochondrial membrane potential detection
Oxidative Stress	ROS	Reactive oxygen species	Reactive molecules reflecting oxidative stress	Intracellular alkalinization induces oxidative stress accumulation	Oxidative stress evaluation
	MitoSOX	Mitochondrial superoxide indicator	Detects mitochondrial superoxide production	Indicates mitochondrial ROS elevation during alkaliptosis	Mitochondrial oxidative stress marker
	SOD2	Superoxide dismutase 2 (MnSOD)	Antioxidant enzyme converting superoxide to hydrogen peroxide	Reflects mitochondrial antioxidant response	Oxidative defense marker
	GPX4	Glutathione peroxidase 4	Removes lipid peroxides and protects against ferroptosis	Used to exclude or compare ferroptotic mechanisms	Ferroptosis exclusion marker
Ion Homeostasis	Ca²⁺	Calcium ion	Essential second messenger regulating cellular functions	Alkalinization disrupts Ca²⁺ homeostasis and contributes to cell stress	Calcium imbalance detection
	K⁺ channel‑related proteins	Potassium channel‑related proteins	Maintain intracellular potassium balance	Altered K⁺ transport contributes to ionic homeostasis disturbance	Ion imbalance evaluation

1.3 Application Scheme

Marker	Significance
p65/RELA	NF‑κB activation
IκBα	NF‑κB liberation
CA9	Core execution molecule of alkaliptosis
pHi (BCECF‑AM)	Intracellular alkalinization phenotype
Annexin V/PI	Cell death outcome

1.4 References

1. Song X, et al. JTC801 Induces pH‑dependent Death Specifically in Cancer Cells and Slows Growth of Tumors in Mice. Gastroenterology. 2018;154(5):1480‑1493.e8.
2. Chen F, Kang R, Liu J, Tang D. Mechanisms of alkaliptosis. Frontiers in Cell and Developmental Biology. 2023;11:1213995