Correction: Meisaprow et al. Caffeine Induces G0/G1 Cell Cycle Arrest and Inhibits Migration through Integrin αv, ß3, and FAK/Akt/c-Myc Signaling Pathway. Molecules 2021, 26, 7659.

In the original publication [...].

N,N'-Diarylurea Derivatives (CTPPU) Inhibited NSCLC Cell Growth and Induced Cell Cycle Arrest through Akt/GSK-3ß/c-Myc Signaling Pathway.

Lung cancer is one of the most common malignancies worldwide. Non-small-cell lung cancer (NSCLC) accounts for more than 80% of lung cancers, shows chemotherapy resistance, metastasis, and relapse. The phosphatidylinositol-3 kinase (PI3K)/Akt pathway has been implicated in the carcinogenesis and disease progression of NSCLC, suggesting that it may be a promising therapeutic target for cancer therapy. Although phenylurea derivatives have been reported as potent multiple kinase inhibitors, novel unsymmetrical N,N'-diarylurea derivatives targeting the PI3K/Akt pathway in NSCLC cells remain unknown. METHODS: N,N'-substituted phenylurea derivatives CTPPU and CT-(4-OH)-PU were investigated for their anticancer proliferative activity against three NSCLC cell lines (H460, A549, and H292) by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide, colony formation, Hoechst33342/PI staining assays, and apoptosis analysis. The protein expressions of Akt pathway-related proteins in response to CTPPU or CT-(4-OH)-PU were detected by Western blot analysis. The Kyoto Encyclopedia of Genes and Genomes mapper was used to identify the possible signaling pathways in NSCLC treated with CTPPU. The cell cycle was analyzed by flow cytometry. Molecular docking was used to investigate the possible binding interaction of CTPPU with Akt, the mammalian target of rapamycin complex 2 (mTORC2), and PI3Ks. Immunofluorescence and Western blot analysis were used to validate our prediction. RESULTS: The cytotoxicity of CTPPU was two-fold higher than that of CT-(4-OH)-PU for all NSCLC cell lines. Similarly, the non-cytotoxic concentration of CTPPU (25 µM) dramatically inhibited the colony formation of NSCLC cells, whereas its relative analog CT-(4-OH)-PU had no effect. Protein analysis revealed that Akt and its downstream effectors, namely, phosphorylated glycogen synthase kinase (GSK)-3ß (Ser9), ß-catenin, and c-Myc, were reduced in response to CTPPU treatment, which suggested the targeting of Akt-dependent pathway, whereas CT-(4-OH)-PU had no effect on such cell growth regulatory signals. CTPPU induced G1/S cell cycle arrest in lung cancer cells. Immunofluorescence revealed that CTPPU decreased p-Akt and total Akt protein levels, which implied the effect of the compound on protein activity and stability. Next, we utilized in silico molecular docking analysis to reveal the potential molecular targets of CTPPU, and the results showed that the compound could specifically bind to the allosteric pocket of Akt and three sites of mTORC2 (catalytic site, A-site, and I-site), with a binding affinity greater than that of reference compounds. The compound cannot bind to PI3K, an upstream regulator of the Akt pathway. The effect of CTPPU on PI3K and Akt was confirmed. This finding indicated that the compound could decrease p-Akt but caused no effect on p-PI3K. CONCLUSIONS: The results indicate that CTPPU significantly inhibits NSCLC cell proliferation by inducing G1/S cell cycle arrest via the Akt/GSK-3ß/c-Myc signaling pathway. Molecular docking revealed that CTPPU could interact with Akt and mTORC2 molecules with a high binding affinity. These data indicate that CTPPU is a potential novel alternative therapeutic approach for NSCLC.

Analysis of Protein-Protein Interactions Identifies NECTIN2 as a Target of N,N-Bis (5-Ethyl-2-hydroxybenzyl) Methylamine for Inhibition of Lung Cancer Metastasis.

BACKGROUND/AIM: Metastasis negatively affects the survival of lung cancer patients, however, relatively few compounds have potential in metastasis suppression. This study investigated the molecular targets of N,N-bis (5-ethyl-2-hydroxybenzyl) methylamine (EMD) for metastatic inhibition. MATERIALS AND METHODS: Proteins were analyzed by proteomic and bioinformatic analyses. Protein-protein interaction (PPI) networks were created with the Search Tool for the Retrieval of Interacting Genes. The Kyoto Encyclopedia of Genes and Genomes database and hub genes were used to determine dominant pathways. Immunofluorescence and western blot analyses validated the proteomic results and investigated signaling pathways in NCI-H23 lung cancer cells. RESULTS: A total of 1,751 proteins were common to the control, EMD and N,N-bis(5-methoxy-2-hydroxybenzyl) methylamine (MeMD) groups; 1,980 different proteins were categorized using metastatic capacity category and analyzed for unique proteins affected by EMD. Fifteen proteins were associated with cell adhesion and six with cell migration. Nectin cell adhesion molecule 2 (NECTIN2) was expressed in the control and MeMD-treated groups but not the EMD-treated group, suggesting NECTIN2 as an EMD target. PPI network showed association of NECTIN2 with proteins regulating cancer metastasis. Kyoto Encyclopedia of Genes and Genomes pathways revealed that NECTIN2 is an upstream target of cytoskeletal regulation via SRC signaling. Western blot and immunofluorescence analyses confirmed that EMD suppressed NECTIN2, and its downstream targets, including p-SRC (Y146 and Y527) and the epithelial-to-mesenchymal transition markers tight junction protein 1, vimentin, ß-catenin, snail family transcriptional repressor 1 (SNAI1), and SNAI2, while increasing E-cadherin. CONCLUSION: EMD suppressed NECTIN2-induced activation of EMT signaling. These data support the development of EMD to prevent metastasis of lung cancer.

Caffeine Induces G0/G1 Cell Cycle Arrest and Inhibits Migration through Integrin αv, ß3, and FAK/Akt/c-Myc Signaling Pathway.

Lung cancer is recognized as a major cause of mortality worldwide owing to its metastatic activity. Given the lack of solid information regarding the possible effects of caffeine, one of the most consumed natural psychoactive substances, on molecular signaling pathways implicated in the aggressive behavior of lung cancer, our study aimed to evaluate the effect and mechanism of caffeine on metastasis-related mechanisms. The results revealed that caffeine treatment at concentrations of 0-500 µM caused no direct cytotoxic effects on NCI-H23 cells. Treatment of cells with caffeine showed good potential to inhibit cell proliferation at 48 h and induced significant cell cycle arrest at the G0/G1 phase. Concerning metastasis, caffeine was shown to reduce filopodia formation, inhibit migration and invasion capability, and reduce the ability of cancer cells to survive and grow in an anchorage-independent manner. Moreover, caffeine could attenuate the formation of 3D tumor spheroids in cancer stem cell (CSC)-enriched populations. With regard to mechanisms, we found that caffeine significantly altered the integrin pattern of the treated cells and caused the downregulation of metastasis-associated integrins, namely, integrins αv and ß3. Subsequently, the downstream signals, including protein signaling and transcription factors, namely, phosphorylated focal adhesion kinase (p-FAK), phosphorylated protein kinase B (p-Akt), cell division cycle 42 (Cdc42), and c-Myc, were significantly decreased in caffeine-exposed cells. Taken together, our novel data on caffeine-inhibiting mechanism in relation to metastasis in lung cancer could provide insights into the impact of caffeine intake on human diseases and conditions.

Pongol Methyl Ether Inhibits Akt and Suppresses Cancer Stem Cell Phenotypes in Lung Cancer Cells.

Cancer stem cells (CSCs) are an important therapeutic target. The therapeutic agents targeting CSCs should lead to improved clinical outcomes. Here we have demonstrated the CSC-suppressing activity of pongol methyl ether (PME), a pure compound from Millettia erythrocalyx. METHODS: CSC-suppressing effects were evaluated by spheroid formation assay and detection of CSC markers. The related CSC cell signals were evaluated by Western blot, immunofluorescence and molecular docking analysis. Proteins affected by PME treatment were subjected to bioinformatic analysis. Protein-protein interaction (PPI) networks were constructed by the Search Tool for Interactions of Chemicals (STITCH). The Kyoto Encyclopedia of Genes and Genomes (KEGG) mapper were used to confirm the underlying pathways. RESULTS: PME (5-25 µM) significantly suppressed the ability of lung cancer cells to form colonies, grow in an anchorage-independent manner and generate tumour spheroids. PME at 25 µM significantly decreased the CSC markers (CD133 and ALDH1A1) and pluripotent transcription factors (Oct4 and Nanog). Akt, the key upstream signal of CSC control, was significantly decreased by the PME treatment. The molecular docking indicated that PME was bound to Akt-1 with a binding affinity of -9.2 kcal/mol greater than the Akt-1 inhibitor (reference compound; CQW). The STITCH network identified a total of 15 proteins interacted in PPI networks, and Akt-1 was identified as a central protein. The KEGG mapper indicated that the selected CSC markers were mostly involved in the 'signalling pathways regulating pluripotency of stem cells' pathway map and Akt, Oct4 and Nanog were the regulatory proteins in the dominant pathway. In addition, PME (10-25 µM) can suppress spheroid formation and reduce CSC-specific marker expression in patient-derived primary lung cancer cells. CONCLUSIONS: Our study revealed a novel pharmacological effect and the underlying mechanism of PME that can attenuate CSC phenotypes in lung cancer cells and may be developed for lung cancer therapy.

Hydroquinone 5-O-Cinnamoyl Ester of Renieramycin M Suppresses Lung Cancer Stem Cells by Targeting Akt and Destabilizes c-Myc.

Cancer stem cells (CSCs) are distinct cancer populations with tumorigenic and self-renewal abilities. CSCs are drivers of cancer initiation, progression, therapeutic failure, and disease recurrence. Thereby, novel compounds targeting CSCs offer a promising way to control cancer. In this study, the hydroquinone 5-O-cinnamoyl ester of renieramycin M (CIN-RM) was demonstrated to suppress lung cancer CSCs. CIN-RM was toxic to lung cancer cells with a half-maximal inhibitory concentration of around 15 µM. CIN-RM suppressed CSCs by inhibiting colony and tumor spheroid formation. In addition, the CSC population was isolated and treated and the CSCs were dispatched in response to CIN-RM within 24 h. CIN-RM was shown to abolish cellular c-Myc, a central survival and stem cell regulatory protein, with the depletion of CSC markers and stem cell transcription factors ALDH1A1, Oct4, Nanog, and Sox2. For up-stream regulation, we found that CIN-RM significantly inhibited Akt and consequently decreased the pluripotent transcription factors. CIN-RM also inhibited mTOR, while slightly decreasing p-GSK3ß (Ser9) but rarely affected the protein kinase C (PKC) signal. Inhibiting Akt/mTOR induced ubiquitination of c-Myc and promoted degradation. The mechanism of how Akt regulates the stability of c-Myc was validated with the Akt inhibitor wortmannin. The computational analysis further confirmed the strong interaction between CIN-RM and the Akt protein with a binding affinity of -10.9 kcal/mol at its critical active site. Taken together, we utilized molecular experiments, the CSC phenotype, and molecular docking methods to reveal the novel suppressing the activity of this compound on CSCs to benefit CSC-targeted therapy for lung cancer treatment.

Millettocalyxin B Inhibits Migratory Behavior of Lung Cancer Cells via Integrin α5 Suppression.

BACKGROUND/AIM: Integrin-targeting compounds have shown clinically significant benefits in many patients. Here, we examined the activity of millettocalyxin B, extracted from the stem bark of Millettia erythrocalyx, in lung cancer cells. MATERIALS AND METHODS: The viability of human lung cancer cells was investigated by the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazoliumbromide (MTT) assay. Migration and invasion assays were performed. Phalloidin-rhodamine staining was used to determine the formation of filopodia. Western blot analysis and immunofluorescence staining were used to identify the signaling proteins involved in migration regulation. RESULTS: Non-toxic concentrations (0-25 µM) of millettocalyxin B reduced migration and invasion of lung cancer A549 cells. Filopodia were significantly reduced in millettocalyxin B-treated cells. The migration regulatory proteins including integrin α5, active FAK, active Akt, and Cdc42 were significantly decreased in Millettocalyxin B-treated cells. CONCLUSION: Our findings revealed a novel anti-migration and anti-invasion effects and the underlying mechanism of millettocalyxin B, which may be exploited for cancer treatment.

Analysis of the Protein-Protein Interaction Network Identifying c-Met as a Target of Gigantol in the Suppression of Lung Cancer Metastasis.

BACKGROUND/AIM: c-Met (mesenchymal-epithelial transition factor) facilitates cancer progression and is recognized as a promising drug target. The molecular target of gigantol from Dendrobium draconis in suppressing cancer metastasis is largely unknown. MATERIALS AND METHODS: Proteins affected by gigantol treatment were subjected to proteomic and bioinformatic analysis. Protein-Protein interaction (PPI) networks were constructed by the Search Tool for the Retrieval of Interacting Genes (STRING). The Kyoto Encyclopedia of Genes and Genomes (KEGG) database and hub gene were used to enrich the dominant pathways. Western blot analysis and immunofluorescence were used to validate the effect of gigantol on the target protein and signaling. RESULTS: Gigantol down-regulates 41 adhesion proteins and 39-migratory proteins, while it up-regulates 30 adhesion-related proteins and 22 proteins controlling cell migration. The key components of our constructed PPI network comprised 41 proteins of cell adhesion enriched in 40 nodes with 25 edges, 39 proteins of cell migration enriched in 39 nodes with 76 edges in down-regulated proteins, 30 proteins of cell adhesion enriched in 30 nodes with 21 edges, and 22 proteins of cell migration enriched in 22 nodes with 22 edges in up-regulated protein. c-Met was identified as a central protein of the PPI network in the largest degree. KEGG mapper further suggested that c-Met, PI3K, and AKT were the regulatory proteins affected by gigantol. To confirm, the effects of gigantol on c-Met, the p-PI3K, PI3K, p-AKT, and AKT proteins were investigated by western blotting and the results showed a consistent effect of gigantol in the suppression of the c-Met/PI3K/AKT signal. Next, immunofluorescence showed a dramatic decrease in c-Met, PI3K and AKT activation in response to gigantol. CONCLUSION: c-Met is an important target of gigantol treatment in lung cancer cells. Gigantol suppresses metastasis-related cell motility through decreasing c-Met resulting in PI3K/AKT signaling disruption.

Integrin as a Molecular Target for Anti-cancer Approaches in Lung Cancer.

Integrins are cell-matrix adhesion molecules providing both mechanical engagement of cell to extracellular matrix, and generation of cellular signals that are implicated in cancer malignancies. The concept that integrins play important roles in cell survival, proliferation, motility, differentiation, and ensuring appropriate cell localization, leads to the hypothesis that inhibition of certain integrins would benefit cancer therapy. In lung cancer, integrins αv, α5, ß1, ß3, and ß5 have been shown to augment survival and metastatic potential of cancer cells. This review presents data suggesting integrins as molecular targets for anti-cancer approaches, and the mechanisms through which integrins confer resistance of lung cancer to chemotherapeutics and metastasis. The better understanding of these key molecules may benefit the discovery of anti-cancer drugs and strategies.

Novel Potential Biomarkers for Opisthorchis viverrini Infection and Associated Cholangiocarcinoma.

BACKGROUND/AIM: Early detection of disease is a pivotal factor for determining prognosis and clinical outcome of patients with cancer. As cholangiocarcinoma (CCA) is currently difficult to detect and most cases of such cancer present with late-stage disease at the time of initial diagnosis, we employed proteomic analysis of the bile to identify potential candidate biomarkers for Opisthorchis viverrini (OV)-associated CCA. MATERIALS AND METHODS: Proteins in pooled bile samples from patients with CCA and OV infection, with CCA without OV infection, with OV infection but no CCA, and with neither OV infection nor CCA were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in-gel trypsin digestion and analyzed by liquid chromatography-tandem mass spectrometry. RESULTS: According to our analysis, three proteins, namely aristaless-like homeobox1 isoform X1 (ALX1), major histocompatibility complex polypeptide-related sequence A (MICA), and uncharacterized protein C14orf105 isoform X12 were found to be potential markers for OV infection, as they were predominantly found in all OV-infected groups. Although these proteins were detected in both OV-infected patients with and without CCA, their abundance was 2.90-, 7.06-and 3.65-fold higher, respectively, in those with CCA. In patients with CCA, potential novel biomarkers wre immunoglobulin heavy chain, translocated in liposarcoma (TLS), visual system homeobox 2 (VSX2) and an unnamed protein product. CONCLUSION: We provided novel information regarding potential biomarkers for OV infection and CCA. These two protein profiles could benefit diagnosis as well as monitoring of CCA.