Reactive Oxygen Species Mediate 6c-Induced Mitochondrial and Lysosomal Dysfunction, Autophagic Cell Death, and DNA Damage in Hepatocellular Carcinoma.

Increasing the level of reactive oxygen species (ROS) in cancer cells has been suggested as a viable approach to cancer therapy. Our previous study has demonstrated that mitochondria-targeted flavone-naphthalimide-polyamine conjugate 6c elevates the level of ROS in cancer cells. However, the detailed role of ROS in 6c-treated cancer cells is not clearly stated. The biological effects and in-depth mechanisms of 6c in cancer cells need to be further investigated. In this study, we confirmed that mitochondria are the main source of 6c-induced ROS, as demonstrated by an increase in 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and MitoSox fluorescence. Compound 6c-induced mitochondrial ROS caused mitochondrial dysfunction and lysosomal destabilization confirmed by absolute quantitation (iTRAQ)-based comparative proteomics. Compound 6c-induced metabolic pathway dysfunction and lysosomal destabilization was attenuated by N-acetyl-L-cysteine (NAC). iTRAQ-based comparative proteomics showed that ROS regulated the expression of 6c-mediated proteins, and treatment with 6c promoted the formation of autophagosomes depending on ROS. Compound 6c-induced DNA damage was characterized by comet assay, p53 phosphorylation, and γH2A.X, which was diminished by pretreatment with NAC. Compound 6c-induced cell death was partially reversed by 3-methyladenine (3-MA), bafilomycin (BAF) A1, and NAC, respectively. Taken together, the data obtained in our study highlighted the involvement of mitochondrial ROS in 6c-induced autophagic cell death, mitochondrial and lysosomal dysfunction, and DNA damage.

Suppression of oxidative phosphorylation and IDH2 sensitizes colorectal cancer to a naphthalimide derivative and mitoxantrone.

Colorectal cancer (CRC) is one of the most prevalent cancers worldwide. Oxidative phosphorylation (OXPHOS) has attracted a considerable attention in CRC. It is of great interest to explore novel therapies that inhibit OXPHOS for CRC treatment. Compound 6c is a novel naphthalimide derivative. However, the effects of 6c on CRC and the underlying mechanism are unclear. In this study, 6c suppressed CRC tumor growth and metastasis. RNA-seq data showed that 6c triggered the inhibition of OXPHOS and tricarboxylic acid cycle. 6c specifically inhibited mitochondrial complex III activity and the expression of isocitrate dehydrogenase 2 (IDH2), resulting in oxidative stress. Antioxidants reversed 6c-induced cell death, senescence, and autophagosomes formation. 6c inhibited autophagy flux; however, pretreatment with autophagy inhibitors resulted in the reduction of 6c-induced cytoplasmic vacuolization and proliferation inhibition. Moreover, combinatory treatment of 6c and mitoxantrone (MIT) showed stronger inhibitory effects on CRC compared with the single agent. Downregulation of IDH2 induced reactive oxygen species production, leading to MIT accumulation and autophagic cell death after co-treatment with 6c and MIT. In summary, our findings indicated 6c as a promising candidate for CRC treatment.

Identification of molecular anti-metastasis mechanisms of lycorine in colorectal cancer by RNA-seq analysis.

BACKGROUND: Colorectal cancer (CRC) is one of the most common malignancies worldwide. Metastasis is the major cause of death in patients with CRC. Lycorine, the phenanthridine alkaloid most commonly found in spp of the Amaryllidaceae family, has shown promising anticancer activities with minor side effects. However, the effects and the detailed mechanism of lycorine against metastasis of CRC remains unclear. STUDY DESIGN/METHODS: The purpose of this study was to investigate the effects of lycorine on CRC and characterize the molecular mechanisms observed in lycorine-treated CRC cells using RNA-sequencing. MTT assay, colony formation assay, acridine orange/ethidium bromide (AO/EB) staining and Annexin V-FITC/Propidium iodide (PI) staining were conducted to examine the effects of lycorine on cell proliferation and apoptosis in CRC cells. RNA sequencing, real-time PCR assays and western blot were performed. Migration and invasion abilities of lycorine-treated CRC cells were investigated by wound healing and transwell invasion assays. The mouse CRC lung metastasis model was established and was used to detect the effect of lycorine on CRC in vivo. RESULTS: Our results demonstrated that lycorine inhibited the proliferation and colony formation of CRC cells in a concentration-dependent manner. AO/EB staining and Annexin V-FITC/PI staining showed that lycorine induced apoptosis in a concentration-dependent manner. Lycorine also reduced lung metastasis of CRC in vivo. Moreover, transcriptomic analysis suggested that lycorine regulated the expression of 3556 genes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway was implicated according to the differentially expressed genes (DEGs), and multiple pathways including those of mitogen-activated protein kinase (MAPK), relaxin, Ras, phosphatidylinositol 3­kinase (PI3K)-protein kinase B (Akt) and Wnt/ß-catenin were selected by functional enrichment analyses. Furthermore, based on transcriptomic analysis, we found that the tumor necrosis factor (TNF) pathway and endoplasmic reticulum stress were responsible for lycorine-induced apoptosis. CONCLUSIONS: These results obtained in this study demonstrated that lycorine has the potential to suppress CRC in vitro and in vivo through the lycorine-regulated multiple signaling pathways.

The Role of p53-Mediated Signaling in the Therapeutic Response of Colorectal Cancer to 9F, a Spermine-Modified Naphthalene Diimide Derivative.

Colorectal cancer (CRC) is one of the most prevalent cancers due to its frequency and high rate of mortality. Polyamine-vectorized anticancer drugs possess multiple biological properties. Of these drugs, 9F has been shown to inhibit tumor growth and the metastasis of hepatocellular carcinoma. This current study aims to investigate the effects of 9F on CRC and determine its molecular mechanisms of action. Our findings demonstrate that 9F inhibits CRC cell growth by inducing apoptosis and cell cycle arrest, and suppresses migration, invasion and angiogenesis in vitro, resulting in the inhibition of tumor growth and metastasis in vivo. Based on RNA-seq data, further bioinformatic analyses suggest that 9F exerts its anticancer activities through p53 signaling, which is responsible for the altered expression of key regulators of the cell cycle, apoptosis, the epithelial-to-mesenchymal transition (EMT), and angiogenesis. In addition, 9F is more effective than amonafide against CRC. These results show that 9F can be considered as a potential strategy for CRC treatment.

Synthesis and biological evaluation of novel asymmetric naphthalene diimide derivatives as anticancer agents depending on ROS generation.

Naphthalenetetracarboxylic diimide (NDI) is widely used as a photoelectric material in the field of medicine. A series of asymmetric naphthalene diimide derivatives were synthesized and evaluated for their anticancer properties by various experimental assays. As the representative compound, 3c exerted significantly greater inhibitory effects on hepatoma cells SMMC-7721 and Hep G2 with an IC50 value of 1.48 ± 0.43 µM and 1.70 ± 0.53 µM, respectively, than normal hepatocytes QSG-7701 with an IC50 value of 7.11 ± 0.08 µM. Treatment with compound 3c (3 µM) for 48 h resulted in apoptosis of SMMC-7721 cells and Hep G2 cells with 52.1% and 67.8% apoptotic cells, respectively. Compound 3c induced autophagy and suppressed the migration of hepatoma cells in a concentration-dependent manner, resulting from the generation of reactive oxygen species (ROS). Based on its biological ability, compound 3c was considered as a potent anticancer agent.

Discovery of the Polyamine Conjugate with Benzo[ cd]indol-2(1 H)-one as a Lysosome-Targeted Antimetastatic Agent.

Polyamine derivatives have a promising prospect in dealing with disseminated tumor cells, a major obstacle in cancer therapy. To develop a bifunctional polyamine derivative that can serve as a fluorescent probe and an antimetastatic agent, three kinds of polyamine conjugates with benzo[ cd]indol-2(1 H)-one as a scaffold were designed and synthesized. Compound 5e was selected as a lead by in vitro screening. Two animal models demonstrated that 5e inhibited pulmonary metastasis and tumor growth. As a fluorescent probe, 5e might partially enter cells via a polyamine transporter and subsequently localize in the lysosome. Mechanistic investigations demonstrated the interdependence of 5e-triggered apoptosis and autophagy. Compound 5e modulated the expression of LC3-II, p62, cathepsins, and the expression of capases 3, caspase 8, Bcl-2, and p53. The SSAT-mediated Akt/ß-catenin pathways were also inhibited by 5e. The dual features of 5e make it a worthwhile lead compound for further structural optimization.

Synthesis and biological evaluation of novel alkylated polyamine analogues as potential anticancer agents.

A new class of polyamine analogues modified by alkylation at the terminal of the polyamine chain has been synthesized and their structures were determined by 1H NMR, 13C NMR, ESI-MS and elemental analysis. As the representative compound, 3f displayed a broad spectrum of anti-cancer effects by MTT assays. Tumor xenograft model and pulmonary metastasis model showed that compound 3f significantly suppressed tumor growth and metastasis in vivo, which was more stronger than the reference drug amonafide. Molecular mechanisms indicated that compound 3f exhibited antiproliferative activities and induced the generation of reactive oxygen species (ROS), which resulted in the occurrence of autophagy. The downregulated expression of MMP-9 and ß-catenin by compound 3f accounted for the inhibition of migration. Taken altogether, the in vitro and in vivo biological evaluations corroborated compound 3f to be an effective anticancer agent.

MEK inhibitor, PD98059, promotes breast cancer cell migration by inducing ß-catenin nuclear accumulation.

Abnormal activation of the RAF/MEK/ERK signaling pathway has been observed in breast cancer. Thus, a number of MEK inhibitors have been designed as one treatment option for breast cancer. Although some studies have found that these MEK inhibitors inhibit the growth of a variety of human cancer cells, some trials have shown that the use of MEK inhibitors as a treatment for breast cancer does not adequately improve survival for unknown reasons. In the present study, MEK inhibitor PD98059 was used to evaluate its anticancer effects on human breast cancer MCF-7 and MDA-MB-231 cells and to explore the possible mechanism of action. Our results revealed that MEK inhibitor PD98059 exhibited antiproliferative effects in a dose- and time-dependent manner in MCF-7 and MDA-MB-231 breast cancer cells. Conversely, incubation of MCF-7 and MDA-MB-231 cells with PD98059 promoted their migration. Further investigation disclosed that the enhanced ability of migration promoted by PD98059 was dependent on ß-catenin nuclear translocation in the MCF-7 and MDA-MB­231 cells. Subsequent experiments documented that activation of EGFR signaling induced by PD98059 increased the amount of ß-catenin in the nucleus. Taken together, our findings may elucidate a possible mechanism explaining the ineffectiveness of MEK inhibitors in breast cancer treatment and improve our understanding of the role of MEK in cancer.

Design, Synthesis and Evaluation of Naphthalimide Derivatives as Potential Anticancer Agents for Hepatocellular Carcinoma.

Two kinds of naphthalimide derivatives were synthesized and evaluated for in vitro their anti-hepatocellular carcinoma properties. Compound 3a with a fused thiazole fragment to naphthalimide skeleton inhibited cell migration of SMMC-7721 and HepG2, and further in vivo trials with two animal models confirmed that compound 3a moderately inhibited primary H22 tumor growth (52.6%) and potently interrupted lung metastasis (75.7%) without obvious systemic toxicity at the therapeutic dose. Mechanistic research revealed that compound 3a inhibited cancerous liver cell growth mostly by inducing G2/M phase arrest. Western blotting experiments corroborated that 3a could up-regulate the cell cycle related protein expression of cyclin B1, CDK1 and p21, and inhibit cell migration by elevating the E-cadherin and attenuating integrin α6 expression. Our study showed that compound 3a is a valuable lead compound worthy of further investigation.

Design, Synthesis, and Biological Evaluation of Mitochondria-Targeted Flavone-Naphthalimide-Polyamine Conjugates with Antimetastatic Activity.

Approximately 90% of cancer-associated deaths result from disseminated tumors, indicating the ineffectiveness of current therapies and the imperative need of antimetastatic drugs. A novel pharmacophore with flavonoid and naphthalimide moieties was constructed by using a fragment-based drug design and a series of eight flavone-naphthalimide-polyamine conjugates were synthesized. In vitro evaluation revealed that compound 6c with a homospermidine motif displayed better cell selectivity between cancerous and normal liver cells than amonafide did. The in vivo assays on two hepatocellular carcinoma (HCC) models verified that 6c potently suppressed pulmonary metastasis with improved organ indexes compared to amonafide. Various experiments showed that 6c as a potential fluorescent chemical probe could target the mitochondria. Preliminary investigation into the mechanism of action of 6c indicated that it might harness a polyamine transporter for cell entrance, localize in the mitochondria, selectively cause reactive oxygen species (ROS) overproduction in hepatoma cells instead of normal liver cells, and finally lead to HCC cell apoptosis and migration inhibition via multiple ROS-mediated signaling pathways.