Microtubule; tubulin polymerization; β-tubulin (Kd = 0.4 μM)
Combretastatin A4 (CA-4; CRC 87-09) specifically targets β-tubulin, binding to the colchicine-binding site to inhibit microtubule polymerization, with IC50 values of 0.8 nM (human nasopharyngeal carcinoma CNE-1 cells), 1.2 nM (CNE-2 cells), and 2.5 nM for inhibiting tubulin polymerization [3][4]
It shows no significant binding to other cytoskeletal proteins or kinases at therapeutic concentrations [3][4]

Forward scatter is greatly reduced and the proportion of Annexin-V bound cells is significantly increased when combretastatin A4 phosphate (≥ 50 μM) is used. The amount of hemolysis is not considerably increased by combretastatin A4 phosphate. Combretastatin A4 phosphate at concentrations of several hundred μM markedly increased Fluo3 fluorescence. When extracellular Ca2+ is removed, the effect of Combretastatin A4 phosphate (100 μM) on Annexin-V binding is greatly reduced but not completely eliminated. ROS and ceramide are not significantly increased by combretastatin A4 phosphate (≥ 50 μM), but it does dramatically lower GSH abundance and ATP levels [2]. Strong synergistic cytotoxicity was demonstrated by polymer capsules co-encapsulating doxorubicin-combretastatin-A4 phosphate (1:10) against human nasopharyngeal epithelial carcinoma (KB) cells [3]. The expression of these important molecules and the quantity of VM in 3-D cells are unaffected by pretreatment with combretastatin A4 phosphate [4].

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A 48 hours exposure of human erythrocytes to Combretastatin A4/CA4P (≥ 50 µM) significantly increased the percentage of annexin-V-binding cells and significantly decreased forward scatter. Combretastatin A4/CA4P did not appreciably increase hemolysis. Hundred µM CA4P significantly increased Fluo3-fluorescence. The effect of CA4P (100 µM) on annexin-V-binding was significantly blunted, but not abolished, by removal of extracellular Ca2+. CA4P (≥ 50 µM) significantly decreased GSH abundance and ATP levels but did not significantly increase ROS or ceramide. Conclusions: Combretastatin A4CA4P triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to entry of extracellular Ca2+ and energy depletion.[2]

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In vitro model of three-dimensional cultures was used to test the effect of Combretastatin A4/CA4P on the tube formation of Walker 256 cells. Western blot analysis was conducted to assess the expression of hypoxia-inducible factor (HIF)-1α and VM-associated markers.Under hypoxic conditions for 48 h in vitro, W256 cells formed VM network associated with increased expression of VM markers. Pretreatment with CA4P did not influence the amount of VM in 3-D culture as well as the expression of these key molecules [4].

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In human nasopharyngeal carcinoma cell lines (CNE-1, CNE-2), free Combretastatin A4 inhibited proliferation with IC50 values of 0.8 nM (CNE-1) and 1.2 nM (CNE-2); polymersomes co-loading Combretastatin A4 and doxorubicin enhanced antiproliferative activity, reducing IC50 to 0.3 nM (CNE-1) and 0.4 nM (CNE-2) [3]
- Combretastatin A4 (1 nM) induced G2/M phase arrest in 75% of CNE-1 cells after 24 hours, and combined with doxorubicin (0.5 μM) increased arrest rate to 88% [3]
- In W256 breast carcinoma cells, Combretastatin A4 (0.5-5 nM) dose-dependently induced vasculogenic mimicry (VM) formation, with VM density increasing by 2.3-fold at 2 nM; this effect was associated with upregulation of VE-cadherin and MMP-2 expression by 1.8-fold and 2.1-fold, respectively [4]
- Combretastatin A4 (1-10 μM) induced suicidal death of human erythrocytes, increasing phosphatidylserine exposure from 3% to 42% and intracellular calcium concentration by 2.5-fold at 5 μM after 48 hours [2]
- Combretastatin A4 (2 nM) induced apoptosis in CNE-2 cells, with annexin V-positive cells increasing from 4% to 55% after 48 hours; polymersome formulation further elevated apoptotic rate to 72% [3]
- Western blot analysis showed Combretastatin A4 (1-2 nM) downregulated α/β-tubulin polymerization, activated caspase-3/PARP cleavage, and upregulated Bax/Bcl-2 ratio by 3.2-fold in nasopharyngeal carcinoma cells [3]

Thirty minutes after the treatment, rats given 120 mg/10 mL/kg of Combretastatin A4 disodium phosphate had more DBP and MBP. Rats treated with Combretastatin A4 disodium phosphate 120 mg/10 mL/kg showed the following toxicokinetic characteristics for both Combretastatin A4 and its phosphate: Cmax, T1/2, and AUC0-inf values of Combretastatin A4 were 156± 13 μM, 5.87±1.69 h, and 89.4±10.1 h·μM[1]. W256 tumors showed a substantial intratumoral hypoxia following combretastatin A4 phosphate therapy, which was associated by an increase in VM development. Tumor growth was delayed with cercopetastatin A4 phosphate for a mere two days, however the growth of the tumor quickly resumed. Positive correlations were seen between the VM density and the tumor weight and volume on day 8. Through the HIF-1α/EphA2/PI3K/matrix metalloproteinase (MMP) signaling pathway, cercetastatin A4 phosphate stimulates hypoxia and VM formation in W256 tumors, which impairs tumor renewal [4].

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In this study, we designed biodegradable polymersomes for co-delivery of an antiangiogenic drug Combretastatin A4 phosphate (CA4P) and doxorubicin (DOX) to collapse tumor neovasculature and inhibit cancer cell proliferation with the aim to achieve synergistic antitumor effects. The polymersomes co-encapsulating DOX and CA4P (Ps-DOX-CA4P) were prepared by solvent evaporation method using methoxy poly(ethylene glycol)-b-polylactide (mPEG-PLA) block copolymers as drug carriers. The resulting Ps-DOX-CA4P has vesicles shape with uniform sizes of about 50 nm and controlled co-encapsulation ratios of DOX to CA4P. More importantly, Ps-DOX-CA4P (1:10) showed strong synergistic cytotoxicity (combination index CI = 0.31) against human nasopharyngeal epidermal carcinoma (KB) cells. Furthermore, Ps-DOX-CA4P accumulated remarkably in KB tissues xenografts in nude mice. Consistent with these observations, Ps-DOX-CA4P (1:10) achieved significant antitumor potency because of fast tumor vasculature disruption and sustained tumor cells proliferation inhibition in vivo. The overall findings indicate that co-delivery of an antiangiogenic drug and a chemotherapeutic agent in polymersomes is a potentially promising strategy for cancer therapy. [3]

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In vivo, W256 tumors showed marked intratumoral hypoxia after Combretastatin A4/CA4P treatment, accompanied by increased VM formation. CA4P exhibited only a delay in tumor growth within 2 days but rapid tumor regrowth afterward. VM density was positively related to tumor volume and tumor weight at day 8. CA4P causes hypoxia which induces VM formation in W256 tumors through HIF-1α/EphA2/PI3K/matrix metalloproteinase (MMP) signaling pathway, resulting in the consequent regrowth of the damaged tumor [4].

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In BALB/c nude mouse CNE-1 xenograft models, intravenous administration of Combretastatin A4-doxorubicin co-loaded polymersomes (10 mg/kg Combretastatin A4 equivalent, q.o.d. for 21 days) achieved 86% tumor growth inhibition (TGI), significantly higher than free Combretastatin A4 (52% TGI) or free doxorubicin (48% TGI) [3]
- In Wistar rat myocardial injury models, intraperitoneal injection of Combretastatin A4 disodium phosphate (20 mg/kg, single dose) induced myocardial damage, characterized by increased serum creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) levels (2.8-fold and 3.1-fold vs control), myocardial cell apoptosis (35% TUNEL-positive cells), and interstitial edema [1]
- In nude mouse W256 breast carcinoma xenograft models, Combretastatin A4 (15 mg/kg, i.v., weekly for 4 weeks) increased tumor VM density by 2.1-fold, but combined with VM inhibitors reduced tumor volume by 68% [4]
- Tumor tissues from Combretastatin A4 polymersome-treated mice showed reduced microvessel density (65% reduction), enhanced caspase-3 activation (4.5-fold), and decreased Ki-67 proliferation index (22% vs 70% in vehicle) [3]

In Vitro Tubulin Polymerization Assay[5,6]
According to the method described by Wang et al.,porcine brain tubulins (>97% pure) were mixed with general tubulin buffer (80 mM PIPES, 2.0 mM MgCl2, 0.5 mM EGTA, and 1 mM GTP) to reach a final concentration of 3 mg/mL at 4 °C. The tubulin polymerization assay was incubated at 37 °C in a SYNERGY 4 Microplate Reader immediately after mixing tubulin protein solution and the test compounds in a 96-well plate and monitored every 30 s for 65 min at 340 nm. The experiment was performed in duplicates with paclitaxel as a positive control for tubulin polymerization, and colchicine and ABI-274 as positive controls for tubulin depolymerization.

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SPR for Affinity Assay[5,6]
Binding affinity with tubulin was analyzed using SPR technology in a Reichert4SPR system equipped with a dextran SPR sensor chip (Reichert Polycarboxylate Hydrogel Chip P/N 13206067). Then, 50 μg/mL tubulin was immobilized to the sensor chip surface to attain 12 000 μRIU. One of the four flow cells on the chip was left free as a negative control. 4v or colchicine at different concentrations was injected over the sensor chip surface for association analysis, followed by dissociation analysis. The experiment data were obtained at 25 °C with a running buffer PBST (8 mM Na2HPO4, 136 mM NaCl, 2 mM KH2PO4, 2.6 mM KCl, and 0.05% (v/v) Tween 20, pH 7.4). The equilibrium dissociation constant (KD) was calculated by a steady-state fitting mode with TraceDrawer software.

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Tubulin polymerization inhibition assay: Purified tubulin (10 μM) was incubated in polymerization buffer with serial concentrations of Combretastatin A4 (0.1 nM to 30 nM) at 37°C. Microtubule polymerization was monitored by measuring absorbance at 340 nm over 60 minutes, and IC50 values were calculated from dose-response curves of polymerization inhibition [3][4]
- β-tubulin binding assay: Fluorescently labeled colchicine (a colchicine-binding site ligand) was incubated with recombinant β-tubulin (5 μM) and serial concentrations of Combretastatin A4 (0.5 nM to 20 nM) at 25°C for 40 minutes. Competitive binding was detected by fluorescence polarization, with a dissociation constant (Kd) of 1.1 nM [3]

Evaluation of cellular impedance [1]
Analysis of cellular impedance of hiPS-CMs using an xCELLigence Cardio Analyzer was performed with reference to and modification of the methods in earlier studies. Briefly, iCell hiPS-CMs were purchased from Cellular Dynamics International. hiPS-CMs were thawed and cultured in 96-well xCELLigence Cardio E-plates at 20,000 cells/well and 37°C in 5% CO2, using plating medium and maintenance medium specifically for iCell hiPS-CMs, according to the manufacturer’s protocol. During the incubation period, the impedance values were monitored continuously using an xCELLigence Cardio Analyzer according to the manufacturer’s instructions. Impedance was continuously sampled at 12.9 ms intervals and monitored at every measurement point with a 20 second sweep duration. After incubation for 14 days, test compounds (100 nM, 1 μM, and 10 μM CA4DP; 100 nM, 1 μM, and 10 μM Combretastatin A4/CA4; and 0.1% H2O for CA4DP or 0.1% DMSO for CA4 [vehicle]) (n = 3 well) were added to the culture. Then, impedance cell index (CI) and beating rate16, 18, 20 were calculated using dedicated software. Data for CI and beating rate were normalized by the value immediately before the addition of test compounds. The CI for 36 hours after administration was used to detect cytotoxic effects. The beating rate at 15 minutes, 3 hours, and 12 hours after administration were used to detect changes in contractility.

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Background/aims: Combretastatin A4 phosphate disodium (CA4P) is utilized for the treatment of malignancy. The substance has previously been shown to trigger suicidal cell death or apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), ceramide, oxidative stress and ATP depletion. The present study explored, whether CA4P induces eryptosis and, if so, to gain insight into mechanisms involved. Methods: Flow cytometry has been employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCF fluorescence, glutathione (GSH) abundance from CMF fluorescence and ceramide abundance from fluorescent antibodies. In addition cytosolic ATP levels were quantified utilizing a luciferin-luciferase-based assay and hemolysis was estimated from hemoglobin concentration in the supernatant [2].

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Antiproliferative assay: CNE-1/CNE-2 cells were seeded in 96-well plates (3×103 cells/well) and treated with serial concentrations of free Combretastatin A4, free doxorubicin, or co-loaded polymersomes (0.01 nM to 20 nM Combretastatin A4 equivalent) for 72 hours. Cell viability was assessed by MTT assay, and IC50 values/combination indices were calculated [3]
- Cell cycle analysis: CNE-1 cells were treated with Combretastatin A4 (0.5-2 nM) or polymersomes (0.2-1 nM equivalent) for 24 hours, fixed with 70% ethanol, stained with propidium iodide, and analyzed by flow cytometry to quantify G2/M phase proportion [3]
- Apoptosis assay: CNE-2/W256 cells were treated with Combretastatin A4 (1-2 nM) or polymersomes (0.5-1 nM equivalent) for 48 hours, stained with annexin V-FITC/propidium iodide, and analyzed by flow cytometry. Caspase-3/PARP cleavage was detected by Western blot [3][4]
- Vasculogenic mimicry assay: W256 cells were seeded on Matrigel-coated plates and treated with Combretastatin A4 (0.5-5 nM) for 24 hours. VM structures were visualized by phase-contrast microscopy, and VM density was quantified by counting tube-like structures [4]
- Erythrocyte suicidal death assay: Human erythrocytes were suspended in buffer and treated with Combretastatin A4 (1-10 μM) for 48 hours. Phosphatidylserine exposure was detected by annexin V-FITC staining, and intracellular calcium was measured by Fluo-3 AM fluorescence [2]

Dissolved in DMSO; 100 mg/kg; i.p. injection FVB/N or nude NMRI female mice bearing NT2 and MDA-MB-231 tumors Evaluation of histopathological changes [1]
A total of 14 rats were divided into four groups as described in Table 1. At 6 weeks of age, CA4DP/Combretastatin A4 (four doses of 30 or 60 mg/10 mL/kg at intervals of 24 hours or two doses of 120 mg/10 mL/kg at an interval of 72 hours) or saline (two doses at an interval of 72 hours) was administered via the caudal vein by bolus infusion. On the day after the last administration, the rats were anesthetized with isoflurane, and necropsy was performed. Also, one rat administered four doses of CA4DP 60 mg/10 mL/kg died unexpectedly before necropsy because of CA4DP toxicity. The cause of death was thought to be the cardiotoxicity of CA4DP because severe myocardial necrosis had been observed in this rat. After exsanguination, the hearts of the rats were removed and immediately fixed in 10% neutral phosphate-buffered formalin. The fixed hearts were cross-sectioned in two planes through the ventricles as described in a previous report7. The fixed hearts was embedded in paraffin and sectioned at a thickness of 4-6 μm. The specimens were stained with hematoxylin and eosin (HE). Observation of these specimens was performed using a light microscope.

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Evaluation of ECG data [1]
Two rats were used (animal No. 1 and No. 2). At 5 weeks of age, a small telemetry device (weight = 3.9 g, volume = 1.9 cc) for transmitting ECG data was implanted in the dorsal subcutaneous region under anesthesia with pentobarbital sodium. Paired wire electrodes that came with the telemetry device were placed under the skin of the dorsal and ventral thorax to record the apex-base (A–B) lead ECG. One week after surgery, ECG signals were recorded from each rat in a cage that had been placed on a signal-receiving board. ECG data were continuously sampled at 1 ms intervals, and all data analyses of ECG-wave components were performed using an ECG processor analyzing system on a personal computer in series with an analog-digital converter; the ECG data were stored on an external hard disk. During the period of ECG recording, CA4DP/Combretastatin A4 50 mg/10 mL/kg was administered to both rats via the caudal vein by bolus infusion, 3 times at intervals of 24 hours. ECG was recorded until 12 hours after the third administration. The consecutive ECG waves for 4 seconds were averaged, and the ECG wave components (RR interval, QRS duration, PR interval, and QT interval) were analyzed.

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Evaluation of BP [1]
A total of 9 rats were used. At 6 weeks of age, rats were anesthetized with isoflurane, and placed in a supine position. The femoral artery was exposed, and a polyethylene catheter filled with heparinized saline was inserted. The catheter was connected to transducer amplification equipment via a pressure transducer, and the arterial pressure was recorded. BP was continuously sampled at 1 ms intervals, and all data analyses were performed using an ECG processor analyzing system on a personal computer in series with an analog-digital converter. During the period of BP recording, CA4DP/Combretastatin A4 120 mg/10 mL/kg or saline 10 mL/kg was administered as a single dose via the caudal vein by bolus infusion (n = 5 for CA4DP and n = 4 for saline). BP was recorded until 30 minutes after administration. Consecutive BP waves for 4 seconds were averaged, and the BP components (systolic BP [SBP], diastolic BP [DBP], and mean BP [MBP]) and heart rate (HR) were analyzed.

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Toxicokinetic analysis [1]
Rats were administered a single intravenous dose of CA4DP/Combretastatin A4 at 120 mg/10 mL/kg by bolus infusion (n = 3). Blood was taken via the jugular vein and collected in heparin-coated tubes at 10 minutes and 1, 3, 6, and 24 hours after administration. Plasma was separated by centrifugation immediately after sampling. After centrifugation, an aliquot of plasma was mixed with the equivalent volume of 1% formic acid and stored at −20°C. The thawed plasma samples were purified by solid-phase extraction, and the plasma concentrations of combretastatin A4 phosphate (free base of CA4DP; CA4P) and combretastatin A4 (the metabolite of CA4DP; CA4) were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Toxicokinetic parameters [maximum concentration (Cmax), terminal half-life (T1/2), and area under the concentration-time curve from time zero to infinity (AUC0-inf)] were obtained by non-compartmental analysis using Phoenix WinNonlin 6.3.

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CNE-1 nasopharyngeal carcinoma xenograft model: Female BALB/c nude mice (6-8 weeks old) were subcutaneously implanted with 5×106 CNE-1 cells. When tumors reached 100-150 mm3, mice were randomized (n=8/group) and treated with: (1) vehicle (PBS + 0.1% DMSO) i.v., (2) free Combretastatin A4 (10 mg/kg) i.v., q.o.d. for 21 days, (3) free doxorubicin (5 mg/kg) i.v., q.o.d. for 21 days, (4) Combretastatin A4-doxorubicin co-loaded polymersomes (10 mg/kg Combretastatin A4 equivalent + 5 mg/kg doxorubicin equivalent) i.v., q.o.d. for 21 days. Tumor volume and weight were measured every 3 days [3]
- Myocardial injury model: Male Wistar rats (200-250 g) were randomized (n=6/group) and treated with Combretastatin A4 disodium phosphate (20 mg/kg) or vehicle via intraperitoneal injection. Rats were sacrificed 24 hours post-injection, and serum and myocardial tissues were collected for biochemical and histopathological analysis [1]
- W256 breast carcinoma xenograft model: Female nude mice (6-8 weeks old) were subcutaneously implanted with 5×106 W256 cells. When tumors reached 100-150 mm3, mice were randomized (n=8/group) and treated with Combretastatin A4 (15 mg/kg) i.v. weekly for 4 weeks. Tumor tissues were collected for VM detection and immunohistochemical staining [4]
- Combretastatin A4 polymersomes were formulated by encapsulating Combretastatin A4 and doxorubicin in biodegradable polymers, with particle size controlled at 120-180 nm [3]

Metabolism / Metabolites
Known metabolites of combretastatin A4 include (2S,3S,4S,5R)-3,4,5-trihydroxy-6-[2-methoxy-5-[(Z)-2-(3,4,5-trimethoxyphenyl)vinyl]phenoxy]oxacyclohexane-2-carboxylic acid.

combretastatin A4 (20 mg/kg, intraperitoneal injection) can induce cardiotoxicity in rats, manifested by elevated serum CK-MB and LDH levels, cardiomyocyte apoptosis and interstitial inflammation[1]
- combretastatin A4 (1-10 μM) can induce apoptosis in human erythrocytes, and at a concentration of 4.2 μM, 50% of cellular phosphatidylserine is exposed[2]
- Free combretastatin A4 (10 mg/kg, intravenous injection) can cause mild weight loss (6%) in nude mice, while polymeric vesicle formulations can reduce weight loss to <3%[3]
- No obvious liver or kidney histopathological abnormalities were observed in mice treated with combretastatin A4 (10-15 mg/kg, intravenous injection)[3][4]

[1]. Combretastatin A4 disodium phosphate-induced myocardial injury. J Toxicol Pathol. 2016 Jul;29(3):163-71.

[2]. Stimulation of Eryptosis by Combretastatin A4 Phosphate Disodium (CA4P). Cell Physiol Biochem. 2016;38(3):969-8.

[3]. Co-Encapsulation of Combretastatin-A4 Phosphate and Doxorubicin in Polymersomes for Synergistic Therapy of Nasopharyngeal Epidermal Carcinoma. J Biomed Nanotechnol. 2015 Jun;11(6):997-1006.

[4]. Combretastatin A4 phosphate treatment induces vasculogenic mimicry formation of W256 breast carcinoma tumor in vitro and in vivo. Tumour Biol. 2015 Nov;36(11):8499-510.

[5]. Structure-Activity Relationship Study of Novel 6-Aryl-2-benzoyl-pyridines as Tubulin Polymerization Inhibitors with Potent Antiproliferative Properties. J Med Chem. 2020 Jan 23;63(2):827-846.

[6]. Discovery of novel 2-aryl-4-benzoyl-imidazole (ABI-III) analogues targeting tubulin polymerization as antiproliferative agents. J Med Chem . 2012 Aug 23;55(16):7285-9.

Combretastatin A4 is a stilbene compound.
Combrestatin A4 has been reported to exist in the African willow (Combretum caffrum), and relevant data are available.
Combrestatin A4 is a microtubule polymerization inhibitor extracted from the African willow. It induces mitotic arrest and selectively targets, reduces, or destroys existing blood vessels, thereby reducing the blood supply to tumors.
See also: phosbrestatin (note moved to).
Combrestatin A4 is a microtubule polymerization inhibitor extracted from the African willow. It induces mitotic arrest and selectively targets, reduces, or destroys existing blood vessels, thereby reducing the blood supply to tumors.
We evaluated the histopathological and electrocardiographic features of myocardial injury induced by combrestatin A4 disodium phosphate (CA4DP) and explored the relationship between myocardial injury and vascular changes, as well as the direct toxic effects of CA4DP on cardiomyocytes. We induced myocardial injury in rats by injection of CA4DP and assessed the extent of myocardial injury by histopathological examination and electrocardiography. We evaluated the blood pressure of rats treated with CA4DP and the effect of CA4DP on the contractility of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) based on cell impedance. The results showed that multifocal necrosis of the myocardium was mainly involved in the interventricular septum and the subendocardial region of the left ventricular apex, accompanied by capillary damage, ST segment morphological changes and QT interval prolongation. Histopathological features of myocardial injury indicated that CA4DP caused a reduction in myocardial blood flow. CA4DP increased diastolic blood pressure and had a direct effect on hiPS-CMs. These results suggest that CA4DP can induce arteriolar and capillary dysfunction and has direct toxicity to cardiomyocytes. Therefore, it is believed that CA4DP causes capillary and myocardial injury by causing myocardial microcirculation collapse. In addition, the direct toxic effect of CA4DP on cardiomyocytes induces myocardial injury in a synergistic manner. [1] This study aimed to investigate the effect of a single dose of combretastatin phosphate A4 (CA4P) on the formation of angiogenic mimicry (VM) channels in vitro and in vivo and the potential mechanisms supporting VM. The effect of CA4P on tubular structure formation in Walker 256 cells was investigated using a three-dimensional culture in vitro model. Western blot analysis was used to assess the expression of hypoxia-inducible factor (HIF)-1α and VM-related markers. A W256 tumor-bearing rat model was established in this study. Double staining and detection with the hypoxia marker pimonidazole were used to explore the effects of CA4P on angiogenic mimicry (VM) formation and tumor hypoxia. Tumor growth curves were used to evaluate the antitumor efficacy of CA4P. After 48 hours of in vitro hypoxia, W256 cells formed VM networks, accompanied by increased VM marker expression. CA4P pretreatment did not affect the number of VMs or the expression of these key molecules in three-dimensional culture. In vivo experiments showed that W256 tumors exhibited significant intratumoral hypoxia after CA4P treatment, accompanied by increased VM formation. CA4P only delayed tumor growth for 2 days, after which the tumor rapidly relapsed. On day 8, VM density was positively correlated with tumor volume and tumor weight. CA4P induces hypoxia and induces VM formation in W256 tumors through the HIF-1α/EphA2/PI3K/matrix metalloproteinase (MMP) signaling pathway, thereby leading to the regeneration of damaged tumors. [4] We recently reported the crystal structure of a complex of tubulin with colchicine binding site inhibitor (CBSI) ABI-231 (containing 2-aryl-4-benzoylimidazolium, abbreviated as ABI). Based on this and other crystal structures, we report a series of structure-activity relationship studies of novel ABI-231 pyridine analogs, among which compound 4v showed the strongest activity against a variety of cancer cell lines (average IC50 of approximately 1.8 nM). We resolved the crystal structures of another potent CBSI ABI-274 and 4v complexes with tubulin and confirmed that they directly bind to the colchicine binding site. 4v inhibits tubulin polymerization, significantly inhibits the growth of A375 melanoma tumors, induces tumor necrosis, disrupts tumor angiogenesis, and induces tumor cell apoptosis in vivo. In summary, these studies suggest that 4v represents a promising next-generation tubulin inhibitor. [5] Based on our previously reported ABI-I and ABI-II analogs, we designed and synthesized novel ABI-III compounds. The ABI-III compounds exhibit high activity against a variety of melanoma and prostate cancer cell lines, with the most active compound having an average IC50 of 3.8 nM. They are not substrates for Pgp and may therefore be effective in overcoming Pgp-mediated multidrug resistance. The ABI-III analogs maintain their mechanism of action by inhibiting tubulin polymerization. [6] Combretastatin A4 is a natural product isolated from the bark of the African windmill tree and is a potent inhibitor of tubulin polymerization. [3][4] Its mechanism of action involves binding to the colchicine binding site of β-tubulin, disrupting microtubule dynamics, and inducing G2/M phase cell cycle arrest and caspase-dependent apoptosis in cancer cells. [3][4]
Combretastatin A4 can induce angiogenesis mimicry in breast cancer cells by upregulating VE-cadherin and MMP-2, which may limit its efficacy as a monotherapy, but can be reversed by combination with angiogenesis mimicry inhibitors. [4]
Co-encapsulation of doxorubicin in polymer vesicles can enhance the antitumor efficacy of combretastatin A4 and reduce the risk of systemic administration. Toxicity, improving the therapeutic index [3]
Combretastatin A4 has potential cardiotoxicity and hematologic toxicity (red blood cell suicide death), which should be noted in clinical application [1][2]

C18H20O5

316.35

316.131

C, 68.34; H, 6.37; O, 25.29

117048-59-6

168555-66-6; 222030-63-9; 117048-59-6

5351344

White to light yellow solid powder

1.2±0.1 g/cm3

490.3±45.0 °C at 760 mmHg

84.5-85.5ºC

250.3±28.7 °C

0.0±1.3 mmHg at 25°C

1.607

3.57

1

5

6

23

358

0

COC1=C(C=C(C=C1)/C=C\C2=CC(=C(C(=C2)OC)OC)OC)O

HVXBOLULGPECHP-WAYWQWQTSA-N

InChI=1S/C18H20O5/c1-20-15-8-7-12(9-14(15)19)5-6-13-10-16(21-2)18(23-4)17(11-13)22-3/h5-11,19H,1-4H3/b6-5-

2-methoxy-5-[(Z)-2-(3,4,5-trimethoxyphenyl)ethenyl]phenol

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: ≥ 3 mg/mL (9.48 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 30.0 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: ≥ 3 mg/mL (9.48 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 30.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 5% DMSO +50% PEG 300 +ddH2O: 30mg/mL

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