Syntheses and evaluation of acridone derivatives as anticancer agents targeting Kras promoter i-motif structure.

Two series of novel acridone derivatives were designed and synthesized, with their anticancer activity evaluated. Most of these compounds showed potent antiproliferative activity against cancer cell lines. Among them, compound C4 with dual 1,2,3-triazol moieties exhibited the most potent activity against Hep-G2 cells with IC50 value determined to be 6.29 ± 0.93 µM. Subsequent experiments showed that C4 could bind to and destabilize Kras gene promoter i-motif structure without significant interaction with its corresponding G-quadruplex. C4 could down-regulate Kras expression in Hep-G2 cells, possibly due to its interaction with the Kras i-motif. Further cellular studies indicated that C4 could induce apoptosis of Hep-G2 cells, possibly related to its effect on mitochondrial dysfunction. These results indicated that C4 could be further developed as a promising anticancer agent.

Development of a Smart Fluorescent Probe Specifically Interacting with C-Myc I-Motif.

I-motifs play key regulatory roles in biological processes, holding great potential as attractive therapeutic targets. In the present study, we developed a novel fluorescent probe G59 with strong and selective binding to the c-myc gene promoter i-motif. G59 had an i-motif-binding carbazole moiety conjugated with naphthalimide fluorescent groups. G59 could differentiate the c-myc i-motif from other DNA structures through selective activation of its fluorescence, with its apparent visualization in solution. The smart probe G59 showed excellent sensitivity, with a low fluorescent detection limit of 154 nM and effective stabilization to the c-myc i-motif. G59 could serve as a rapid and sensitive probe for label-free screening of selective c-myc i-motif binding ligands under neutral crowding conditions. To the best of our knowledge, G59 is the first fluorescent probe with high sensitivity for recognizing the i-motif structure and screening for selective binding ligands.

High anticancer activity and apoptosis- and autophagy-inducing properties of novel lanthanide(III) complexes bearing 8-hydroxyquinoline-N-oxide and 1,10-phenanthroline.

In the quest for rare earth metal complexes with enhanced cancer chemotherapeutic properties, the discovery of seven lanthanide(iii) complexes bearing 8-hydroxyquinoline-N-oxide (NQ) and 1,10-phenanthroline (phen) ligands, i.e., [SmIII(NQ)(phen)(H2O)Cl2] (Ln1), [EuII(NQ)(phen)(H2O)Cl2] (Ln2), [GdIII(NQ)(phen)(H2O)Cl2] (Ln3), [DyIII(NQ)(phen)(H2O)Cl2] (Ln4), [HoIII(NQ)(phen)(H2O)Cl2] (Ln5), [ErIII(NQ)(phen)(H2O)Cl2] (Ln6), and [YbIII(NQ)(phen)(H2O)Cl2] (Ln7), as potential anticancer drugs is described. Complexes Ln1-Ln7 exhibit high antiproliferative activity against cisplatin-resistant A549/DDP cells (IC50 = 0.025-0.097 µM) and low toxicity to normal HL-7702 cells. Moreover, complex Ln1, and to a lesser extent Ln7, can upregulate the expression of LC3 and Beclin1 and downregulate p62 to induce apoptosis in cisplatin-resistant A549/DDP cell lines, which is related to the cell autophagy-inducing properties of Ln1 and Ln7. Furthermore, in vivo assays suggest that Ln1 significantly inhibits A549/DDP xenograft tumor growth (56.5%). These results indicate that lanthanide(iii) complex Ln1 is a promising candidate as an anticancer drug against cisplatin-resistant A549/DDP cells.

Copper(II) and iron(III) complexes of chiral dehydroabietic acid derived from natural rosin: metal effect on structure and cytotoxicity.

A novel optically pure dinuclear copper(II) complex of a rosin derivative dehydroabietic acid (DHA, HL) was synthesized and fully characterized. The in vitro antitumor activities of the copper(II) complex Cu2(µ2-O)(L)4(DMF)2 (1) were explored and compared with those of a trinuclear iron(III) complex [Fe3(µ3-O)(L)6(CH3OH)2(CH3O)]·H2O (2). 1 was more cytotoxic than 2, and the in vitro cytotoxicity of 1 was comparable to that of cisplatin and oxaliplatin. The metal coordination improved the cytotoxicity of DHA. 1 could arrest cycle in G1 phase and induce apoptosis in MCF-7 cell. 1 increased reactive oxygen species level, GSSG/GSH ratio, and Ca2+ production, and caused the loss of mitochondrial membrane potential (Δψm) in MCF-7 cells. The up-regulated Bax and down-regulated Bcl-2 expression levels, caspase-9/caspase-3 activation, and the release of Cyt c demonstrate that 1 triggered mitochondria-mediated intrinsic apoptosis in MCF-7 cells. Caspase-8/caspase-4 activation and up-regulated Fas expression indicate that death receptor-mediated extrinsic apoptosis was included. Comet assay and up-regulated γ-H2AX and p53 expressions confirmed that 1 caused DNA damage in MCF-7 cells. Moreover, 1 led to enhancement of the biomarker of lipid peroxidation and the indicator of protein carbonylation in MCF-7 cells. All the results suggest that 1 could kill MCF-7 cells by generating oxidative stress, impairing DNA, promoting lipid peroxidation and protein carbonylation, and inducing apoptosis and autophagy. Furthermore, 1 also displayed antimetastatic activities with inhibition of cell invasion and migration, together with antiangiogenesis properties. On the whole, copper complex based on rosin derivatives is worth developing as metal-based antitumor drugs.

Transcriptional regulation of telomeric repeat-containing RNA by acridine derivatives.

Telomere is a specialized DNA-protein complex that plays an important role in maintaining chromosomal integrity. Shelterin is a protein complex formed by six different proteins, with telomeric repeat factors 1 (TRF1) and 2 (TRF2) binding to double-strand telomeric DNA. Telomeric DNA consists of complementary G-rich and C-rich repeats, which could form G-quadruplex and intercalated motif (i-motif), respectively, during cell cycle. Its G-rich transcription product, telomeric repeat-containing RNA (TERRA), is essential for telomere stability and heterochromatin formation. After extensive screening, we found that acridine derivative 2c and acridine dimer DI26 could selectively interact with TRF1 and telomeric i-motif, respectively. Compound 2c blocked the binding of TRF1 with telomeric duplex DNA, resulting in up-regulation of TERRA. Accumulated TERRA could bind with TRF1 at its allosteric site and further destabilize its binding with telomeric DNA. In contrast, DI26 could destabilize telomeric i-motif, resulting in down-regulation of TERRA. Both compounds exhibited anti-tumour activity for A549 cells, but induced different DNA damage pathways. Compound 2c significantly suppressed tumour growth in A549 xenograft mouse model. The function of telomeric i-motif structure was first studied with a selective binding ligand, which could play an important role in regulating TERRA transcription. Our results showed that appropriate level of TERRA transcript could be important for stability of telomere, and acridine derivatives could be further developed as anti-cancer agents targeting telomere. This research increased understanding for biological roles of telomeric i-motif, TRF1 and TERRA, as potential anti-cancer drug targets.

Syntheses and evaluation of acridone-naphthalimide derivatives for regulating oncogene PDGFR-ß expression.

Upregulation of platelet-derived growth factor receptor ß (PDGFR-ß) has been found to be associated with development of various types of cancers, which has become an attractive target for anti-tumor treatment. Previously, we have synthesized and studied an acridone derivative B19, which can selectively bind to and stabilize oncogene c-myc promoter i-motif, resulting in down-regulation of c-myc transcription and translation, however its effect on tumor cells apoptosis requires improvement. In the present study, we synthesized a variety of B19 derivatives containing a known anti-cancer fluorescent chromophore naphthalimide for the purpose of enhancing anti-cancer activity. After screening, we found that acridone-naphthalimide derivative WZZ02 could selectively stabilize PDGFR-ß promoter G-quadruplex and destabilize its corresponding i-motif structure, without significant interaction to other oncogenes promoter G-quadruplex and i-motif. WZZ02 down-regulated PDGFR-ß gene transcription and translation in a dose-dependent manner, possibly due to above interactions. WZZ02 could significantly inhibit cancer cell proliferation, and induce cell apoptosis and cycle arrest. WZZ02 exhibited tumor growth inhibition activity in MCF-7 xenograft tumor model, which could be due to its binding interactions with PDGFR-ß promoter G-quadruplex and i-motif. Our results suggested that WZZ02 as a dual G-quadruplex/i-motif binder could be effective on both oncogene replication and transcription, which could become a promising lead compound for further development with improved potency and selectivity. The wide properties for the derivatives of 1,8-naphthalimide could facilitate further in-depth mechanistic studies of WZZ02 through various fluorescent physical and chemical methods, which could help to further understand the function of PDGFR-ß gene promoter G-quadruplex and i-motif.

Mitochondria-localizing curcumin-cryptolepine Zn(II) complexes and their antitumor activity.

Many metal complexes are potent candidates as mitochondrial-targeting agents. In this study, four novel Zn(II) complexes, [Zn(BPQA)Cl2] (Zn1), [Zn(BPQA)(Curc)]Cl (Zn2), [Zn(PQA)Cl2] (Zn3), and [Zn(PQA)(Curc)]Cl (Zn4), containing N,N-bis(pyridin-2-ylmethyl)benzofuro[3,2-b]quinolin-11-amine (BPQA), N-(pyridin-2-ylmethyl)benzofuro[3,2-b]quinolin-11-amine (PQA), and curcumin (H-Curc) were synthesized. An MTT assay showed that Zn1-Zn4 had strong anticancer activities against SK-OV-3/DDP and T-24 tumor cells with IC50 values of 0.03-6.19 µM. Importantly, Zn1 and Zn2 displayed low toxicities against normal HL-7702 cells. Mechanism experiments demonstrated that probe Zn2 showed appreciable fluorescence in the red region of the spectrum, and substantial accumulation of Zn2 occurred in the mitochondria after treatment, indicating increases in Ca2+ and reactive oxygen species levels, loss of the mitochondrial membrane potential, and consequent induction of mitochondrial dysfunction at low concentrations. In addition, the probe Zn2 effectively (50.7%) inhibited the growth of T-24 bladder tumor cells in vivo. The probe Zn2 shows potential for use in cancer therapy while retaining the H-Curc as an imaging probe.

Syntheses and Evaluation of New Bisacridine Derivatives for Dual Binding of G-Quadruplex and i-Motif in Regulating Oncogene c-myc Expression.

The c-myc oncogene is an important regulator for cell growth and differentiation, and its aberrant overexpression is closely related to the occurrence and development of various cancers. Thus, the suppression of c-myc transcription and expression has been investigated for cancer treatment. In this study, various new bisacridine derivatives were synthesized and evaluated for their binding with c-myc promoter G-quadruplex and i-motif. We found that a9 could bind to and stabilize both G-quadruplex and i-motif, resulting in the downregulation of c-myc gene transcription. a9 could inhibit cancer cell proliferation and induce SiHa cell apoptosis and cycle arrest. a9 exhibited tumor growth inhibition activity in a SiHa xenograft tumor model, which might be related to its binding with c-myc promoter G-quadruplex and i-motif. Our results suggested that a9 as a dual G-quadruplex/i-motif binder could be effective in both oncogene replication and transcription and become a promising lead compound for further development with improved potency and selectivity.

Upregulation of BCL-2 by acridone derivative through gene promoter i-motif for alleviating liver damage of NAFLD/NASH.

Nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH) are global epidemic public health problems with pathogenesis incompletely understood. Hepatocyte excessive apoptosis is a significant symbol for NAFLD/NASH patients, and therefore anti-apoptosis therapy could be used for NAFLD/NASH treatment. Up-regulation of BCL-2 has been found to be closely related with anti-apoptosis. BCL-2 gene promoter region has a C-rich sequence, which can form i-motif structure and play important role in regulating gene transcription. In this study, after extensive screening and evaluation, we found that acridone derivative A22 could up-regulate BCL-2 transcription and translation in vitro and in cells through selective binding to and stabilizing BCL-2 gene promoter i-motif. Our further experiments showed that A22 could reduce hepatocyte apoptosis in NAFLD/NASH model possibly through up-regulating BCL-2 expression. A22 could reduce inflammation, endoplasmic reticulum stress and cirrhosis in high-fat diet-fed mice liver model. Our findings provide a potentially new approach of anti-apoptosis for NAFLD/NASH treatment, and A22 could be further developed as a lead compound for NAFLD/NASH therapy. Our present study first demonstrated that gene promoter i-motif could be targeted for gene up-regulation for extended treatment of other important diseases besides cancer.

Imaging and therapeutic applications of Zn(ii)-cryptolepine-curcumin molecular probes in cell apoptosis detection and photodynamic therapy.

Novel red Zn(ii) complex-based fluorescent probes featuring cryptolepine-curcumin derivatives, namely, [Zn(BQ)Cl2] (BQ-Zn) and [Zn(BQ)(Cur)]Cl (BQCur-Zn), were developed for the simple and fluorescent label-free detection of apoptosis, an important biological process. The probes could synergistically promote mitochondrion-mediated apoptosis and enhance tumor therapeutic effects in vitro and vivo.

Biological evaluation of optically pure chiral binuclear copper(ii) complexes based on a rosin derivative as highly potential anticancer agents.

Two optically pure chiral binuclear copper(ii) complexes [Cu2(µ-Cl)2L2]·CH2Cl2 (1) and Cu2L4 (2) based on the natural product rosin derivative N-(5-dehydroabietyl-1,3,4-thiadiazole)-2-substituted pyridinecarboxamide (HL) were prepared, fully characterized and their biological activities were evaluated. The circular dichroism (CD), fluorescence spectroscopy, and DNA melting studies indicate that 1 and 2 interact with calf thymus DNA (CT DNA) via intercalation. It can be concluded that 1 and 2 have a strong affinity to bovine serum albumin (BSA) based on the fluorescence and CD spectral evidence. The MTT assay illustrates that the selective cytotoxic activity of 1 is better than that of HL, 2, cisplatin and oxaliplatin. The exposure of 1 to MCF-7 cells resulted in cell cycle arrest in the G1 phase, apoptosis, mitochondrial dysfunction and an elevated ROS level. The western blot analysis results indicate that 1 might induce apoptosis through intrinsic and extrinsic pathways, autophagy and DNA damage in MCF-7 cells. Furthermore, the down-regulated VEGFR2, MMP-2 and MMP-9 expression levels indicate that 1 should have the ability to resist metastasis and angiogenesis. Thus, based on the above described results 1 has high potential value for anticancer applications.

Optically pure chiral copper(II) complexes of rosin derivative as attractive anticancer agents with potential anti-metastatic and anti-angiogenic activities.

The development of optically pure drugs is the trend of new drugs research. Searching for optically pure metallodrugs against cancer has not been taken seriously. [CuL4Cl]Cl·2CH2Cl2·H2O (1) and [CuL4Br]Br·2CH2Cl2 (2) (L = 2-amino-5-dehydroabietyl-1,3,4-thiadiazole), two rosin-derivative based optically pure chiral copper(II) complexes, are rationally synthesized as potential anticancer agents. 1 exhibits effective in vitro and in vivo anticancer activities and tolerable toxicities. 1 promotes MCF-7 cell death by combination of cell arrest at G1 phase, apoptosis (both extrinsic and intrinsic apoptotic pathways), anti-metastasis, anti-angiogenesis, damage of DNA, protein and lipid, and autophagy mediated by the oxidative stress which is confirmed by ROS generation and intracellular glutathione depletion assays. 1 can be identified as a lead anticancer molecule of therapeutic importance. This work may offer insights into the design and mechanism study of multifunctional optically pure metal-based anticancer candidates derived from natural products.

5-Bromo-oxoisoaporphine platinum(II) complexes exhibit tumor cell cytotoxcicity via inhibition of telomerase activity and disruption of c-myc G-quadruplex DNA and mitochondrial functions.

Two platinum(II) complexes [Pt(L)(DMSO)Cl] (1) and [Pt(L)(pn)]Cl (2) with 5-bromo-oxoisoaporphine (H-L) were synthesized. We found that the two new platinum(II) complexes were more selective for Hep-G2 tumor cells than for normal cells (HL-7702, WI-38 and L-o2 cell lines). 5-Bromine-oxoisoaporphine platinum(II) complex 2 was a telomerase inhibitor targeting c-myc G4, and it triggered Hep-G2 cell apoptosis more potently than complex 1. Moreover, they induced cell apoptosis via disruption of mitochondrial functions. Significantly increased ROS level, loss of Δψ, decrease of bcl-2 level, and increase of some of the mitochondria-initiated apoptosis protein levels (including bax, Cyt C, caspase-3, caspase-9, and apaf-1) were observed in Hep-G2 cells. In brief, complexes 1 and 2 triggered Hep-G2 cell apoptosis mainly through inhibiting telomerase activity by interacting with c-myc promoter elements and disruption of mitochondrial pathway. Our results also showed the effects of second ligands on the in vitro antitumor activity in the order of pn > Cl and DMSO.

Preparation of 6/8/11-Amino/Chloro-Oxoisoaporphine and Group-10 Metal Complexes and Evaluation of Their in Vitro and in Vivo Antitumor Activity.

A series of group-10 metal complexes 1-14 of oxoisoaporphine derivatives were designed and synthesized. 1-14 were more selectively cytotoxic to Hep-G2 cells comparing with normal liver cells. In vitro cytotoxicity results showed that complexes 1-6, 7, 8, 10 and 11, especially 3, were telomerase inhibitors targeting c-myc, telomeric, and bcl-2 G4s and triggered cell senescence and apoptosis; they also caused telomere/DNA damage and S phase arrest. In addition, 1-6 also caused mitochondrial dysfunction. Notably, 3 with 6-amino substituted ligand La exhibited less side effects than 6 with 8-amino substituted ligand Lb and cisplatin, but similar tumor growth inhibition efficacy in BEL-7402 xenograft model. Complex 3 has the potential to be developed as an effective anticancer agent.

Oxoisoaporphine as Potent Telomerase Inhibitor.

Two compounds previously isolated from traditional Chinese medicine, Menispermum dauricum (DC), 6-hydroxyl-oxoisoaporphine (H-La), and 4,6-di(2-pyridinyl)benzo[h]isoindolo[4,5,6-de]quinolin-8(5H)-one (H-Lb), were known to have in vitro antitumor activity and to selectively bind human telomeric, c-myc, and bcl-2 G-quadruplexes (G4s). In this study, the binding properties of these two compounds to telomerase were investigated through molecular docking and telomeric repeat amplication protocol and silver staining assay (TRAP-silver staining assay). The binding energies bound to human telomerase RNA were calculated by molecular docking to be -6.43 and -9.76 kcal/mol for H-La and H-Lb, respectively. Compared with H-La, the ligand H-Lb more strongly inhibited telomerase activity in the SK-OV-3 cells model.

High in vivo antitumor activity of cobalt oxoisoaporphine complexes by targeting G-quadruplex DNA, telomerase and disrupting mitochondrial functions.

Two G-quadruplex ligands: [Co(H-La)2Cl2] (Co1) and [Co(Lb)2][CoCl4]⋅2H2O (Co2) have been synthesized and characterized. Two cobalt oxoisoaporphine complexes exhibited selective cytotoxicity to SK-OV-3/DDP cells than for HL-7702 cells. Cytotoxic mechanism studies indicated that both Co1 and Co2 were telomerase inhibitor targeting c-myc, telomere, and bcl-2 G4s, and triggering cell senescence and apoptosis, which caused S phase arrest. They also induced mitochondrial dysfunction. The better antitumor activity of Co2, which should be correlated with a moiety of 2-[5-(2-pyridinyl)-1H-pyrrol-2-yl]pyridine in the Lb. Importantly, Co2 at high doses showed at least the same level of tumor growth inhibition efficacy compared to that of cisplatin, and better in vivo safety profile.

Stabilization of c-myc G-Quadruplex DNA, inhibition of telomerase activity, disruption of mitochondrial functions and tumor cell apoptosis by platinum(II) complex with 9-amino-oxoisoaporphine.

[Pd(L)(DMSO)Cl2] (1) and [Pt(L)(DMSO)Cl2] (2) with 9-amino-oxoisoaporphine (L), were synthesized and characterized. 1 and 2 are more selectively cytotoxic to Hep-G2 cells versus normal liver cells (HL-7702). Various experiments showed that 2 acted as telomerase inhibitors targeting G4-DNA and triggered cell apoptosis by interacting with c-myc G4-DNA. Furthermore, 2 significantly induced cell cycle arrest at both G2/M and S phase, which leading to the down-regulation of cdc25 A, cyclin D, cyclin B, cyclin A and CDK2 and the up-regulation of p53, p27, p21,chk1 and chk2. In addition, 2 also caused mitochondrial dysfunction. Taken together, we found that 2 exerted its cytotoxic activity mainly via inhibiting telomerase by interaction with c-myc G4-DNA and disruption of mitochondrial function.