(4-Methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone inhibits tubulin polymerization,induces G2/M arrest,and triggers apoptosis in human leukemia HL-60 cells

(4-Methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone (PHT) is a known cytotoxic compound belonging to the phenstatin family. However, the exact mechanism of action of PHT-induced cell death remains to be determined. The aim of this study was to investigate the mechanisms underlying PHT-induced cytotoxicity. We found that PHT displayed potent cytotoxicity in different tumor cell lines, showing IC₅₀ values in the nanomolar range. Cell cycle arrest in G₂/M phase along with the augmented metaphase cells was found. Cells treated with PHT also showed typical hallmarks of apoptosis such as cell shrinkage, chromatin condensation, phosphatidylserine exposure, increase of the caspase 3/7 and 8 activation, loss of mitochondrial membrane potential, and internucleosomal DNA fragmentation without affecting membrane integrity. Studies conducted with isolated tubulin and docking models confirmed that PHT binds to the colchicine site and interferes in the polymerization of microtubules. These results demonstrated that PHT inhibits tubulin polymerization, arrests cancer cells in G₂/M phase of the cell cycle, and induces their apoptosis, exhibiting promising anticancer therapeutic potential.     

Heterocyclic organobismuth(III) induces apoptosis of human promyelocytic leukemic cells through activation of caspases and mitochondrial perturbation

We have synthesized novel heterocyclic organobismuth compounds that have potent antibacterial properties. In this study, we examined their anticancer activity and addressed the cellular mechanisms involved. Heterocyclic organobismuth compounds showed anticancer activities in various human cancer cell lines. These compounds have particularly potent anticancer activities against leukemia cell lines. One of them, bi-chlorodibenzo [c,f][1,5] thiabismocine (compound 3), inhibited the growth of the human promyelocytic leukemia cell line HL-60 at a concentration of 0.22 μM. Low concentrations of compound 3 (0.22-0.44 μM) induced apoptosis, whereas at a higher concentration (>1.1 μM) it causes acute necrosis. During the apoptosis, caspase-3, -8, and -9 were activated but caspase-12 was not. A broad caspase inhibitor (z-VAD-fmk), and caspase-3 (z-DEVD-fmk) and caspase-9 (z-LEHD-fmk) inhibitors suppressed the compound 3-induced apoptosis, but a caspase-8 inhibitor (z-IETD-fmk) was less effective, suggesting that the caspase-8 activity only partially participates in the apoptosis. In the apoptotic cells, cytochrome c was released from mitochondria to cytosol and a loss of mitochondrial transmembrane potential (ΔΨ_m) was detected. Compound 3-induced apoptosis was associated with enhanced generation of intracellular reactive oxygen species (ROS). Pretreatment of the cells with N-acetyl-l-cysteine or catalase suppressed the apoptosis. On the other hand, buthionine sulfoximine enhanced the compound 3-induced collapse of ΔΨ_m and apoptosis. Taken together, these results indicate that compound 3 is a potent inducer of apoptosis, triggering a caspase-3-mediated mechanism via the generation of ROS and release of cytochrome c from mitochondria, suggesting a potential mechanism for the anticancer activity of compound 3.     

Cell cycle arrest through inhibition of tubulin polymerization by withaphysalin F,a bioactive compound isolated from <Emphasis Type="Italic">Acnistus arborescens</Emphasis>

Many compounds used in the treatment of cancer possess tubulin-interacting properties that lead to mitotic arrest. Withaphysalins are potent cytotoxic compounds that are commonly found in plants belonging to the Solanaceae family, such as Acnistus arborescens; however, the cytotoxic mechanisms or molecular targets of these compounds remain unknown. Thus, the aim of this study was to evaluate the effects of whitaphysalins on cancer cell cycle progression and tubulin interaction. In this report, we show the antiproliferative activity of withaphysalin F and its effect in arresting cells in the G₂/M phase of the cell cycle. These two effects are the result of the interference of withaphysalin F in the polymerization of microtubules. Withaphysalin F also induced DNA fragmentation, which can be related to an increase in mitochondrial membrane depolarization. These results suggest that interference of withaphysalin F in microtubule polymerization may induce cell cycle arrest in the G₂/M phase and therefore contribute to growth inhibition of tumor cells in vitro. Taken together, these studies indicate that withaphysalin F could potentially be used as an anticancer drug.     

Ligand modulation of a dinuclear platinum compound leads to mechanistic differences in cell cycle progression and arrest

Despite similar structures and DNA binding profiles, two recently synthesized dinuclear platinum compounds are shown to elicit highly divergent effects on cell cycle progression. In colorectal HCT116 cells, BBR3610 shows a classical G₂/M arrest with initial accumulation in S phase, but the derivative compound BBR3610-DACH, formed by introduction of the 1,2-diaminocyclohexane (DACH) as carrier ligand, results in severe G₁/S as well as G₂/M phase arrest, with nearly complete S phase depletion. The origin of this unique effect was studied. Cellular interstrand crosslinking as assayed by comet analysis was similar for both compounds, confirming previous in vitro results obtained on plasmid DNA. Immunoblotting revealed a stabilization of p53 and concomitant transient increases in p21 and p27 proteins after treatment with BBR3610-DACH. Cell viability assays and cytometric analysis of p53 and p21 null cells indicated that BBR3610-DACH-induced cell cycle arrest was p21-dependent and partially p53-dependent. However, an increase in the levels of cyclin E was observed with steady state levels of CDK2 and Cdc25A, suggesting that the G₁ block occurs downstream of CDK/cyclin complex formation. The G₂/M block was corroborated with decreased levels of cyclin A and cyclin B1. Surprisingly, BBR3610-DACH-induced G₁ block was independent of ATM and ATR. Finally, both compounds induced apoptosis, with BBR3610-DACH showing a robust PARP-1 cleavage that was not associated with caspase-3/7 cleavage. In summary, BBR3610-DACH is a DNA binding platinum agent with unique inhibitory effects on cell cycle progression that could be further developed as a chemotherapeutic agent complementary to cisplatin and oxaliplatin.     

Induction of G2/M arrest and apoptosis through mitochondria pathway by a dimer sesquiterpene lactone from Smallanthus sonchifolius in HeLa cells

Dimer sesquiterpene lactones (SLs), uvedafolin and enhydrofolin, against four monomer SLs isolated from yacon, Smallanthus sonchifolius, leaf were the most cytotoxic substances on HeLa cells (IC₅₀ values 2.96–3.17 μM at 24 hours). However, the cytotoxic mechanism of dimer SL has not been elucidated yet. Therefore, in this study, we clarified the in vitro cytotoxic mechanism of uvedafolin on the HeLa cells, and evaluated the cytotoxicity against NIH/3T3 cells which were used as normal cells. In consequence, the dimer SLs had low toxicity for the NIH/3T3 cells (IC₅₀ 4.81–4.98 μM at 24 hours) and then the uvedafolin mediated cell cycle arrest at the G₂/M phase and induced apoptosis on the HeLa cells evidenced by appearance of a subG1 peak. Uvedafolin induced apoptosis was attributed to caspase-9 and caspase-3/7 activities. An effectively induced apoptosis pathway was demonstrated from mitochondria membrane potential change and cytochrome c release to cytosol. These results reveal that uvedafolin induced apoptosis via the mitochondria pathway. The present results indicate the potential of uvedafolin as a leading compound of new anticancer agents.     

Cardiac glycosides from the seeds of Thevetia peruviana and their pro-apoptotic activity toward cancer cells

Phytochemical investigation of the seeds of Thevetia peruviana resulted in the isolation of seven cardiac glycosides (1–7), including two new compounds (1 and 2). Cytotoxicity of them toward cancer cell lines P15 (human lung cancer cell), MGC-803 (human gastric cancer cells), SW1990 (human pancreatic cancer cells), and normal hepatocyte cell LO2 suggested that compound 1 could selectively inhibit the proliferation of cancer cell lines with IC₅₀ from 0.05 to 0.15 μM. Pro-apoptotic activity revealed that it induced the apoptosis of MGC-803 cancer cells in a dose-dependent manner. Meanwhile, treatment of MGC-803 cancer cells with 1 resulted in diminution of pro-caspases 3 and 9 and activation of caspases 3 and 9, while it increased the Bax/Bcl-2 ratio in a dose-dependent manner. These meant that 1 induced the apoptosis of cancer cells by involving the intrinsic apoptotic pathway. In addition, the cell cycle distribution of MGC-803 cancer cells treated by 1 revealed that it could lead to cell cycle arrest at the G2/M phase. Altogether, this study suggested that compound 1 may exhibit anticancer activity by its capability of induction of intrinsic apoptosis and cell cycle arrest at G2/M phase.     

Synthesis,molecular docking and biological evaluation of 1‐phenylsulphonyl‐2‐(1‐methylindol‐3‐yl)‐benzimidazole derivatives as novel potential tubulin assembling inhibitors

A series of new 1‐phenylsulphonyl‐2‐(1‐methylindol‐3‐yl)‐benzimidazole derivatives were designed, synthesized and evaluated as potential inhibitors of tubulin polymerization and anthropic cancer cell lines. Among them, compound 33 displayed the most potent tubulin polymerization inhibitory activity in vitro (IC₅₀ = 1.41 μM) and strong antiproliferative activities against A549, Hela, HepG2 and MCF‐7 cell lines in vitro with GI₅₀ value of 1.6, 2.7, 2.9 and 4.3 μM, respectively, comparable with the positive control colchicine (GI₅₀ value of 4.1, 7.2, 9.5 and 14.5 μM, respectively) and CA‐4 (GI₅₀ value of 2.2, 4.3, 6.4 and 11.4 μM, respectively). Simultaneously, we evaluated that compound 33 could effectively induce apoptosis of A549 associated with G2/M phase cell cycle arrest. Immunofluorescence microscopy also clearly indicated compound 33 a potent antimicrotubule agent. Docking simulation showed that compound 33 could bind tightly with the colchicine‐binding site and act as a tubulin inhibitor. Three‐dimensional‐QSAR model was also built to provide more pharmacophore understanding that could be used to design new agents with more potent tubulin assembling inhibitory activity in the future.     

Dihydroptychantol A, a macrocyclic bisbibenzyl derivative, induces autophagy and following apoptosis associated with p53 pathway in human osteosarcoma U2OS cells

Dihydroptychantol A (DHA), a novel macrocyclic bisbibenzyl compound extracted from liverwort Asterella angusta, has antifungal and multi-drug resistance reversal properties. Here, the chemically synthesized DHA was employed to test its anti-cancer activities in human osteosarcoma U2OS cells. Our results demonstrated that DHA induced autophagy followed by apoptotic cell death accompanied with G₂/M-phase cell cycle arrest in U2OS cells. DHA-induced autophagy was morphologically characterized by the formation of double membrane-bound autophagic vacuoles recognizable at the ultrastructural level. DHA also increased the levels of LC3-II, a marker of autophagy. Surprisingly, DHA-mediated apoptotic cell death was potentiated by the autophagy inhibitor 3-methyladenine, suggesting that autophagy may play a protective role that impedes the eventual cell death. Furthermore, p53 was shown to be involved in DHA-meditated autophagy and apoptosis. In this connection, DHA increased nuclear expression of p53, induced p53 phosphorylation, and upregulated p53 target gene p21^Waf1/Cip1. In contrast, cytoplasmic p53 was reduced by DHA, which contributed to the stimulation of autophagy. In relation to the cell cycle, DHA decreased the expression of cyclin B₁, a cyclin required for progression through the G₂/M phase. Taken together, DHA induces G₂/M-phase cell cycle arrest and apoptosis in U2OS cells. DHA-induced apoptosis was preceded by the induction of protective autophagy. DHA-mediated autophagy and apoptosis are associated with the cytoplasmic and nuclear functions of p53.     

Synthesis and biological evaluation of quinoxaline derivatives as tubulin polymerization inhibitors that elevate intracellular ROS and triggers apoptosis via mitochondrial pathway

A series of novel quinoxaline derivatives were synthesized and evaluated for their antiproliferative activity in three human cancer cell lines. Compound 12 exhibited the most potent antiproliferative activity with IC₅₀ in the range of 0.19–0.51 μM. The compound inhibited tubulin polymerization and disrupted the microtubule network, leading to G2/M phase arrest. Furthermore, compound 12 induced ROS production and malfunction of mitochondrial membrane potential. Compound 12 led to cancer cells apoptosis in a dose‐dependent manner. Western blot analysis showed that compound 12 induced up‐regulation of p21 and affected the expression of cell cycle‐related proteins. The binding mode was also probed by molecular docking.     

Normalizing effect of plant-originated glycoprotein (116 kDa) on G0/G1 arrest in cadmium chloride-induced primary cultured mouse myelocytes

Cadmium chloride is a well-known carcinogenic and immunotoxic metal chemical, which is commonly found in cigarette smoke and industrial effluent and which is able to cause cell cycle arrest in various cell lines. This study demonstrated that glycoprotein (116 kDa) isolated from Ulmus Davidiana Nakai (UDN) is able to normalize cell cycle arrest caused by cadmium chloride (10 μM, for indicated treatment time in the each experiment) in primary cultured mouse myelocytes. To assess cell cycle arrest, the parameters that are related to the cell cycle evaluated included cytotoxicity, production of intracellular reactive oxygen species (ROS), intracellular Ca²⁺ mobilization, the activities of cell cycle-related proteins (p53, p21, and p27), and cyclin D1/cell cyclin-dependent kinase 4 (CDK4) using immunoblot analysis and fluorescence-activated cell sorter analysis. The results in this study showed that UDN glycoprotein (50 μg/ml) inhibits the cytotoxicity, production of intracellular reactive oxygen species (ROS), and intracellular Ca²⁺ mobilization brought about by cadmium chloride. With regard to cell cycle-related proteins, UDN glycoprotein (50 μg/ml) significantly suppressed the expression of p53, p21, and p27, whereas it enhanced activity of cyclin D1/CDK4. Taken together, these findings suggest that UDN glycoprotein (50 μg/ml) significantly normalizes arrest of G₀/G₁ in the cell cycle. Thus, UDN glycoprotein appears to be one compound derived from natural products that is able normalize the calcium chloride-mediated arrest of cell cycle (G₀/G₁) in immune cells.     

Dependency of 2-methoxyestradiol-induced mitochondrial apoptosis on mitotic spindle network impairment and prometaphase arrest in human Jurkat T cells

The present study sought to determine the correlation between 2-methoxyestradiol (2-MeO-E₂)-induced cell cycle arrest and 2-MeO-E₂-induced apoptosis. Exposure of Jurkat T cell clone (JT/Neo) to 2-MeO-E₂ (0.5–1.0 μM) caused G₂/M arrest, Bak activation, Δψm loss, caspase-9 and -3 activation, PARP cleavage, intracellular ROS accumulation, and apoptotic DNA fragmentation, whereas none of these events except for G₂/M arrest were induced in Jurkat T cells overexpressing Bcl-2 (JT/Bcl-2). Under these conditions, Cdk1 phosphorylation at Thr-161 and dephosphorylation at Tyr-15, up-regulation of cyclin B1 expression, histone H1 phosphorylation, Cdc25C phosphorylation at Thr-48, Bcl-2 phosphorylation at Thr-56 and Ser-70, Mcl-1 phosphorylation at Ser-159/Thr-163, and Bim phosphorylation were detected irrespective of Bcl-2 overexpression. Concomitant treatment of JT/Neo cells with 2-MeO-E₂ and the G₁/S blocking agent aphidicolin resulted in G₁/S arrest and abrogation of all apoptotic events, including Cdk1 activation, phosphorylation of Bcl-2, Mcl-1 and Bim, and ROS accumulation. The 2-MeO-E₂-induced phosphorylation of Bcl-2 family proteins and mitochondrial apoptotic events were suppressed by a Cdk1 inhibitor, but not by an Aurora A kinase (AURKA), Aurora B kinase (AURKB), JNK, or p38 MAPK inhibitor. Immunofluorescence microscopic analysis revealed that 2-MeO-E₂-induced mitotic arrest was caused by mitotic spindle network impairment and prometaphase arrest. Whereas 10–20 μM 2-MeO-E₂ reduced the proportion of intracellular polymeric tubulin to monomeric tubulin, 0.5–5.0 μM 2-MeO-E₂ increased it. These results demonstrate that the apoptogenic effect of 2-MeO-E₂ (0.5–1.0 μM) was attributable to mitotic spindle defect-mediated prometaphase arrest, Cdk1 activation, phosphorylation of Bcl-2, Mcl-1, and Bim, and activation of Bak and mitochondria-dependent caspase cascade.     

Molecular Cytotoxic Mechanisms of 1-(3,4,5-Trihydroxyphenyl)-dodecylbenzoate in Human Leukemia Cell Lines

Recent studies have shown that gallic acid and its alkylesters induce apoptosis in different cell lines. Since new compounds with biological activity and less cytotoxicity to normal cells are necessary for cancer therapy, the aim of this study was to evaluate the cytotoxic effect of 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate on human acute myeloid leukemia K562 cells and on human acute lymphoblastic leukemia Jurkat cells. The cell viability was determined by MTT method. The apoptosis induction was assessed by bromide and acridine orange staining and by Annexin V-FITC Apoptosis Detection kit. The cell cycle analysis was carried out by flow cytometry using propidium iodide. Cytometric analysis was also performed to evaluate the expression of the following proteins: AIF, p53, Bcl-2 and Bax. The mitochondrial potential was also assessed by flow cytometry using MitoView633 kit. The results showed that the compound significantly reduced the cell viability of K562 and Jurkat cells in a concentration and time dependent manner (IC₅₀ of 30 μM). The compound induced cell cycle arrest in G₀/G₁phase and significantly increased the proportion of cells in the sub-G₀/G₁phase. Apoptosis was confirmed by the sight of morphological characteristics of apoptosis and by phosphatidylserine externalization (73.47±5.71% of cells expressing annexin). The results also showed that the compound promotes a modification in Bax:Bcl-2 ratio and increases p53 expression. Thus, it is possible to conclude that 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate induces apoptosis by inhibiting the antiapoptotic protein Bcl-2 and by increasing the release of AIF, Bax and p53. In addition, it blocks the cell cycle at G₀/G₁, stopping cell proliferation. So far, the results suggest that this compound may have a potential therapeutic effect against leukemia cells.     

Induction of G<Subscript>1</Subscript>/S phase arrest and apoptosis by quercetin in human osteosarcoma cells

Quercetin (3,3′,4′,5,7-pentahydroxyflavone) is a polyphenolic flavonoid compound and is found in a variety of plants. Potential biological activities including antioxidant and anticarcinogenesis have been reported. The antiproliferative effect and apoptosis inducing effect of quercetin in human osteosarcoma cells was evaluated in this study. The IC₅₀ values were 290 μM and 160 μM at 24 h and 48 h incubation, respectively. Antiproliferative action of quercetin appeared to be linked to apoptotic cell death based on increase in the sub-G₁ apoptotic cell population analyzed by flow cytometric analysis. Prior to apoptosis induction, quercetin caused cell cycle arrest at G₁/S phase. The G₁/S phase arrest was accompanied by down regulation of cyclin D1, one of the cyclins required for advance from G₁ to S. Subsequent apoptosis was induced by the gradual activation of caspase-3 and the cleavage of PARP.     

dentification of DW532 as a novel anti-tumor agent targeting both kinases and tubulin

Aim： 7,8-Dihydroxy-4-（3-hydroxy-4-methoxyphenyl）-2H-chromen-2-one （DW532） is one of simplified analogues of hematoxylin that has shown broad-spectrum inhibition on tyrosine kinases and in vitro anti-cancer activities. The aim of this study was to identify DW532 as a agent targeting both kinases and tubulin, and to investigate its anti-cancer and anti-angiogenesis activities. Methods： In vitro tyrosine kinases activity was examined with ELISA, and tyrosine kinases activity in cells was evaluated with Western blot analysis. Tubulin turbidity assay, surface plasmon resonance and immunofluorescence technique were used to characterize the tubulin inhibitory activity. Cell proliferation was examined with SRB assay, and cell apoptosis and cell cycle distribution were analyzed with Annexin-V/PI staining and flow cytometry. Tube formation, aortic ring and chick chorioallantoic membrane assays were used to evaluate the anti-angiogenesis efficacy. Results： DW532 inhibited EGFR and VEGFR2 in vitro kinase activity （the ICso values were 4.9 and 5.5 pmol/L, respectively）, and suppressed their downstream signaling. DW532 dose-dependently inhibited tubulin polymerization via direct binding to tubulin, thus disrupting the mitotic spindle assembly and leading to abnormal cell division. In a panel of human cancer cells, DW532 （1 and 10 pmol/L） induced G2/M phase arrest and cell apoptosis, which subsequently resulted in cytotoxicity. Knockdown of BubR1 or Mpsl, the two core proteins of the spindle assembly checkpoint dramatically decreased DW532-induced cell cycle arrest in MDA-MB-468 cells. Moreover, treatment with DW532 potently and dose-dependently suppressed angiogenesis in vitro and in vivo. Conclusion： DW532 is a dual inhibitor against tubulin and tyrosine kinases, and deserves further development as a novel anti-cancer agent.     

Novel microtubule-targeted agent 6-chloro-4-(methoxyphenyl) coumarin induces G2-M arrest and apoptosis in HeLa cells

Aim:

To identify a novel coumarin analogue with the highest anticancer activity and to further investigate its anticancer mechanisms.

Methods:

The viability of cancer cells was investigated using the MTT assay. The cell cycle progression was evaluated using both flow cytometric and Western blotting analysis. Microtubule depolymerization was observed with immunocytochemistry in vivo and a tubulin depolymerization assay in vitro. Apoptosis was demonstrated using Annexin V/Propidium Iodide (PI) double-staining and sub-G₁ analysis.

Results:

Among 36 analogues of coumarin, 6-chloro-4-(methoxyphenyl) coumarin showed the best anticancer activity (IC₅₀ value about 200 nmol/L) in HCT116 cells. The compound had a broad spectrum of anticancer activity against 9 cancer cell lines derived from colon cancer, breast cancer, liver cancer, cervical cancer, leukemia, epidermoid cancer with IC₅₀ value of 75 nmol/L–1.57 μmol/L but with low cytotocitity against WI-38 human lung fibroblasts (IC₅₀ value of 12.128 μmol/L). The compound (0.04–10 μmol/L) induced G₂-M phase arrest in HeLa cells in a dose-dependent manner, which was reversible after the compound was removed. The compound (10–300 μmol/L) induced the depolymerization of purified porcine tubulin in vitro. Finally, the compound (0.04–2.5 μmol/L) induced apoptosis of HeLa cells in dose- and time-dependent manners.

Conclusion:

6-Chloro-4-(methoxyphenyl) coumarin is a novel microtubule-targeting agent that induces G₂–M arrest and apoptosis in HeLa cells.     

Benzyl isothiocyanate promotes apoptosis of oral cancer cells via an acute redox stress-mediated DNA damage response

Benzyl isothiocyanate (BITC) is a cruciferous vegetable-derived compound with anticancer properties in human cancer cells. However, its anticancer potential and underlying mechanisms remain absent in human oral cancer cells. Results indicate that BITC inhibits growth, promotes G₂/M phase arrest and triggers apoptosis of OC2 cells with a minimal toxicity to normal cells. BITC-induced cell death was completely prevented by pretreatment with thiol-containing redox compounds including N-acetyl-l-cysteine (NAC), glutathione (GSH), dithiothreitol, and 2-mercaptoethanol, but not free radical scavengers mito-TEMPO, catalase, apocynin, l-NAME and mannitol. BITC rapidly produced reactive oxygen species and nitric oxide, triggered oxidative DNA damage. BITC effectively decreased the intracellular GSH and GSH/GSSG ratio and redox balance recovery by thiol-containing redox compounds, but not by free radical scavengers. Accordingly, redox stresses-DNA damage response (DDR) activated ATM, Chk2, p53, and p21 and subsequently resulted in G₂/M phase arrest by inhibiting Cdc2 and cyclin B1. Notably, BITC-induced apoptosis was associated with reduced Mcl-1 and Bcl-2 expression, diminished mitochondrial membrane potential (ΔΨm), and increased PARP cleavage. These BITC-induced redox stress-mediated DDR and apoptosis could be blocked by NAC and GSH. Therefore, BITC can be a rational drug candidate for oral cancer and acted via a redox-dependent pathway.     

Celecoxib in combination with retinoid CD437 inhibits melanoma A375 cell in vitro

This study aimed to investigate the effects of celecoxib, synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylicacid (CD437) and the combination of the two on cell proliferation, apoptosis, and cycle arrest of human malignant melanoma A375 cells. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay (MTT assay) was applied to determine the anti-proliferative effects of the drugs on human malignant melanoma A375 cells. Flow cytometry was performed to investigate the influence of the drugs on cell cycle and cell apoptosis. Both celecoxib and CD437 could inhibit the growth of human malignant melanoma A375 cells in a dose-dependent manner. Celecoxib at 80 μmol/L inhibited proliferation, induced apoptosis and G₂/M cell cycle arrest of human malignant melanoma A375 cells after treatment for 24 h [proliferation inhibiting rate: (50.2 ± 2.51)%, apoptosis rate: (35.91 ± 1.80)%]. CD437 at 10 μmol/L inhibited proliferation, induced apoptosis and G₀/G₁ cell cycle arrest of human malignant melanoma A375 cells after treatment for 24 h [proliferation inhibiting rate: (58.6 ± 2.38)%, apoptosis rate: (28.03 ± 0.77)%]. Celecoxib in combination with CD437 could significantly enhance the effects of inhibiting proliferation and inducing apoptosis of human malignant melanoma A375 cells 24 h after treatment compared with the drug alone [proliferation inhibiting rate: (68.92 ± 1.72)%, apoptosis rate: (42.09 ± 1.05)%, both P<0.05] and decrease the proportion of the S phase in the cell cycle. Celecoxib could inhibit the growth of human malignant melanoma A375 cells by inducing apoptosis and G₂/M cycle arrest. CD437 could inhibit the growth of human malignant melanoma A375 cells by inducing apoptosis and G₀/G₁ cycle arrest. Celecoxib exhibited additive effects with CD437 on retarding the growth and inducing apoptosis of human malignant melanoma A375 cells. Celecoxib in combination with CD437 may become an effective method for prevention and treatment of human melanoma.     

WTC-01, a novel synthetic oxime-flavone compound,destabilizes microtubules in human nasopharyngeal carcinoma cells in vitro and in vivo

Background and Purpose

Dynamic polymerization of microtubules is essential for cancer cell growth and metastasis, and microtubule-disrupting agents have become the most successful anti-cancer agents in clinical use. Besides their antioxidant properties, flavonoids also exhibit strong microtubule-disrupting activity and inhibit tumour growth. We have designed, synthesized and tested a series of oxime/amide-containing flavone derivatives. Here we report the evaluation of one compound, WTC-01 for its anti-proliferative effects in human cancer cells.

Experimental approach

We used a range of cancer cell lines including two human nasopharyngeal carcinoma (NPC) cell lines, measuring proliferation, cell cycle and apoptosis, along with caspase levels and mitochondrial membrane potentials. Assays of tubulin polymerisation in vitro and computer modelling of the colchicine binding site in tubulin were also used. In mice, pharmacokinetics and growth of NPC-derived tumours were studied.

Key Results

WTC-01 was most potent against proliferation of NPC cells (IC₅₀ = 0.45 μM), inducing accumulation of cells in G₂/M and increasing apoptosis, time- and concentration-dependently. The colchicine competition-binding experiments and computer modelling results suggested that WTC-01 causes microtubule disruption via binding to the colchicine-binding site of tubulin resulting in mitochondrial membrane damage and cell apoptosis via activation of caspase-9/-3 without noticeable activation of the caspase-8. Notably, our in vivo studies demonstrated that at doses of 25 and 50 mg·kg⁻¹, WTC-01 exhibited good pharmacokinetic properties and completely inhibited the growth of NPC-TW01 cells in a xenograft nude mouse model.

Conclusions and Implications

WTC-01, a new synthetic oxime-containing flavone, exhibited potent anti-tumour activity against NPC cells and merits further investigation.     

Ochratoxin A induces G2 phase arrest in human gastric epithelium GES-1 cells in vitro

Ochratoxin A (OTA) is a mycotoxin commonly found in several food commodities worldwide. OTA exposure was involved in the nephrotoxicity, hepatotoxicity as well as immunotoxicity in experimental model. Our previous study showed that the high level of OTA contamination in wheat might be related to the high incidence of gastric carcinoma in rural area of China. However, there were no available data regarding the gastric toxicity of OTA up to now. In the present study, we explored the toxicity of OTA in human gastric epithelium immortalized cells (GES-1) by analyzing the regulation of the cell cycle, apoptosis and its molecular mechanism. We found that OTA could induce GES-1 cells arrested in G₂/M phase. Among these cycle-arrested cells, the proportion of cells in M phase was down-regulated after OTA treatment by the mitotic index and the level of phospho-histone H3. Thus, it was clear that OTA exerted a major influence on G₂ phase arrest instead of M phase. We further detected the expression of the key factors which are critical to the G₂/M phase transmission such as Cdc25C, Cdc2 and cyclinB1. The cyclinB1–Cdc2 complex was reduced and the expression of Cdc25C, Cdc2 and cyclinB1 were significantly decreased by OTA treatment both at protein and mRNA level, respectively. Considering that the cells may undergo apoptosis or death due to the cell cycle arrest, so we next detected the apoptosis of cells by OTA treatment. The results confirmed that OTA did induce apoptosis of GES-1 cells and activate the cleavage of capase-3. In conclusion, cell apoptosis and G₂ phase arrest mediated by Cdc25C, Cdc2 and cyclinB1 may be the initiating event in the gastric toxicity of OTA.