Transcriptomics, molecular docking, and cross-resistance profiling of nobiletin in cancer cells and synergistic interaction with doxorubicin upon SOX5 transfection.

BACKGROUND: Nobiletin is a polymethoxylated flavone from citrus fruit peels. Among other bioactivities, it acts antioxidative, anti-inflammatory, neuroprotective, and cardiovascular-protective. Nobiletin exerts profound anticancer activity in vitro and in vivo but the underlying mechanisms are not well understood. PURPOSE: The aim was to unravel the multiple modes of action against cancer cells by bioinformatic and transcriptomic techniques and their verification by molecular pharmacological methods. METHODS: The in silico methods used were COMPARE analysis of transcriptomic data, signaling pathway analysis, transcription factor binding motif analysis in promoter sequences of target genes, and molecular docking. The in vitro methods used were resazurin assay, isobologram analysis, generation of stably SOX5-tranfected cells, and Western blotting. RESULTS: Nobiletin was cytotoxic against a wide range of cell lines from different tumor types, including diverse phenotypes to established anticancer drugs (e.g., P-glycoprotein, ABCB5, p53, EGFR). Cross-resistance profiling with 83 standard anticancer drugs revealed a correlation to antihormonal anticancer drugs, which can be explained by the phytoestrogenic features of nobiletin. Transcriptomic analysis showed that the responsiveness of tumor cells was predictable by their specific mRNA expression profile. Nobiletin bound to the transcription factor SOX5 in silico. SOX5 conferred resistance to the control drug doxorubicin but collateral sensitivity to nobiletin in HEK293 cells transfected with a lentiviral GFP-tagged pLOCORF-SOX5 vector. The combination of nobiletin and doxorubicin synergistically killed HEK293-SOX5 cells in isobologram analyses, implying attractive new treatment options. CONCLUSION: Nobiletin represents an interesting candidate for cancer therapy with broad-spectrum activity and multiple modes of action. The identification of novel targets (i.e., SOX5) may allow its use for targeted tumor therapy in individualized treatment protocols.

Cytotoxicity and apoptosis induction by Fumaria officinalis extracts in leukemia and multiple myeloma cell lines.

ETHNOPHARMACOLOGICAL RELEVANCE: Fumaria officinalis (Fumariaceae) is recorded in the Kurdish ethnobotany for various health problems. AIM OF THE STUDY: In this study, the cytotoxic activity of F. officinalis extracts on two leukemia and nine multiple myeloma (MM) cell lines was investigated. MATERIALS AND METHODS: The cytotoxic and ferroptotic activity were examined by resazurin reduction assay. Flow cytometry, immunoblotting assay and fluorescence microscopy were used to measure cell cycle distribution, apoptosis, induction of reactive oxygen species (ROS), loss integrity of mitochondrial membrane potential (MMP) and autophagy. LC-ESI/MS was used to identify chemical constituents present in F. officinalis. RESULTS: Chloroform (CF) and ethyl acetate (EF) fractions showed drastic cytotoxic effect on CCRF-CEM and CEM/ADR 5000 cells. NCI-H929 cell line exhibited higher sensitivity against CF, while EF demonstrated its higher cytotoxicity on OPM-2 cells with IC50 value 14.80 ± 1.70 and 28.13 ± 1.38 µg/mL respectively. Flow cytometric and morphological studies confirmed that CF and EF induced apoptosis in NCI-H929 cells by loss of MMP, generation of ROS and obvious morphological variations. In DNA histograms, up to 50% of the cells were accumulated by CF and 44% by EF in the sub-G0/G1 phase following 72 h treatment. EF induced autophagic cell death, while CF stimulated iron-dependent cell death. Moreover, two isoquinoline alkaloids and four flavonoids were identified in the active fractions. CONCLUSION: To our knowledge, this is the first report demonstrating the cytotoxicity of F. officinalis extracts in MM cell lines. CF and EF fractions inhibited MM cell proliferation through various modes of actions.

Cytotoxicity of apigenin toward multiple myeloma cell lines and suppression of iNOS and COX-2 expression in STAT1-transfected HEK293 cells.

BACKGROUND: Apigenin is one of the most abundant dietary flavonoids that possesses multiple bio-functions. PURPOSE: This study was designed to determine the influence of apigenin on gene expressions, cancer cells, as well as STAT1/COX-2/iNOS pathway mediated inflammation and tumorigenesis in HEK293-STAT1 cells. Furthermore, the cytotoxic activity toward multiple myeloma (MM) cell lines was investigated. METHODS: Bioinformatic analyses were used to predict the sensitivity and resistance of tumor cells toward apigenin and to determine cellular pathways influenced by this compound. The cytotoxic and ferroptotic activity of apigenin was examined by the resazurin reduction assay. Additionally, we evaluated apoptosis, and cell cycle distribution, induction of reactive oxygen species (ROS) and loss of integrity of mitochondrial membrane (MMP) by using the flow cytometry analysis. DAPI staining was used to detect characteristic apoptotic features. Furthermore, we verified its anti-inflammatory and additional mechanism of cell death by western blotting. RESULTS: COMPARE and hierarchical cluster analyses exhibited that 29 of 55 tumor cell lines were sensitive against apigenin (p < 0.001). The Ingenuity Pathway Analysis data showed that important bio-functions affected by apigenin were: gene expression, cancer, hematological system development and function, inflammatory response, and cell cycle. The STAT1 transcription factor was chosen as target protein on the basis of gene promoter binding motif analyses. Apigenin blocked cell proliferation of wild-type HEK293 and STAT1 reporter cells (HEK293-STAT1), promoted STAT1 suppression and subsequent COX-2 and iNOS inhibition. Apigenin also exhibited synergistic activity in combination with doxorubicin toward HEK293-STAT1 cells. Apigenin exerted excellent growth-inhibitory activity against MM cells in a concentration-dependent manner with the greatest activity toward NCI-H929 (IC50 value: 10.73 ± 3.21 µM). Apigenin induced apoptosis, cell cycle arrest, ferroptosis and autophagy in NCI-H929 cells. CONCLUSION: Apigenin may be a suitable candidate for MM treatment. The inhibition of the STAT1/COX-2/iNOS signaling pathway by apigenin is an important mechanism not only in the suppression of inflammation but also in induction of apoptosis.