ABSTRACT
3,4,5,4'-Trans-tetramethoxystilbene (Synonyms: DMU-212) is a resveratrol analogue with stronger antiproliferative activity and more bioavailability. However, the metabolite characterization of this component remains insufficient. An efficient strategy was proposed for the comprehensive in vivo metabolite profiling of DMU-212 after oral administration in ApcMin/+ mice based on the effectiveness of the medicine. Ultra-high performance liquid chromatography-quadrupole/orbitrap/linear ion trap mass spectrometry (UHPLC-Q/Orbitrap/LTQ MS) in the AcquireXTM intelligent data acquisition mode, combining the exact mass and structural information, was established for the profiling and identification of the metabolites of DMU-212 in vivo, and the possible metabolic pathways were subsequently proposed after the oral dose of 240mg/kg for 3 weeks in the colorectal adenoma (CRA) spontaneous model ApcMin/+ mice. A total of 63 metabolites of DMU-212 were tentatively identified, including 48, 48, 34 and 28 metabolites in the ApcMin/+ mice's intestinal contents, liver, serum, and colorectal tissues, respectively. The metabolic pathways, including demethylation, oxidation, desaturation, methylation, acetylation, glucuronide and cysteine conjugation were involved in the metabolism. Additionally, further verification of the representative active metabolites was employed using molecular docking analysis. This study provides important information for the further investigation of the active constituents of DMU-212 and its action mechanisms for CRA prevention.
Subject(s)
Colorectal Neoplasms , Mice , Animals , Chromatography, High Pressure Liquid/methods , Molecular Docking Simulation , Mass Spectrometry/methodsABSTRACT
PURPOSE: Steroid hormone secretion is one of the key functions of granulosa cells (GCs). Resveratrol is a natural polyphenol, known for its beneficial health effects, such as improving reproductive health. However, its application is limited due to poor bioavailability. The methoxy derivative of resveratrol (DMU-212) was demonstrated to be more lipophilic, and therefore of greater bioavailability. However, since the addition of methoxy groups to the stilbene scaffold was found to make the molecule insoluble in water, DMU-212 was loaded into liposomes. This study aimed to evaluate how the liposomal formulation of DMU-212 (lipDMU-212) alters estradiol and progesterone secretion of human ovarian GCs in a primary three-dimensional cell culture model. METHODS: DMU-212-loaded liposomes were prepared by thin film hydration followed by extrusion. Cell viability was measured after exposure of GCs spheroids to the liposomal formulation of DMU-212 using CellTiter-Glo® 3D Cell Viability Assay. The secretion of estradiol and progesterone was determined using commercial ELISA kits. RT-qPCR was conducted to analyze the expression of steroidogenesis-related genes. Finally, the western blot technique was used to analyze the effect of lipDMU-212 and FSH treatments on CYP11A1 and HSD3B1 protein levels. RESULTS: lipDMU-212 was found to significantly increase estradiol and progesterone secretion in a dose-dependent manner by enhancing the expression of CYP11A1, HSD3B1, StAR, CYP17A1, CYP19A1, and HSD17B1 genes. We have also shown that lipDMU-212, used alone and in combination with FSH, significantly increased the expression of the HSD3B1 and CYP11A1 proteins in GCs. Furthermore, our study suggests that lipDMU-212 increases FSH activity. CONCLUSIONS: This is the first study to describe the steroidogenic activity of liposomal formulation of DMU-212, possibly through increasing the StAR and CYP19A1 expression. These findings suggest that lipDMU-212 might have a beneficial effect in the treatment of disorders related to estrogen deficiency and hyperandrogenism, such as PCOS.
Subject(s)
Progesterone , Stilbenes , Female , Humans , Resveratrol/pharmacology , Resveratrol/metabolism , Progesterone/pharmacology , Cholesterol Side-Chain Cleavage Enzyme/metabolism , Liposomes/metabolism , Liposomes/pharmacology , Stilbenes/pharmacology , Stilbenes/metabolism , Estradiol/pharmacology , Follicle Stimulating Hormone/metabolism , Granulosa Cells/metabolism , Multienzyme Complexes/metabolism , Multienzyme Complexes/pharmacologyABSTRACT
Although some important advances have been achieved in clinical and diagnosis in the past few years, the management of non-small cell lung cancer (NSCLC) is ultimately dissatisfactory due to the low overall cure and survival rates. Epidermal growth factor (EGFR) has been recognized as a carcinogenic driver and is a crucial pharmacological target for NSCLC. DMU-212, an analog of resveratrol, has been reported to have significant inhibitory effects on several types of cancer. However, the effect of DMU-212 on lung cancer remains unclear. Therefore, this study aims to determine the effects and underlying mechanism of DMU-212 on EGFR-mutant NSCLC cells. The data found that the cytotoxicity of DMU-212 on three EGFR-mutant NSCLC cell lines was significantly higher than that of normal lung epithelial cell. Further study showed that DMU-212 can regulate the expression of cell cycle-related proteins including p21 and cyclin B1 to induce G2/M phase arrest in both H1975 and PC9 cells. Moreover, treatment with DMU-212 significantly promoted the activation of AMPK and simultaneously down-regulated the expression of EGFR and the phosphorylation of PI3K, Akt and ERK. In conclusion, our study suggested that DMU-212 inhibited the growth of NSCLCs via targeting of AMPK and EGFR.
ABSTRACT
The concept of a scaffold concerns many aspects at different steps on the drug development path. In medicinal chemistry, the choice of relevant "drug-likeness" scaffold is a starting point for the design of the structure dedicated to specific molecular targets. For many years, the chemical uniqueness of the stilbene structure has inspired scientists from different fields such as chemistry, biology, pharmacy, and medicine. In this review, we present the outstanding potential of the stilbene-based derivatives. Naturally occurring stilbenes, together with powerful synthetic chemistry possibilities, may offer an excellent approach for discovering new structures and identifying their therapeutic targets. With the development of scientific tools, sophisticated equipment, and a better understanding of the disease pathogenesis at the molecular level, the stilbene scaffold has moved innovation in science. This paper mainly focuses on the stilbene-based compounds beyond resveratrol, which are particularly attractive due to their biological activity. Given the "fresh outlook" about different stilbene-based compounds starting from stilbenoids with particular regard to isorhapontigenin and methoxy- and hydroxyl- analogues, the update about the combretastatins, and the very often overlooked and underestimated benzanilide analogues, we present a new story about this remarkable structure.
Subject(s)
Stilbenes/chemistry , Stilbenes/pharmacology , Animals , Bibenzyls/chemistry , Bibenzyls/pharmacology , Drug Discovery , Humans , Resveratrol/chemistry , Resveratrol/pharmacologyABSTRACT
BACKGROUND: Naphthalene is a good structural replacement for the isovanillin moiety (i.e. the 3- hydroxy-4-methoxyphenyl unit) in the combretastatin A-4 molecule, a natural product structurally related to resveratrol, which consistently led to the generation of highly cytotoxic naphthalene analogues when combined with a 3,4,5-trimethoxyphenyl or related aromatic system. Also, the naphthalene ring system is present in many current drug molecules that are utilized for anti-tumor, anti-arrhythmia and antioxidant therapy. OBJECTIVE: In our continuing quest to improve the potencies of naturally occurring anti-cancer molecules through chemical modification, we have now synthesized a small library of 2-naphthaleno trans- stilbenes and cyanostilbenes that are structurally related to both resveratrol and DMU-212, and have evaluated these novel analogs against a panel of 54 human tumor cell lines. METHOD: A series of 2-naphthaleno-containing trans-stilbenes 3a-3h (Scheme 1) were synthesized by Wittig reaction of a variety of aromatic substituted benzyl-triphenylphosphonium bromide reactants with 2- naphthaldehyde using n-BuLi as a base in THF. A second series of 2-naphthaleno trans-cyanostilbenes analogs 5a-5h was synthesized by reaction of 2-naphthaldehyde (2; 1 mmol) with an appropriately substituted 2- phenylacrylonitrile 4a-4h; 1 mmol) in 5% sodium methoxide/methanol. The reaction mixture was stirred at room temperature for 2-3 hours and the reaction allowed to go to completion (TLC monitoring), during which time the desired product precipitated out of the solution as a solid. The resulting precipitate was filtered off, washed with water and dried to yield the desired compound in yields ranging from 70-95% (Scheme 2). RESULTS: The percentage growth inhibition of 54 human cancer cell lines in a primary NCI screen after exposure to compounds 3a, 3d, 5b and 5c was carried out. The results showed that only compounds 5b and 5c met the criteria for subsequent testing to determine growth inhibition values (GI50) in dose-response studies. At 10-5 M concentration, compounds 5b and 5c exhibited cytotoxic activity against leukemia cell lines HL-60(TB) and SR, lung cancer cell line NCI-H522, colon cancer cell lines COLO 205 and HCT-116, CNS-cancer cell line SF-539, melanoma cell line MDA-MB-435, and breast cancer cell line BT-549. The naphthalene trans-stilbene analogue 3a, exhibited significant growth inhibition against only one cell line, melanoma cell line MDA-MB-435 (96 % growth inhibition). Compound 3d was inactive in the 10-5 M single dose screen. CONCLUSION: We have synthesized a small set of novel 2-naphthaleno stilbenes and cyanostilbenes and evaluated several of these compounds for their anticancer properties against a panel of 54 human tumor cell lines. The most active analogs, 5b and 5c, showed significantly improved growth inhibition against the human cancer cells in the NCI panel when compared to DMU-212. Of these compounds, analog 5c was found to be the most potent anticancer agent and exhibited significant growth inhibitory effects against COLO 205, CNS SF 539 and melanoma SK-MEL 5 and MDA-MB-435 cell lines with GI50 values ≤ 25 nM. Analog 5b also exhibited GI50 values in the range 25-41 nM against CNS SF 295 and melanoma MDA-MB-435 and UACC-62 cell lines. Compounds 5b and 5c were also cytotoxic towards the MV4-11 leukemia cell line with LD50 value of 450 nM and 200 nM, respectively, and demonstrated >50% inhibition of tubulin polymerization at concentrations below their LD50 values in these cells. In silico docking studies suggest that compounds 5b and 5c bind favorably at the colchicine- binding pocket of the tubulin dimer, indicating that both 5b and 5c may inhibit tubulin polymerization through a mechanism similar to that exhibited by colchicine. Derivative 5c demonstrated more favorable binding based on the docking score and buried surface area, as compared to compound 5b, in agreement with the higher observed potency of 5c against a broader range of tumor cell lines. Based on these results, analog 5c is considered to be a lead compound for further optimization as a clinical candidate for treating a variety of cancers.
Subject(s)
Antineoplastic Agents/pharmacology , Naphthalenes/pharmacology , Stilbenes/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Molecular Structure , Naphthalenes/chemistry , Stilbenes/chemical synthesis , Stilbenes/chemistry , Structure-Activity RelationshipABSTRACT
The role of CYP1A1 and CYP1B1 enzymes in the biotransformation and biological activity of the methylated resveratrol analogue, 3,4,5,4'-tetramethoxystilbene (DMU-212) is still elusive. Our recently published data have shown that one of the metabolites of DMU-212, 3'-hydroxy-3,4,5,4'-tetramethoxystilbene (DMU-214) exerts more potent cytotoxic effects in A-2780 ovarian cancer cell line, as compared to the parent compound. Hence, this study aims to elucidate whether the biological activity of DMU-212 is related to its biotransformation to DMU-214. Furthermore, we aimed to assess which enzymes of CYP1 family are involved in the biotransformation of DMU-212. The human ovarian cancer cell lines A-2780, A-2780CYP1A1(-) and non-cancerous human ovarian surface epithelial (HOSE) cells were employed in the present study. In contrary to other authors' suggestions we have found that CYP1A1 is the major enzyme of CYP1 family involved in the metabolic activation of DMU-212. Since the distinctly weaker anti-proliferative effects of DMU-212 against HOSE and A-2780CYP1A1(-) cells have been associated with the lack of the expression of CYP1A1, we suggest that the biological activity of the parent compound may be related to its metabolic activation to DMU-214 and the level of this enzyme.
Subject(s)
Antineoplastic Agents/pharmacology , Cytochrome P-450 CYP1A1/metabolism , Epithelial Cells/drug effects , Ovarian Neoplasms/drug therapy , Stilbenes/pharmacology , Activation, Metabolic , Antineoplastic Agents/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cytochrome P-450 CYP1A1/genetics , Cytochrome P-450 CYP1B1/genetics , Cytochrome P-450 CYP1B1/metabolism , Dose-Response Relationship, Drug , Epithelial Cells/enzymology , Epithelial Cells/pathology , Female , Humans , Hydroxylation , Ovarian Neoplasms/enzymology , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , RNA Interference , Stilbenes/metabolism , Time Factors , TransfectionABSTRACT
The aim of the study was to examine whether antioxidant properties of 3,4,4',5-tetramethoxystilbene (DMU-212) contribute to its anticarcinogenic activity and whether DMU-212 affects the expression of apoptosis-related genes. Two-stage model of hepatocarcinogenesis was used; male Wistar rats were challenged with N-nitrosodiethylamine (NDEA), 200 mg/kg body weight (b.w.), intraperitoneal, then phenobarbital (PB) in drinking water (0.05%) was administered. Simultaneously, DMU-212 was given per os at a dose 20 or 50 mg/kg b.w. two times a week for 16 weeks. DMU-212 caused a moderate decrease in hepatic thiobarbituric acid reactive substances and protein carbonyls concentration elevated in rats treated with NDEA/PB. The activity of antioxidant enzymes examined reduced by NDEA/PB treatment was not restored in rats coadministered with DMU-212. Effects of DMU-212 on messenger RNA (mRNA) expression of antioxidant enzymes in rats challenged with NDEA/PB were diversified; no changes in their protein expression were noted in any of the groups. The expression of 17,000 genes was analyzed by Affymetrix® Rat Gene 1.1 ST Array; 15 apoptosis-related genes were selected and validated by RT-q PCR. The combined treatment with NDEA/PB and DMU-212 increased the mRNA level of some genes driving mitochondria-mediated apoptosis, whereas the mRNA expression of some anti-apoptotic genes triggering receptor-mediated apoptosis was reduced. The expression of genes encoding caspases-4, -8, -9, and -12 was also increased in rats treated with DMU-212. Although antioxidant effect of DMU-212 in rats challenged with NDEA/PB was moderate, its potential anticarcinogenic properties were demonstrated as evidenced by modulation of apoptosis-related genes.
Subject(s)
Antioxidants/pharmacology , Apoptosis/genetics , Liver Neoplasms, Experimental/genetics , Stilbenes/chemistry , Stilbenes/pharmacology , Animals , Male , Oxidative Stress/drug effects , Rats , Rats, Wistar , ResveratrolABSTRACT
DMU-212 has been shown to evoke a mitochondrial apoptotic pathway in transformed fibroblasts and breast cancer. However, recently published data indicated the ability of DMU-212 to evoke apoptosis in both mitochondria- and receptor-mediated manner in two ovarian cancer cell lines, namely A-2780 and SKOV-3, which showed varied sensitivity to the compound tested. The pronounced cytotoxic effects of DMU-212 observed in A-2780 cells were related to the execution of extracellular apoptosis pathway and cell cycle arrest in G2/M phase. In view of the great anticancer potential of DMU-212 against A-2780 cell line, the aim of the current study was to assess antiproliferative activity of DMU-212 in xenograft model of ovarian cancer. To evaluate in vitro metabolic properties of cells that were to be injected into SCID mice, uptake and decline of DMU-212 in A-2780 ovarian cancer cell line was investigated. It was found that the concentration of the test compound in A-2780 cells was growing within first eight hours, and then the gradual decline was observed. A-2780 cells stably transfected with pcDNA3.1/Zeo(-)-Luc vector were subcutaneously inoculated into the right flanks of SCID mice. After seven days of the treatment with DMU-212 (50mg/kg b.w), tumor growth appeared to be suppressed in the animals treated with the compound tested. At day 14 of the experiment, tumor burden in mice treated with DMU-212 was significantly lower, as compared to untreated controls. Our findings suggest that DMU-212 might be considered as a potential anticancer agent used in ovarian cancer therapy.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Proliferation/drug effects , Ovarian Neoplasms/drug therapy , Stilbenes/pharmacology , Animals , Apoptosis/drug effects , Cell Line, Tumor , Female , Humans , Mice , Mice, SCID , Time Factors , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
The cytotoxic activity of DMU-212 has been shown to vary in cell lines derived from the same type of cancer, i.e. ovarian, breast and colorectal ones. However, the molecular mechanism of DMU-212 cytotoxicity has not been clarified in colon cancer cells. This study aims to elucidate the mechanism of antitumor effects of DMU-212 in two human colon cancer cell lines, DLD-1 and LOVO. We showed the stronger cytotoxic activity in DLD-1 cells in which DMU-212 evoked a greater pro-apoptotic effect as compared to that of LOVO cells. The analysis of the expression pattern of 84 apoptosis-related genes indicated transcripts specific to the mitochondria-mediated apoptosis pathway in both colon cancer cell lines used. We found that DMU-212 caused up-regulation of pro-apoptotic Bak1, Bok, Bik, Noxa, Bad, Bax, p53 and Apaf1 transcripts level in DLD-1 cell line, whereas anti-apoptotic Bcl-2, Bcl-xL and Bag1 mRNA expression was decreased. Changes in apoptosis-related genes expression were less pronounced in LOVO cells which did not express CYP1B1 protein and showed lower expression of CYP1A1 protein level than that in DLD-1 cells. Our results suggest that anticancer activity of DMU-212 is closely related to its biotransformation catalysed by these cytochrome P450 isoenzymes.