Macroautophagy/autophagy[1]
In A549 cells and HUVEC, CA-5f (0-40 μM, 6 hours) elevates the levels of SQSTM1 protein and LC3B-II (an autophagy monitoring marker) in a concentration- and time-dependent manner [1]. A549 cells and HUVECs treated with CA-5f (20 μM, 6 hours) or in combination with Bafilomycin A1 (100 nM) or Chloroquine (30 μM) do not exhibit autophagosome degradation [1]. Neither the number of lysosomes nor the hydrolytic function are impacted by CA-5f (20 μM) [1]. CA-5f (20 μM, 96 hours) is less harmful to normal HUVECs and suppresses the development of A549 cells [1].

In A549 cells and HUVEC, CA-5f (0-40 μM, 6 hours) elevates the levels of SQSTM1 protein and LC3B-II (an autophagy monitoring marker) in a concentration- and time-dependent manner [1]. A549 cells and HUVECs treated with CA-5f (20 μM, 6 hours) or in combination with Bafilomycin A1 (100 nM) or Chloroquine (30 μM) do not exhibit autophagosome degradation [1]. Neither the number of lysosomes nor the hydrolytic function are impacted by CA-5f (20 μM) [1]. CA-5f (20 μM, 96 hours) is less harmful to normal HUVECs and suppresses the development of A549 cells [1].

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CA-5f induces cytoplasmic vacuolization in both A549 non-small cell lung cancer (NSCLC) cells and human umbilical vein endothelial cells (HUVECs) in a concentration- and time-dependent manner (0-40 μM, 3-24 hours).[1]
CA-5f elevates the protein levels of the autophagy marker LC3B-II and the selective autophagy substrate SQSTM1/p62 in A549 cells and HUVECs in a concentration- (0-40 μM) and time- (3-24 hours) dependent manner, while SQSTM1 mRNA levels remain unchanged, suggesting inhibition of autophagic flux rather than induction.[1]
CA-5f significantly increases the number of GFP-LC3B puncta (autophagosomes) in A549 cells and HUVECs, as observed by fluorescence microscopy.[1]
Transmission electron microscopy (TEM) reveals an increased number of vesicular structures and double-membrane autophagosomes in CA-5f-treated A549 cells and HUVECs.[1]
The accumulation of LC3B-II induced by CA-5f is not abolished by co-treatment with early-stage autophagy inhibitors 3-methyladenine (3-MA) or wortmannin. Co-treatment with late-stage inhibitors chloroquine (CQ) or bafilomycin A1 does not produce an additive effect on LC3B-II accumulation compared to CA-5f alone, confirming CA-5f acts as a late-stage autophagy inhibitor.[1]
Using the tandem mRFP-GFP-LC3B reporter, CA-5f treatment leads to a marked increase in yellow puncta (autophagosomes) and a decrease in red-only puncta (autolysosomes) in both A549 cells and HUVECs, indicating impaired autophagic flux. This effect is similar to but more potent than CQ.[1]
CA-5f treatment disrupts the colocalization of GFP-LC3B with LysoTracker Red, and of endogenous LC3B with the lysosomal marker LAMP1, demonstrating that it suppresses the fusion of autophagosomes with lysosomes.[1]
CA-5f does not affect lysosomal pH, as evidenced by sustained red fluorescence in acridine orange (AO) and LysoTracker Red staining, unlike bafilomycin A1. It also does not impair the hydrolytic function of lysosomes, as the activities of acid phosphatase (ACP), cathepsin B (CTSB), and cathepsin D (CTSD) remain unchanged in treated cells.[1]
An iTRAQ-based proteomic screen in HUVECs treated with CA-5f for 1 hour identified downregulation of proteins associated with cytoskeleton and membrane vesicle trafficking, including LM07, ACTB, DNM2, SEPT8, SNX18, SNX5, and SYN1. Western blot validation confirmed these decreases in both HUVECs and A549 cells.[1]
CA-5f significantly increases mitochondrial-derived reactive oxygen species (ROS) production and decreases mitochondrial membrane potential (MMP) in A549 cells and HUVECs. This ROS increase can be reversed by the antioxidant N-acetyl cysteine (NAC).[1]
Real-time cell analysis (RTCA) shows that 20 μM CA-5f initially arrests growth of both A549 cells and HUVECs within 12 hours. However, HUVECs recover and resume growth, whereas A549 cells do not, indicating greater cytotoxicity against cancer cells.[1]
CA-5f induces apoptosis in A549 cells and HUVECs, as shown by Annexin V/PI staining, increased PARP1 cleavage, and ultrastructural features like nuclear pyknosis and fragmentation. It also induces necrosis in A549 cells, evidenced by elevated lactate dehydrogenase (LDH) activity in a dose-dependent manner (0-40 μM).[1]
The cytotoxicity of CA-5f (apoptosis, necrosis, reduced cell viability) is dependent on ROS generation, as co-treatment with NAC significantly reverses these effects.[1]
The cytotoxic effect of CA-5f is weaker in A549 cells where autophagy initiation is suppressed by siRNA against ATG5 or PIK3C3, suggesting that constitutive autophagy contributes to CA-5f-induced cell death.[1]

In nude mice containing A549 lung cancer cells, CA-5f (40 mg/kg, intraperitoneal injection, once every two days, up to 30 days) is well tolerated and can successfully stop tumor growth [1]. Using A549 lung cancer cells, CA-5f (40 mg/kg, ip) suppresses autophagy flux and causes apoptosis in nude mice [1].

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In nude mice bearing subcutaneous A549 lung cancer xenografts, intravenous injection of CA-5f (40 mg/kg) every 2 days for 30 days significantly suppressed tumor volume and weight growth compared to the vehicle control group, without causing significant body weight loss or obvious toxicity.[1]
Western blot analysis of tumor tissues from CA-5f-treated mice showed increased accumulation of LC3B-II and SQSTM1 proteins.[1]
Immunofluorescence staining of tumor sections from CA-5f-treated mice revealed a punctate pattern of LC3B and SQSTM1, contrasting with the diffuse pattern in controls, confirming autophagy inhibition in vivo. No obvious changes in CTSB and CTSD levels were observed.[1]
TUNEL staining of tumor sections showed a significant increase in apoptotic cells in the CA-5f-treated group compared to the control.[1]

The activity of acid phosphatase (ACP), a lysosomal marker enzyme, was measured in cell lysates from A549 cells and HUVECs treated with CA-5f (20 μM) for various time points. The absorbance of the reaction mixture was measured at 405 nm using a spectrophotometer.[1]
The enzymatic activities of cathepsin B (CTSB) and cathepsin D (CTSD) were measured in cell lysates from A549 cells and HUVECs treated with CA-5f (20 μM) for various time points using commercial fluorometric assay kits. Fluorescence was read with specific excitation/emission wavelengths (400/505 nm for CTSB, 328/460 nm for CTSD) in a microplate reader.[1]

Cell Viability Assay[1]
Cell Types: A549, HUVECs
Tested Concentrations: 20 μM
Incubation Duration: 96 hrs (hours)
Experimental Results: demonstrated more cytotoxicity against A549 cells compared with normal HUVECs.

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Western Blot Analysis[1]
Cell Types: A549 cells and HUVECs
Tested Concentrations: 0 -40 μM
Incubation Duration: 6 hrs (hours)
Experimental Results: Elevated LC3B-II (a marker to monitor autophagy) and SQSTM1 protein levels in a concentration- and time-dependent manner.

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For western blot analysis of autophagy markers, cells were treated with CA-5f, harvested, and lysed. Equal amounts of protein were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against LC3B, SQSTM1, and other targets. Signals were detected using fluorescence-labeled or HRP-conjugated secondary antibodies and quantified.[1]
For GFP-LC3B puncta analysis, A549 cells or HUVECs were transfected with a GFP-LC3B plasmid. After treatment with CA-5f, cells were fixed, nuclei stained, and imaged by fluorescence microscopy. Cells with GFP-LC3B puncta were quantified.[1]
For the mRFP-GFP-LC3B tandem reporter assay, cells were transfected with the reporter construct. After treatment with CA-5f, cells were imaged live or fixed. Yellow (autophagosomes) and red-only (autolysosomes) puncta were counted per cell.[1]
For colocalization studies, cells transfected with GFP-LC3B were stained with LysoTracker Red. Alternatively, cells were fixed and immunostained for endogenous LC3B and LAMP1. Images were acquired by confocal microscopy to assess colocalization.[1]
For lysosomal pH assessment, cells were stained with acridine orange (AO) or LysoTracker Red after CA-5f treatment and imaged by fluorescence microscopy.[1]
For cell viability and cytotoxicity assays, real-time cell analysis (RTCA) was performed by seeding cells in special plates and monitoring impedance (cell index) continuously after CA-5f addition. Alternatively, cell viability was assessed by MTT assay after 24-hour treatment.[1]
Apoptosis was assessed by flow cytometry using Annexin V-FITC/PI double staining after treating cells with CA-5f for 24 hours.[1]
Lactate dehydrogenase (LDH) activity in the cell culture supernatant was measured spectrophotometrically at 340 nm after treating cells with CA-5f for 24 hours as an indicator of necrosis.[1]
For proteomic analysis, HUVECs were treated with CA-5f (20 μM) for 1 hour. Cells were harvested, proteins were extracted, digested, labeled with iTRAQ reagents, and analyzed by two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D LC-MS/MS). Differentially expressed proteins were identified based on fold change and statistical significance.[1]
Mitochondrial ROS levels were measured using MitoSOX Red staining and flow cytometry or fluorescence microscopy. Mitochondrial membrane potential was assessed using the JC-1 dye kit.[1]
Cellular ROS levels were measured using a fluorescent ROS assay kit.[1]

Animal/Disease Models: Nude mice bearing A549 lung cancer cells[1]
Doses: 40 mg/kg
Route of Administration: Injected via caudal vein, every 2 days for up to 30 days
Experimental Results: Dramatically suppressed tumor volume and weight in mice, increased the number of apoptotic cells in mice.

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Four-week-old BALB/c nude mice were subcutaneously injected with A549 cells (1 x 10^6 cells) at the right scapula. When palpable tumors formed (around day 8 post-implantation), mice were randomly divided into control and treatment groups (n=5 per group). CA-5f was dissolved in DMSO as a stock solution and diluted for injection. The treatment group received CA-5f (40 mg/kg body weight) via caudal vein injection every 2 days for a total of 30 days. The control group received vehicle (DMSO) on the same schedule. Tumor dimensions were measured every 2 days to calculate volume. Mice were sacrificed at the end of the experiment, tumors were resected, weighed, and processed for further analysis (western blot, immunofluorescence, TUNEL).[1]

In the A549 xenograft mouse model, intravenous administration of CA-5f (40 mg/kg, every 2 days for 30 days) was well tolerated, with no obvious toxicity observed, and there was no significant difference in average body weight compared to the control group. [1]
In vitro experiments showed that CA-5f had lower cytotoxicity to normal human umbilical vein endothelial cells (HUVECs) compared to A549 non-small cell lung cancer cells, because HUVECs were able to recover from initial growth arrest, while A549 cells were not. [1]

[1]. Identification of compound CA-5f as a novel late-stage autophagy inhibitor with potent anti-tumor effect against non-small cell lung cancer. Autophagy. 2019 Mar;15(3):391-406.

CA-5f (chemical name: (3E,5E)-3-(3,4-dimethoxybenzyl)-5-[(1H-indol-3-yl)methylene]-1-methylpiperidin-4-one) is a synthetic curcumin analogue. [1] It is considered a novel, potent late autophagy inhibitor that blocks the fusion of autophagosomes with lysosomes without affecting the pH or hydrolytic function of lysosomes. [1] Its mechanism of action is thought to involve the downregulation of cytoskeletal proteins (e.g., LM07, ACTB, DNM2) and membrane vesicle transport proteins (e.g., SNX18, SNX5), which are essential for the motility and fusion of autophagosomes. [1] CA-5f can induce the production of mitochondrial-derived reactive oxygen species (ROS), which contributes to its cytotoxic effects, especially in cancer. CA-5f exhibits selective cytotoxicity against non-small cell lung cancer (NSCLC) cells (such as A549) but no significant cytotoxicity against normal endothelial cells (HUVECs) and other epithelial cells. [1] As a single drug, CA-5f showed significant antitumor activity in an A549 xenograft mouse model, suggesting its potential as a therapeutic agent for NSCLC. [1]

C24H24N2O3

388.46

388.178

1370032-19-1

57341148

Light yellow to yellow solid powder

3.7

1

4

4

29

659

0

C(/C1=CNC2=CC=CC=C12)=C1\C(/C(/CN(C)C\1)=C/C1C=CC(OC)=C(OC)C=1)=O

JYOLPDWVAMBMQN-UBIAKTOFSA-N

InChI=1S/C24H24N2O3/c1-26-14-18(10-16-8-9-22(28-2)23(11-16)29-3)24(27)19(15-26)12-17-13-25-21-7-5-4-6-20(17)21/h4-13,25H,14-15H2,1-3H3/b18-10+,19-12+

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.35 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 (5.35 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 (5.35 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.)