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Inflammatory response plays an important role not only in the normal physiology but also in the pathology such as cancers. 7b, a novel naphthalimide-based DNA intercalator, has exhibited anti-inflammatory effects in phorbol12-myristate 13-acetate/phytohemagglutinin (PMA/PHA)-induced inflammatory responses of Jurkat T cells in our previous study. Here, we tried to further investigate its anti-inflammatory potential and the possible underlying mechanisms in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and primary mouse macrophages. In our current study, ELISA and Real-time PCR revealed that non-toxic doses of 7b reduced the production and expression of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in LPS-induced RAW264.7 cells and primary mouse macrophages. Moreover, 7b dose-dependently suppressed the production of prostaglandin E2 (PGE2), nitric oxide (NO). Except for COX-1, non-toxic doses of 7b exhibited parallel inhibition of LPS-induced expression of COX-2 and iNOS at both mRNA and protein levels. The molecular mechanism was associated with inhibition of the phosphorylation/degradation of IκB-α and nuclear translocation of the NF-κB p65. Further analysis of upstream mechanisms showed that blocking of NF-κB activation by 7b was mediated by inhibiting TAK1-downstream extracellular signal-regulated kinase (ERK1/2) and p38 kinase signal pathway. Taken together, these results indicated that 7b exhibited anti-inflammatory effects by targeting inhibiting TAK1, leading to ERK1/2- and p38 MAPK-mediated inactivation of NF-κB in LPS-stimulated RAW264.7 cells, and this would make 7b a strong candidate for further study as anti-inflammatory agent., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In this study, a novel naphthalimide derivative 7c was designed which is topo II inhibiting though owning weak DNA binders. It was shown that 7c could induce cancer cells apoptosis and have less cytotoxicity in normal human cell. Further investigations on Hela cells revealed that 7c could also induce ROS generation, lysosome rupture as well as cathepsin B release. Subsequent mitochondrial damages including mitochondrial membrane permeabilization and the release of cytochrome c were also found in 7c when treating with Hela cells. According to our data, 7c may act as a lead compound for potential anticancer drugs. The idea of naphthalimides modification may also provide a novel strategy for naphthalimides design., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Previous studies have reported that 8-c [6-(2-(2-(dimethylamino)ethylamino)ethylamino)-2-octyl-1H-benzo[de]isoquinoline-1,3(2H)-dione], a novel amonafide analogue, was generated as a new anticancer candidate. However, little is known about its activity in chemoresistant cells. In this study, the antitumor effects of 8-c on the multi-drug-resistant human colorectal carcinoma cancer cell lines HCT-116/L-OHP and HCT-8/VCR have been investigated for the first time. 8-c showed similar concentration-dependent inhibitory activities against multi-drug-resistant cells and corresponding parental cell lines by the MTT assay after 48 h of treatment. 8-c treatment resulted in the induction of apoptosis, as evidenced by fluorescent staining analysis, comet assay data, and the increase in the number of apoptotic cells as detected by flow cytometry. Western blot, qPCR, and siRNA techniques were used to elucidate the molecular mechanism. Our study suggested that the apoptotic effect of 8-c can be attributed to the upregulation of p53, caspase-3, and cleaved poly(ADP-ribose) polymerase (PARP) and the downregulation of Bcl-2. Furthermore, ERCC1 is essential for nucleotide excision repair. ERCC1 expression was correlated with sensitivity to chemotherapy in various colon cancer cell lines. It is intriguing that decreases in ERCC1 protein and mRNA levels were also observed in the HCT-116/L-OHP and HCT-8/VCR cells after exposure to 8-c. Further transient transfection of multi-drug-resistant cells with ERCC1 siRNA enhanced 8-c-induced cytotoxicity. In contrast, epidermal growth factor-induced increase in ERCC1 protein levels was shown to rescue cell viability upon 8-c treatment. These findings suggest that 8-c has a strong potential to be developed as a new antitumor agent for the treatment of multi-drug-resistant colon cancer cells, and is worthy of further studies.

7b, a novel amonafide analog, has shown high antitumor activity against Raji B-cell lymphoma. We report here that 7b also shows high cytotoxicity against various T lymphoma cells, with the highest IC(50) (concentration for 50% cytotoxicity) value in Jurkat cells. In a previous study, p53-mutant Raji cells were sensitive to 7b treatment. In the present study, the Jurkat T lymphoma cells were characterized as p53-null. Additional assays showed that 7b could induce G1/S phase arrest and mitochondrial apoptosis in Jurkat cells, suggesting 7b as a potential drug candidate for treatment of T-cell lymphoma. This action was not affected by p53 status. Further analysis of molecular mechanisms revealed that up-regulation of p21 and the Bak/Bcl-2 ratio and down-regulation of UHRF1 and c-Myc were attributed to p73 activation. In turn, up-regulation of p73 was initiated by DNA damage-induced reactive oxygen species (ROS) formation. Interestingly, at non-toxic drug concentrations, 7b could also inhibit phorbol 12-myristate 13-acetate/phytohemagglutinin (PMA/PHA)-induced inflammatory responses of Jurkat T cells owing to the suppression of nuclear factor-κB (NF-κB) DNA-binding. Indeed, electrophoretic mobility shift assay and NF-κB binding assay showed that NF-κB DNA-binding was inhibited by 7b, and correspondingly, proinflammatory cytokine production was also decreased. In conclusion, 7b exhibits both antiproliferative and anti-inflammatory activities in T lymphoma cells.

The E2F1 gene well known is its pivotal role in regulating the entry from G1 to S phase, while the salvage antitumoral pathway which implicates it, especially in the absence of p53, is not fully characterized. We therefore attempted to identify the up- and down-stream events involved in the activation of the E2F1-dependent pro-apoptotic pathway. For this purpose, a amonafide analogue, 7-d (2-(3-(2-(Dimethylamino)ethylamino)propyl)-6-(dodecylamino)-1H-benzo[de]isoquinoline-1,3(2H)-dione) was screened, which exhibited high antitumor activity against p53-deficient human Chronic Myelogenous Leukemia (CML) K562 cells. Analysis of flow cytometry and western blots of K562 cells treated with 7-d revealed an appreciable G2/M cycle arrest and apoptosis in a dose and time-dependent manner via p53-independent pathway. A striking increase in "Comet tail" formation and γ-H2AX expression showed that DNA double strand breaks (DSB) were caused by 7-d treatment. ATM/ATR signaling was reported to connect E2F1 induction with apoptosis in response to DNA damage. Indeed, 7-d-induced G2/M arrest and apoptosis were antagonized by ATM/ATR signaling inhibitor, Caffeine, which suggested that ATM/ATR signaling was activated by 7-d treatment. Furthermore, the increased expression of E2F1, p73, and Apaf-1 and p73 dissociation from HDM2 was induced by 7-d treatment, however, knockout of E2F1 expression reversed p73, Apaf-1, and p21(Cip1/WAF1) expression, reactivated cell cycle progression, and inhibited 7-d-induced apoptosis. Altogether our results for the first time indicate that 7-d mediates its growth inhibitory effects on CML p53-deficient cells via the activation of an E2F1-dependent mitochondrial and cell cycle checkpoint signaling pathway which subsequently targets p73, Apaf-1, and p21(Cip1/WAF1)., (Copyright © 2012 Wiley Periodicals, Inc.)

Selenium analogues (4b-4h, and 4j) of two known sulfur compounds were synthesized and tested their anticancer activities. The selenium compound 4b had comparable activity with its sulfur analogue 4a, while DNA-binding study showed these two compounds had similar interaction with ct-DNA, the K(b) was 8.23 and 2.36, respectively. The primary results showed that most compounds had moderate anticancer activities with IC(50) values between 10(-6) and 10(-5) M. Another selenium analogue 4j showed the highest activity with the IC(50) values around 5.3 μM against K562 and MCF-7 cell lines. More importantly, the organochalcogen compounds exhibited stronger anticancer activities against K562 cell line than the other cell lines tested., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

The Bcl-2 family contains a panel of proteins which are conserved regulators of apoptosis in mammalian cells, like the anti-apoptotic protein Bcl-2. According to its significant role in altering susceptibility to apoptosis, the deciphering of the mechanism of Bcl-2 expression modulation may be crucial for identifying therapeutics strategies for cancer. Treatment with naphthalimide-based DNA intercalators, including M2-A and R16, generally leads to a decrease in Bcl-2 intracellular amounts. Whereas the interest for these chemotherapeutics is accompanied by advances in the fundamental understanding of their anticancer properties, the molecular mechanism underlying changes in Bcl-2 expression remains poorly understood. We report here that p53 contributes to Bcl-2 down-regulation induced by B1, a novel naphthalimide-based DNA intercalating agent. Indeed, the decrease in Bcl-2 protein levels observed during B1-induced apoptosis was correlated to the decrease in mRNA levels, as a result of the inhibition of Bcl-2 transcription and promoter activity. In this context, we evaluated p53 contribution in the Bcl-2 transcriptional down-regulation. We found a significant increase of p53 binding to P(2) promoter TATA box in MCF7 cells by chromatin immunoprecipitation. These data suggest that B1-induced caspase-independent apoptosis in MCF-7 cells is associated with the activation of p53 and the down-regulation of Bcl-2. Our study strengthens the links between p53 and Bcl-2 at a transcriptional level, upon naphthalimide-based DNA intercalator treatment., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In the course of screening for novel anticancer compounds, B1 (N-(2-(Dimethylamino)ethyl)-2-aminothiazonaphthalimide), a novel naphthalimide-based DNA intercalator, was generated as a new anticancer candidate. For the first time, our investigation demonstrates that B1 inhibited the growth of HeLa cells by the induction of cell cycle arrest and apoptosis. Analysis of flow cytometry and western blots of HeLa cells treated with B1 revealed an appreciable cell cycle arrest and apoptotic induction in dose and time-dependent manner via the p53-dependent pathway. Furthermore, the release of cytochrome c from mitochondria was detected using confocal microscopy in HeLa cells treated with B1. Accordingly, these data demonstrate that the anticancer activity of B1 is associated with the activation of p53 and the release of cytochrome c, which suggest that B1 might have therapeutic potential against cervix carcinoma as an effective lead compound.

A series of novel aliphatic N-oxide of naphthalimides (A1-A5) were designed and prepared. The N-O group was firstly introduced into the amine side chain tailed to planar naphthalimide chromophore as hypoxic bioreductive marker. Fluorescence image analysis showed that the compounds could be used as potential markers for hypoxic cells (V79) in vitro especially for A1 with 17 times hypoxic-oxic fluorescence differential, which was probably due to the bis-bioreduction mechanism., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Previous studies have shown that 7-b (6-(dodecylamino)-2-(3-(4-methylpiperazin-1-yl)propyl)-1H-benzo-[de]isoquinoline-1,3(2H)-dione), a novel amonafide-based DNA intercalator, was generated as a new anticancer candidate. However, the effects induced by 7-b and the molecular mechanisms involved remain poorly understood in Burkitt's lymphoma. To shed light on these issues, we have investigated the effects of 7-b on proliferation, cell cycle progression, apoptosis activity and oxidative stress levels of lymphoma Raji cells in vitro. Our results showed that 7-b inhibited the proliferation of Raji cells and induced G1 cell cycle arrest in a dose-dependent manner. Moreover, 7-b treatment triggered programmed cell death, production of reactive oxygen species (ROS) and alteration of the mitochondrial membrane potential (Δψm). Altogether our results showed that 7-b mediated its growth inhibitory effects on Raji cells via the activation of a ROS-mediated mitochondrial pathway and cell cycle checkpoint signaling pathway which subsequently targeted p21., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Amonafide is a naphthalimide derivative with antitumor activity and has failed to enter clinical phase III, because of its high-variable and unpredictable toxicity. In order to develop selective, efficient, and safe drugs, applying the 'nonfused' aromatic system strategy, a series of 5-non-amino aromatic substituted naphthalimides as replacement for amonafide were designed and were synthesized from naphthalic anhydride by three steps including bromination, amination, and Pd(PPh₃)₄ catalyzed Suzuki reaction. These new naphthalimide derivatives, except 4b, not only exhibited better activity than amonafide against HeLa and P388D1 cell lines in vitro under the same experimental conditions, but also could avoid the side effect of amonafide due to their structure, which lacks an easy acetylated arylamine at the 5 position. The DNA-binding behavior of the naphthalimide derivatives was also investigated, and the results suggested that they bind to DNA via intercalation and 4a and 4g intercalated into DNA in different fashion., (Copyright © 2010. Published by Elsevier Ltd.)

In the course of screening for novel anticancer compounds, B1 [N-(2-(dimethylamino)ethyl)-2-aminothiazonaphthalimide], a novel amonafide analogue, was generated as a new anticancer candidate. In the present study, B1 displayed stronger antitumor effects than amonafide in HL60 cells. We further examined whether B1 overcomes the resistance conferred by Bcl-2, as overcoming the resistance conferred by Bcl-2 represents an attractive therapeutic strategy against cancer. Our viability assay showed that B1 overcomes the resistance conferred by Bcl-2 in human promyelocytic leukemia HL60 cells. Various apoptosis assessment assays showed that B1 overcomes the resistance conferred by Bcl-2 in HL60 cells by inducing apoptosis. Noticeably, we elucidated the marked downregulation of 14-3-3σ protein by B1, indicating that B1 overcomes the resistance conferred by Bcl-2 in HL60 cells via 14-3-3σ. The analysis of chromatin immunoprecipitation assay indicated that MBD2 was associated with the methylated 14-3-3σ promoter-associated CpG island and thus interfered with transcriptional activity of the methylated promoter. Furthermore, knockdown of MBD2, using siRNA transfection, inhibited B1-induced apoptosis and overcame the resistance conferred by Bcl-2. Accordingly, these data showed the involvement of MBD2 in B1-induced apoptosis and overcoming the resistance conferred by Bcl-2, which suggested that MBD2 might guide the development of future anticancer agents., (©2010 AACR.)

Based on the advantages of multitarget drugs for cancer treatment, a new class of naphthalimides was designed, synthesized, and proved to inhibit topoisomerase II (topo II), induced lysosomal membrane permeabilization (LMP), and ultimately caused apoptosis and cell death. The majority of compounds 7a-d and 8a-d potently inhibited the growth of the five tested cancer cell lines with IC(50) values ranging from 2 to 10 microM and are more active than amonafide, a naphthalimide that was in phase III clinical trials. These compounds were tested for their interactions with DNA and their cell-free topo II inhibition activities, which demonstrated these compounds were weak DNA binders but modest topo II inhibitors. Furthermore, compounds 7b-d were found to notably induce LMP and exhibited better antiproliferative activity compared with their single-target analogues. All of the newly synthesized compounds were demonstrated to efficiently induce apoptosis via a mitochondrial pathway. Accordingly, a new paradigm was suggested for the design of novel multitarget anticancer drugs.

A series of novel naphthalimide derivatives with flexible alkyl/aryl moieties were designed and synthesized. Their antitumor activities were evaluated against HeLa, A549, P388, HL-60, MCF-7, HCT-8 and A375 cancer cell lines in vitro. The preliminary results showed that most of the derivatives had comparable antitumor activities over Amonafide with the IC(50) values of 10(-6) to 10(-5)M. More importantly, flow cytometric analysis indicated that the derivatives could effectively induce G(2)/M arrest and progress to apoptosis in HL-60 cell line after double staining with annexin V-FITC and propidium iodide. The present work provided a novel class of naphthalimide-based derivatives with potent apoptosis-inducing and antitumor activities for further optimization.

Amonafide, a naphthalimide derivative, although selected for exploratory clinical trials for its potent anticancer activity, has long been challenged by its unpredictable side effects. In the present study, a novel amonafide analogue, M(2)-A 2-(2-(dimethylamino)ethyl)-6-(thiophene-2-ylmethylamino)-1H-benzo[de]isoquinoline-1,3(2H)-dione was ascribed to its potent effects on topoisomerase IIalpha. Moreover, our investigation indicates that M(2)-A induces G(2)/M phase growth arrest through inhibiting PI3K/Akt pathway. M(2)-A inhibits proliferation of HeLa, HL60, HCT-8, A375, MCF-7 and MRC-5 cells, especially inhibits proliferation of HL60 with an IC(50) value of 18.86 microM. M(2)-A can not only induce DNA fragmentation, but also enhance Annexin V-FITC binding of the cells. On the one hand the expression levels of protein Cyclin B1, Cdk1 changed in response to M(2)-A treatment in HL60 cells. On the other hand we observed the inhibition of NF-kappaB nuclear translocation, up-regulation of Bax and down-regulation of Bcl-2, the caspase -3, -9 activity increase in HL60 cells after treated with M(2)-A, which indicated that the mitochondrial pathway was involved in the apoptosis signal pathway. Our results showed that the phosphorylation of p85/PI3K and Akt decreased following M(2)-A treatment. In summary, M(2)-A displayed a significant anti-tumor effect through cell cycle arrest and apoptotic induction in HL60 cells, which suggested that M(2)-A might have therapeutic potential against leukaemia.

A series of 5-alkylamino substituted amonafide analogues were synthesized from naphthalic anhydride by three steps including bromization, amination and CuI/proline catalyzed coupling reaction. The CuI/L-proline catalyzed coupling reaction was first applied to the naphthalimide system. These new amonafide analogues showed potential anticancer activities against HeLa and P388D1 cell lines in vitro, and 4a, 4b, and 4h exhibited better activity than amonafide against HeLa cell under the same experimental conditions. More importantly, the new analogues could avoid the side effect of amonafide due to their structure, in which lacks a primary amine at the 5 position. Moreover, the DNA-binding of the analogues was also investigated.

A series of sulfur-substituted naphthalimides (1-5) was prepared and investigated as antitumor drugs. Initial DNA interaction studies (by the fluorescence quenching method, UV/vis and CD spectroscopy, thermal denaturation, topoisomerase Western blot analysis, and DNA photocleavage experiments) expectedly suggested the DNA and topoisomerase as main targets of the agents. Fluorescence spectroscopic and microscopic experiments indicated a significant sensitivity of the emission intensities of 3 and 5 to the cellular environment and confirmed the cellular uptake and biodistribution into cell compartments for 1-3 and 5. A comparative evaluation of the antiproliferative effects under different experimental setups (concerning drug exposure period and an additional short-time UV irradiation) revealed significant phototoxic effects for the environmentally sensitive compounds 3 and 5 and strongly suggested the further development of sulfur-substituted naphthalimides for potential use in photodynamic tumor therapy.

Novel tertiary amine N-oxides of naphthalimides were designed and synthesized as potential anticancer agents against hypoxic solid tumors. Although their ctDNA-binding affinities and cytotoxic activities against usual tumor cell lines were lower than those of corresponding amines, the N-oxides A1 and A4 showed hypoxia preference activities against A375 cells in vitro and might be used as interesting candidates of prodrug leads in hypoxic tumor cells.

Abstract: Novel naphthalimides with two heterocyclic side chains of 2-nitroimidazole for bioreductive binding were designed, synthesized, and used as fluorescent markers for hypoxic cells. Their evaluation for imaging tumor hypoxia was carried out in V79 cells, CHO cells, and 95D cells in vitro by using fluorescence scan ascent. A 2 and A 4 showed a very large differential fluorescence between hypoxic and oxic cells (V79 cells) in vitro and are promising candidate markers for hypoxic cells. [Copyright &y& Elsevier]