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Background: The upgrade of natural products for cancer treatment is essential since current anticancer drugs still pose severe side effects. Cymensifin A (Cym A) isolated from an orchid Cymbidium ensifolium has shown its potential to induce the death of several cancer cells; however, its underlying molecular mechanisms are hitherto unknown.Methods: Here, we conducted a set of in vitro preliminary tests to assess the cytotoxic effects of Cym A on non-small-cell lung cancer (NSCLC) cells (A549, H23, H292, and H460). A flow cytometry system and Western blot analyses were employed to unveil molecular mechanisms underlying cancer cell apoptosis caused by Cym A.Results: Cym A at 25–50 μM caused the death of all NSCLC cells tested, and its cytotoxicity was comparable to cisplatin, a currently used anticancer drug. The compound induced apoptosis of all NSCLC cells in a dose-dependent manner (5–50 μM), proven by flow cytometry, but H460 cells showed more resistance compared to other cells tested. Cym A-treated H460 cells demonstrated increased reactive oxygen species (ROS) and downregulated antioxidants (catalase, superoxide dismutase, and thioredoxin). The compound also upregulated the tumor suppressor P53 and the pro-apoptotic protein BAX but downregulated pro-survival proteins (BCL-2 and MCL-1) and deactivated survival signals (AKT and ERK) in H460 cells. Cym A was proven to trigger cellular ROS formation, but P53 and BAX were 2-fold more activated by Cym A compared to those treated with hydrogen peroxide. Our findings also supported that Cym A exerted its roles in the downregulation of nuclear factor erythroid 2–related factor 2 (a regulator of cellular antioxidant activity) and the increased levels of cleaved poly (ADP-ribose) polymerase (PARP) and cleaved caspase 3/7 during apoptosis.Conclusion: We propose that Cym A induces lung cancer cell death via ROS-mediated apoptosis, while the modulation of cellular ROS/antioxidant activity, the upregulation of P53 and BAX, the downregulation or deactivation of BCL-2, MCL-1, AKT, and ERK, and the increased cleavage of PARP and caspase 3/7, were the elucidated underlying molecular mechanisms of this phytochemical. The compound can be a promising candidate for future anticancer drug development.

Abstract The semisynthesis of 5-O-ester derivatives of renieramycin T was accomplished through the photoredox reaction of renieramycin M (1), a bistetrahydroisoquinolinequinone alkaloid isolated from the Thai blue sponge Xestospongia sp. This process led to the conversion of compound 1 to renieramycin T (2), which was subsequently subjected to Steglich esterification with appropriate acylating agents containing linear alkyl, N-tert-butoxycarbonyl-L-amino, and heterocyclic aromatic substituent. Notably, the one-pot transformation, combining the photoredox reaction and esterification led to the formation of 7-O-ester derivatives of renieramycin S due to hydrolysis. Subsequently, the in vitro cytotoxicity of the 17 semisynthesized derivatives against human non-small-cell lung cancer (NSCLC) cells in parallel with normal cell lines was evaluated. Among the tested compounds, 5-O-(3-propanoyl) ester of renieramycin T (3b) exhibited potent cytotoxic activity with half-maximal inhibitory concentration (IC50) values at 33.44 and 33.88 nM against H292 and H460 cell lines, respectively. These values were within the same range as compound 1 (IC50 = 34.43 and 35.63 nM) and displayed twofold higher cytotoxicity compared to compound 2 (IC50 = 72.85 and 83.95 nM). The steric characteristics and aromatic orientation of the 5-O-ester substituents played significant roles in their cytotoxicity. Notably, derivative 3b induced apoptosis with minimal necrosis, in contrast to the parental compound 1. Hence, the relationship between the structure and cytotoxicity of renieramycin–ecteinascidin hybrid alkaloids was investigated. This study emphasizes the potential of the series of 5-O-ester derivatives of renieramycin T as promising leads for the further development of potential anti-NSCLC agents.

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Constitutive activation of STAT3 contributes to tumor development and metastasis, making it a promising target for cancer therapy. (1R,4R,5S)-10-hydroxy-9-methoxy-8,11-dimethyl-3-(naphthalen-2-ylmethyl)-1,2,3,4,5,6-hexahydro-1,5-epiminobenzo[d]azocine-4-carbonitrile, DH_31, a new derivative of the marine natural product Renieramycin T, showed potent activity against H292 and H460 cells, with IC50 values of 5.54 ± 1.04 µM and 2.9 ± 0.58 µM, respectively. Structure–activity relationship (SAR) analysis suggests that adding a naphthalene ring with methyl linkers to ring C and a hydroxyl group to ring E enhances the cytotoxic effect of DH_31. At 1–2.5 µM, DH_31 significantly inhibited EMT phenotypes such as migration, and sensitized cells to anoikis. Consistent with the upregulation of ZO1 and the downregulation of Snail, Slug, N-cadherin, and Vimentin at both mRNA and protein levels, in silico prediction identified STAT3 as a target, validated by protein analysis showing that DH_31 significantly decreases STAT3 levels through ubiquitin-proteasomal degradation. Immunofluorescence and Western blot analysis confirmed that DH_31 significantly decreased STAT3 and EMT markers. Additionally, molecular docking suggests a covalent interaction between the cyano group of DH_31 and Cys-468 in the DNA-binding domain of STAT3 (binding affinity = −7.630 kcal/mol), leading to destabilization thereafter. In conclusion, DH_31, a novel RT derivative, demonstrates potential as a STAT3-targeting drug that significantly contribute to understanding of the development of new targeted therapy.

Abstract Background Malignant cells adopt anoikis resistance to survive anchorage-free stresses and initiate cancer metastasis. It is still unknown how varying periods of anchorage loss contribute to anoikis resistance, cell migration, and metabolic reprogramming of cancerous cells. Results Our study demonstrated that prolonging the anchorage-free lifetime of non-small-cell lung cancer NCI-H460 cells for 7 days strengthened anoikis resistance, as shown by higher half-life and capability to survive and grow without anchorage, compared to wild-type cells or those losing anchorage for 3 days. While the prolonged anchorage-free lifetime was responsible for the increased aggressive feature of survival cells to perform rapid 3-dimensional migration during the first 3 h of a transwell assay, no significant influence was observed with 2-dimensional surface migration detected at 12 and 24 h by a wound-healing method. Metabolomics analysis revealed significant alteration in the intracellular levels of six (oxalic acid, cholesterol, 1-ethylpyrrolidine, 1-(3-methylbutyl)-2,3,4,6-tetramethylbenzene, β-alanine, and putrescine) among all 37 identified metabolites during 7 days without anchorage. Based on significance values, enrichment ratios, and impact scores of all metabolites and their associated pathways, three principal metabolic activities (non-standard amino acid metabolism, cell membrane biosynthesis, and oxidative stress response) offered potential links with anoikis resistance. Conclusions These findings further our insights into the evolution of anoikis resistance in lung cancer cells and identify promising biomarkers for early lung cancer diagnosis.

Abstract Background Compound with cancer stem cell (CSC)-suppressing activity is promising for the improvement of lung cancer clinical outcomes. Toward this goal, we discovered the CSC-targeting activity of resveratrol (RES) analog moscatilin (MOS). With slight structural modification from RES, MOS shows dominant cytotoxicity and CSC-suppressive effect. Methods Three human lung cancer cell lines, namely H23, H292, and A549, were used to compare the effects of RES and MOS. Cell viability and apoptosis were determined by the MTT assay and Hoechst33342/PI double staining. Anti-proliferative activity was determined by colony formation assay and cell cycle analysis. Intracellular reactive oxygen species (ROS) were measured by fluorescence microscopy using DCFH2-DA staining. CSC-rich populations of A549 cells were generated, and CSC markers, and Akt signaling were determined by Western blot analysis and immunofluorescence. Molecular docking and molecular dynamics (MD) simulations were used to predict the possible binding of the compound to Akt protein. Results In this study, we evaluated the effects of RES and MOS on lung cancer and its anti-CSC potential. Compared with RES, its analog MOS more effectively inhibited cell viability, colony formation, and induced apoptosis in all lung cancer cell lines (H23, H292, and A549). We further investigated the anti-CSC effects on A549 CSC-rich populations and cancer adherent cells (A549 and H23). MOS possesses the ability to suppress CSC-like phenotype of lung cancer cells more potent than RES. Both MOS and RES repressed lung CSCs by inhibiting the viability, proliferation, and lung CSC-related marker CD133. However, only MOS inhibits the CSC marker CD133 in both CSC-rich population and adherent cells. Mechanistically, MOS exerted its anti-CSC effects by inhibiting Akt and consequently restored the activation of glycogen synthase kinase 3β (GSK-3β) and decreased the pluripotent transcription factors (Sox2 and c-Myc). Thus, MOS inhibits CSC-like properties through the repression of the Akt/GSK-3β/c-Myc pathway. Moreover, the superior inhibitory effects of MOS compared to RES were associated with the improved activation of various mechanism, such as cell cycle arrest at G2/M phase, production of ROS-mediated apoptosis, and inhibition of Akt activation. Notably, the computational analysis confirmed the strong interaction between MOS and Akt protein. MD simulations revealed that the binding between MOS and Akt1 was more stable than RES, with MM/GBSA binding free energy of − 32.8245 kcal/mol at its allosteric site. In addition, MOS interacts with Trp80 and Tyr272, which was a key residue in allosteric inhibitor binding and can potentially alter Akt activity. Conclusions Knowledge about the effect of MOS as a CSC-targeting compound and its interaction with Akt is important for the development of drugs for the treatment of CSC-driven cancer including lung cancer.

In the original publication [...]

This study aimed to isolate and purify resveratrol and oxyresveratrol from the heartwoods of Maclura cochinchinensis, and to evaluate their inhibitory effects on melanogenesis in B16F10 murine melanoma cells. A methanol maceration process yielded a crude extract comprising 24.86% of the initial mass, which was subsequently analyzed through HPTLC, HPLC, and LC-MS/MS. These analyses revealed the presence of resveratrol and oxyresveratrol at concentrations of 4.32 mg/g and 33.6 mg/g in the extract, respectively. Initial purification employing food-grade silica gel column chromatography separated the extract into two fractions: FA, exhibiting potent inhibition of both tyrosinase activity and melanogenesis, and FM, showing no such inhibitory activity. Further purification processes led to the isolation of fractions Y11 and Gn12 with enhanced concentrations of resveratrol (94.9 and 110.21 mg/g, respectively) and fractions Gn15 and Gn16 with elevated levels of oxyresveratrol (321.93 and 274.59 mg/g, respectively), all of which significantly reduced melanin synthesis. These outcomes affirm the substantial presence of resveratrol and oxyresveratrol in the heartwood of M. cochinchinensis, indicating their promising role as natural agents for skin lightening.

Abstract Background The leading cause of cancer-related mortality worldwide is lung cancer, and its clinical outcome and prognosis are still unsatisfactory. The understanding of potential molecular targets is necessary for clinical implications in precision diagnostic and/or therapeutic purposes. Histone deacetylase 6 (HDAC6), a major deacetylase enzyme, is a promising target for cancer therapy; however, the molecular mechanism regulating cancer pathogenesis is largely unknown. Methods The clinical relevance of HDAC6 expression levels and their correlation with the overall survival rate were analyzed based on the TCGA and GEO databases. HDAC6 expression in clinical samples obtained from lung cancer tissues and patient-derived primary lung cancer cells was evaluated using qRT–PCR and Western blot analysis. The potential regulatory mechanism of HDAC6 was identified by proteomic analysis and validated by immunoblotting, immunofluorescence, microtubule sedimentation, and immunoprecipitation-mass spectrometry (IP-MS) assays using a specific inhibitor of HDAC6, trichostatin A (TSA) and RNA interference to HDAC6 (siHDAC6). Lung cancer cell growth was assessed by an in vitro 2-dimensional (2D) cell proliferation assay and 3D tumor spheroid formation using patient-derived lung cancer cells. Results HDAC6 was upregulated in lung cancer specimens and significantly correlated with poor prognosis. Inhibition of HDAC6 by TSA and siHDAC6 caused downregulation of phosphorylated extracellular signal-regulated kinase (p-ERK), which was dependent on the tubulin acetylation status. Tubulin acetylation induced by TSA and siHDAC6 mediated the dissociation of p-ERK on microtubules, causing p-ERK destabilization. The proteomic analysis demonstrated that the molecular chaperone glucose-regulated protein 78 (GRP78) was an important scaffolder required for p-ERK localization on microtubules, and this phenomenon was significantly inhibited by either TSA, siHDAC6, or siGRP78. In addition, suppression of HDAC6 strongly attenuated an in vitro 2D lung cancer cell growth and an in vitro 3D patient derived-lung cancer spheroid growth. Conclusions HDAC6 inhibition led to upregulate tubulin acetylation, causing GRP78-p-ERK dissociation from microtubules. As a result, p-ERK levels were decreased, and lung cancer cell growth was subsequently suppressed. This study reveals the intriguing role and molecular mechanism of HDAC6 as a tumor promoter, and its inhibition represents a promising approach for anticancer therapy.

Context Endoplasmic reticulum (ER) stress contributes to endothelium pathological conditions. Chitooligosaccharides (COS) have health benefits, but their effect on endothelial cells is unknown. We demonstrate for the first time a protective effect of COS against ER-induced endothelial cell damage.Objective To evaluate the protective effect of COS on ER stress-induced apoptosis in endothelial cells.Material and methods Endothelial (EA.hy926) cells were pre-treated with COS (250 or 500 μg/mL) for 24 h, and then treated with 0.16 μg/mL of Tg for 24 h and compared to the untreated control. Apoptosis and necrosis were detected by Annexin V-FITC/propidium iodide co-staining. Reactive oxygen species (ROS) were measured with the DCFH2-DA and DHE probes. The protective pathway and ER stress markers were evaluated by reverse transcription-polymerase chain reaction, western blot, and immunofluorescence analyses.Results COS attenuated ER stress-induced cell death. The viability of EA.hy926 cells treated with Tg alone was 44.97 ± 1% but the COS pre-treatment increased cells viability to 74.74 ± 3.95% in the 250 μg/mL COS and 75.34 ± 2.4% in the 500 μg/mL COS treatments. Tg induced ER stress and ROS, which were associated with ER stress-mediated death. Interestingly, COS reduced ROS by upregulating nuclear factor-E2-related factor 2 (Nrf2), and the oxidative enzymes, superoxide dismutase1 (SOD1) and catalase. COS also suppressed up-regulation of the ER-related apoptosis protein, CHOP induced by Tg.Conclusions COS protected against ER stress-induced apoptosis in endothelial cells by suppressing ROS and up-regulation Nrf2 and SOD1. These findings support the use of COS to protect endothelial cells.

Context Sericin, a protein found in wastewater from the silk industry, was shown to contain a variety of biological activities, including antioxidant. The enzymatic conditions have been continuously modified to improve antioxidant effect and scavenging capacity against various free radicals of silk sericin protein. Objective Variables in enzymatic reactions, including pH, temperature and enzyme/substrate ratio were analysed to discover the optimum conditions for antioxidant activity of sericin hydrolysates. Materials and methods Hydrolysis reaction catalysed by Alcalase® was optimized through response surface methodology (RSM) in order to generate sericin hydrolysates possessing potency for % inhibition on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, ferric-reducing power and peroxyl scavenging capacity. Flow cytometry was performed to evaluate cellular ROS level in human HaCaT keratinocytes and melanin-generating MNT1 cells pre-treated either with 20 mg/mL RSM-optimized sericin hydrolysates or 5 mM N-acetyl cysteine (NAC) for 60 min prior exposure with 1 mM hydrogen peroxide (H2O2). Results Among these three variables, response surface plots demonstrate the major role of temperature on scavenging capacity of sericin hydrolysates. Sericin hydrolysates prepared by using Alcalase® at RSM-optimized condition (enzyme/substrate ratio: 1.5, pH: 7.5, temperature: 70 °C) possessed % inhibition against H2O2 at 99.11 ± 0.54% and 73.25 ± 8.32% in HaCaT and MNT1 cells, respectively, while pre-treatment with NAC indicated the % inhibition only at 30.26 ± 7.62% in HaCaT and 51.05 ± 7.14% in MNT1 cells. Discussion and conclusions The acquired RSM information would be of benefit for further developing antioxidant peptide from diverse resources, especially the recycling of waste products from silk industry.

Shrimp is a rich source of bioactive molecules that provide health benefits. However, the high cholesterol content in shrimp oil may pose a risk. We utilized the cholesterol elimination method to obtain cholesterol-free shrimp lipids (CLs) and investigated their anticancer potential, focusing on cancer stem cells (CSCs) and epithelial-to-mesenchymal transition (EMT). Our study focused on CSCs and EMT, as these factors are known to contribute to cancer metastasis. The results showed that treatment with CLs at doses ranging from 0 to 500 µg/mL significantly suppressed the cell migration ability of human lung cancer (H460 and H292) cells, indicating its potential to inhibit cancer metastasis. The CLs at such concentrations did not cause cytotoxicity to normal human keratinocytes. Additionally, CL treatment was found to significantly reduce the levels of Snail, Slug, and Vimentin, which are markers of EMT. Furthermore, we investigated the effect of CLs on CSC-like phenotypes and found that CLs could significantly suppress the formation of a three-dimensional (3D) tumor spheroid in lung cancer cells. Furthermore, CLs induced apoptosis in the CSC-rich population and significantly depleted the levels of CSC markers CD133, CD44, and Sox2. A mechanistic investigation demonstrated that exposing lung cancer cells to CLs downregulated the phosphorylation of Akt and mTOR, as well as c-Myc expression. Based on these findings, we believe that CLs may have beneficial effects on health as they potentially suppress EMT and CSCs, as well as the cancer-potentiating pathway of Akt/mTOR/c-Myc.

Abstract Background Akt and mTOR are aberrantly activated in cancers and targeting these proteins are interesting for cancer drug discovery. Napabucasin (NB), a phytochemical compound, has been reported as potential anti-cancer agent, however, Akt and mTOR targeting mechanisms remain unclear. Method Apoptosis induction was investigated by Hoechst 33342/PI double staining and annexin V/PI staining with flowcytometry. Autophagy was evaluated by monodansylcadaverine staining and Western blot analysis. Binding affinity of NB and essential signaling proteins (PI3K, Akt, and mTOR) was investigated using molecular docking and confirmed by Western blot analysis. Result A structure modification from changing methyl moiety of acetyl group of NB to hydroxyl moiety of carboxyl group of NB derivative (napabucasin-acid or NB-acid) greatly affected the compound activities. NB showed more potent anti-cancer activity. NB reduced cell viability with an approximately 20 times lower IC50 and inhibited the colony formation capacity much more than NB-acid treated cells. NB induced cell apoptosis, which was accompanied by decrease Bcl‑2 and Mcl-1 and clevage of PARP, while NB-acid show lesser effect on Mcl-1. NB was found to strongly induce autophagy indicated by acidic vesicle staining and the LC3B conversion. Interestingly, computational molecular docking analysis further demonstrated that NB directly bound to Akt and mTOR (complex 1 and 2) proteins at their critical sites indicating that NB targets the upstream regulators of apoptosis and autophagy. The docking results were confirmed by decrease of p-Akt/Akt, p-mTOR/mTOR, and c-Myc a downstream target of Akt protein levels. Conclusion Results show for the first time that NB exerts an anti-cancer activity through the direct interaction to Akt and mTOR proteins. The methyl moiety of acetyl group of NB is required for its potent anti-cancer activities. These data encourage further development of NB compounds for Akt and mTOR driven cancers.

Abstract Background Crinum latifolium L. (Amaryllidaceae) has been used in Southeast Asian traditional medicine to alleviate the symptoms of benign prostatic hyperplasia (BPH). The pathological mechanism of BPH is associated with the induction of prostate stromal cell proliferation through transforming growth factor-beta (TGF-β). Standardization as well as investigation of the potential anti-BPH activity of C. latifolium extract could benefit the further development of BPH-related analyses and provide evidence to support the application of this extract for BPH treatment. This study aimed to standardize and investigate the antiproliferative activity of the ethanolic extract of C. latifolium leaves. The major alkaloids isolated from C. latifolium were also explored for their potential use as bioactive markers. Methods Two major alkaloids were isolated from the ethanolic extract of C. latifolium leaves by chromatographic techniques, identified by NMR and MS, and quantified by a validated UHPLC method. Their antiproliferative activity was studied in human prostate stromal cells (WPMY-1) induced by TGF-β. The synergistic effect of combining the two major isolated alkaloids was analyzed by the zero interaction potency (ZIP) model. Results Two alkaloids, lycorine (1) and 6α-hydroxybuphanidrine (2), were isolated from the ethanolic leaf extract of C. latifolium. A UHPLC method for the quantification of (1) and (2) was developed and validated in terms of linearity, precision, and accuracy. The C. latifolium leaf extract contained 0.279 ± 0.003% (1) and 0.232 ± 0.004% (2). The crude extract was more potent than either (1) and (2) alone against TGF-β-treated WPMY-1 cell proliferation. The drug combination study revealed that the greatest synergistic effect of (1) and (2) was achieved at a 1:1 ratio. Conclusions The results of this study support the anti-BPH activity of C. latifolium in traditional medicine and suggest that these the two isolated alkaloids may promote the efficacy of the C. latifolium extract. Additionally, major alkaloids (1) and (2) can be used as bioactive markers for the standardization of C. latifolium extracts.

Cancer stem cells (CSCs) found within cancer tissue play a pivotal role in its resistance to therapy and its potential to metastasize, contributing to elevated mortality rates among patients. Significant strides in understanding the molecular foundations of CSCs have led to preclinical investigations and clinical trials focused on CSC regulator β-catenin signaling targeted interventions in malignancies. As part of the ongoing advancements in marine-organism-derived compound development, it was observed that among the six analogs of Renieramycin T (RT), a potential lead alkaloid from the blue sponge Xestospongia sp., the compound DH_32, displayed the most robust anti-cancer activity in lung cancer A549, H23, and H292 cells. In various lung cancer cell lines, DH_32 exhibited the highest efficacy, with IC50 values of 4.06 ± 0.24 μM, 2.07 ± 0.11 μM, and 1.46 ± 0.06 μM in A549, H23, and H292 cells, respectively. In contrast, parental RT compounds had IC50 values of 5.76 ± 0.23 μM, 2.93 ± 0.07 μM, and 1.52 ± 0.05 μM in the same order. Furthermore, at a dosage of 25 nM, DH_32 showed a stronger ability to inhibit colony formation compared to the lead compound, RT. DH_32 was capable of inducing apoptosis in lung cancer cells, as demonstrated by increased PARP cleavage and reduced levels of the proapoptotic protein Bcl2. Our discovery confirms that DH_32 treatment of lung cancer cells led to a reduced level of CD133, which is associated with the suppression of stem-cell-related transcription factors like OCT4. Moreover, DH_32 significantly suppressed the ability of tumor spheroids to form compared to the original RT compound. Additionally, DH_32 inhibited CSCs by promoting the degradation of β-catenin through ubiquitin–proteasomal pathways. In computational molecular docking, a high-affinity interaction was observed between DH_32 (grid score = −35.559 kcal/mol) and β-catenin, indicating a stronger binding interaction compared to the reference compound R9Q (grid score = −29.044 kcal/mol). In summary, DH_32, a newly developed derivative of the right-half analog of RT, effectively inhibited the initiation of lung cancer spheroids and the self-renewal of lung cancer cells through the upstream process of β-catenin ubiquitin–proteasomal degradation.

Abstract Background Cellular senescence is an aging‐related process found in cancer cells that contributes to irreversible growth arrest and tumor aggressiveness. Recently, calmodulin‐regulated spectrin‐associated protein 3 (CAMSAP3), a minus‐end microtubule‐stabilizing protein, has received increasing attention in cancer cell biology. However, the biological role of CAMSAP3 on senescence in human lung cancer remains incompletely understood. Methods The function of CAMSAP3 on the regulation of cellular senescence‐associated phenotypes in human non‐small cell lung cancer H460 cells were determined in CAMSAP3 deletion (H460/C3ko) cells. The effects of CAMSAP3 on cell proliferation were investigated using MTT and colony formation assays. The cell cycle activity was evaluated by flow cytometry and the senescence‐associated phenotypes were observed by SA‐β‐Gal staining. Quantitative RT‐PCR and westen blot were used to evaluate the expression of cell cycle and senescence markers. Moreover, the interaction of CAMSAP3‐ERK1/2 and possible partner protein was quantified using immunoprecipitation/mass spectrometry and immunofluorescence. Lastly, an xenograft model were performed. Results CAMSAP3 knockout promotes lung cancer cell senescence‐associated phenotypes and induces G1 cell cycle arrest. Mechanistic investigation revealed that phosphorylated ERK (p‐ERK) was markedly downregulated in CAMSAP3‐deleted cells, suppressing cyclin D1 expression levels, and full‐length CAMSAP3 abrogated these phenotypes. Proteomic analysis demonstrated that vimentin, an intermediate filament protein, is required as a scaffold for CAMSAP3‐modulating ERK signaling. Furthermore, an in vivo tumor xenograft experiment showed that tumor initiation is potentially delayed in CAMSAP3 knockout tumors with the downregulation of p‐ERK and cyclin D1, resulting in a senescence‐like phenotype. Conclusion This study is the first to report an intriguing role of CAMSAP3 in lung carcinoma cell senescence‐associated phenotypes via the modulation of p‐ERK/cyclin D1 signaling.

Abstract Cisplatin (CDDP) induces senescence characterized by senescence-associated secretory phenotypes (SASP) and the unfolded protein response (UPR). In this study, we investigated the proteins related to the UPR during the senescence cell fate. Strikingly, we found that one of the critical ER-resident proteins, GRP78/BiP, was significantly altered. Here we show that GRP78 levels differentially expressed depending on non-small lung cancer subtypes. GRP78 indeed regulates the evasion of senescence in adenocarcinoma A549 cells, in which the increased GRP78 levels enable them to re-proliferate after CDDP removal. Conversely, GRP78 is downregulated in the senescence H460 cells, making them lacking senescence evasion capability. We observed that the translational regulation critically contributed to the GRP78 protein levels in CDDP-induces senescence. Furthermore, the increased GRP78 level during senescence confers resistance to senolytic drug, Bortezomib, as observed by a twofold increase in IC50 in A549 senescence cells compared to the wild-type. This observation is also consistent in the cells that have undergone genetic manipulation by transfection with pcDNA3.1(+)-GRP78/BiP plasmids and pSpCas9(BB)-2A-Puro containing guide RNA sequence targeting GRP78 exon 3 to induce the overexpression and downregulation of GRP78 in H460 cells, respectively. Our findings reveal a unique role of GRP78 on the senescence evasion cell fate and senolytic drug resistance after cisplatin-based chemotherapy.

Non-small cell lung cancer (NSCLC) poses a significant global health burden with unsatisfactory survival rates, despite advancements in diagnostic and therapeutic modalities. Novel therapeutic approaches are urgently required to improve patient outcomes. Pharmacological ascorbate (P-AscH−; ascorbate at millimolar concentration in plasma) emerged as a potential candidate for cancer therapy for recent decades. In this present study, we explore the anti-cancer effects of P-AscH− on NSCLC and elucidate its underlying mechanisms. P-AscH− treatment induces formation of cellular oxidative distress; disrupts cellular bioenergetics; and leads to induction of apoptotic cell death and ultimately reduction in clonogenic survival. Remarkably, DNA and DNA damage response machineries are identified as vulnerable targets for P-AscH− in NSCLC therapy. Treatments with P-AscH− increase the formation of DNA damage and replication stress markers while inducing mislocalization of DNA repair machineries. The cytotoxic and genotoxic effects of P-AscH− on NSCLC were reversed by co-treatment with catalase, highlighting the roles of extracellular hydrogen peroxide in anti-cancer activities of P-AscH−. The data from this current research advance our understanding of P-AscH− in cancer treatment and support its potential clinical use as a therapeutic option for NSCLC therapy.

The semisynthesis of renieramycin-type derivatives was achieved under mild and facile conditions by attaching a 1,3-dioxole-bridged phenolic moiety onto ring A of the renieramycin structure and adding a 4′-pyridinecarbonyl ester substituent at its C-5 or C-22 position. These were accomplished through a light-induced intramolecular photoredox reaction using blue light (4 W) and Steglich esterification, respectively. Renieramycin M (4), a bis-tetrahydroisoquinolinequinone compound isolated from the Thai blue sponge (Xestospongia sp.), served as the starting material. The cytotoxicity of the 10 natural and semisynthesized renieramycins against non-small-cell lung cancer (NSCLC) cell lines was evaluated. The 5-O-(4′-pyridinecarbonyl) renieramycin T (11) compound exhibited high cytotoxicity with half-maximal inhibitory concentration (IC50) values of 35.27 ± 1.09 and 34.77 ± 2.19 nM against H290 and H460 cells, respectively. Notably, the potency of compound 11 was 2-fold more than that of renieramycin T (7) and equal to those of 4 and doxorubicin. Interestingly, the renieramycin-type derivatives with a hydroxyl group at C-5 and C-22 exhibited weak cytotoxicity. In silico molecular docking and dynamics studies confirmed that the mitogen-activated proteins, kinase 1 and 3 (MAPK1 and MAPK3), are suitable targets for 11. Thus, the structure–cytotoxicity study of renieramycins was extended to facilitate the development of potential anticancer agents for NSCLC cells.

Stem cells have demonstrated significant potential for tissue engineering and repair, anti-aging, and rejuvenation. Hair follicle stem cells can be found in the dermal papilla at the base of the follicle and the bulge region, and they have garnered increased attention because of their potential to regenerate hair as well as their application for tissue repair. In recent years, these cells have been shown to affect hair restoration and prevent hair loss. These stem cells are endowed with mesenchymal characteristics and exhibit self-renewal and can differentiate into diverse cell types. As research in this field continues, it is probable that insights regarding stem cell maintenance, as well as their self-renewal and differentiation abilities, will benefit the application of these cells. In addition, an in-depth discussion is required regarding the molecular basis of cellular signaling and the influence of nature-derived compounds in stimulating the stemness properties of dermal papilla stem cells. This review summarizes (i) the potential of the mesenchymal cells component of the hair follicle as a target for drug action; (ii) the molecular mechanism of dermal papilla stem cells for maintenance of their stem cell function; and (iii) the positive effects of the natural product compounds in stimulating stemness in dermal papilla stem cells. Together, these insights may help facilitate the development of novel effective hair loss prevention and treatment.

Chitooligosaccharide (COS), found in both insects and marine sources, has several bioactivities, such as anti-inflammation and antioxidant activities. However, the mechanism of shrimp shell COS on retardation of inflammatory and antioxidant effects is limited. Therefore, the aim of this study is to examine the mechanism of the aforementioned activities of COS in LPS-activated RAW264.7 macrophage cells. COS significantly improved cell viability in LPS-activated cells. COS at the level of 500 µg/mL could reduce the TNF-α, NO and IL-6 generations in LPS-activated cells (p < 0.05). Furthermore, COS could reduce ROS formation, NF-κB overactivation, phosphorylation of Erk1/2 and Akt and Nrf2/HO-1 in LPS-exposed cells. These results indicate that COS manifests anti-inflammatory activity and antioxidant action via NF-κB, Erk1/2, Akt and Nrf2/HO-1 signaling with an increasing relevance for inflammatory disorders.

Abstract Background Accumulated evidence demonstrates cisplatin, a recommended chemotherapy, modulating pro-survival autophagic response that contributes to treatment failure in lung cancer patients. However, distinct mechanisms involved in cisplatin-induced autophagy in human lung cancer cells are still unclear. Results Herein, role of autophagy in cisplatin resistance was indicated by a decreased cell viability and increased apoptosis in lung cancer H460 cells pre-incubated with wortmannin, an autophagy inhibitor, prior to treatment with 50 µM cisplatin for 24 h. The elevated level of hydroxyl radicals detected via flow-cytometry corresponded to autophagic response, as evidenced by the formation of autophagosomes and autolysosomes in cisplatin-treated cells. Interestingly, apoptosis resistance, autophagosome formation, and the alteration of the autophagic markers, LC3-II/LC3-I and p62, as well as autophagy-regulating proteins Atg7 and Atg3, induced by cisplatin was abrogated by pretreatment of H460 cells with deferoxamine, a specific hydroxyl radical scavenger. The modulations in autophagic response were also indicated in the cells treated with hydroxyl radicals generated via Fenton reaction, and likewise inhibited by pretreatment with deferoxamine. Conclusions In summary, the possible role of hydroxyl radicals as a key mediator in the autophagic response to cisplatin treatment, which was firstly revealed in this study would benefit for the further development of novel therapies for lung cancer.

Abstract Background Intratumour heterogeneous gene expression among cancer and cancer stem cells (CSCs) can cause failure of current targeted therapies because each drug aims to target the function of a single gene. Long mononucleotide A-T repeats are cis-regulatory transcriptional elements that control many genes, increasing the expression of numerous genes in various cancers, including lung cancer. Therefore, targeting A-T repeats may dysregulate many genes driving cancer development. Here, we tested a peptide nucleic acid (PNA) oligo containing a long A-repeat sequence [A(15)] to disrupt the transcriptional control of the A-T repeat in lung cancer and CSCs. Methods First, we separated CSCs from parental lung cancer cell lines. Then, we evaluated the role of A-T repeat gene regulation by counting the number of repeats in differentially regulated genes between CSCs and the parental cells of the CSCs. After testing the dosage and effect of PNA-A15 on normal and cancer cell toxicity and CSC phenotypes, we analysed genome-wide expression to identify dysregulated genes in CSCs. Results The number of A-T repeats in genes differentially regulated between CSCs and parental cells differed. PNA-A15 was toxic to lung cancer cells and CSCs but not to noncancer cells. Finally, PNA-A15 dysregulated a number of genes in lung CSCs. Conclusion PNA-A15 is a promising novel targeted therapy agent that targets the transcriptional control activity of multiple genes in lung CSCs.

Abstract The posttranslational modifications (PTMs) of microtubules have been reported to play an important role in cancer aggressiveness, including apoptosis resistance. In this study, we aimed to investigate the biological role of microtubule PTMs in the regulation of paclitaxel responsiveness. The acetylated tubulin (Ace-tub) level was strongly associated with paclitaxel sensitivity, as observed in patient-derived primary lung cancer cells and xenografted immunodeficient mice. We showed that paclitaxel-resistant H460 lung cancer cells, generated by a stepwise increase in paclitaxel, exhibited markedly increased tubulin acetylation and consequently acquired paclitaxel resistance. Upregulation of tubulin acetylation by overexpression of α-tubulin acetyltransferase 1 wild-type (αTAT1wt), an enzyme required for acetylation, or by treatment with trichostatin A (TSA), a histone deacetylase 6 (HDAC6) inhibitor, significantly attenuated paclitaxel-induced apoptosis. Investigation of the underlying mechanism revealed that the levels of antiapoptotic Mcl-1 appeared to increase in αTAT1wt-overexpressing and TSA-treated cells compared to control cells, whereas the levels of other antiapoptotic regulatory proteins were unchanged. On the other hand, decreased tubulin acetylation by αTAT1 RNA interference downregulated Mcl-1 expression in patient-derived primary lung cancer and paclitaxel-resistant lung cancer cells. A microtubule sedimentation assay demonstrated that Mcl-1 binds to microtubules preferentially at Ace-type, which prolongs the Mcl-1 half-life (T1/2). Furthermore, immunoprecipitation analysis revealed that polyubiquitination of Mcl-1 was extensively decreased in response to TSA treatment. These data indicate that tubulin acetylation enhances the resistance to paclitaxel-induced cell death by stabilizing Mcl-1 and protecting it from ubiquitin–proteasome-mediated degradation.

Abstract The limitations of cisplatin, a standard chemotherapy for lung cancer, have been documented with serious adverse effects and drug resistance. To address the need for novel therapy, this study firstly reveals the potential of peptide from Lentinus squarrosulus (Mont.) as a chemotherapeutic adjuvant for cisplatin treatment. The purified peptide from L. squarrosulus aqueous extracts was obtained after eluting with 0.4 M NaCl through FPLC equipped with anion exchange column. Preincubation for 24 h with 5 µg/mL of the peptide at prior to treatment with 5 µM cisplatin significantly diminished %cell viability in various human lung cancer cells but not in human dermal papilla and proximal renal cells. Flow cytometry indicated the augmentation of cisplatin-induced apoptosis in lung cancer cells pretreated with peptide from L. squarrosulus. Preculture with the peptide dramatically inhibited colony formation in lung cancer cells derived after cisplatin treatment. Strong suppression on integrin-mediated survival was evidenced with the diminution of integrins (β1, β3, β5, α5, αV) and down-stream signals (p-FAK/FAK, p-Src/Src, p-Akt/Akt) consequence with alteration of p53, Bax, Blc-2 and Mcl-1 in cisplatin-treated lung cancer cells preincubated with peptide from L. squarrosulus. These results support the development of L. squarrosulus peptide as a novel combined chemotherapy with cisplatin for lung cancer treatment.

Colicin N (ColN) is a bacteriocin secreted by Escherichia coli (E. coli) to kill other Gram-negative bacteria by forcefully generating ion channels in the inner membrane. In addition to its bactericidal activity, ColN have been reported to selectively induce apoptosis in human lung cancer cells via the suppression of integrin modulated survival pathway. However, ColN showed mild toxicity against human lung cancer cells which could be improved for further applications. The protein resurfacing strategy was chosen to engineer ColN by extensive mutagenesis at solvent­-exposed residues on ColN. The highly accessible Asp and Glu on wild­type ColN (ColNWT) were replaced by Lys to create polycationic ColN (ColN+12). Previous studies have shown that increase of positive charges on proteins leads to the enhancement of mammalian cell penetration as well as increased interaction with negatively charged surface of cancer cells. Those solvent­-exposed residues of ColN were identified by Rosetta and AvNAPSA (Average number of Neighboring Atoms Per Side­chain Atom) approaches. The findings revealed that the structural features and stability of ColN+12 determined by circular dichroism were similar to ColNWT. Furthermore, the toxicity of ColN+12 was cancer ­selective. Human lung cancer cells, H460 and H23, were sensitive to ColN but human dermal papilla cells were not. ColN+12 also showed more potent toxicity than ColNWT in cancer cells. This confirmed that polycationic resurfacing method has enabled us to improve the anticancer activity of ColN towards human lung cancer cells.

Alpha folate receptor (FRα) is currently under investigation as a target for the treatment of patients with non-small-cell lung cancer (NSCLC), since it is highly expressed in tumor cells but is largely absent in normal tissue. In this study, a novel human variable domain of a heavy-chain (VH) antibody fragment specific to FRα was enriched and selected by phage bio-planning. The positive phage clone (3A102 VH) specifically bound to FRα and also cross-reacted with FRβ, as tested by ELISA. Clone 3A102 VH was then successfully expressed as a soluble protein in an E. coli shuffle strain. The obtained soluble 3A102 VH demonstrated a high affinity for FRα with affinity constants (Kaff) values around 7.77 ± 0.25 × 107 M−1, with specific binding against both FRα expressing NSCLC cells and NSCLC patient-derived primary cancer cells, as tested by cell ELISA. In addition, soluble 3A102 VH showed the potential desired property of a targeting molecule by being internalized into FRα-expressing cells, as observed by confocal microscopy. This study inspires the use of phage display to develop human VH antibody (Ab) fragments that might be well suited for drug targeted therapy of NSCLC and other FRα-positive cancer cells.

Abstract Background Cyanidin-3-glucoside (C3G), a major anthocyanin present in berries, exhibits a strong antioxidant and has been shown to possess a neuroprotection. Prolonged exposure to glutamate will lead to oxidative damage and endoplasmic reticulum stress which could play a key detrimental role in the development of neurodegenerative disorders (NDs). In the present study, we investigated the neuroprotective effect and underlying mechanisms of C3G on the reduction of oxidative/ER stress-induced apoptosis by glutamate in HT22 mouse hippocampal neuronal cells. Method Cells were pre-treated with C3G in various concentrations, followed by glutamate. Cell viability and toxicity were examined using MTT and LDH assays. The apoptotic and necrotic cell death were carried out by Annexin V-FITC/propidium iodide co-staining assays. Generation of intracellular reactive oxygen species (ROS) in cells was measured by flow cytometry using DCFH-DA probe. Expression of antioxidant genes was evaluated by Real-time polymerase chain reaction analysis. The possible signaling pathways and proteins involved were subsequently demonstrated by Western blot analysis. Result The pretreatment of the HT22 cells with C3G protected cell death from oxidative toxicity induced by glutamate. We demonstrated that treatment cells with glutamate caused several radical forms of ROS formation, and they were abolished by specific ROS inhibitors. Interestingly, C3G directly scavenged radical activity and inhibited intracellular ROS generation in our cell-based system. In addition, C3G pretreatment suppressed the up-regulation of specific ER proteins namely calpain, caspase-12 and C/EBP homologous proteins (CHOP) induced by glutamate-mediated oxidative and ER stress signal by up-regulating the expressions of survival proteins, including extracellular regulated protein kinase (ERK) and nuclear factor E2-related factor 2 (Nrf2). Furthermore, dramatically activated gene expression of endogenous antioxidant enzymes (i.e. superoxide dismutases (SODs), catalase (CAT) and glutathione peroxidase (GPx)), and phase II enzymes (glutathione-S-transferases (GSTs)) was found in C3G-treated with cells. Conclusions Our finding suggest that C3G could be a promising neuroprotectant via inhibition of glutamate-induced oxidative and ER stress signal and activation of ERK/Nrf2 antioxidant mechanism pathways.

Triple-negative breast cancer (TNBC) is characterized by excessive accumulation of tumor-infiltrating immune cells, including tumor-associated macrophages (TAMs). TAMs consist of a heterogeneous population with high plasticity and are associated with tumor aggressiveness and poor prognosis. Moreover, breast cancer cells can secrete factors that influence TAM polarization. Therefore, this study aimed to evaluate the crosstalk between cancer cells and macrophages in the context of TNBC. Cytokine-polarized M2 macrophage were used as control. Distinct from the classical M2 macrophage, TAMs generated from TNBC-conditioned media upregulated both M1- and M2-associated genes, and secreted both the anti-inflammatory cytokine interleukin IL-10 and the proinflammatory cytokine IL-6 and tumor necrosis factor- α. Theses TNBC-induced TAMs exert aggressive behavior of TNBC cells. Consistently, TCGA and MTABRIC analyses of human breast cancer revealed upregulation of M1- associated genes in TNBC comparing with non-TNBC. Among these M1-associated genes, CXCL10 and IL1B were revealed to be independent prognostic factors for disease progression. In conclusion, TNBC cells induce macrophage polarization with a mixture of M1 and M2 phenotypes. These cancer-induced TAMs further enhance tumor cell growth and aggressiveness.

Lung cancer metastasis is a multifaceted process that accounts for 90% of cancer deaths. According to several studies, the epithelial–mesenchymal transition (EMT) plays an essential role in lung cancer metastasis. Therefore, this study aimed to investigate the potential pharmacological effect of cycloartocarpin on the suppression of metastasis-related behaviors and EMT. An MTT assay was used to examine cell viability. Cell migration was determined using a wound healing assay. Anchorage-independent cell growth was also performed. Western blot analysis was used to identify the key signaling proteins involved in the regulation of EMT and migration. The results found that non-toxic concentrations of cycloartocarpin (10–20 μM) effectively suppressed cell migration and attenuated anchorage-independent growth in H292, A549, and H460 cells. Interestingly, these effects were consistent with the findings of Western blot analysis, which revealed that the level of phosphorylated focal adhesion kinase (p-FAK), phosphorylated ATP-dependent tyrosine kinase (p-AKT), and cell division cycle 42 (Cdc42) were significantly reduced, resulting in the inhibition of the EMT process, as evidenced by decreased N-cadherin, vimentin, and slug expression. Taken together, the results suggest that cycloartocarpin inhibits EMT by suppressing the FAK/AKT signaling pathway, which is involved in Cdc42 attenuation. Our findings demonstrated that cycloartocarpin has antimetastatic potential for further research and development in lung cancer therapy.

The Akt-mTOR signal is important for the survival and proliferation of cancer cells and has become an interesting drug target. In this study, five resveratrol derivatives were evaluated for anticancer activity and Akt/mTOR targeting activity in non-small lung cancer cell lines. The effects of resveratrol derivatives on cell proliferation were assessed by 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, nucleus staining, and colony formation assay. Furthermore, the effect of resveratrol derivatives on proliferation-related protein expression was analyzed by immunofluorescence and Western blotting. For the structure–activity relationship (SAR), results reveal that two derivatives of resveratrol which are 4,4′-(ethane-1,2-diyl) bis(2-methoxyphenol) (RD2) and the 4-(3-hydroxy-4-methoxyphenethyl)-2-methoxyphenol (RD3) had very similar structures but exerted different cytotoxicity. The IC50 of RD2 and RD3 were 108.6 ± 10.82 and more than 200 µM in the A549 cell line and 103.5 ± 6.08 and more than 200 µM in H23 cells, respectively. RD2 inhibited cell proliferation and induced apoptosis when compared with the control, while RD3 caused minimal effects. Cells treated with RD2 exhibited apoptotic nuclei in a concomitant with the reduction of cellular p-Akt and p-mTOR. RD3 had minimal effects on such proteins. According to these results, molecular docking analysis revealed a high-affinity interaction between RD2 and an Akt molecule at the ATP-binding and the allosteric sites, indicating this RD2 as a potential Akt inhibitor. This study provides useful information of resveratrol derivatives RD2 for treating lung cancer via Akt/mTOR inhibition.

The most prevalent lung cancer is non-small cell lung cancer (NSCLC). This lung cancer type often develops other organ-specific metastases that are critical burdens in the treatment process. Orchid species in the genus Vanda have shown their potential in folkloric medication of diverse diseases but not all its species have been investigated, and little is known about their anticancer activities against NSCLC. Here, we firstly profiled the specialized metabolites of Vanda bensonii and examined their capability to inhibit growth and metastasis of NSCLC using NCI-H460 cells as a study model. Four phytochemicals, including phloretic acid methyl ester (1), cymbinodin-A (2), ephemeranthoquinone B (3), and protocatechuic acid (4), were isolated from the whole plant methanolic extract of V. bensonii. The most distinguished cytotoxic effect on NCI-H460 cells was observed in the treatments with crude methanolic extract and compound 2 with the half maximal inhibitory concentrations of 40.39 μg mL−1 and 50.82 μM, respectively. At non-cytotoxic doses (10 μg mL−1 or 10 μM), only compound 1 could significantly limit NCI-H460 cell proliferation when treated for 48 h, while others excluding compound 4 showed significant reduction in cell proliferation after treating for 72 h. Compound 1 also significantly decreased the migration rate of NCI-H460 cells examined through a wound-healing assay. Additionally, the crude extract and compound 1 strongly affected survival and growth of NCI-H460 cells under anchorage-independent conditions. Our findings proved that natural products from V. bensonii could be promising candidates for the future pharmacotherapy of NSCLC.

Cellular senescence is linked with chemotherapy resistance. Based on previous studies, GRP78 is a signal transducer in senescent cells. However, the association between GRP78 and stem cell phenotype remains unknown. Cisplatin treatment was clarified to induce cellular senescence leading to stemness induction via GRP78/Akt signal transduction. H460 cells were treated with 5 μM of cisplatin for 6 days to develop senescence. The colony formation assay and cell cycle analysis were performed. SA-β-galactosidase staining indicated senescence. Western blot analysis and RT-PCR were operated. Immunoprecipitation (IP) and immunocytochemistry assays (ICC) were also performed. Colony-forming activity was completely inhibited, and 87.07% of the cell population was arrested in the G2 phase of the cell cycle. mRNA of p21 and p53 increased approximately by 15.91- and 19.32-fold, respectively. The protein level of p21 and p53 was elevated by 9.57- and 5.9-fold, respectively. In addition, the c-Myc protein level was decreased by 0.2-fold when compared with the non-treatment control. Even though, the total of GRP78 protein was downregulated after cisplatin treatment, but the MTJ1 and downstream regulator, p-Akt/Akt ratio were upregulated by approximately 3.38 and 1.44-fold, respectively. GRP78 and MTJ1 were found at the cell surface membrane. Results showed that the GRP78/MTJ1 complex and stemness markers, including CD44, CD133, Nanog, Oct4, and Sox2, were concomitantly increased in senescent cells. MTJ1 anchored GRP78, facilitating the signal transduction of stem-like phenotypes. The strategy that could interrupt the binding between these crucial proteins or inhibit the translocation of GRP78 might beuseful for cancer therapy.

Shrimp contains a fat that benefits cardiovascular function and may help in the prevention of diseases. The stress of essential cellular organelle endoplasmic reticulum (ER) is linked to endothelial dysfunction and damage. This research aimed at investigating the effect of shrimp lipid (SL) on endothelial cells in response to ER stress, as well as the underlying mechanisms. Human endothelial cells were pretreated with SL (250 and 500 μg/mL) for 24 h, and treated with 0.16 μg/mL of Thapsigargin (Tg) for 24 h. The apoptosis and necrosis were detected by Hoechst 33342/propidium iodide (PI) co-staining. Cellular signaling pathways and ER stress markers were evaluated by Western blot analysis and immunofluorescence. SL protected against ER-induced endothelial cell apoptosis. According to the results, the viability of EA.hy926 cells treated with Tg alone was 44.97 ± 1%, but SL (250 μg/mL) pretreatment increased cell viability to 77.26 ± 3.9%, and SL (500 μg/mL) increased to 72.42 ± 4.3%. SL suppressed the increase in ER stress regulator glucose-regulated protein 78 (GRP78) and attenuated the RNA-dependent protein kinase-like ER eukaryotic initiation factor-2α kinase (PERK) and inositol-requiring ER-to-nucleus signaling protein 1 (IRE1) pathways. SL could inhibit cell damage by reducing the ER-related apoptosis protein, C/EBPα-homologous protein (CHOP), induced by ER stress. Taken together, we found the protective effect and mechanism of SL in protecting ER stress-induced endothelial cell apoptosis through suppression of the ER stress pathway. The findings may support the potential use of SL as an approach with a protective effect on endothelial cells.

Autophagy is the multistep mechanism for the elimination of damaged organelles and misfolded proteins. This mechanism is preceded and may induce other program cell deaths such as apoptosis. This study unraveled the potential pharmacological effect of 24MD in inducing the autophagy of lung cancer cells. Results showed that 24MD was concomitant with autophagy induction, indicating by autophagosome staining and the induction of ATG5, ATG7 and ubiquitinated protein, p62 expression after 12-h treatment. LC3-I was strongly conversed to LC3-II, and p62 was downregulated after 24-h treatment. The apoptosis-inducing activity was found after 48-h treatment as indicated by annexin V-FITC/propidium iodide staining and the activation of caspase-3. From a mechanistic perspective, 24-h treatment of 24MD at 60 μM substantially downregulated p-mTOR. Meanwhile, p-PI3K and p-Akt were also suppressed by 24MD at concentrations of 80 and 100 μM, respectively. We further confirmed m-TOR-mediated autophagic activity by comparing the effect of 24MD with rapamycin, a potent standard mTOR1 inhibitor through Western blot and immunofluorescence assays. Although 24MD could not suppress p-mTOR as much as rapamycin, the combination of rapamycin and 24MD could increase the mTOR suppressive activity and LC3 activation. Changing the substituent groups (R groups) from dimethylphenol to ethylphenol in EMD or changing methylazanedyl to cyclohexylazanedyl in 24CD could only induce apoptosis activity but not autophagic inducing activity. We identified 24MD as a novel compound targeting autophagic cell death by affecting mTOR-mediated autophagy.

In searching for novel targeted therapeutic agents for lung cancer treatment, norcycloartocarpin from Artocarpus gomezianus was reported in this study to promisingly interacted with Akt and exerted the apoptosis induction and epithelial-to-mesenchymal transition suppression. Selective cytotoxic profile of norcycloartocarpin was evidenced with approximately 2-fold higher IC50 in normal dermal papilla cells (DPCs) compared with human lung cancer A549, H460, H23, and H292 cells. We found that norcycloartocarpin suppressed anchorage-independent growth, cell migration, invasion, filopodia formation, and decreased EMT in a dose-dependent manner at 24 h, which were correlated with reduced protein levels of N-cadherin, Vimentin, Slug, p-FAK, p-Akt, as well as Cdc42. In addition, norcycloartocarpin activated apoptosis caspase cascade associating with restoration of p53, down-regulated Bcl-2 and augmented Bax in A549 and H460 cells. Interestingly, norcycloartocarpin showed potential inhibitory role on protein kinase B (Akt) the up-stream dominant molecule controlling EMT and apoptosis. Computational molecular docking analysis further confirmed that norcycloartocarpin has the best binding affinity of -12.52 kcal/mol with Akt protein at its critical active site. As Akt has recently recognized as an attractive molecular target for therapeutic approaches, these findings support its use as a plant-derived anticancer agent in cancer therapy.

Targeted therapies that selectively inhibit certain molecules in cancer cells have been considered promising for cancer treatment. In lung cancer, evidence has suggested that mesenchymal-epithelial transition factor (c-Met) oncoprotein drives cancer progression through its signaling transduction pathway. In this paper, we report the downregulation of c-Met by artonin F, a flavonoid isolated from Artocarpus gomezianus. Artonin F was found to be dominantly toxic to lung cancer cells by mediating apoptosis. With regard to its mechanism of action, artonin F downregulated c-Met expression, consequently suppressed the phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin signaling, increased Bax expression, decreased Bcl-2 expression, and activated caspase-3. The depletion of c-Met was mediated by ubiquitin-proteasomal degradation following co-treatment with artonin F, with the proteasome inhibitor MG132 reversing its c-Met-targeting effect. The immunoprecipitation analysis revealed that artonin F significantly promoted the formation of the c-Met–ubiquitin complex. Given that ubiquitin-specific protease 8 (USP8) prevents c-Met degradation by deubiquitination, we performed a preliminary in silico molecular docking and observed that artonin F blocked the catalytic site of USP8. In addition, artonin F interacted with the catalytic residues of palmitoylating enzymes. By acting as a competitive inhibitor, artonin F could reduce the degree of palmitoylation of c-Met, which affected its stability and activity. In conclusion, c-Met is critical for cancer cell survival and the failure of chemotherapeutic regimens. This novel information on the c-Met downregulating effect of artonin F will be beneficial for the development of efficient anticancer strategies or targeted therapies.

From the aerial parts of Cymbidium ensifolium, three new dihydrophenanthrene derivatives, namely, cymensifins A, B, and C (1–3) were isolated, together with two known compounds, cypripedin (4) and gigantol (5). Their structures were elucidated by analysis of their spectroscopic data. The anticancer potential against various types of human cancer cells, including lung, breast, and colon cancers as well as toxicity to normal dermal papilla cells were assessed via cell viability and nuclear staining assays. Despite lower cytotoxicity in lung cancer H460 cells, the higher % apoptosis and lower % cell viability were presented in breast cancer MCF7 and colon cancer CaCo2 cells treated with 50 µM cymensifin A (1) for 24 h compared with the treatment of 50 µM cisplatin, an available chemotherapeutic drug. Intriguingly, the half-maximum inhibitory concentration (IC50) of cymensifin A in dermal papilla cells at >200 µM suggested its selective anticancer activity. The obtained information supports the further development of a dihydrophenanthrene derivative from C. ensifolium as an effective chemotherapy with a high safety profile for the treatment of various cancers.

Cancer stem cells (CSCs) drive aggressiveness and metastasis by utilizing stem cell-related signals. In this study, 5-O-(N-Boc-l-alanine)-renieramycin T (OBA-RT) was demonstrated to suppress CSC signals and induce apoptosis. OBA-RT exerted cytotoxic effects with a half-maximal inhibitory concentration of approximately 7 µM and mediated apoptosis as detected by annexin V/propidium iodide using flow cytometry and nuclear staining assays. Mechanistically, OBA-RT exerted dual roles, activating p53-dependent apoptosis and concomitantly suppressing CSC signals. A p53-dependent pathway was indicated by the induction of p53 and the depletion of anti-apoptotic Myeloid leukemia 1 (Mcl-1) and B-cell lymphoma 2 (Bcl-2) proteins. Cleaved poly (ADP-ribose) polymerase (Cleaved-PARP) was detected in OBA-RT-treated cells. Interestingly, OBA-RT exerted strong CSC-suppressing activity, reducing the ability to form tumor spheroids. In addition, OBA-RT could induce apoptosis in CSC-rich populations and tumor spheroid collapse. CSC markers, including prominin-1 (CD133), Octamer-binding transcription factor 4 (Oct4), and Nanog Homeobox (Nanog), were notably decreased after OBA-RT treatment. Upstream CSCs regulating active Akt and c-Myc were significantly decreased; indicating that Akt may be a potential target of action. Computational molecular modeling revealed a high-affinity interaction between OBA-RT and an Akt molecule. This study has revealed a novel CSC inhibitory effect of OBA-RT via Akt inhibition, which may improve cancer therapy.

Abstract Background During metastasis, cancer cells require anokis resistant mechanism to survive until reach the distant secondary tissues. As anoikis sensitization may benefit for cancer therapy, this study demonstrated the potential of avicequinone B, a natural furanonaphthoquinone found in mangrove tree (Avicenniaceae) to sensitize anoikis in human lung cancer cells. Methods Anoikis inducing effect was investigated in human lung cancer H460, H292 and H23 cells that were cultured in ultra-low attachment plate with non-cytotoxic concentrations of avicequinone B. Viability of detached cells was evaluated by XTT assay at 0–24 h of incubation time. Soft agar assay was performed to investigate the inhibitory effect of avicequinone B on anchorage-independent growth. The alteration of anoikis regulating molecules including survival and apoptosis proteins were elucidated by western blot analysis. Results Avicequinone B at 4 μM significantly induced anoikis and inhibited proliferation under detachment condition in various human lung cancer cells. The reduction of anti-apoptotic proteins including anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) and myeloid cell leukemia 1 (Mcl-1) associating with the diminution of integrin/focal adhesion kinase (FAK)/Proto-oncogene tyrosine-protein kinase (Src) signals were detected in avicequinone B-treated cells. Conclusions Avicequinone B sensitized anoikis in human lung cancer cells through down-regulation of anti-apoptosis proteins and integrin-mediated survival signaling.

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 β3. 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.

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.

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-GSK3β (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.

Metal nanomaterials can enhance the efficacy of current cancer therapies. Here, we show that Ti0.8O2 nanosheets cause cytotoxicity in several lung cancer cells but not in normal cells. The nanosheet-treated cells showed certain apoptosis characteristics. Protein analysis further indicated the activation of the p53-dependent death mechanism. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analyses revealed the cellular uptake of the nanosheets and the induction of cell morphological change. The nanosheets also exhibited a substantial apoptosis effect on drug-resistant metastatic primary lung cancer cells, and it was found that the potency of the nanosheets was dramatically higher than standard drugs. Ti0.8O2 nanosheets induce apoptosis through a molecular mechanism involving peroxynitrite (ONOO−) generation. As peroxynitrite is known to be a potent inducer of S-nitrosylation, we further found that the nanosheets mediated the S-nitrosylation of p53 at C182, resulting in higher protein-protein complex stability, and this was likely to induce the surrounding residues, located in the interface region, to bind more strongly to each other. Molecular dynamics analysis revealed that S-nitrosylation stabilized the p53 dimer with a ΔGbindresidue of S-nitrosylation of the p53 protein, emphasizing the mechanism of action of nanomaterials for cancer therapy.

Abstract Background The conversion of the epithelial phenotype of cancer cells into cells with a mesenchymal phenotype-so-called epithelial–mesenchymal transition (EMT)-has been shown to enhance the capacity of the cells to disseminate throughout the body. EMT is therefore becoming a potential target for anti-cancer drug discovery. Here, we showed that phoyunnanin E, a compound isolated from Dendrobium venustum, possesses anti-migration activity and addressed its mechanism of action. Methods The cytotoxic and proliferative effects of phoyunnanin E on human non-small cell lung cancer-derived H460, H292, and A549 cells and human keratinocyte HaCaT cells were investigated by MTT assay. The effect of phoyunnanin E on EMT was evaluated by determining the colony formation and EMT markers. The migration and invasion of H460, H292, A549 and HaCaT cells was evaluated by wound healing assay and transwell invasion assay, respectively. EMT markers, integrins and migration-associated proteins were examined by western blot analysis. Results Phoyunnanin E at the concentrations of 5 and 10 μM, which are non-toxic to H460, H292, A549 and HaCaT cells showed good potential to inhibit the migratory activity of three types of human lung cancer cells. The anti-migration effect of phoyunnanin E was shown to relate to the suppressed EMT phenotypes, including growth in anchorage-independent condition, cell motility, and EMT-specific protein markers (N-cadherin, vimentin, slug, and snail). In addition to EMT suppression, we found that phoyunnanin E treatment with 5 and 10 μM could decrease the cellular level of integrin αv and integrin β3, these integrins are frequently up-regulated in highly metastatic tumor cells. We further characterized the regulatory proteins in cell migration and found that the cells treated with phoyunnanin E exhibited a significantly lower level of phosphorylated focal adhesion kinase (p-FAK) and phosphorylated ATP-dependent tyrosine kinase (p-AKT), and their downstream effectors (including Ras-related C3 botulinum (Rac-GTP); Cell division cycle 42 (Cdc42); and Ras homolog gene family, member A (Rho-GTP)) in comparison to those of the non-treated control. Conclusions We have determined for the first time that phoyunnanin E could inhibit the motility of lung cancer cells via the suppression of EMT and metastasis-related integrins. This new information could support further development of this compound for anti-metastasis approaches.

Context: Lentinus squarrosulus Mont. (Polyporaceae) is an interesting source of diverse bioactive compounds. Objective: This is the first study of the anticancer activity and underlying mechanism of peptides extracted from Lentinus squarrosuls. Materials and methods: Peptides were isolated from the aqueous extract of L. squarrosulus by employing solid ammonium sulphate precipitation. They were further purified by ion-exchange chromatography on diethylaminoethanol (DEAE)-cellulose and gel filtration chromatography on Sephadex G25. Anticancer activity was investigated in human lung cancer H460, H292 and H23 cells cultured with 0–40 μg/mL of peptide extracts for 24 h. Cell viability and mode of cell death were evaluated by MTT and nuclear staining assay, respectively. Western blotting was used to investigate the alteration of apoptosis-regulating proteins in lung cancer cells treated with peptide extracts (0–20 μg/mL) for 24 h. Results: The cytotoxicity of partially-purified peptide extracts from L. squarrosulus was indicated with IC50 of ∼26.84 ± 2.84, 2.80 ± 2.14 and 18.84 ± 0.30 μg/mL in lung cancer H460, H292 and H23 cells, respectively. The extracts at 20 μg/mL induced apoptosis through the reduction of anti-apoptotic Bcl-2 protein (∼0.5-fold reduction) and up-regulation of BAX (∼4.5-fold induction), a pro-apoptotic protein. Furthermore, L. squarrosulus peptide extracts (20 μg/mL) also decreased the cellular level of death receptor inhibitor c-FLIP (∼0.6-fold reduction). Conclusions and discussion: This study provides the novel anticancer activity and mechanism of L. squarrosulus peptide extracts, which encourage further investigation and development of the extracts for anticancer use.

It has been recognized that cancer stem-like cells (CSCs) in tumor tissue crucially contribute to therapeutic failure, resulting in a high mortality rate in lung cancer patients. Due to their stem-like features of self-renewal and tumor formation, CSCs can lead to drug resistance and tumor recurrence. Herein, the suppressive effect of jorunnamycin A, a bistetrahydroisoquinolinequinone isolated from Thai blue sponge Xestospongia sp., on cancer spheroid initiation and self-renewal in the CSCs of human lung cancer cells is revealed. The depletion of stemness transcription factors, including Nanog, Oct-4, and Sox2 in the lung CSC-enriched population treated with jorunnamycin A (0.5 μM), resulted from the activation of GSK-3β and the consequent downregulation of β-catenin. Interestingly, pretreatment with jorunnamycin A at 0.5 μM for 24 h considerably sensitized lung CSCs to cisplatin-induced apoptosis, as evidenced by upregulated p53 and decreased Bcl-2 in jorunnamycin A-pretreated CSC-enriched spheroids. Moreover, the combination treatment of jorunnamycin A (0.5 μM) and cisplatin (25 μM) also diminished CD133-overexpresssing cells presented in CSC-enriched spheroids. Thus, evidence on the regulatory functions of jorunnamycin A may facilitate the development of this marine-derived compound as a novel chemotherapy agent that targets CSCs in lung cancer treatment.

Focal adhesion kinase (FAK) controls several cancer aggressive potentials of cell movement and dissemination. As epithelial–mesenchymal transition (EMT) and the migratory-associated integrins, known influencers of metastasis, have been found to be linked with FAK activity, this study unraveled the potential pharmacological effect of artocarpin in targeting FAK resulting in the suppression of EMT and migratory behaviors of lung cancer cells. Treatment with artocarpin was applied at concentrations of 0–10 μM, and the results showed non-cytotoxicity in lung cancer cell lines (A549 and H460), normal lung (BEAS-2B) cells and primary metastatic lung cancer cells (ELC12, ELC16, and ELC20). We also found that artocarpin (0–10 µM) had no effect on cell viability, proliferation, and migration in BEAS-2B cells. For metastasis-related approaches, artocarpin significantly inhibited cell migration, invasion, and filopodia formation. Artocarpin also dramatically suppressed anchorage-independent growth, cancer stem cell (CSC) spheroid formation, and viability of CSC-rich spheroids. For molecular targets of artocarpin action, computational molecular docking revealed that artocarpin had the best binding affinity of −8.0 kcal/mol with FAK protein. Consistently, FAK-downstream proteins, namely active Akt (phosphorylated Akt), active mTOR (phosphorylated mTOR), and Cdc42, and EMT marker and transcription factor (N-cadherin, Vimentin, and Slug), were found to be significantly depleted in response to artocarpin treatment. Furthermore, we found the decrease of Caveolin-1 (Cav-1) accompanied by the reduction of integrin-αν and integrin-β3. Taken together, these findings support the anti-metastasis potentials of the compound to be further developed for cancer therapy.