The Synthesis of Chalcones as Anticancer Prodrugs and their Bioactivation in CYP1 Expressing Breast Cancer Cells.

BACKGROUND: Although the expression levels of many P450s differ between tumour and corresponding normal tissue, CYP1B1 is one of the few CYP subfamilies which is significantly and consistently overexpressed in tumours. CYP1B1 has been shown to be active within tumours and is capable of metabolising a structurally diverse range of anticancer drugs. Because of this, and its role in the activation of procarcinogens, CYP1B1 is seen as an important target for anticancer drug development. OBJECTIVE: To synthesise a series of chalcone derivatives based on the chemopreventative agent DMU-135 and investigate their antiproliferative activities in human breast cancer cell lines which express CYP1B1 and CYP1A1. METHOD: A series of chalcones were synthesised in yields of 43-94% using the Claisen-Schmidt condensation reaction. These were screened using a MTT assay against a panel of breast cancer cell lines which have been characterised for CYP1 expression. RESULT: A number of derivatives showed promising antiproliferative activities in human breast cancer cell lines which express CYP1B1 and CYP1A1, while showing significantly lower toxicity towards a non-tumour breast cell line with no CYP expression. Experiments using the CYP1 inhibitors acacetin and α-naphthoflavone provided supporting evidence for the involvement of CYP1 enzymes in the bioactivation of these compounds. CONCLUSION: Chalcones show promise as anticancer agents with evidence suggesting that CYP1 activation of these compounds may be involved.

Synthesis of different heterocycles-linked chalcone conjugates as cytotoxic agents and tubulin polymerization inhibitors.

A series of new heterocycles-linked chalcone conjugates has been designed and synthesized by varying different alkane spacers. These conjugates were tested for their in vitro cytotoxic potential against a panel of selected human cancer cell lines namely, lung (A549 and NCI-H460), prostate (DU-145 and PC-3), colon (HCT-15 and HCT-116), and brain (U-87 glioblastoma) by MTT assay. Notably, among all the tested compounds, 4a exhibited potent cytotoxicity on NCI-H460 (lung cancer) cells with IC50 of 1.48±0.19µM. The compound 4a showed significant inhibition of tubulin polymerization and disruption of the formation of microtubules (IC50 of 9.66±0.06µM). Moreover, phase contrast microscopy and DAPI staining studies indicated that compound 4a can induce apoptosis in NCI-H460 cells. Further, the flow-cytometry analysis revealed that compound 4a arrests NCI-H460 cells in the G2/M phase of the cell cycle. In addition, molecular docking studies of the most active compounds 4a and 4b into the colchicine site of the tubulin, revealed the possible mode of interaction by these new conjugates.

Design, synthesis and cytotoxic activity of certain novel chalcone analogous compounds.

A series of chalcone analogous compounds were designed and synthesized. Replacing/substituting the enone or ethylenic bridge of the parent chalcone with rigid heterocyclic moieties or substituted aromatic amines gave nineteen target compounds. Their cytotoxic activities were screened against both breast and liver cancer cells as well as breast and liver normal cells. Target compounds were also evaluated for their inhibition activity of tubulin beta polymerization. Target compound 2e, 3a, 3b, 3c, 4a-4d, 5a, 5b and 6 showed broad spectrum excellent anticancer activity against both MCF-7 and HepG2. Compound 4a showed the most TUBb inhibition activity.

Design, synthesis and antiproliferative activity studies of novel dithiocarbamate-chalcone derivates.

A series of novel dithiocarbamate-chalcone derivates were designed, synthesized and evaluated for antiproliferative activity against three selected cancer cell lines (EC-109, SK-N-SH and MGC-803). Majority of the synthesized compounds exhibited moderate to potent activity against all the cancer cell lines assayed. Particularly, compounds II2 and II5 exhibited the excellent growth inhibition against SK-N-SH with IC50 values of 2.03µM and 2.46µM, respectively. Further mechanism studies revealed that compound II2 could obviously inhibit the proliferation of SK-N-SH cells by inducing apoptosis and arresting the cell cycle at G0/G1 phase.

Flavokawain A inhibits Cytochrome P450 in in vitro metabolic and inhibitory investigations.

ETHNOPHARMACOLOGICAL RELEVANCE: Flavokawain A, the major chalcone in kava extracts, was served as beverages for informal social occasions and traditional ceremonials in most South Pacific islands. It exhibited strong antiproliferative and apoptotic effects against human prostate and urinary bladder cancer cells. AIM OF THE STUDY: The current study was purposed to investigate the interaction between Flavokawain A and Cytochrome P450, including the inhibitory effects of Flavokawain A on predominant CYP450 isotypes and further clarified the inhibitory mechanism of FKA on CYP450 enzymes. Besides, study about identifying the key CYP450 isotypes responsible for the metabolism of FKA was also performed. MATERIALS AND METHODS: In this study, probe-based assays with rat liver microsome system were used to characterize the inhibitory effects of FKA. Molecular docking study was performed to further explore the binding site of FKA on CYP450 isoforms. In addition, chemical inhibition experiments using specific inhibitors (a-naphthoflavone, quinidine, sulfamethoxazde, ketoconazole, omeprazole) were performed to clarify the individual CYP450 isoform that are responsible for the metabolism of FKA. RESULTS: FKA showed significant inhibition on CYP1A2, CYP2D1, CYP2C6 and CYP3A2 activities with IC50 values of 102.23, 20.39, 69.95, 60.22µmol/L, respectively. The inhibition model was competitive, mixed-inhibition, uncompetitive, and noncompetitive for CYP1A2, CYP2D1, CYP2C6 and CYP3A2 enzymes. Molecular docking study indicated the ligand-binding conformation of FKA in the active site of CYP450 isoforms. The chemical inhibition experiments showed that the metabolic clearance rate of Flavokawain A decreased to 19.84%, 50.38%, and 67.02% of the control in the presence of ketoconazole, sulfamethoxazde and a-naphthoflavone. CONCLUSION: The study showed that Flavokawain A has varying inhibitory effect on CYP450 enzymes and CYP3A2 was the principal CYP isoform contributing to the metabolism of Flavokawain A. Besides, CYP2C6 and CYP1A2 isoforms also play important roles in the metabolism of FKA. Our results provided a basis for better understanding the biotransformation of FKA and prediction of drug-drug interaction of FKA.

Flavokawains A and B from kava (Piper methysticum) activate heat shock and antioxidant responses and protect against hydrogen peroxide-induced cell death in HepG2 hepatocytes.

Context Flavokawains are secondary metabolites from the kava plant (Piper methysticum Forst. f., Piperaceae) that have anticancer properties and demonstrated oral efficacy in murine cancer models. However, flavokawains also have suspected roles in rare cases of kava-induced hepatotoxicity. Objective To compare the toxicity flavokawains A and B (FKA, FKB) and monitor the resulting transcriptional responses and cellular adaptation in the human hepatocyte cell line, HepG2. Materials and methods HepG2 were treated with 2-100 µM FKA or FKB for 24-48 h. Cellular viability was measured with calcein-AM and changes in signalling and gene expression were monitored by luciferase reporter assay, real-time PCR and Western blot of both total and nuclear protein extracts. To test for subsequent resistance to oxidative stress, cells were pretreated with 50 µM FKA, 10 µM FKB or 10 µM sulphoraphane (SFN) for 24 h, followed by 0.4-2.8 mM H2O2 for 48 h, and then viability was assessed. Results FKA (≤100 µM) was not toxic to HepG2, whereas FKB caused significant cell death (IC50=23.2 ± 0.8 µM). Both flavokawains activated Nrf2, increasing HMOX1 and GCLC expression and enhancing total glutathione levels over 2-fold (p < 0.05). FKA and FKB also activated HSF1, increasing HSPA1A and DNAJA4 expression. Also, flavokawain pretreatment mitigated cell death after a subsequent challenge with H2O2, with FKA being more effective than FKB, and similar to SFN. Conclusions Flavokawains promote an adaptive cellular response that protects hepatocytes against oxidative stress. We propose that FKA has potential as a chemopreventative or chemotherapeutic agent.

Flavokawains a and B in kava, not dihydromethysticin, potentiate acetaminophen-induced hepatotoxicity in C57BL/6 mice.

Anxiolytic kava products have been associated with rare but severe hepatotoxicity in humans. This adverse potential has never been captured in animal models, and the responsible compound(s) remains to be determined. The lack of such knowledge greatly hinders the preparation of a safer kava product and limits its beneficial applications. In this study we evaluated the toxicity of kava as a single entity or in combination with acetaminophen (APAP) in C57BL/6 mice. Kava alone revealed no adverse effects for long-term usage even at a dose of 500 mg/kg bodyweight. On the contrary a three-day kava pretreatment potentiated APAP-induced hepatotoxicity, resulted in an increase in serum ALT and AST, and increased severity of liver lesions. Chalcone-based flavokawains A (FKA) and B (FKB) in kava recapitulated its hepatotoxic synergism with APAP while dihydromethysticin (DHM, a representative kavalactone and a potential lung cancer chemopreventive agent) had no such effect. These results, for the first time, demonstrate the hepatotoxic risk of kava and its chalcone-based FKA and FKB in vivo and suggest that herb-drug interaction may account for the rare hepatotoxicity associated with anxiolytic kava usage in humans.

Cytotoxic, antimutagenic, and antioxidant activities of methanolic extract and chalcone dimers (lophirones B and C) derived from Lophira alata (Van Tiegh. Ex Keay) stem bark.

The cytotoxic, antimutagenic, and antioxidant activities of methanolic extract and lophirones B and C derived from Lophira alata stem bark were evaluated. The extract and lophirones B and C significantly (P < .05) reduced the viability of Ehrlich ascites carcinoma cells. There were concentration-dependent reduction in 4-nitro-o-aminophenylenediamine and benzo[a]pyrene-induced frame shift mutation as well as aflatoxin B1-induced base pair substitution by the extract and lophirones B and C. The extract and lophirones B and C concentration dependently scavenged DPPH radical, superoxide anion radical, hydrogen peroxide, hydroxyl radicals, and reduced ferric ion in the potassium hexacyanoferrate III reducing system. The results obtained from this study revealed that methanolic extract and lophirones B and C derived from Lophira alata stem bark posses anticancer, antimutagenic, and antioxidant activities, with lophirone C producing the best anticancer, antimutagenic, and antioxidant activities. The acclaimed anticancer activity of Lophira alata may be attributed to lophirones B and C.

Cytotoxic and apoptotic effects of chalcone derivatives of 2-acetyl thiophene on human colon adenocarcinoma cells.

Recent studies report that chalcones exhibit cytotoxicity to human cancer cell lines. Typically, the form of cell death induced by these compounds is apoptosis. In the context of the discovery of new anticancer agents and in light of the antitumour potential of several chalcone derivatives, in the present study, we synthesized and tested the cytotoxicity of six chalcone derivatives on human colon adenocarcinoma cells. Six derivatives of 3-phenyl-1-(thiophen-2-yl) prop-2-en-1-one were prepared and characterized on the basis of their (1) H and (13) C NMR spectra. HT-29 cells were treated with synthesized chalcones on two concentrations by three different incubation times. Cells were evaluated by cell morphology, Tetrazolium dye (MTT) colorimetric assay, live/dead, flow cytometry (annexin V) and gene expression analyses to determine the cytotoxic way. Chalcones 3-(4-bromophenyl)-1-(thiophen-2-yl)prop-2-en-1-one (C06) and 3-(2-nitrophenyl)-1-(thiophen-2-yl)prop-2-en-1-one (C09) demonstrated higher cytotoxicity than other chalcones as shown by cell morphology, live/dead and MTT assays. In addition, C06 induced apoptosis on flow cytometry annexin V assay. These data were confirmed by a decreased expression of anti-apoptotic genes and increased pro-apoptotic genes. Our findings indicate in summary that the cytotoxic activity of chalcone C06 on colorectal carcinoma cells occurs by apoptosis.

Targeting death receptor TRAIL-R2 by chalcones for TRAIL-induced apoptosis in cancer cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in cancer cells without toxicity to normal cells. TRAIL binds to death receptors, TRAIL-R1 (DR4) and TRAIL-R2 (DR5) expressed on cancer cell surface and activates apoptotic pathways. Endogenous TRAIL plays an important role in immune surveillance and defense against cancer cells. However, as more tumor cells are reported to be resistant to TRAIL mediated death, it is important to search for and develop new strategies to overcome this resistance. Chalcones can sensitize cancer cells to TRAIL-induced apoptosis. We examined the cytotoxic and apoptotic effects of TRAIL in combination with four chalcones: chalcone, isobavachalcone, licochalcone A and xanthohumol on HeLa cancer cells. The cytotoxicity was measured by MTT and LDH assays. The apoptosis was detected using annexin V-FITC staining by flow cytometry and fluorescence microscopy. Death receptor expression was analyzed using flow cytometry. The decreased expression of death receptors in cancer cells may be the cause of TRAIL-resistance. Chalcones enhance TRAIL-induced apoptosis in HeLa cells through increased expression of TRAIL-R2. Our study has indicated that chalcones augment the antitumor activity of TRAIL and confirm their cancer chemopreventive properties.

Synthesis and selective cytotoxic activity of novel hybrid chalcones against prostate cancer cells.

A new class of hybrid chalcones (17a-l &18a-l) was synthesized by Claisen-Schmidt condensation. All compounds were characterized by (1)H NMR, IR and mass spectral analysis and tested for their cytotoxic activity against PC-3 (prostate cancer), HT-29 (colon cancer), B-16 (mouse macrophages) and NCI-H460 (lung cancer) cell lines. Three compounds 18i, 18j and 18l (IC(50)=8.4, 7.9 & 5.9 µM) showed significant activity against PC-3 cell line.

Synthesis of a natural chalcone and its prenyl analogs--evaluation of tumor cell growth-inhibitory activities, and effects on cell cycle and apoptosis.

Six prenyl (=3-methylbut-2-en-1-yl) chalcones (=1,3-diphenylprop-2-en-1-ones), 2-7, and one natural non-prenylated chalcone, 1, have been synthesized and evaluated for their in vitro growth-inhibitory activity against three human tumor cell lines. A pronounced dose-dependent growth-inhibitory effect was observed for all prenylated derivatives, except for 7. The chalcone possessing one prenyloxy group at C(2'), i.e., 2, was the most active derivative against the three human tumor cell lines (5.9

In vitro inhibitory properties of ferrocene-substituted chalcones and aurones on bacterial and human cell cultures.

Two series of ten chalcones and ten aurones, where ferrocene replaces the C ring and with diverse substituents on the A ring were synthesized. The compounds were tested against two antibiotic-sensitive bacterial strains, E. coli ATCC 25922 and S. aureus ATCC 25923, and two antibiotic-resistant strains, S. aureus SA-1199B and S. epidermidis IPF896. The unsubstituted compound and those with methoxy substitution showed an inhibitory effect on all bacterial strains at minimum inhibitory concentrations ranging between 2 and 32 mg L(-1). For four of these compounds, the effect was bactericidal, as opposed to bacteriostatic. The corresponding organic aurones did not show growth inhibition, underscoring the role of the ferrocene group. The methoxy-substituted aurones and the unsubstituted aurone also showed low micromolar (IC(50)) activity against MRC-5 non-tumoral lung cells and MDA-MB-231 breast cancer cells, suggesting non-specific toxicity.

Synthesis and topoisomerase II inhibitory and cytotoxic activity of oxiranylmethoxy- and thiiranylmethoxy-chalcone derivatives.

In order to find potential anticancer drug candidate targeting topoisomerases enzyme, we have designed and synthesized oxiranylmethoxy- and thiiranylmethoxy-retrochalcone derivatives and evaluated their pharmacological activity including topoisomerases inhibitory and cytotoxic activity. Of the compounds prepared compound 25 showed comparable or better cytotoxic activity against cancer cell lines tested. Compound 25 inhibited MCF7 (IC(50): 0.49 ± 0.21 µM) and HCT15 (IC(50): 0.23 ± 0.02 µM) carcinoma cell growth more efficiently than references. In the topoisomerases inhibition test, all the compounds were inactive to topoisomerase I but moderate inhibitors to topoisomerase II enzyme. Especially, compound 25 inhibited topoisomerase II activity with comparable extent to etoposide at 100 µM concentrations. Correlation between cytotoxicity and topoisomerase II inhibitory activity implies that compound 25 can be a possible lead compound for anticancer drug impeding the topoisomerase II function.

Naphthylchalcones induce apoptosis and caspase activation in a leukemia cell line: The relationship between mitochondrial damage, oxidative stress, and cell death.

In this study, we investigated the effects of 24 chalcone derivatives from 2-naphthylacetophenone toward a lymphoblastic leukemia cell line (L1210). Three compounds, called R7, R13, and R15, presented concentration- and time-dependent cytotoxicity and induced cellular death by apoptosis via mitochondrial injury and oxidative stress. The effects of these compounds appear to occur through different mechanisms because R13 and R7 induced a greater disturbance of mitochondrial potential, and all compounds induced disturbances of cellular ATP content and increased caspase-3 activity before cellular death. These compounds also interfered with antioxidant enzymes activities and GSH content through different mechanisms.

Synthesis, characterization, electrochemical studies and antitumor activity of some new chalcone analogues containing ferrocenyl pyrazole moiety.

A series of new alpha,beta-unsaturated conjugated ketones containing ferrocenyl pyrazole unit were synthesized and fully characterized by IR and NMR spectroscopy. Electrochemical characterization of subject compounds was performed by means of cyclic voltametry. The in vitro cytotoxic activity of all the synthesized compounds was studied against cervix adenocarcinoma HeLa, melanoma Fem-x and myelogenous leukemia K562 cell lines by the MTT method. Derivative 1l containing 3-pyridyl moiety exhibited a better cytotoxic activity in the cell growth inhibition of K562 cell lines in comparison with cisplatin as a reference compound.

Combretastatin-chalcone hybrids: synthesis and cytotoxicity.

A series of all-trans-1-aryl-4-aryl-5-aryl-2,4-pentanediene-1-one (3), a hybridized form of chalcone and combretastatin, was synthesized and evaluated against a panel of cancer cell lines, including B16, murine melanoma; HCT116, colon cancer; A431, human epidermoid carcinoma; and human umbilical venous endothelial cells (HUVEC). Structure-activity relationships analysis of this series revealed that a 2,5-dihydroxyphenyl at position 1 of the 2,4-pentanediene-1-one was essential for cytotoxicity. all-trans-1-(2,5-Dihydroxyphenyl)-5-(4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-2,4-pentanediene-1-one (3a) was the most potent compound from this series.

In vivo antitumor activity by 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone in a solid human carcinoma xenograft model.

Previously, we showed that 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC), isolated from the buds of Cleistocalyx operculatus, significantly inhibited the growth of human liver cancer SMMC-7721 cells and could induce SMMC-7721 cells apoptosis in vitro. Here, we report the antitumor effects of DMC in vivo, using a solid human tumor xenograft model with a human liver cancer SMMC-7721 cell line. Our results revealed that the average tumor weight in a control group and a 150-mg/kg DMC injection group was 1.42+/-0.11 g and 0.59+/-0.12 g, respectively. Flow cytometric analysis of the tumor cell population demonstrated the existence of an aneuploid peak (representing 33.60+/-0.80% of the total in the 150-mg/kg DMC injection group). To our knowledge, this is the first time that chalcone compounds were applied to a human tumor xenograft model.

Hydroxychalcones exhibit differential effects on XRE transactivation.

Chalcones are phenolic compounds that can be isolated from plants. Previous studies have described some pharmacological applications for these compounds. Making use of our established reporter gene system, we determined the effect of five hydroxychalcones--2-hydroxychalcone, 2'-hydroxychalcone, 4-hydroxychalcone, 4,2',4'-trihydroxychalcone, and 3,4,2',4'-tetrahydroxychalcone--on the cellular xenobiotic responsive element (XRE)-transactivation. The interference of chalcones acting against polycyclic aromatic hydrocarbon (PAH)-DNA binding was also examined. Enzyme inhibition assays of cytochrome P450 (CYP) 1A1 and CYP1B1 were initially performed on recombinant protein expressed in insect microsomes. 2'-Hydroxychalcone and 2-hydroxychalcone were the most effective among the tested hydroxychalcones. The two hydroxychalcones had comparable IC50 values for CYP1A1 and CYP1B1, which were determined to be at the micromolar and submicromolar range, respectively. However, reporter gene assays indicated that 2'-hydroxychalcone suppressed XRE-transactivation, whereas 2-hydroxychalcone induced it when 7,12-dimethylbenz[a]anthracene (DMBA) was co-administered. In the absence of DMBA, 10 microM 2-hydroxychalcone and 2'-hydroxychalcone increased XRE-transactivation by 18- and 2.5-fold, respectively, while other chalcones did not significantly alter the response. Cultures treated with the two hydroxychalcones also displayed separate trends in ethoxyresorufin-O-deethylase (EROD) activity and DMBA-DNA covalent binding. In summary, the present study illustrated that the inhibition of hydroxychalcone on CYP1 enzymes and XRE-transactivation was affected by the position and number of hydroxyl groups in its structure.

Cytotoxic 4'-aminochalcones and related compounds.

A series of 4'-aminochalcones 1 and related maleamic acids 2 and Schiff bases 3 were designed and synthesized as candidate cytotoxic agents. The atomic charges on different atoms of representative compounds were calculated. Evaluation of the enones 1-3 against human Molt 4/C8 and CEM T-lymphocytes as well as murine P388 and L1210 leukemic cells revealed that approximately 40% of the IC50 values generated were less than 10 microM. In some cases cytotoxicity was correlated with the Hammett sigma values of the aryl substituents and less frequently with the aryl Hansch pi values. Evidence was obtained that in general these compounds displayed selective toxicity for certain malignant cells and were well tolerated in mice. This study has revealed various directions whereby the project may be amplified in the future with a view to finding compounds with increased cytotoxicity to tumour cells.