Mono- and Dimeric Xanthones with Anti-Glioma and Anti-Inflammatory Activities from the Ascidian-Derived Fungus Diaporthe sp. SYSU-MS4722

Seven new xanthones, diaporthones A−G (1−7), together with 13 known analogues, including five mono- (8−14) and six dimeric xanthones (15−20), were obtained from the ascidian-derived fungus Diaporthe sp. SYSU-MS4722. Their planar structures were established by extensive spectroscopic analyses, including 1D and 2D NMR and high-resolution mass spectrometry (HR-ESIMS). The absolute configurations of 1−7 were clearly identified by X-ray crystallographic analysis and calculation of the ECD Spectra. Compounds 15−20 showed significant anti-inflammatory activity with IC50 values between 6.3 and 8.0 μM. In addition, dimeric xanthones (15−20) showed selective cytotoxicity against T98G cell lines with IC50 values ranging from 19.5 to 78.0 μM.

EXTH-32. NOVEL THIORIDAZINE DERIVATES: ANTIPROLIFERATIVE AND APOPTOSIS-INDUCING ACTIVITY ON GLIOBLASTOMA CELLS IN VITRO

BACKGROUND Although withdrawn from the market due to cardiotoxicity, we showed that the antipsychotic drug Thioridazine shows chemosensitizing effects in combination with Temozolomide (TMZ) for the treatment of glioblastoma multiforme (GBM). Based on our prior observations, the aim of this study was through medicinal chemistry, to design and synthesize new compounds based on Thioridazines tricyclic structure, and to determine their therapeutic potential. METHODS Fourteen compounds were synthesized where variations were made within the tricyclic side chains. The newly synthesized compounds were screened for therapeutic efficacy with or without TMZ using a WST-1 cell viability assay and real-time imaging system (IncuCyte). Tests were performed on both monolayer cell cultures, as well as on glioma stem cell spheroids (GSC). The therapeutic effects were also studied on human astrocytes (NHA) as well as on rat brain organoids (BO). Annexin V/propidium iodide (PI) double staining followed by flow cytometric analysis was performed after 48 hours of treatment. RESULTS Following an extensive screening, we identified two novel compounds (EA01 and EA02) that at concentrations of 4 and 9.5 µM showed a strong cytotoxicity on GBM cell lines (U-87 MG p< 0,0001, U251 p< 0,0001, LN18 p= 0,0004) as well as on glioma stem cells (GSC) (P3 p< 0,0001) compared to NHA and BOs respectively. Also, when BOs were confronted with GSC spheres in an invasion assay, a selective cytotoxicity was observed in the GSCs. Mechanistically, we show that both compounds induce apoptosis in GBM cells. Moreover, intravenous delivery of increasing concentrations of EA01 and EA02 revealed no toxicity in animals at concentrations up to 21 mg/kg. CONCLUSION We developed two new tricyclic therapeutic compounds that show a strong selective cytotoxicity in GBM cells with limited systemic toxicity in animals. Ongoing studies are investigating the therapeutic potential of EA01 and EA02 in orthotopic xenografts in vivo.

P13.08 Novel Thioridazine derivates: Antiproliferative and apoptosis-inducing activity on glioblastoma cells in vitro

BACKGROUND Although withdrawn from the market due to cardiotoxicity, we have shown that the antipsychotic drug Thioridazine shows chemosensitizing effects in combination with Temozolomide (TMZ) for the treatment of glioblastoma multiforme (GBM). Based on our prior observations, the aim of the presented project was through medicinal chemistry, to design and synthesize new compounds based on Thioridazines tricyclic structure, and to determine their therapeutic potential. MATERIAL AND METHODS Fourteen compounds were synthesized where variations were made within the tricyclic side chains. The newly synthesized compounds were screened for therapeutic efficacy with or without TMZ using a WST-1 cell viability assay as well as a real-time imaging system (IncuCyte). Tests were performed on both monolayer cell cultures, as well as on glioma stem cell spheroids (GSC). The therapeutic effects were also studied on human astrocytes (NHA) as well as on rat brain organoids (BO). Annexin V/propidium iodide (PI) double staining followed by flow cytometric analysis was performed after 48 hours of treatment. RESULTS Following an extensive screening, we identified two novel compounds (EA01 and EA02) that at concentrations of 4 and 9.5 µM showed a strong cytotoxicity on GBM cell lines (U-87 MG p<0,0001, U251 p<0,0001, LN18 p=0,0004) as well as on glioma stem cells (GSC) (P3 p<0,0001) compared to NHA and BOs respectively. Also, when BOs were confronted with GSC spheres in an invasion assay, a selective cytotoxicity was observed in the GSCs. Mechanistically, we show that both compounds induce apoptosis in the GBM cells. Moreover, intravenous delivery of increasing concentrations of EA01 and EA02 revealed no toxicity in animals at concentrations up to 21 mg/kg. CONCLUSION We have developed two new tricyclic therapeutic compounds that show a strong selective cytotoxicity in GBM cells with limited systemic toxicity in animals. Ongoing studies are investigating the therapeutic potential of EA01 and EA02 in orthotopic xenografts in vivo.

Cinobufagin-induced DNA damage response activates G2/M checkpoint and apoptosis to cause selective cytotoxicity in cancer cells

Background Processed extracts from toad skin and parotoid gland have long been used to treat various illnesses including cancer in many Asian countries. Recent studies have uncovered a family of bufadienolides as the responsible pharmacological compounds, and the two major molecules, cinobufagin and bufalin, have been shown to possess robust antitumor activity; however, the underlying mechanisms remain poorly understood. Methods Intracellular reactive oxygen species (ROS) were measured by DCFH-DA staining and flow cytometry, and DNA damage was analyzed by immunofluorescent staining and the alkaline comet assay. Cytotoxicity was measured by MTT as well as colony formation assays, and cell cycle and apoptosis were analyzed by flow cytometry. In addition, apoptosis was further characterized by TUNEL and mitochondrial membrane potential assays. Results Here we showed that sublethal doses of cinobufagin suppressed the viability of many cancer but not noncancerous cell lines. This tumor-selective cytotoxicity was preceded by a rapid, cancer-specific increase in cellular ROS and was significantly reduced by the ROS inhibitor N-acetyl cysteine (NAC), indicating oxidative stress as the primary source of cinobufagin-induced cancer cell toxicity. Sublethal cinobufagin-induced ROS overload resulted in oxidative DNA damage and intense replication stress in cancer cells, leading to strong DNA damage response (DDR) signaling. Subsequent phosphorylation of CDC25C and stabilization of p53 downstream of DDR resulted in activation of the G2/M checkpoint followed by induction of apoptosis. These data indicate that cinobufagin suppresses cancer cell viability via DDR-mediated G2 arrest and apoptosis. Conclusion As elevated oxidative pressure is shared by most cancer cells that renders them sensitive to further oxidative insult, these studies suggest that nontoxic doses of cinobufagin can be used to exploit a cancer vulnerability for induction of cancer-specific cytotoxicity.

Salinomycin-Based Drug Delivery Systems: Overcoming the Hurdles in Cancer Therapy

Cancer stem cells (CSCs) are reportedly responsible for the initiation and propagation of cancer. Since CSCs are highly resistant to conventional chemo- and radiotherapy, they are considered the main cause of cancer relapse and metastasis. Salinomycin (Sali), an anticoccidial polyether antibiotic, has emerged as a promising new candidate for cancer therapy, with selective cytotoxicity against CSCs in various malignancies. Nanotechnology provides an efficient means of delivering Sali to tumors in view of reducing collateral damage to healthy tissues and enhancing the therapeutic outcome. This review offers an insight into the most recent advances in cancer therapy using Sali-based nanocarriers.