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Chemical Structure| 161401-82-7 Chemical Structure| 161401-82-7

Structure of Z-VAD-FMK
CAS No.: 161401-82-7

Chemical Structure| 161401-82-7

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Z-VAD-FMK (Z-VAD(OH)-FMK) is a pan caspase inhibitor. Z-VAD-FMK does not inhibit the activity of ubiquitin C-terminal hydrolase L1 (UCHL1) even at concentrations up to 440 μM.

Synonyms: Z-Val-Ala-Asp-(OH)-Fluoromethyl Ketone; Caspase Inhibitor VI; Z-VAD(OH)-FMK

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Li, Xiao-huan ; Lv, Feng ; Li, Si-qi ; Deng, Hao-wei ; Tang, Li-ping ; Li, Ke-zhi , et al.

Abstract: Carnitine palmitoyltransferase 1A (CPT1A) has been implicated in the development of colorectal cancer (CRC), yet its role in ferroptosis remains to be fully understood. In this study, we found that CPT1A expression was associated with metastasis of CRC by gene datasets analysis and immunohistochemical staining of clinical samples, and it was upregulated in CRC cells compared with normal colon cell. The CCK-8, Transwell, and wound healing assays demonstrated that overexpression of CPT1A enhanced the viability, invasion, and migratory capacity of CRC cells. CPT1A expression was reduced following induction of ferroptosis in CRC cells, and this downregulation could be reversed by a ferroptosis inhibitor. Moreover, CPT1A overexpression inhibited ferroptosis in CRC cells. Nrf2, a well-known negative regulator of ferroptosis, was found to colocalize with CPT1A in CRC cells. Molecular docking, Co-IP assay and Ch-IP assay further confirmed an interaction between CPT1A and Nrf2. Notably, Nrf2 overexpression upregulated CPT1A expression, whereas Nrf2 knockdown produced the opposite effect. CPT1A overexpression led to activation of PI3K/AKT pathway in CRC cells. Inactivation of the PI3K/AKT pathway by the inhibitor partially reversed the anti-ferroptosis effect of CPT1A overexpression. Furthermore, inhibition of PI3K/AKT pathway suppressed Nrf2 expression, and reduce nuclear translocation of Nrf2, whereas activation of this pathway enhanced Nrf2 expression and nuclear translocation. In vivo experiments corroborated these findings, showing that CPT1A overexpression promoted Nrf2 expression, suppress ferroptosis, facilitated tumor growth, inactivated PI3K/AKT pathway. Taken together, our data suggest that CPT1A associates with metastasis of CRC, and inhibits ferroptosis through a regulatory feedback loop involving Nrf2 and PI3K/AKT pathway.

Keywords: Colorectal cancer ; CPT1A ; Ferroptosis ; Feedback loop

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Pan, Shaoshan ; Jia, Haiying ; Fang, Xue ; Liu, Qian ; Wang, Qian ; Luo, Tianzhi

Abstract: Accurate and early discrimination of regulated cell death (RCD) modalities, such as ferroptosis, apoptosis, and cuproptosis, remains a critical challenge for advancing disease understanding and therapy. Existing analytical approaches often lack the capabilities for real-time, non-invasive, and discriminative monitoring. Here, we introduce TFM-CRIM, an innovative nanomechanical profiling platform. It synergistically integrates traction force microscopy (TFM) for visualizing and profiling cellular nanomechanics with confocal reflection interference microscopy (CRIM), ensuring precise cell boundary definition for enhanced accuracy in force quantification. This platform enables label-free, quantitative, and dynamic assessment of nanomechanical responses in early RCD. Real-time TFM-CRIM monitoring revealed unique kinetic signatures discriminating RCD modalities: staurosporine-induced apoptosis exhibited an extremely rapid force collapse to near-zero by ∼20–30 min (initiating within 10 min); Elesclomol-CuCl2-induced cuproptosis displayed a swift, sustained force decrease from ∼10 to 20 min, stabilizing at a low level by ∼40–60 min. These profiles were markedly different from the slower ferroptosis kinetics: RSL3-induction initiated a sustained force reduction ∼20–40 min (over ∼3 h), while Erastin-induction, after an early subtle decrease from ∼20 min, showed a delayed major decline from ∼120 min (spanning ∼6 h). Critically, TFM-CRIM demonstrated significantly superior sensitivity and temporal resolution compared to nanoindentation (delayed/non-continuous) and cellular impedance measurements (slower, less distinct kinetics). These distinct nanomechanical signatures, correlated with alterations in cytoskeletal actin fibres, enable robust RCD classification. This work establishes TFM-CRIM-based nanomechanical profiling as a rapid, quantitative, and highly sensitive analytical method for early RCD characterization, poised to accelerate mechanistic insights and drug screening.

Keywords: Nanomechanical profiling ; Regulated cell death ; Cell traction force ; Label-free analysis ; Dynamic response ; Actin cytoskeleton

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Alternative Products

Product Details of Z-VAD-FMK

CAS No. :161401-82-7
Formula : C21H28FN3O7
M.W : 453.46
SMILES Code : O=C(O)C[C@@H](C(CF)=O)NC([C@H](C)NC([C@H](C(C)C)NC(OCC1=CC=CC=C1)=O)=O)=O
Synonyms :
Z-Val-Ala-Asp-(OH)-Fluoromethyl Ketone; Caspase Inhibitor VI; Z-VAD(OH)-FMK
MDL No. :MFCD00016489
InChI Key :SUUHZYLYARUNIA-YEWWUXTCSA-N
Pubchem ID :5497171

Safety of Z-VAD-FMK

GHS Pictogram:
Signal Word:Warning
Hazard Statements:H302-H315-H319-H335
Precautionary Statements:P261-P305+P351+P338

Related Pathways of Z-VAD-FMK

pyroptosis

Isoform Comparison

Biological Activity

Description
Z-VAD-FMK, known as Z-VAD(OH)-FMK, is a renowned pan-caspase inhibitor, remarkable for its specificity in that it does not inhibit the activity of ubiquitin carboxy-terminal hydrolase L1 (UCHL1) even at concentrations up to 440 μM[1].
Target
  • Caspase

In Vitro:

Cell Line
Concentration Treated Time Description References
WILL-2 cells 50 µM 1 hour To evaluate the effect of Z-VAD-FMK on VEN-induced phagocytosis, results showed that Z-VAD-FMK did not affect the phagocytosis of VEN/DARA combination. Blood Adv. 2022 Aug 23;6(16):4847-4858.
CARNAVAL cells 50 µM 1 hour To evaluate the effect of Z-VAD-FMK on VEN-induced phagocytosis, results showed that Z-VAD-FMK did not affect the phagocytosis of VEN/RTX combination. Blood Adv. 2022 Aug 23;6(16):4847-4858.
RKO cells 50 μM 12 h Inhibited caspase-dependent apoptosis, partially rescued Sur-X-induced cell death J Exp Clin Cancer Res. 2020 May 7;39(1):82.
HCT116 cells 50 μM 12 h Inhibited caspase-dependent apoptosis, partially rescued Sur-X-induced cell death J Exp Clin Cancer Res. 2020 May 7;39(1):82.
HEK293 cells 20 µM 1 hour To test the protective effect of Z-VAD-FMK against LLO-induced cell death, the results showed that Z-VAD-FMK had no significant effect on LLO-induced cell lysis. Front Cell Infect Microbiol. 2017 May 3;7:154.
iBMDM cells 30 μM 1 hour inhibited pyroptosis and IL-1β secretion Nat Immunol. 2020 Jul;21(7):736-745.
THP-1 cells 30 μM 1 hour inhibited pyroptosis and IL-1β secretion Nat Immunol. 2020 Jul;21(7):736-745.
MDA-MB-231 cells 50 μM Inhibited caspase-3 activation and significantly reduced cell death induced by blockade of the Arf6 –AMAP1 pathway. Nat Commun. 2018 Jul 11;9(1):2682.
fetal liver megakaryocytes 100 µM 1 day Inhibited caspase-3 activity, reducing IL-1α-induced megakaryocyte rupture and platelet-like particle release. J Cell Biol. 2015 May 11;209(3):453-66.
PMN-MDSC 10 μM 24 h Z-VAD-FMK significantly improved the survival of spleen PMN-MDSC, confirming the contribution of apoptosis to PMN-MDSC cell death. Nature. 2022 Dec;612(7939):338-346.

In Vivo:

Species
Animal Model
Administration Dosage Frequency Description References
Mice LPS-induced sepsis model Intraperitoneal injection 50 mg/kg 24 and 4 hours before challenge, followed by daily injections Inhibited LPS-induced sepsis Nat Immunol. 2020 Jul;21(7):736-745.
Mice C57BL/6J mice Intraperitoneal injection 3 mg/kg Once daily for 5 days Inhibited caspase-3 activity, reducing IL-1α-induced megakaryocyte rupture and platelet production. J Cell Biol. 2015 May 11;209(3):453-66.

Protocol

Bio Calculators
Preparing Stock Solutions 1mg 5mg 10mg

1 mM

5 mM

10 mM

2.21mL

0.44mL

0.22mL

11.03mL

2.21mL

1.10mL

22.05mL

4.41mL

2.21mL

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