Emodin azide methyl anthraquinone derivative induced G0/ G1 arrest in HER2/neu-overexpressing MDA-MB-453 breast cancer cells.

PURPOSE: Our previous data have shown that emodin azide methyl anthraquinone derivative (AMAD) triggered mitochondrial- dependent cell apoptosis involving caspase-8-mediated Bid cleavage, and induced proteasomal degradation of HER2/neu by blocking Her2/neu binding to Hsp90. In the present study, we futher investigated the effect of this compound on the cell cycle and related molecular mechanisms in HER2/neu-overexpressing MDA-MB-453 breast cancer cells. METHODS: The cell cycle distribution was tested by flow cytometry. The expression of cell cycle-related proteins was determined by Western blot analysis; DNA agarose gel electrophoresis was used to examine the apoptosis of MDAMB- 453 cells induced by emodin AMAD. RESULTS: After MDA-MB-453 cells were treated with different concentrations of emodin AMAD for 24 hrs, cells were arrested in G0/G1 phase, and the expression of G0/G1 related proteins c/Myc, Cyclin D1, CDK4 and p-Rb changed. DNA fragmentation appeared on the agarose gel in a concentration- dependent manner. CONCLUSION: Emodin AMAD induced G0/G1 arrest in Her2/ neu-overexpressing MDA-MB-453 cancer cells. This G0/G1 arrest was associated with decreasing protein expression of c-Myc, Cyclin D1, CDK4, and p-Rb.

Saracatinib (AZD0530) is a potent modulator of ABCB1-mediated multidrug resistance in vitro and in vivo.

Saracatinib, a highly selective, dual Src/Abl kinase inhibitor, is currently in a Phase II clinical trial for the treatment of ovarian cancer. In our study, we investigated the effect of saracatinib on the reversal of multidrug resistance (MDR) induced by ATP-binding cassette (ABC) transporters in vitro and in vivo. Our results showed that saracatinib significantly enhanced the cytotoxicity of ABCB1 substrate drugs in ABCB1 overexpressing HeLa/v200, MCF-7/adr and HEK293/ABCB1 cells, an effect that was stronger than that of gefitinib, whereas it had no effect on the cytotoxicity of the substrates in ABCC1 overexpressing HL-60/adr cells and its parental sensitive cells. Additionally, saracatinib significantly increased the doxorubicin (Dox) and Rho 123 accumulation in HeLa/v200 and MCF-7/adr cells, whereas it had no effect on HeLa and MCF-7 cells. Furthermore, saracatinib stimulated the ATPase activity and inhibited photolabeling of ABCB1 with [(125)I]-iodoarylazidoprazosin in a concentration-dependent manner. In addition, the homology modeling predicted the binding conformation of saracatinib within the large hydrophobic drug-binding cavity of human ABCB1. However, neither the expression level of ABCB1 nor the phosphorylation level of Akt was altered at the reversal concentrations of saracatinib. Importantly, saracatinib significantly enhanced the effect of paclitaxel against ABCB1-overexpressing HeLa/v200 cancer cell xenografts in nude mice. In conclusion, saracatinib reverses ABCB1-mediated MDR in vitro and in vivo by directly inhibiting ABCB1 transport function, without altering ABCB1 expression or AKT phosphorylation. These findings may be helpful to attenuate the effect of MDR by combining saracatinib with other chemotherapeutic drugs in the clinic.

Neratinib reverses ATP-binding cassette B1-mediated chemotherapeutic drug resistance in vitro, in vivo, and ex vivo.

Neratinib, an irreversible inhibitor of epidermal growth factor receptor and human epidermal receptor 2, is in phase III clinical trials for patients with human epidermal receptor 2-positive, locally advanced or metastatic breast cancer. The objective of this study was to explore the ability of neratinib to reverse tumor multidrug resistance attributable to overexpression of ATP-binding cassette (ABC) transporters. Our results showed that neratinib remarkably enhanced the sensitivity of ABCB1-overexpressing cells to ABCB1 substrates. It is noteworthy that neratinib augmented the effect of chemotherapeutic agents in inhibiting the growth of ABCB1-overexpressing primary leukemia blasts and KBv200 cell xenografts in nude mice. Furthermore, neratinib increased doxorubicin accumulation in ABCB1-overexpressing cell lines and Rhodamine 123 accumulation in ABCB1-overexpressing cell lines and primary leukemia blasts. Neratinib stimulated the ATPase activity of ABCB1 at low concentrations but inhibited it at high concentrations. Likewise, neratinib inhibited the photolabeling of ABCB1 with [(125)I]iodoarylazidoprazosin in a concentration-dependent manner (IC(50) = 0.24 µM). Neither the expression of ABCB1 at the mRNA and protein levels nor the phosphorylation of Akt was affected by neratinib at reversal concentrations. Docking simulation results were consistent with the binding conformation of neratinib within the large cavity of the transmembrane region of ABCB1, which provides computational support for the cross-reactivity of tyrosine kinase inhibitors with human ABCB1. In conclusion, neratinib can reverse ABCB1-mediated multidrug resistance in vitro, ex vivo, and in vivo by inhibiting its transport function.

Axitinib targeted cancer stemlike cells to enhance efficacy of chemotherapeutic drugs via inhibiting the drug transport function of ABCG2.

Stemlike cells have been isolated by their ability to efflux Hoechst 33342 dye and are called the side population (SP). We evaluated the effect of axitinib on targeting cancer stemlike cells and enhancing the efficacy of chemotherapeutical agents. We found that axitinib enhanced the cytotoxicity of topotecan and mitoxantrone in SP cells sorted from human lung cancer A549 cells and increased cell apoptosis induced by chemotherapeutical agents. Moreover, axitinib particularly inhibited the function of adenosine triphosphate (ATP)-binding cassette subfamily G member 2 (ABCG2) and reversed ABCG2-mediated multidrug resistance (MDR) in vitro. However, no significant reversal effect was observed in ABCB1-, ABCC1- or lung resistance-related protein (LRP)-mediated MDR. Furthermore, in both sensitive and MDR cancer cells axitinib neither altered the expression of ABCG2 at the mRNA or protein levels nor blocked the phosphorylation of AKT and extracellular signal-regulated kinase (ERK)1/2. In nude mice bearing ABCG2-overexpressing S1-M1-80 xenografts, axitinib significantly enhanced the antitumor activity of topotecan without causing additional toxicity. Taken together, these data suggest that axitinib particularly targets cancer stemlike cells and reverses ABCG2-mediated drug resistance by inhibiting the transporter activity of ABCG2.

Enhancing chemosensitivity in ABCB1- and ABCG2-overexpressing cells and cancer stem-like cells by an Aurora kinase inhibitor CCT129202.

Imidazopyridine CCT129202 is an inhibitor of Aurora kinase activity and displays a favorable antineoplastic effect in preclinical studies. Here, we investigated the enhanced effect of CCT129202 on the cytotoxicity of chemotherapeutic drugs in multidrug resistant (MDR) cells with overexpression of ATP-binding cassette (ABC) transporters and cancer stem-like cells. CCT129202 of more than 90% cell survival concentration significantly enhanced the cytotoxicity of substrate drugs and increased the intracellular accumulations of doxorubicin and rhodamine 123 in ABCB1 and ABCG2 overexpressing cells, while no effect was found on parental sensitive cells. Interestingly, CCT129202 also potentiated the sensitivity of cancer stem-like cells to doxorubicin. Importantly, CCT129202 increased the inhibitory effect of vincristine and paclitaxel on ABCB1 overexpressing KBv200 cell xenografts in nude mice and human esophageal cancer tissue overexpressing ABCB1 ex vivo, respectively. Furthermore, the ATPase activity of ABCB1 was inhibited by CCT129202. Homology modeling predicted the binding conformation of CCT129202 within the large hydrophobic cavity of ABCB1. On the other hand, CCT129202 neither apparently altered the expression levels of ABCB1 and ABCG2 nor inhibited the activity of Aurora kinases in MDR cells under the concentration of reversal MDR. In conclusion, CCT129202 significantly reversed ABCB1- and ABCG2-mediated MDR in vitro, in vivo and ex vivo by inhibiting the function of their transporters and enhanced the eradication of cancer stem-like cells by chemotherapeutic agents. CCT129202 may be a candidate as MDR reversal agent for antineoplastic combination therapy and merits further clinical investigation.

FG020326 sensitized multidrug resistant cancer cells to docetaxel-mediated apoptosis via enhancement of caspases activation.

Apoptotic resistance is the main obstacle for treating cancer patients with chemotherapeutic drugs. Multidrug resistance (MDR) is often characterized by the expression of P-glycoprotein (P-gp), a 170-KD ATP-dependent drug efflux protein. Functional P-gp can confer resistance to activate caspase-8 and -3 dependent apoptosis induced by a range of different stimuli, including tumor necrosis and chemotherapeutic drugs such as docetaxel and vincristine. We demonstrated here that comparison of sensitive KB cells, P-gp positive (P-gp(+ve)) KBv200 cells were extremely resistant to apoptosis induced by docetaxel. FG020326, a pharmacological inhibitor of P-gp function, could enhance concentration-dependently the effect of docetaxel on cell apoptosis and sensitize caspase-8, -9 and -3 activation in P-gp overexpressing KBv200 cells, but not in KB cells. Therefore, the enhancement of caspase-8, -9 and -3 activation induced by docetaxel may be one of the key mechanisms of the reversal of P-gp mediated docetaxel resistance by FG020326.

ABCG2-overexpressing S1-M1-80 cell xenografts in nude mice keep original biochemistry and cell biological properties.

S1-M1-80 cells, derived from human colon carcinoma S1 cells, are mitoxantrone-selected ABCG2-overexpressing cells and are widely used in in vitro studies of multidrug resistance(MDR). In this study, S1-M1-80 cell xenografts were established to investigate whether the MDR phenotype and cell biological properties were maintained in vivo. Our results showed that the proliferation, cell cycle, and ABCG2 expression level in S1-M1-80 cells were similar to those in cells isolated from S1-M1-80 cell xenografts (named xS1-M1-80 cells). Consistently, xS1-M1-80 cells exhibited high levels of resistance to ABCG2 substrates such as mitoxantrone and topotecan, but remained sensitive to the non-ABCG2 substrate cisplatin. Furthermore, the specific ABCG2 inhibitor Ko143 potently sensitized xS1-M1-80 cells to mitoxantrone and topotecan. These results suggest that S1-M1-80 cell xenografts in nude mice retain their original cytological characteristics at 9 weeks. Thus, this model could serve as a good system for further investigation of ABCG2-mediated MDR.

Euphorbia factor L1 reverses ABCB1-mediated multidrug resistance involving interaction with ABCB1 independent of ABCB1 downregualtion.

Euphorbia factor L1 (EFL1) belongs to diterpenoids of genus Euphorbia. In this article, its reversal activity against ABCB1-mediated MDR in KBv200 and MCF-7/adr cells was reported. However, EFL1 did not alter the sensitivity of KB and MCF-7 cells to chemotherapeutic agents. Meanwhile, EFL1 significantly increased accumulation of doxorubicin and rhodamine 123 in KBv200 and MCF-7/adr cells, showing no significant influence on that of KB and MCF-7 cells. Furthermore, EFL1 could enhance the ATP hydrolysis activity of ABCB1 stimulated by verapamil. At the same time, EFL1 inhibited the efflux of ABCB1 in KBv200 and MCF-7/adr cells. In addition, EFL1 did not downregulate expression of ABCB1 in KBv200 and MCF-7/adr cells either in mRNA or protein level.

Blockade of Her2/neu binding to Hsp90 by emodin azide methyl anthraquinone derivative induces proteasomal degradation of Her2/neu.

Overexpression of HER2/neu, a transmembrane tyrosine kinase acting as a coreceptor for other EGFR family members, is well-known to be associated with a poor prognosis in cancer. In the present study, we observed that emodin AMAD, a novel emodin azide methyl anthraquinone derivative, extracted from nature's giant knotweed rhizome of traditional Chinese herbs, potently decreased Her2/neu protein in dose- and time-dependent manners and also inhibited the downstream MAPK and PI3K-Akt signaling pathway. Intriguingly, reverse transcription-PCR and protein turnover assay revealed that the decrease of Her2/neu was independent of mRNA level but primarily owing to its protein stability. Meanwhile, proteasome inhibitor MG132 but not lysosome inhibitor chloroquine could restore Her2/neu and polyubiquitination of Her2/neu was augmented during emodin AMAD treatment. Furthermore, immunofluorescence study with anti-Her2/neu antibody showed that emodin AMAD disturbed the subcellular distribution of Her2/neu, with decreased location in the plasma membrane. Molecular docking studies predicted that AMAD can interact with the ATP-binding pocket of both Hsp90 and Her2/neu. Importantly, coimmunoprecipitation and immunofluorescence study revealed that emodin AMAD markedly impaired the binding between Hsp90 and Her2/neu and could bind to both Hsp90 and Her2/neu as reinforced by molecular modeling studies. In addition, combination of emodin AMAD treatment and siRNA against Her2 synergistically inhibited proliferation and induced apoptosis. Taken together, these data suggest that blockade of Her2/neu binding to Hsp90 and following proteasomal degradation of Her2/neu were involved in emodin AMAD-induced apoptosis in Her2/neu-overexpressing cancer cells. Our results provide suggestions that emodin AMAD could be promising as a new targeting therapeutic strategy in the treatment of Her2/neu-overexpressing cancers.

Synthesis and cytotoxicity of 8-cyano-3-substitutedalkyl-5-methyl-4-methylene-7-methoxy-3,4-dihydropyrido[4,3-d]pyrimidines.

Synthesis and cytotoxicity of 11 4-methylene pyrido[4,3-d]pyrimidines 5a-k were described. Cytotoxicity assay results showed that some compounds had much stronger antitumor activity than Fluorouracil against KB cell lines. The most active compound 5i exhibited high potency against KB, CNE2, MGC-803 cell lines with IC(50) values of 0.48, 0.15, 0.59 µM, respectively. The preliminary structure-activity relationships indicated that the introduction of benzyl groups bearing electron-donating function groups is favorable for the activity.

Synthesis and biological evaluation of novel (thio)urea derivatives as potential antitumor agents.

A series of novel (thio)ureas containing the pyrimidinyl group was designed and synthesized. Their in-vitro antitumor activity against different human tumor cells was examined. Some of the compounds showed potential antitumor activity, which provided some hints for further studies on structure modification.

Structure identification of Euphorbia factor L3 and its induction of apoptosis through the mitochondrial pathway.

In this article, we have focused on the structure identification of Euphorbia factor L3 belonging to the lathyrane diterpenoids isolated from Caper Euphorbia Seed. Its anticancer activity in vitro against lung cancer A549 cells was also investigated and the IC(50) values were 34.04 ± 3.99 µM. Furthermore, Euphorbia factor L3 could induce apoptosis in A549 cells via the mitochondrial pathway including loss of mitochondrial potential and release of cytochrome c.

Anticancer effect and structure-activity analysis of marine products isolated from metabolites of mangrove fungi in the South China Sea.

Marine-derived fungi provide plenty of structurally unique and biologically active secondary metabolites. We screened 87 marine products from mangrove fungi in the South China Sea for anticancer activity by MTT assay. 14% of the compounds (11/86) exhibited a potent activity against cancer in vitro. Importantly, some compounds such as compounds 78 and 81 appeared to be promising for treating cancer patients with multidrug resistance, which should encourage more efforts to isolate promising candidates for further development as clinically useful chemotherapeutic drugs. Furthermore, DNA intercalation was not involved in their anticancer activities, as determined by DNA binding assay. On the other hand, the structure-activity analysis indicated that the hydroxyl group was important for their cytotoxic activity and that bulky functional groups such as phenyl rings could result in a loss of biological activity, which will direct the further development of marine product-based derivatives.

Anthracenedione derivatives as anticancer agents isolated from secondary metabolites of the mangrove endophytic fungi.

In this article, we report anticancer activity of 14 anthracenedione derivatives separated from the secondary metabolites of the mangrove endophytic fungi Halorosellinia sp. (No. 1403) and Guignardia sp. (No. 4382). Some of them inhibited potently the growth of KB and KBv200 cells, among which compound 6 displayed strong cytotoxicity with IC(50) values of 3.17 and 3.21 microM to KB and KBv200 cells, respectively. Furthermore, we demonstrate that the mechanism involved in the apoptosis induced by compound 6 is probably related to mitochondrial dysfunction. Additionally, the structure-activity relationships of these compounds are discussed.

A cell-based screen for anticancer activity of 13 pyrazolone derivatives.

BACKGROUND AND OBJECTIVE: Pyrazolone derivatives were reported to have a potent cytotoxicity against some tumor cells. In the present study, we evaluated the cytotoxic activity of a series of pyrazolone derivatives against four human tumor cell lines including HepG2, OVCAR3, KB, and multidrug resistance (MDR) KBv200 cell lines in vitro and in vivo. Additionally, the structure-activity relationships of these compounds were discussed. METHODS: To analyze the antiproliferative potential of the synthesized compounds against several human tumor cell lines, the 50% inhibitory concentration (IC50) values were determined by MTT assay. Besides, the KBv200 cell xenograft experimental model was established and the sensitivity to the pyrazolone compounds was compared between drug-sensitive parental KB cells and MDR KBv200 cells. RESULTS: Of 13 compounds screened, compound 9 presented remarkable anticancer effects, of which IC50 values were (3.24 ± 0.28), (2.58 ± 0.61), (3.81 ± 0.02), and (3.45 ± 0.03) µg/mL in HepG2, OVCAR3, KB and MDR KBv200 cells, respectively (P > 0.05). Furthermore, compound 9 effectively inhibited tumor growth of KBv200 cell xenografts in vivo, the inhibition ratio was 25.37%, 38.43%, and 47.50% for 1.5 mg/kg, 3 mg/kg, and 6 mg/kg of compound 9 groups, respectively. CONCLUSION: Compound 9 was the most promising antitumor agent in this study.

Bullatacin triggered ABCB1-overexpressing cell apoptosis via the mitochondrial-dependent pathway.

This paper was to explore bullatacin-mediated multidrug-resistant cell apoptosis at extremely low concentration. To investigate its precise mechanisms, the pathway of cell apoptosis induced by bullatacin was examined. Bullatacin causes an upregulation of ROS and a downregulation of DeltaPsi(m) in a concentration-dependent manner in ABCB1-overexpressing KBv200 cells. In addition, cleavers of caspase-9, caspase-3, and PARP were observed following the release of cytochrome c from mitochondria after bullatacin treatment. However, neither cleavage of caspase-8 nor change of expression level of bcl-2, bax and Fas was observed by the same treatment. Pretreating KBv200 cells with N-acetylcysteine, an antioxidant modulator, resulted in a significant reduction of ROS generation and cell apoptosis induced by bullatacin. Bullatacin-induced apoptosis was antagonized by z-LEHD-fmk, a caspase-9 inhibitor, but not by z-IETD-fmk, a caspase-8 inhibitor. These implied that apoptosis of KBv200 cells induced by bullatacin was associated with the mitochondria-dependent pathway that was limited to activation of apical caspase-9.

A facile synthesis and biological activity of novel tetrahydrobenzo[4',5']thieno[3',2':5,6]pyrido[4,3-d]pyrimidin-4(3H)-ones.

The synthesis and biological activity of 2-substituted-8,9,10,11-tetrahydrobenzo[4',5']thieno[3',2':5,6] pyrido[4,3-d]pyrimidin-4(3H)-ones are described. Bioassay results indicated that these compounds have antifungal activity against Botrytis cinerea at a concentration of 50mg/L. In addition, compounds 5m and 5n were effective to both KB cells and their parent multidrug resistant KBv200 cells with the overexpression of ABCB1. For example, compound 5m showed the best inhibition against KB and KBv200 cells with IC(50) values of 17.4 and 25.4 microM, respectively.

Efficient synthesis and biological evaluation of 1,2,9-trisubstituted 1,9-dihydro-6H-purin-6-ones.

The carbodiimides 4, obtained from aza-Wittig reactions of iminophosphorane 3 with aromatic isocyanates, reacted with amines in the presence of a catalytic amount of RO(-)Na(+) to give the 1,2,9-trisubstituted 1,9-dihydro-6H-purin-6-ones 6 in good yields. Compound 6 exhibited cytotoxicity against various cancer cells. For example, compounds 6b showed the best inhibition activities against KB, HepG2 and OVCAR3 with IC(50) 9.5, 20.4 and 10.0 microM.

Four new cytotoxic oligosaccharidic derivatives of 12-oleanene from Lysimachia heterogenea Klatt.

Cytotoxicity-guided phytochemical analysis on the extract of Lysimachia heterogenea Klatt led to the isolation of 3beta,16beta-12-oleanene-3,16,23,28-tetrol (1) and its four new oligosaccharidic derivatives heterogenosides A, B, C, and D (2-5). Their structural elucidation was mainly based on NMR and mass spectral data. The time course experimental results indicated that unlike the likely lysis activity of heterogenosides B-D, heterogenoside A showed a significantly time-dependent cytotoxicity.

Lgf-YL-9 induces apoptosis in human epidermoid carcinoma KB cells and multidrug resistant KBv200 cells via reactive oxygen species-independent mitochondrial pathway.

Pyrazolon derivatives were reported to have cytotoxicity to some tumour cells. In the present study, we investigated the effect of Lgf-YL-9 on cytotoxicity and cell apoptosis in human epidermoid carcinoma drug-sensitive parental KB cells and multidrug resistant (MDR) KBv200 cells. Lgf-YL-9 exhibited potent cytotoxicity not only to KB cells but also to KBv200 cells, and the IC(50) were 3.81 and 3.45 microg/mL in KB cells and KBv200 cells, respectively. Importantly, Lgf-YL-9 effectively inhibited tumour growth of KB cell xenografts in nude mice. Lgf-YL-9-induced cell apoptosis was confirmed by chromatin condensation, DNA fragmentation, Annexin-V and propidium iodide (PI) double-staining assay and poly(ADP-ribose) polymerase (PARP) cleavage. Furthermore, Lgf-YL-9-mediated apoptosis in KB cells and KBv200 cells was accompanied by the loss of mitochondrial membrane potential (DeltaPsi(m)), the release of cytochrome c, and the activation of caspases-3, -7, and -9, but not by intercalating to DNA. Although Lgf-YL-9-induced apoptosis was associated with the decrease of DeltaPsi(m), reactive oxygen species (ROS) reduction was interestingly observed in both cell lines. The data suggest that Lgf-YL-9 has similar cytotoxicity to drug-sensitive parental KB cells and MDR KBv200 cells. Lgf-YL-9-induced apoptosis is involved in a new ROS-independent mitochondrial dysfunction pathway, but not in intercalating to DNA.