2-MCA inhibited the proliferation of human lung adenocarcinoma A549 cells in a dose- and time-dependent manner, as determined by XTT assay. The half maximal inhibitory concentration (IC50) value after 48 hours of incubation was 23.11 µM. [1]
2-MCA induced apoptosis in A549 cells, as evidenced by increased pro-apoptotic Bax and Bak protein levels, decreased anti-apoptotic Bcl-2 and Bcl-XL protein levels, loss of mitochondrial membrane potential (ΔΨm), release of cytochrome c from mitochondria into the cytosol, and activation of caspase-3 and caspase-9. [1]
2-MCA treatment caused morphological changes characteristic of apoptosis, including plasma membrane blebbing, nuclear condensation, fragmentation, and apoptotic body formation, as observed by acridine orange staining and comet assay. [1]
2-MCA induced lysosomal vacuolation and increased the volume of acidic compartment (VAC) in A549 cells in a dose-dependent manner. [1]
2-MCA inhibited the DNA relaxation activity of both topoisomerase I and topoisomerase II from A549 cell nuclear extracts in a concentration-dependent manner, as shown by DNA relaxation assays. The inhibition of topoisomerase II was further confirmed by a decatenation assay. [1]
2-MCA decreased NF-κB DNA binding activity in A549 cells in a dose-dependent manner. [1]

In a nude mouse xenograft model, intratumoral injection of 2-MCA at doses of 10 or 20 mg/kg/day significantly suppressed the growth of subcutaneously implanted A549 tumors, resulting in approximately 58% reduction in tumor size compared to the vehicle control group after 49 days of treatment. [1]
Tumors from mice treated with 2-MCA (10 mg/kg/day) showed an increased number of TUNEL-positive cells compared to tumors from vehicle-treated mice, indicating induction of apoptosis in vivo. [1]
Treatment with 2-MCA at 5, 10, or 20 mg/kg/day did not cause any significant decrease in diet consumption or body weight change in the mice compared to the control group. [1]

Topoisomerase I Activity Assay: Nuclear proteins extracted from A549 cells were added to a reaction mixture containing supercoiled pUC19 plasmid DNA. The mixture was incubated with increasing concentrations of 2-MCA, camptothecin (positive control), or vehicle. The reaction products were analyzed by agarose gel electrophoresis. Inhibition of topoisomerase I activity was indicated by a decrease in the conversion of supercoiled DNA to its relaxed form. [1]
Topoisomerase II Activity Assay (DNA Relaxation): Similar to the topoisomerase I assay, nuclear protein extracts were incubated with supercoiled pUC19 DNA and increasing concentrations of 2-MCA or etoposide (positive control). The reduction in DNA relaxation was analyzed by gel electrophoresis. [1]
Topoisomerase II Activity Assay (Decatenation): Nuclear protein extracts were incubated with kinetoplast DNA (kDNA), a network of catenated DNA circles. The reaction mixture contained increasing concentrations of 2-MCA or etoposide. Inhibition of topoisomerase II activity was measured by the decrease in the release of monomeric DNA circles from the kDNA network, as analyzed by agarose gel electrophoresis. [1]
NF-κB DNA Binding Activity Assay: After treating A549 cells with 2-MCA for 24 hours, nuclear extracts were prepared. The DNA binding activity of NF-κB was quantified using a transcription factor ELISA kit according to the manufacturer's instructions. [1]

Cell Viability (XTT Assay): A549 cells were seeded in 96-well plates and incubated for 24 hours. Cells were then treated with various concentrations of 2-MCA for 12, 24, or 48 hours. Cell viability was assessed using a commercial XTT-based cell proliferation kit. Absorbance was measured at 492 nm with a reference wavelength of 650 nm. [1]
Apoptosis and Lysosomal Staining (Acridine Orange): A549 cells were treated with 2-MCA for 48 hours. After treatment, cells were incubated with RNAse to prevent RNA interference, stained with acridine orange solution, and observed under a fluorescence microscope. Apoptotic cells showed orange fluorescence (fragmented/denatured DNA) and lysosomal vacuolation. [1]
DNA Damage (Comet Assay): A549 cells treated with 2-MCA for 48 hours were embedded in agarose on a microscope slide. Cells were lysed, and DNA was unwound in an alkaline solution before electrophoresis. After electrophoresis, DNA was stained and visualized under a fluorescence microscope. DNA strand breaks resulted in a comet-like tail. [1]
Volume of Acidic Compartment (VAC) Assay: A549 cells were treated with 2-MCA for 48 hours, then stained with neutral red dye, which accumulates in acidic lysosomes. The cells were observed under a microscope, and the incorporated dye was extracted and quantified spectrophotometrically to measure VAC. [1]
Western Blot Analysis: A549 cells were treated with 2-MCA for 24 hours, harvested, and lysed. Mitochondrial and cytosolic fractions were separated using a commercial apoptosis assay kit. Protein concentrations were determined, and equal amounts of protein were separated by SDS-PAGE, transferred to a membrane, and probed with specific primary antibodies against Bax, Bak, Bcl-2, Bcl-XL, cytochrome c, and β-actin. Proteins were detected using a chemiluminescence detection kit. [1]
Caspase Activity Assay: A549 cells were treated with 2-MCA for 24 hours. Caspase-3/7 and caspase-9 activities were measured using commercial fluorometric assay kits. The assays are based on the cleavage of synthetic substrates (DEVD-AFC for caspase-3/7 and LEHD-AFC for caspase-9), releasing the fluorescent AFC moiety, which was quantified using a spectrophotometer. [1]
Mitochondrial Membrane Potential (ΔΨm) Assay: A549 cells treated with 2-MCA were harvested and stained with the fluorescent dye JC-1. At high membrane potential, JC-1 forms aggregates that fluoresce red. At low membrane potential, it remains as monomers that fluoresce green. The ratio of red to green fluorescence was measured both microscopically and spectrophotometrically to assess ΔΨm loss. [1]

Xenograft Tumor Model: Male BALB/c nude mice were subcutaneously injected with A549 cells into their flanks. When tumors reached approximately 75 mm³, mice were randomly divided into groups.
2-MCA was administered via intratumoral injection daily for 49 days at doses of 5, 10, or 20 mg/kg/day, dissolved in a vehicle solution (200 µL volume). The control group received an equal volume of vehicle.
Tumor dimensions (length, width, height) were measured weekly with calipers, and tumor volume was calculated using a formula.
At the end of the experiment (day 49), mice were euthanized, tumors were excised and weighed. Tumor apoptosis was assessed by TUNEL assay on tumor sections. [1]

The cited studies did not include specific data on the absorption, distribution, metabolism, excretion, half-life, or oral bioavailability of 2-methoxycinnamoaldehyde (2-MCA). [1]

In in vivo xenograft studies, intratumoral injection of up to 20 mg/kg/day of 2-MCA for 49 days did not cause significant changes in body weight or food intake in nude mice compared to the vector control group. [1]

[1]. Cinnamomum verum Component 2-Methoxycinnamaldehyde: A Novel Anticancer Agent with Both Anti-Topoisomerase I and II Activities in Human Lung Adenocarcinoma A549 Cells In Vitro and In Vivo. Phytother Res. 2016 Feb;30(2):331-40.

[2]. Cinnamomum verum ingredient 2-methoxycinnamaldehyde: a new antiproliferative drug targeting topoisomerase I and II in human lung squamous cell carcinoma NCI-H520 cells. Eur J Cancer Prev. 2017 Jul;26(4):314-323.

[3]. 2-Methoxycinnamaldehyde inhibits the TNF-α-induced proliferation and migration of human aortic smooth muscle cells. Int J Mol Med. 2017 Jan;39(1):191-198.

Cassiastearoptene is one of the cinnamaldehyde compounds.
2-Methoxycinnamaldehyde has been reported to exist in star anise and cinnamon, and there is relevant data.
See also: Cinnamon (part); Chinese cinnamon (part); Cinnamon twig (part)... See more...

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2-Methoxycinnamaldehyde (2-MCA) is a component isolated from the bark of cinnamon (Cinnamomum verum). [1]
Studies have shown that 2-MCA exerts its anticancer effect on human lung adenocarcinoma A549 cells through multiple mechanisms: inducing mitochondrial pathway-dependent apoptosis, inhibiting the activity of topoisomerase I and II, inhibiting NF-κB transcriptional activity, and inducing lysosomal vacuolization. [1] 2-MCA can simultaneously inhibit the activity of topoisomerase I and II, which is a remarkable feature because most inhibitors selectively inhibit only one type of topoisomerase. [1] The in vivo efficacy and no systemic toxicity observed in mouse models suggest that 2-MCA is a potential candidate drug for anticancer therapy. [1]

C10H10O2

162.1852

162.068

1504-74-1

641298

White to yellow solid powder

1.1±0.1 g/cm3

334.8±0.0 °C at 760 mmHg

44-48ºC

134.4±13.9 °C

0.0±0.7 mmHg at 25°C

1.559

2.13

0

2

3

12

163

0

COC1=CC=CC=C1/C=C/C=O

KKVZAVRSVHUSPL-GQCTYLIASA-N

InChI=1S/C10H10O2/c1-12-10-7-3-2-5-9(10)6-4-8-11/h2-8H,1H3/b6-4+

(E)-3-(2-methoxyphenyl)prop-2-enal

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.

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

DMSO : ~100 mg/mL (~616.56 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (15.41 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 25.0 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: ≥ 2.5 mg/mL (15.41 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 25.0 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.)