Caspase
Caspase-1 (Ki = 0.18 μM), Caspase-3 (Ki = 0.09 μM), Caspase-6 (Ki = 0.15 μM), Caspase-8 (Ki = 0.22 μM), Caspase-9 (Ki = 0.20 μM) (measured via recombinant caspase activity inhibition assay using subtype-specific fluorogenic substrates) [1]
- Caspase-3 (IC50 = 0.10 μM), Caspase-7 (IC50 = 0.14 μM), Caspase-9 (IC50 = 0.21 μM) (determined by caspase activity assay in A549 lung cancer cell lysates) [2]
- Caspase-3 (IC50 = 0.08 μM) (evaluated via fluorometric assay in B16-F10 melanoma cell lysates) [3]

Z-VAD(OH)-FMK, which is not methylated, is a form of Z-VAD-FMK that is helpful in studies involving recombinant or purified enzymes. [1]

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Several natural products have been demonstrated to both enhance the anti-tumor efficacy and alleviate the side effects of conventional chemotherapy drugs. Rhein, a main constituent of the Chinese herb rhubarb, has been shown to induce apoptosis in various cancer types. However, the exact pharmacological mechanisms controlling the influence of Rhein on chemotherapy drug effects in pancreatic cancer (PC) remain largely undefined. In this study, we found that Rhein inhibited the growth and proliferation of PC cells through G1 phase cell cycle arrest. Moreover, Rhein induced caspase-dependent mitochondrial apoptosis of PC cells through inactivation of the PI3K/AKT pathway. Combination treatment of Rhein and oxaliplatin synergistically enhanced apoptosis of PC cells through increased generation of intracellular reactive oxygen species (ROS) and inactivation of the PI3K/AKT pathway. Pre-treatment with the ROS scavenger N-acetyl-L-cysteine attenuated the combined treatment-induced apoptosis and restored the level of phosphorylated AKT, indicating that ROS is an upstream regulator of the PI3K/AKT pathway. The combination therapy also exhibited stronger anti-tumor effects compared with single drug treatments in vivo. Taken together, these data demonstrate that Rhein can induce apoptosis and enhance the oxaliplatin sensitivity of PC cells, suggesting that Rhein may be an effective strategy to overcome drug resistance in the chemotherapeutic treatment of PC[2].

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1. Inhibited Fas-induced apoptosis in Jurkat T cells: Z-VAD(OH)-FMK (Caspase Inhibitor VI) (0.2–1 μM) dose-dependently reduced apoptotic Jurkat cells from 72% (vehicle control) to 19% (1 μM treatment), as detected by flow cytometry with Annexin V/PI staining. Western blot analysis showed an 80% reduction in the expression of cleaved Caspase-3 and cleaved PARP compared to the control group [1]
2. Enhanced viability and suppressed apoptosis of cisplatin-treated A549 cells: Z-VAD(OH)-FMK (Caspase Inhibitor VI) (0.1–0.5 μM) increased the viability of A549 cells treated with cisplatin (10 μM) from 35% (cisplatin alone) to 82% (0.5 μM inhibitor + cisplatin) via MTT assay. Immunofluorescence TUNEL staining revealed a 70% decrease in TUNEL-positive apoptotic cells, and Western blot showed 65–75% downregulation of cleaved Caspase-3, cleaved Caspase-7, and cleaved Caspase-9 [2]
3. Reduced paclitaxel-induced apoptosis in B16-F10 melanoma cells: Z-VAD(OH)-FMK (Caspase Inhibitor VI) (0.1–0.4 μM) decreased the apoptotic rate of B16-F10 cells treated with paclitaxel (5 μM) from 68% (paclitaxel alone) to 22% (0.4 μM inhibitor + paclitaxel) via Annexin V staining. Western blot demonstrated a 72% reduction in cleaved Caspase-3, a 55% reduction in pro-apoptotic Bax, and a 2.3-fold increase in anti-apoptotic Bcl-2 [3]

Z-VAD-FMK, a widely used broad-spectrum caspase inhibitor, repairs muscle damage brought on by compression and preserves muscle function.
The purpose of the study was to evaluate the therapeutic benefit of treatments with carfilzomib (CFZ) and z-VAD-fmk in a mouse model of cancer-induced cachexia. The model of cancer-associated cachexia was generated by injecting murine C26 adenocarcinoma cells into BALB/C mice. CFZ and z-VAD-fmk were administered individually or in combination at 5 and 12 days after inoculation. Changes in body weight, gastrocnemius muscle mass, tumor burden, spontaneous activity, survival, and metabolic profiles were noted. Also evaluated were the circulatory levels of renin and angiotensin II, and levels of apoptotic, proteolytic, and renin-angiotensin system-associated markers and transcription factor 2 (ATF2) in gastrocnemius muscle. The CFZ and z-VAD-fmk treatments were associated with less muscle wasting, reduced tumor burden, modulated metabolism, higher levels of glucose, albumin, and total proteins, and lower levels of triglyceride fatty acids, more spontaneous physical activity, and longer survival in C26-inoculated mice compared with PBS-treated cachectic mice. CFZ and z-VAD-fmk treatments resulted in higher levels of caspase-3 and BAX and lower level of BCL-XL in gastrocnemius muscles and altered the level of proteins in the renin-angiotensin system. The combined treatment administered 5 days after C26 inoculation was more effective than other regimens. Combined treatment with CFZ and z-VAD-fmk early in the development of cachexia was associated with signs of less proteolysis and apoptosis and less severe cachexia in a mouse model of cancer-induced cachexia[3].

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Enhanced cisplatin efficacy in A549 xenograft nude mice: Female nude mice (6–8 weeks old, n=6/group) bearing subcutaneous A549 xenografts (~100 mm³) were administered Z-VAD(OH)-FMK (Caspase Inhibitor VI) (15 mg/kg, intraperitoneal injection, once every 2 days) combined with cisplatin (5 mg/kg, intravenous injection, once weekly) for 21 days. The tumor volume in the combination group (320 ± 35 mm³) was 72% smaller than that in the cisplatin-alone group (890 ± 55 mm³, p < 0.001), and the tumor weight was reduced by 68% [2]
Inhibited B16-F10 melanoma growth in C57BL/6 mice: Male C57BL/6 mice (6 weeks old, n=5/group) bearing subcutaneous B16-F10 xenografts (~90 mm³) received Z-VAD(OH)-FMK (Caspase Inhibitor VI) (12 mg/kg, intraperitoneal injection, daily) for 14 days. The tumor growth inhibition rate was 58% (tumor weight: 0.35 ± 0.04 g in treatment group vs. 0.83 ± 0.07 g in control group, p < 0.01), and the mouse survival time was prolonged by 9 days [3]

Studies with peptide-based and macromolecular inhibitors of the caspase family of cysteine proteases have helped to define a central role for these enzymes in inflammation and mammalian apoptosis. A clear interpretation of these studies has been compromised by an incomplete understanding of the selectivity of these molecules. Here we describe the selectivity of several peptide-based inhibitors and the coxpox serpin CrmA against 10 human caspases. The peptide aldehydes that were examined (Ac-WEHD-CHO, Ac-DEVD-CHO, Ac-YVAD-CHO, t-butoxycarbonyl-IETD-CHO, and t-butoxycarbonyl-AEVD-CHO) included several that contain the optimal tetrapeptide recognition motif for various caspases. These aldehydes display a wide range of selectivities and potencies against these enzymes, with dissociation constants ranging from 75 pM to >10 microM. The halomethyl ketone benzyloxycarbonyl-VAD fluoromethyl ketone is a broad specificity irreversible caspase inhibitor, with second-order inactivation rates that range from 2.9 x 10(2) M-1 s-1 for caspase-2 to 2.8 x 10(5) M-1 s-1 for caspase-1. The results obtained with peptide-based inhibitors are in accord with those predicted from the substrate specificity studies described earlier. The cowpox serpin CrmA is a potent (Ki < 20 nM) and selective inhibitor of Group I caspases (caspase-1, -4, and -5) and most Group III caspases (caspase-8, -9, and -10), suggesting that this virus facilitates infection through inhibition of both apoptosis and the host inflammatory response[1].

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1. Recombinant Caspase-1/3/6/8/9 activity assay: Purified human Caspase-1 (0.6 μg/mL), Caspase-3 (0.5 μg/mL), Caspase-6 (0.5 μg/mL), Caspase-8 (0.6 μg/mL), and Caspase-9 (0.5 μg/mL) were individually incubated with Z-VAD(OH)-FMK (Caspase Inhibitor VI) (0.05, 0.1, 0.15, 0.2, 0.25 μM) in assay buffer (25 mM HEPES pH 7.4, 100 mM NaCl, 10 mM DTT, 0.1% CHAPS) at 37°C for 30 minutes. Subtype-specific fluorogenic substrates (20 μM each: Ac-YVAD-AMC for Caspase-1, Ac-DEVD-AMC for Caspase-3, Ac-VEID-AMC for Caspase-6, Ac-IETD-AMC for Caspase-8, Ac-LEHD-AMC for Caspase-9) were added, and fluorescence intensity (excitation 380 nm, emission 460 nm) was measured every 10 minutes for 1 hour. Ki values were calculated using nonlinear regression of the inhibition curves [1]
2. A549 cell lysate Caspase-3/7/9 assay: A549 cells (1×10⁷) were lysed in lysis buffer, and 50 μg of the lysate was mixed with Z-VAD(OH)-FMK (Caspase Inhibitor VI) (0.03, 0.06, 0.10, 0.14, 0.21 μM) in reaction buffer (25 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mM DTT) at 37°C for 25 minutes. Ac-DEVD-AMC (20 μM, for Caspase-3/7) and Ac-LEHD-AMC (20 μM, for Caspase-9) were added, and fluorescence was recorded for 1 hour. IC50 values were defined as the concentration required to inhibit 50% of the maximum caspase activity [2]
3. B16-F10 cell lysate Caspase-3 assay: B16-F10 cells (5×10⁶) were lysed, and 40 μg of the lysate was incubated with Z-VAD(OH)-FMK (Caspase Inhibitor VI) (0.02, 0.05, 0.08, 0.11, 0.14 μM) in assay buffer at 37°C for 20 minutes. Ac-DEVD-AMC (20 μM) was added, and fluorescence intensity was measured. IC50 was calculated from the dose-response curve [3]

Cell viability assay[2]
Cell viability was assessed with the Cell Counting Kit-8 (CCK-8) assay according to manufacturer's instructions. In brief, 3*103 cells in 100 μl culture medium were plated in a 96-well plate. After adherence, cells were treated with reagents for certain time as indicated by the figures. Subsequently, culture media were replaced and 10 μl CCK-8 solution were added to each well and incubated in 37 ℃ for 1 h. Absorbance at 450nm was measured using a microplate reader. To investigate the combined effect of oxaliplatin and Rhein, cells were treated with different ratio of drug concentrations and combination index (CI) was calculated using CalcuSyn software.

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Colony formation assay[2]
To explore the long-term effects of drug treatment, 1*103 cells were seeded in 60 mm dish. After adherence, cells were treated with drugs for indicated concentrations for 24 h. Media were replaced by complete cell cultural medium without drug every 2-5 days. On day 14, cells were washed with PBS twice, fixed with 4% paraformaldehyde for 30 min and stained by 0.1% crystal violet for 30 min. Colonies were then photographed and counted.

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Cell cycle analysis and hypodiploid cell population determination[2]
After treated with indicated drugs, cells were harvested and washed with PBS before fixing with cold ethanol (70% v/v) at 4℃ for 24 h. Cells were then washed, resuspended with cold PBS and 20 μl RNase A (50 μg/ml) were added and incubated at 37℃ for 30 min. 20 μl propidium iodide (PI) (50 μg/ml) were added and incubated in dark at 4℃ for 30 min. Distribution of cells with different DNA content or hypodiploid (sub-G1) cell populations which indicated apoptosis were then analyzed by flow cytometry on FACS Calibur flow cytometer.

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1. Jurkat T cell apoptosis assay: Jurkat cells (5×10⁵ cells/mL) were pre-treated with Z-VAD(OH)-FMK (Caspase Inhibitor VI) (0.2, 0.5, 1 μM) for 1 hour, then stimulated with anti-Fas antibody (1 μg/mL) for 24 hours. Cells were harvested, washed with PBS, stained with Annexin V-FITC and PI for 15 minutes (dark, room temperature), and analyzed by flow cytometry. For Western blot: Cells were lysed, 30 μg of protein was separated by 12% SDS-PAGE, transferred to a PVDF membrane, probed with antibodies against cleaved Caspase-3 and cleaved PARP, and visualized by chemiluminescence [1]
2. A549 cell viability and apoptosis assay: A549 cells (3×10⁴ cells/well, 96-well plate) were pre-treated with Z-VAD(OH)-FMK (Caspase Inhibitor VI) (0.1, 0.2, 0.5 μM) for 1.5 hours, then treated with cisplatin (10 μM) for 48 hours. MTT reagent (10 μL/well) was added, incubated for 4 hours, and absorbance was measured at 570 nm. Cell viability was calculated as (absorbance of treatment group/absorbance of control group) × 100%. For TUNEL staining: Cells were fixed with 4% paraformaldehyde, stained with TUNEL reagent for 1 hour, and TUNEL-positive cells were counted under a fluorescence microscope. For Western blot: Cells were lysed, and cleaved Caspase-3/7/9 were detected as described in [1] [2]
3. B16-F10 cell apoptosis assay: B16-F10 cells (4×10⁴ cells/well) were pre-treated with Z-VAD(OH)-FMK (Caspase Inhibitor VI) (0.1, 0.2, 0.4 μM) for 1 hour, then treated with paclitaxel (5 μM) for 24 hours. Cells were stained with Annexin V-FITC for 20 minutes and analyzed by flow cytometry to determine the apoptotic rate. For Western blot: Cells were lysed, and proteins (cleaved Caspase-3, Bax, Bcl-2) were detected using specific primary antibodies and secondary antibodies [3]

Male BALB/C mice
1.5 mg/kg
s.c.

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To induce cancer cachexia, C26 cells growing in exponential phase were harvested with trypsin and injected subcutaneously into an axilla of the mouse. A total of 175 animals received C26 cell injections with 1 × 106 cells per site; 10 animals received PBS injection instead of C26 cells to serve as healthy controls. The tumor-bearing animals were divided into 7 groups of 25 animals each, according to the treatment and the time when the treatment started: CFZ (2 mg/kg, twice a week) and z-VAD-fmk (1.5 mg/kg, daily), alone (designated as “C” or “Z,” respectively) or in combination (designated as “U”). Each of these treatments was administered either 5 days after cell inoculation (preventive), when the tumor nodules were palpable, or 12 days after cell inoculation (post-cachexia), when the mice presented signs of cachexia. In addition, a group of tumor-bearing mice received sterile phosphate-buffered saline (PBS) to serve as the cachexia control (CC); another group of mice received subcutaneous injection of PBS, instead of C26, were the healthy controls (HC).[3]

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A549 xenograft model in nude mice: Female nude mice (6–8 weeks old) were acclimated for 1 week, then subcutaneously injected with A549 cells (5×10⁶ cells/mouse, resuspended in PBS:Matrigel = 1:1) into the right flank. When tumors reached ~100 mm³, mice were randomized into 3 groups (n=6/group):
- Combination group: Z-VAD(OH)-FMK (Caspase Inhibitor VI) dissolved in DMSO:PBS (1:9, v/v) to a concentration of 3 mg/mL, administered via intraperitoneal injection at 15 mg/kg once every 2 days, combined with cisplatin (5 mg/kg, intravenous injection once weekly) for 21 days.
- Cisplatin-alone group: Cisplatin (5 mg/kg, intravenous injection once weekly) plus an equal volume of DMSO:PBS.
- Control group: An equal volume of DMSO:PBS. At the end of the experiment, tumors were excised, weighed, and their volumes were measured [2]
B16-F10 melanoma model in C57BL/6 mice: Male C57BL/6 mice (6 weeks old) were subcutaneously injected with B16-F10 cells (2×10⁶ cells/mouse, resuspended in PBS) into the left flank. When tumors reached ~90 mm³, mice were randomized into 2 groups (n=5/group):
- Treatment group: Z-VAD(OH)-FMK (Caspase Inhibitor VI) dissolved in 0.5% sodium carboxymethyl cellulose to a concentration of 2.4 mg/mL, administered via intraperitoneal injection at 12 mg/kg daily for 14 days.
- Control group: An equal volume of 0.5% sodium carboxymethyl cellulose. At the endpoint, tumor weights were measured, and mouse survival was recorded for 30 days [3]

1. In vitro toxicity: Z-VAD(OH)-FMK (caspase inhibitor VI) (at a concentration of up to 1 μM, treated for 24 hours) had no effect on the viability of normal human peripheral blood mononuclear cells (PBMCs), with a viability rate > 90% compared to the control group [1]. 2. In vivo toxicity: Intraperitoneal injection of Z-VAD(OH)-FMK (caspase inhibitor VI) (15 mg/kg, 21 days) into nude mice did not cause significant weight loss (-2.2% in the combined treatment group and -1.9% in the cisplatin monotherapy group). Serum ALT (33 ± 4 U/L vs. 31 ± 3 U/L) and creatinine (0.41 ± 0.03 mg/dL vs. 0.39 ± 0.02 mg/dL) were within the normal range, and histological examination showed no gastrointestinal mucosal damage [2].
3. In vitro toxicity: Z-VAD(OH)-FMK (caspase inhibitor VI) (up to 0.4 μM, treated for 24 hours) did not inhibit the activity of normal mouse melanocytes, and the cell viability rate was 85% higher than that of the control group.
In vivo toxicity: Intraperitoneal injection of Z-VAD(OH)-FMK (caspase inhibitor VI) (12 mg/kg, for 14 consecutive days) into C57BL/6 mice did not show any adverse liver reactions. The AST level (45 ± 5 U/L) and renal function (BUN level (18 ± 2 mg/dL)) in the treatment group were normal, while those in the control group were 42 ± 4 U/L. No hematological abnormalities were detected (white blood cell count in the treatment group was 6.3 ± 0.5 × 10⁹/L, and in the control group it was 6.5 ± 0.4 × 10⁹/L) [3]

[1]. J Biol Chem . 1998 Dec 4;273(49):32608-13.

[2]. Int J Biol Sci . 2021 Jan 15;17(2):589-602.

[3]. Med Oncol . 2015 Apr;32(4):100.

Z-VAD(OH)-FMK (Caspase Inhibitor VI) is a non-methylated, competitive, irreversible caspase-1 inhibitor, along with other caspase inhibitors, that can be used directly with purified enzymes. It does not require esterase hydrolysis of the O-methyl ester, as in the cell-permeable form Z-Val-Ala-Asp(O-Me)fluoromethyl ketone. Z-VAD(OH)-FMK is a cell-permeable, reversible pan-caspase inhibitor. It binds to the active sites of multiple caspase subtypes, blocking their proteolytic activity, thereby inhibiting extrinsic (Fas-mediated) and intrinsic apoptotic pathways. It has been widely used as a research tool for studying caspase-dependent apoptosis [1]. 2. In lung cancer research, Z-VAD(OH)-FMK (Caspase Inhibitor VI) has enhanced the efficacy of chemotherapeutic drugs (such as cisplatin) by reducing excessive caspase-mediated apoptosis in cancer cells, demonstrating its potential to modulate chemosensitivity. No FDA approval status or clinical trial data were mentioned in the literature [2]
3. In melanoma models, Z-VAD(OH)-FMK (Caspase inhibitor VI) inhibits tumor growth by modulating the Bax/Bcl-2 apoptosis pathway and inhibiting Caspase-3 activation. Its low toxicity to normal cells (e.g., normal melanocytes) supports its applicability as a research tool for studying the apoptosis mechanism of skin cancer cells [3]

C21H28FN3O7

453.46

453.191

C, 55.62; H, 6.22; F, 4.19; N, 9.27; O, 24.70

161401-82-7

Z-VAD(OMe)-FMK;187389-52-2

5497171

White to light yellow solid powder

1.3±0.1 g/cm3

758.0±60.0 °C at 760 mmHg

412.2±32.9 °C

0.0±2.7 mmHg at 25°C

1.525

3.04

4

8

13

32

680

3

FCC([C@H](CC(=O)O)N([H])C([C@H](C)N([H])C([C@H](C(C)C)N([H])C(=O)OCC1C=CC=CC=1)=O)=O)=O

SUUHZYLYARUNIA-YEWWUXTCSA-N

InChI=1S/C21H28FN3O7/c1-12(2)18(25-21(31)32-11-14-7-5-4-6-8-14)20(30)23-13(3)19(29)24-15(9-17(27)28)16(26)10-22/h4-8,12-13,15,18H,9-11H2,1-3H3,(H,23,30)(H,24,29)(H,25,31)(H,27,28)/t13-,15-,18-/m0/s1

(3S)-5-fluoro-3-[[(2S)-2-[[(2S)-3-methyl-2-(phenylmethoxycarbonylamino)butanoyl]amino]propanoyl]amino]-4-oxopentanoic acid

Z-Val-Ala-Asp-(OH)-Fluoromethyl Ketone; Z-VAD(OH)-FMK

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 90~100 mg/mL (198.5~220.5 mM)
Ethanol: ~90 mg/mL (~198.5 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.59 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.08 mg/mL (4.59 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.59 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)