Quantification of putative ovarian cancer serum protein biomarkers using a multiplexed targeted mass spectrometry assay.

BACKGROUND: Ovarian cancer is the most lethal gynecologic malignancy in women, and high-grade serous ovarian cancer (HGSOC) is the most common subtype. Currently, no clinical test has been approved by the FDA to screen the general population for ovarian cancer. This underscores the critical need for the development of a robust methodology combined with novel technology to detect diagnostic biomarkers for HGSOC in the sera of women. Targeted mass spectrometry (MS) can be used to identify and quantify specific peptides/proteins in complex biological samples with high accuracy, sensitivity, and reproducibility. In this study, we sought to develop and conduct analytical validation of a multiplexed Tier 2 targeted MS parallel reaction monitoring (PRM) assay for the relative quantification of 23 putative ovarian cancer protein biomarkers in sera. METHODS: To develop a PRM method for our target peptides in sera, we followed nationally recognized consensus guidelines for validating fit-for-purpose Tier 2 targeted MS assays. The endogenous target peptide concentrations were calculated using the calibration curves in serum for each target peptide. Receiver operating characteristic (ROC) curves were analyzed to evaluate the diagnostic performance of the biomarker candidates. RESULTS: We describe an effort to develop and analytically validate a multiplexed Tier 2 targeted PRM MS assay to quantify candidate ovarian cancer protein biomarkers in sera. Among the 64 peptides corresponding to 23 proteins in our PRM assay, 24 peptides corresponding to 16 proteins passed the assay validation acceptability criteria. A total of 6 of these peptides from insulin-like growth factor-binding protein 2 (IBP2), sex hormone-binding globulin (SHBG), and TIMP metalloproteinase inhibitor 1 (TIMP1) were quantified in sera from a cohort of 69 patients with early-stage HGSOC, late-stage HGSOC, benign ovarian conditions, and healthy (non-cancer) controls. Confirming the results from previously published studies using orthogonal analytical approaches, IBP2 was identified as a diagnostic biomarker candidate based on its significantly increased abundance in the late-stage HGSOC patient sera compared to the healthy controls and patients with benign ovarian conditions. CONCLUSIONS: A multiplexed targeted PRM MS assay was applied to detect candidate diagnostic biomarkers in HGSOC sera. To evaluate the clinical utility of the IBP2 PRM assay for HGSOC detection, further studies need to be performed using a larger patient cohort.

Fyn-mediated phosphorylation of Menin disrupts telomere maintenance in stem cells.

Telomeres protect chromosome ends and determine the replication potential of dividing cells. The canonical telomere sequence TTAGGG is synthesized by telomerase holoenzyme, which maintains telomere length in proliferative stem cells. Although the core components of telomerase are well-defined, mechanisms of telomerase regulation are still under investigation. We report a novel role for the Src family kinase Fyn, which disrupts telomere maintenance in stem cells by phosphorylating the scaffold protein Menin. We found that Fyn knockdown prevented telomere erosion in human and mouse stem cells, validating the results with four telomere measurement techniques. We show that Fyn phosphorylates Menin at tyrosine 603 (Y603), which increases Menin's SUMO1 modification, C-terminal stability, and importantly, its association with the telomerase RNA component (TR). Using mass spectrometry, immunoprecipitation, and immunofluorescence experiments we found that SUMO1-Menin decreases TR's association with telomerase subunit Dyskerin, suggesting that Fyn's phosphorylation of Menin induces telomerase subunit mislocalization and may compromise telomerase function at telomeres. Importantly, we find that Fyn inhibition reduces accelerated telomere shortening in human iPSCs harboring mutations for dyskeratosis congenita.

Akt-mediated Ephexin1-Ras interaction promotes oncogenic Ras signaling and colorectal and lung cancer cell proliferation.

ABSTRCT: Ephexin1 was reported to be highly upregulated by oncogenic Ras, but the functional consequences of this remain poorly understood. Here, we show that Ephexin1 is highly expressed in colorectal cancer (CRC) and lung cancer (LC) patient tissues. Knockdown of Ephexin1 markedly inhibited the cell growth of CRC and LC cells with oncogenic Ras mutations. Ephexin1 contributes to the positive regulation of Ras-mediated downstream target genes and promotes Ras-induced skin tumorigenesis. Mechanically, Akt phosphorylates Ephexin1 at Ser16 and Ser18 (pSer16/18) and pSer16/18 Ephexin1 then interacts with oncogenic K-Ras to promote downstream MAPK signaling, facilitating tumorigenesis. Furthermore, pSer16/18 Ephexin1 is associated with both an increased tumor grade and metastatic cases of CRC and LC, and those that highly express pSer16/18 exhibit poor overall survival rates. These data indicate that Ephexin1 plays a critical role in the Ras-mediated CRC and LC and pSer16/18 Ephexin1 might be an effective therapeutic target for CRC and LC.

Quantitative Mass Spectrometry-Based Proteomics for Biomarker Development in Ovarian Cancer.

Ovarian cancer is the most lethal gynecologic malignancy among women. Approximately 70-80% of patients with advanced ovarian cancer experience relapse within five years and develop platinum-resistance. The short life expectancy of patients with platinum-resistant or platinum-refractory disease underscores the need to develop new and more effective treatment strategies. Early detection is a critical step in mitigating the risk of disease progression from early to an advanced stage disease, and protein biomarkers have an integral role in this process. The best biological diagnostic tool for ovarian cancer will likely be a combination of biomarkers. Targeted proteomics methods, including mass spectrometry-based approaches, have emerged as robust methods that can address the chasm between initial biomarker discovery and the successful verification and validation of these biomarkers enabling their clinical translation due to the robust sensitivity, specificity, and reproducibility of these versatile methods. In this review, we provide background information on the fundamental principles of biomarkers and the need for improved treatment strategies in ovarian cancer. We also provide insight into the ways in which mass spectrometry-based targeted proteomics approaches can provide greatly needed solutions to many of the challenges related to ovarian cancer biomarker development.

GSK3ß-Mediated Expression of CUG-Translated WT1 Is Critical for Tumor Progression.

The Wilms' tumor 1 (WT1) gene is well known as a chameleon gene. It plays a role as a tumor suppressor in Wilms' tumor but also acts as an oncogene in other cancers. Previously, our group reported that a canonical AUG starting site for the WT1 protein (augWT1) acts as a tumor suppressor, whereas a CUG starting site for the WT1 protein (cugWT1) functions as an oncogene. In this study, we report an oncogenic role of cugWT1 in the AOM/DSS-induced colon cancer mouse model and in a urethane-induced lung cancer model in mice lacking cugWT1. Development of chemically-induced tumors was significantly depressed in cugWT1-deficient mice. Moreover, glycogen synthase kinase 3ß promoted phosphorylation of cugWT1 at S64, resulting in ubiquitination and degradation of the cugWT1 associated with the F-box-/- WD repeat-containing protein 8. Overall, our findings suggest that inhibition of cugWT1 expression provides a potential candidate target for therapy. SIGNIFICANCE: These findings demonstrate that CUG-translated WT1 plays an oncogenic role in vivo, and GSK3ß-mediated phosphorylation of cugWT1 induces its ubiquitination and degradation in concert with FBXW8.

Estrogen-related receptor alpha directly binds to p53 and cooperatively controls colon cancer growth through the regulation of mitochondrial biogenesis and function.

BACKGROUND: Of the genes that control mitochondrial biogenesis and function, ERRα emerges as a druggable metabolic target to be exploited for cancer therapy. Of the genes mutated in cancer, TP53 remains the most elusive to target. A clear understanding of how mitochondrial druggable targets can be accessed to exploit the underlying mechanism(s) explaining how p53-deficient tumors promote cell survival remains elusive. METHODS: We performed protein-protein interaction studies to demonstrate that ERRα binds to p53. Moreover, we used gene silencing and pharmacological approaches in tandem with quantitative proteomics analysis by SWATH-MS to investigate the role of the ERRα/p53 complex in mitochondrial biogenesis and function in colon cancer. Finally, we designed in vitro and in vivo studies to investigate the possibility of targeting colon cancers that exhibit defects in p53. RESULTS: Here, we are the first to identify a direct protein-protein interaction between the ligand-binding domain (LBD) of ERRα and the C-terminal domain (CTD) of p53. ERRα binds to p53 regardless of p53 mutational status. Furthermore, we show that the ERRα and p53 complex cooperatively control mitochondrial biogenesis and function. Targeting ERRα creates mitochondrial metabolic stresses, such as production of reactive oxygen species (ROS) and mitochondrial membrane permeabilization (MMP), leading to a greater cytotoxic effect that is dependent on the presence of p53. Pharmacological inhibition of ERRα impairs the growth of p53-deficient cells and of p53 mutant patient-derived colon xenografts (PDX). CONCLUSIONS: Therefore, our data suggest that by using the status of the p53 protein as a selection criterion, the ERRα/p53 transcriptional axis can be exploited as a metabolic vulnerability.

ARC Is a Critical Protector against Inflammatory Bowel Disease (IBD) and IBD-Associated Colorectal Tumorigenesis.

The key functional molecules involved in inflammatory bowel disease (IBD) and IBD-induced colorectal tumorigenesis remain unclear. In this study, we found that the apoptosis repressor with caspase recruitment domain (ARC) protein plays critical roles in IBD. ARC-deficient mice exhibited substantially higher susceptibility to dextran sulfate sodium (DSS)-induced IBD compared with wild-type mice. The inflammatory burden induced in ARC-deficient conditions was inversely correlated with CCL5 and CXCL5 levels in immune cells, especially CD4-positive T cells. Pathologically, ARC expression in immune cells was significantly decreased in clinical biopsy specimens from patients with IBD compared with normal subjects. In addition, ARC levels inversely correlated with CCL5 and CXCL5 levels in human biopsy specimens. ARC interacted with TNF receptor associated factor (TRAF) 6, regulating ubiquitination of TRAF6, which was associated with NF-κB signaling. Importantly, we identified a novel ubiquitination site at lysine 461, which was critical in the function of ARC in IBD. ARC played a critical role in IBD and IBD-associated colon cancer in a bone marrow transplantation model and azoxymethane/DSS-induced colitis cancer mouse models. Overall, these findings reveal that ARC is critically involved in the maintenance of intestinal homeostasis and protection against IBD through its ubiquitination of TRAF6 and subsequent modulation of NF-κB activation in T cells. SIGNIFICANCE: This study uncovers a crucial role of ARC in the immune system and IBD, giving rise to a novel strategy for IBD and IBD-associated colon cancer therapy.

Fluvastatin Inhibits HMG-CoA Reductase and Prevents Non-Small Cell Lung Carcinogenesis.

Lung cancer is the leading cause of cancer-related death worldwide. However, promising agents for lung cancer prevention are still very limited. Identification of preventive targets and novel effective preventive agents is urgently needed for clinical applications. In this study, we found that fluvastatin targeted 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGCR), which a rate-limiting enzyme in the mevalonate pathway, and inhibited non-small cell lung cancer (NSCLC) tumorigenesis. Initially, we demonstrated that HMGCR is overexpressed in human lung adenocarcinoma tissues compared with normal tissues. Knockdown of HMGCR in NSCLC cells attenuated growth and induced apoptosis in vitro and in vivo Furthermore, we found that fluvastatin, an inhibitor of HMGCR, suppressed NSCLC cell growth and induced apoptosis. Intriguingly, fluvastastin functions by inhibiting the HMGCR-driven Braf/MEK/ERK1/2 and Akt signaling pathways. Notably, fluvastatin attenuated tumor growth in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis and in a patient-derived xenograft lung tumor model. Overall, our findings suggest that fluvastatin might be promising chemopreventive or potential therapeutic drug against NSCLC tumorigenesis, providing hope for rapid clinical translation.

A proteomic analysis of differentiating dopamine neurons derived from human embryonic stem cells.

Human embryonic stem cells (hESC) are being exploited for potential use in cell transplantation due to their capacity for self-renewal and pluripotency. Dopamine (DA) neurons derived from hESC represent a promising source of cell replacement therapy for Parkinson's disease (PD). While gene expression on the transcriptome level has been extensively studied, limited information is available for the proteome-level changes associated with DA neuron differentiation. Here we analyzed the proteome of differentiating DA neurons to search for the potential biomarkers to assess the efficiency of differentiation. Although the proteome profile of DA neurons did not exhibit significant changes, a number of cytoskeletal proteins including nuclear lamin, tropomyosin 1, and myosin light chain 1 were specifically up-regulated during differentiation. Expression analysis of the respective genes was also consistent with the proteome results. In addition, these differentially expressed proteins form protein interaction network with several PD-related proteins suggesting that they may play roles in PD pathogenesis as well as the maturation of DA neurons.

Dual-specificity phosphatase 18 modulates the SUMOylation and aggregation of Ataxin-1.

We previously reported that SUMOylation promotes the aggregation of ataxin-1 and JNK is involved in the process. Here we show that dual-specificity phosphatase 18 (DUSP18), a member of protein tyrosine phosphatases, exerts the opposite effects on ataxin-1. DUSP18 associated with ataxin-1 and suppressed JNK activated by ataxin-1. Interestingly DUSP18, but not the other DUSPs interacting with ataxin-1, caused the mobility shift of ataxin-1. De-phosphorylation by DUSP18 was initially suspected as a cause for such an effect; however, the phosphorylation of ataxin-1 was unchanged. Instead DUSP18 inhibited SUMOylation and reduced ataxin-1 aggregation. The catalytic mutant of DUSP18 failed to reduce the SUMOylation and aggregation of ataxin-1 indicating that the phosphatase activity is indispensable for the effects. Moreover, DUSP18 disrupted the co-localization of ataxin-1 with the PML component Sp100. These results together implicate that JNK and DUSP18 reciprocally modulate the SUMOylation, which plays a regulatory role in the aggregation of ataxin-1.

RSK2 is required for TRAF6 phosphorylation-mediated colon inflammation.

Inflammation is a complex biological host reaction to tissue damage, infection and trauma. Extensive study of the inflammatory response has led to the identification of several protein kinases that are essential for signaling and could be potential therapeutic targets. The RSK family of kinases has multiple cellular functions. In our study, we found that RSK2 is a mediator for inflammation signaling and interacts with TRAF6. In vitro kinase assay results indicated that RSK2 strongly phosphorylates TRAF6 at serines 46, 47 and 48. Ectopic overexpression of TRAF6 or knocking down RSK2 expression confirmed that RSK2 is a positive regulator of TRAF6 K63 ubiquitination. TRAF6 is also required for RSK2 ubiquitination. TRAF6 bridges the TNF receptor superfamily and intracellular signaling for the induction of proinflammatory cytokines. We developed a colon inflammation model using RSK2 wild type (WT) and knockout (KO) mice. As expected, F4/80 and CD3 infiltration were significantly upregulated in WT mice compared to RSK2 KO mice. Furthermore, inflammation signaling, including Ikkα/ß, p38 and JNKs, was dramatically upregulated in WT mice. Colon tissue immunoprecipitation results further confirmed that TRAF6 K63 ubiquitination was lower in RSK2 KO mice. Overall, these results indicate that phosphorylation of TRAF6 (S46, 47, 48) by RSK2 is required for TRAF6 K63 ubiquitination and inflammation signaling.

Serine 347 Phosphorylation by JNKs Negatively Regulates OCT4 Protein Stability in Mouse Embryonic Stem Cells.

The POU transcription factor OCT4 is critical for maintaining the undifferentiated state of embryonic stem cells (ESCs) and generating induced pluripotent stem cells (iPSCs), but its precise mechanisms of action remain poorly understood. Here, we investigated the role of OCT4 phosphorylation in the biological functions of ESCs. We observed that c-Jun N-terminal kinases (JNKs) directly interacted with and phosphorylated OCT4 at serine 347, which inhibited the transcriptional activity of OCT4. Moreover, phosphorylation of OCT4 induced binding of FBXW8, which reduced OCT4 protein stability and enhanced its proteasomal degradation. We also found that the mutant OCT4 (S347A) might delay the differentiation process of mouse ESCs and enhance the efficiency of generating iPSCs. These results demonstrated that OCT4 phosphorylation on serine 347 by JNKs plays an important role in its stability, transcriptional activities, and self-renewal of mouse ESCs.

RSK2 phosphorylates T-bet to attenuate colon cancer metastasis and growth.

Metastasis is a major cause of cancer-related deaths. Approximately 80% of patients with colorectal cancer develop liver metastasis and 20% develop lung metastasis. We found that at different stages of colon cancer, IFNγ secretion from peripheral blood mononuclear cells was decreased compared with healthy controls. The ribosomal S6 kinase (RSK) family of kinases has multiple cellular functions, and we examined their roles in this observed IFNγ decrease. Flow cytometry analysis of wild-type (WT) and RSK2 knockout (KO) mice revealed significantly lower levels of IFNγ in the RSK2 KO mice compared with the WT mice. Since IFNγ is a component of immunity, which contributes to protection against metastatic carcinomas, we conducted a colon cancer liver metastasis experiment. We found significantly greater metastasis in RSK2 KO mice compared with WT mice. Transcription factor T-bet can directly activate Ifnγ gene transcription. In vitro kinase assay results showed that RSK2 phosphorylated T-bet at serines 498 and 502. We show that phosphorylation of T-bet by RSK2 is required for IFNγ expression, because knockdown of RSK2 expression or overexpression of mutant T-bet reduces IFNγ mRNA expression. To verify the function of the phosphorylation sites, we overexpressed a constitutively active mutant T-bet (S498E/S502E) in bone marrow. Mutant T-bet restored the IFNγ mRNA levels and dramatically reduced the metastasis rate in these mice. Overall, these results indicate that phosphorylation of T-bet is required for the inhibition of colon cancer metastasis and growth through a positive regulation of RSK2/T-bet/IFNγ signaling.

A Small Molecule Inhibitor of the ß-Catenin-TCF4 Interaction Suppresses Colorectal Cancer Growth In Vitro and In Vivo.

Colorectal cancer is associated with aberrant activation of the Wnt pathway. ß-Catenin plays essential roles in the Wnt pathway by interacting with T-cell factor 4 (TCF4) to transcribe oncogenes. We synthesized a small molecule, referred to as HI-B1, and evaluated signaling changes and biological consequences induced by the compound. HI-B1 inhibited ß-catenin/TCF4 luciferase activity and preferentially caused apoptosis of cancer cells in which the survival is dependent on ß-catenin. The formation of the ß-catenin/TCF4 complex was disrupted by HI-B1 due to the direct interaction of HI-B1 with ß-catenin. Colon cancer patient-derived xenograft (PDX) studies showed that a tumor with higher levels of ß-catenin expression was more sensitive to HI-B1 treatment, compared to a tumor with lower expression levels of ß-catenin. The different sensitivities of PDX tumors to HI-B1 were dependent on the ß-catenin expression level and potentially could be further exploited for biomarker development and therapeutic applications against colon cancer.

The CUG-translated WT1, not AUG-WT1, is an oncogene.

The Wilms' tumor 1 (WT1) gene is believed to act as a canonical tumor suppressor. However, it has also been reported to function as an oncogene. Germline WT1 deletion is associated with Wilms' tumor, and exogenous WT1 cDNA introduction into cells induces the transcriptional suppression of its oncogenic target genes. In contrast, high WT1 expression is associated with poor prognosis in patients with various cancers. Why WT1 acts as a tumor suppressor under certain conditions but as an oncogene under other conditions is unknown. Here, we report that CUG initiation site for WT1 protein synthesis (CUG)-translated WT1 (cugWT1), an N-terminally extended form of canonical AUG initiation site for WT1 protein synthesis (AUG)-translated WT1 (augWT1), was overexpressed in most cancer cell lines and cancer tissues and functioned as an oncogene, whereas the classical augWT1 acted as a tumor suppressor as reported previously and inhibited the function of cugWT1. Translation of cugWT1 is initiated from a CUG codon upstream and in-frame with the coding region of augWT1. cugWT1 induced cell transformation and increased the gene expression of c-myc, bcl-2 and egfr, whereas overexpression of augWT1 repressed colony formation of cancer cells and inhibited the expression of the same target genes by recruiting histone deacetylase 1 (HDAC1). In addition, we found that protein kinase B (AKT)-phosphorylated cugWT1 on Ser62 and protected cugWT1 from proteasomal degradation induced by the F-box/WD repeat-containing protein 8 (FBXW8). These results provide an important breakthrough in the field of cancer biology and contribute significantly to the resolution of the chameleon function of WT1.

ADA-07 Suppresses Solar Ultraviolet-Induced Skin Carcinogenesis by Directly Inhibiting TOPK.

Cumulative exposure to solar ultraviolet (SUV) irradiation is regarded as the major etiologic factor in the development of skin cancer. The activation of the MAPK cascades occurs rapidly and is vital in the regulation of SUV-induced cellular responses. The T-LAK cell-originated protein kinase (TOPK), an upstream activator of MAPKs, is heavily involved in inflammation, DNA damage, and tumor development. However, the chemopreventive and therapeutic effects of specific TOPK inhibitors in SUV-induced skin cancer have not yet been elucidated. In the current study, ADA-07, a novel TOPK inhibitor, was synthesized and characterized. Pull-down assay results, ATP competition, and in vitro kinase assay data revealed that ADA-07 interacted with TOPK at the ATP-binding pocket and inhibited its kinase activity. Western blot analysis showed that ADA-07 suppressed SUV-induced phosphorylation of ERK1/2, p38, and JNKs and subsequently inhibited AP-1 activity. Importantly, topical treatment with ADA-07 dramatically attenuated tumor incidence, multiplicity, and volume in SKH-1 hairless mice exposed to chronic SUV. Our findings suggest that ADA-07 is a promising chemopreventive or potential therapeutic agent against SUV-induced skin carcinogenesis that acts by specifically targeting TOPK. Mol Cancer Ther; 16(9); 1843-54. ©2017 AACR.

TRAF1 Is Critical for DMBA/Solar UVR-Induced Skin Carcinogenesis.

TRAF1 is a member of the TRAF protein family, which regulates the canonical and noncanonical NF-κB signaling cascades. Although aberrant TRAF1 expression in tumors has been reported, the role of TRAF1 remains elusive. Here, we report that TRAF1 is required for solar UV-induced skin carcinogenesis. Immunohistochemical analysis showed that TRAF1 expression is up-regulated in human actinic keratosis and squamous cell carcinoma. In vivo studies indicated that TRAF1 expression levels in mouse skin are induced by short-term solar UV irradiation, and a long-term skin carcinogenesis study showed that deletion of TRAF1 in mice results in a significant inhibition of skin tumor formation. Moreover, we show that TRAF1 is required for solar UV-induced extracellular signal-regulated kinase-5 (ERK5) phosphorylation and the expression of AP-1 family members (c-Fos/c-Jun). Mechanistic studies showed that TRAF1 expression enhances the ubiquitination of ERK5 on lysine 184, which is necessary for its kinase activity and AP-1 activation. Overall, our results suggest that TRAF1 mediates ERK5 activity by regulating the upstream effectors of ERK5 and also by modulating its ubiquitination status. Targeting TRAF1 function might lead to strategies for preventing and treating skin cancer.

244-MPT overcomes gefitinib resistance in non-small cell lung cancer cells.

The epidermal growth factor receptor (EGFR) is known to play a critical role in non-small cell lung cancer(NSCLC). Several EGFR tyrosine kinase inhibitors(TKIs), such as gefitinib, have been used as effective clinical therapies for patients with NSCLC. Unfortunately, acquired resistance to gefitinib commonly occurs after 6-12 months of treatment. The resistance is associated with the appearance of the L858R/T790M double mutation of the EGFR. In our present study, we discovered a compound,referred to as 244-MPT, which could suppress either gefitinib-sensitive or -resistant lung cancer cell growth and colony formation, and also suppressed the kinase activity of both wildtype and double mutant (L858R/T790M) EGFR. The underlying mechanism reveals that 244-MPT could interact with either the wildtype or double-mutant EGFR in an ATP-competitive manner and inhibit activity. Treatment with 244-MPT could substantially reduce the phosphorylation of EGFR and its downstream signaling pathways, including Akt and ERK1/2 in gefitinib-sensitive and -resistant cell lines. It was equally effective in suppressing EGFR phosphorylation and downstream signaling in NL20 cells transfected with wildtype, single-mutant (L858R) or mutant (L858R/T790M) EGFR. 244-MPT could also induce apoptosis in a gefitinib-resistant cell line and strongly suppress gefitinib-resistant NSCLC tumor growth in a xenograft mouse model. In addition, 244-MPT could effectively reduce the size of tumors in a gefitinib-resistant NSCLC patient-derived xenograft (PDX) SCID mouse model. Overall, 244-MPT could overcome gefitinib-resistance by directly targeting the EGFR.

Multifunctional effects of honokiol as an anti-inflammatory and anti-cancer drug in human oral squamous cancer cells and xenograft.

The aim of this study was to investigate anti-inflammatory and anti-cancer effects of honokiol (HK) in two oral squamous cancer cell carcinoma (OSCC) cell lines, HN22 and HSC4, through the regulation of inducible nitric oxide synthase (iNOS) and endoplasmic reticulum resident protein 44 (ERp44). Griess assay, zymography, and quantitative PCR were performed to study iNOS expression and subsequent nitric oxide (NO) production in OSCC cell lines. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic analysis was used to elucidate the proteins associated with ER stress and cellular cytotoxic response induced by HK. Pull-down assay and molecular modeling were performed to better understand how HK interacts with ERp44. In vitro and in vivo experiments in which ERp44 expression was knocked down were performed to better understand the effects of ERp44 on a cellular level and anti-cancer effects of HK. Expression levels of iNOS and subsequent NO secretion were reduced in OSCC cell lines treated with HK. ERp44 was significantly decreased in OSCC cell lines by HK treatment. HK directly bound to ERp44, and ERp44 knock-down significantly inhibited oral cancer cell proliferation and colony formation. Moreover, HK treatment effectively inhibited tumor growth and ERp44 levels in BALB/c nude mice bearing HN22 cell xenografts. Our findings suggest that HK inhibited inflammation and induced apoptosis by suppressing both iNOS/NO and ERp44 expression in HN22 and HSC4 cells and xenograft tumors, and thus could be a potent anti-inflammatory and anti-cancer drug candidate for human oral cancer treatment.

(+)-2-(1-Hydroxyl-4-oxocyclohexyl) ethyl caffeate suppresses solar UV-induced skin carcinogenesis by targeting PI3K, ERK1/2, and p38.

For decades, skin cancer incidence has increased, mainly because of oncogenic signaling pathways activated by solar ultraviolet (UV) irradiation (i.e., sun exposure). Solar UV induces multiple signaling pathways that are critical in the development of skin cancer, and therefore the development of compounds capable of targeting multiple molecules for chemoprevention of skin carcinogenesis is urgently needed. Herein, we examined the chemopreventive effects and the molecular mechanism of (+)-2-(1-hydroxyl-4-oxocyclohexyl) ethyl caffeate (HOEC), isolated from Incarvillea mairei var. grandiflora (Wehrhahn) Grierson. HOEC strongly inhibited neoplastic transformation of JB6 Cl41 cells without toxicity. PI3K, ERK1/2, and p38 kinase activities were suppressed by direct binding with HOEC in vitro. Our in silico docking data showed that HOEC binds at the ATP-binding site of each kinase. The inhibition of solar UV-induced PI3K, ERK1/2, and p38 kinase activities resulted in suppression of their downstream signaling pathways and AP1 and NF-κB transactivation in JB6 cells. Furthermore, topical application of HOEC reduced skin cancer incidence and tumor volume in SKH-1 hairless mice chronically exposed to solar UV. In summary, our results show that HOEC exerts inhibitory effects on multiple kinase targets and their downstream pathways activated by solar UV in vitro and in vivo. These findings suggest that HOEC is a potent chemopreventive compound against skin carcinogenesis caused by solar UV exposure.