Translation Initiation Factor-2S2 (eIF2S2) Contributes to Cervical Carcinogenesis by Inhibiting the TGF-ß/SMAD4 Signaling Pathway.

The deregulation of protein translational machinery and the oncogenic role of several translation initiation factors have been extensively investigated. This study aimed to investigate the role of eukaryotic translation initiation factor 2S2 (eIF2S2, also known as eIF2ß) in cervical carcinogenesis. Immunohistochemical analysis of human cervical carcinoma tissues revealed a stage-specific increase in eIF2S2 expression. The knockdown of eIF2S2 in human cervical cancer (SiHa) cells significantly reduced growth and migration properties, whereas its overexpression demonstrated the opposite effect. Immunoprecipitation and Bimolecular fluorescence complementation (BiFC) assay confirmed the previous photo array finding of the interaction between eIF2S2 and SMAD4 to understand the tumorigenic mechanism of eIF2S2. The results indicated that the N-terminus of eIF2S2 interacts with the MH-1 domain of SMAD4. The interaction effect between eIF2S2 and SMAD4 was further evaluated. The knockdown of eIF2S2 increased SMAD4 expression in cervical cancer cells without changing SMAD4 mRNA expression, whereas transient eIF2S2 overexpression reduced SMAD4 expression. This indicates the possibility of post-translational regulation of SMAD4 expression by eIF2S2. Additionally, eIF2S2 overexpression was confirmed to weaken the expression and/or promoter activity of p15 and p27, which are SMAD4-regulated antiproliferative proteins, by reducing SMAD4 levels. Therefore, our study indicated the pro-tumorigenic role of eIF2S2, which diminishes both SMAD4 expression and function as a transcriptional factor in cervical carcinogenesis.

EP4 receptor agonist CAY10598 upregulates ROS-dependent Hsp90 cleavage in colorectal cancer cells.

Prostaglandin E2 (PGE2) interacts with four specific G protein-coupled receptors, namely EP1, EP2, EP3, and EP4, playing a pivotal role in determining the fate of cells. Our previous findings highlighted that stimulating the EP4 receptor with its agonist, CAY10598, triggers apoptosis in colon cancer HCT116 cells via the production of reactive oxygen species (ROS). This process also reduces the phosphorylation of the oncogenic protein JAK2 and leads to its degradation in these cells. In this study, our goal was to explore the pathways through which CAY10598 leads to JAK2 degradation. We focused on Hsp90, a heat shock protein family member known for its role as a molecular chaperone maintaining the stability of several key proteins including EGFR, MET, Akt, and JAK2. Our results show that CAY10598 decreases the levels of client proteins of Hsp90 in HCT116 cells, an effect reversible by pretreatment with the ROS scavenger N-acetyl cysteine (NAC) or the proteasome inhibitor MG132, indicating that the degradation is likely driven by ROS. Furthermore, we observed that CAY10598 cleaves both α and ß isoforms of Hsp90, the process inhibited by NAC. Inhibition of EP4 with the antagonist GW627368x not only prevented the degradation of Hsp90 client proteins but also the cleavage of Hsp90 itself in CAY10598-treated HCT116 cells. Additionally, CAY10598 suppressed the growth of HCT116 cells implanted in mice. Our findings reveal that CAY10598 induces apoptosis in cancer cells by a novel mechanism involving the ROS-dependent cleavage of Hsp90, thereby inhibiting the function of crucial Hsp90 client proteins.

Domperidone, a Dopamine Receptor D2 Antagonist, Induces Apoptosis by Inhibiting the ERK/STAT3-Mediated Pathway in Human Colon Cancer HCT116 Cells.

Colorectal cancer (CRC) continues to demonstrate high incidence and mortality rates, emphasizing that implementing strategic measures for prevention and treatment is crucial. Recently, the dopamine receptor D2 (DRD2), a G protein-coupled receptor, has been reported to play multiple roles in growth of tumor cells. This study investigated the anticancer potential of domperidone, a dopamine receptor D2 antagonist, in HCT116 human CRC cells. Domperidone demonstrated concentration- and time-dependent reductions in cell viability, thereby inducing apoptosis. The molecular mechanism revealed that domperidone modulated the mitochondrial pathway, decreasing mitochondrial Bcl-2 levels, elevating cytosolic cytochrome C expression, and triggering caspase- 3, -7, and -9 cleavage. Domperidone decreased in formation of ß-arrestin2/MEK complex, which contributing to inhibition of ERK activation. Additionally, treatment with domperidone diminished JAK2 and STAT3 activation. Treatment of U0126, the MEK inhibitor, resulted in reduced phosphorylation of MEK, ERK, and STAT3 without alteration of JAK2 activation, indicating that domperidone targeted both MEK-ERK-STAT3 and JAK2-STAT3 signaling pathways, respectively. Immunoblot analysis revealed that domperidone also downregulated DRD2 expression. Domperidone-induced reactive oxygen species (ROS) generation and N-acetylcysteine treatment mitigated ROS levels and restored cell viability. An in vivo xenograft study verified the significant antitumor effects of domperidone. These results emphasize the multifaceted anticancer effects of domperidone, highlighting its potential as a promising therapeutic agent for human CRC.

Domperidone Exerts Antitumor Activity in Triple-Negative Breast Cancer Cells by Modulating Reactive Oxygen Species and JAK/STAT3 Signaling.

The lack of molecular targets hampers the treatment of triple-negative breast cancer (TNBC). In this study, we determined the cytotoxicity of domperidone, a dopamine D2 receptor (DRD2) antagonist in human TNBC BT-549 and CAL-51 cells. Domperidone inhibited cell growth in a dose- and time-dependent manner. The annexin V/propidium iodide staining showed that domperidone induced apoptosis. The domperidone-induced apoptosis was accompanied by the generation of mitochondrial superoxide and the down-regulation of cyclins and CDKs. The apoptotic effect of domperidone on TNBC cells was prevented by pre-treatment with Mito-TEMPO, a mitochondria-specific antioxidant. The prevention of apoptosis with Mito-TEMPO even at concentrations as low as 100 nM, implies that the generation of mitochondrial ROS mediated the domperidone-induced apoptosis. Immunoblot analysis showed that domperidone-induced apoptosis occurred through the down-regulation of the phosphorylation of JAK2 and STAT3. Moreover, domperidone downregulated the levels of D2-like dopamine receptors including DRD2, regardless of their mRNA levels. Our results support further development of DRD2 antagonists as potential therapeutic strategy treating TNBC.

MS-5, a Naphthalene Derivative, Induces Apoptosis in Human Pancreatic Cancer BxPC-3 Cells by Modulating Reactive Oxygen Species.

Pancreatic cancer is one of the most fatal cancers with a poor prognosis. Standard chemotherapies have proven largely ineffective because of their toxicity and the development of resistance. Therefore, there is an urgent need to develop novel therapies. In this study, we investigated the antitumor activity of MS-5, a naphthalene derivative, on BxPC-3, a human pancreatic cancer cell line. We observed that MS-5 was cytotoxic to BxPC-3 cells, as well as inhibited the growth of cells in a concentration- and time- dependent manner. Flow cytometry analysis revealed that the percentage of annexin V-positive cells increased after MS-5 treatment. We also observed cleavage of caspases and poly (ADP-ribose) polymerase, and downregulation of Bcl-xL protein. Flow cytometry analysis of intracellular levels of reactive oxygen species (ROS) and mitochondrial superoxide suggested that MS-5 induced the generation of mitochondrial superoxide while lowering the overall intracellular ROS levels. Thus, MS-5 may be potential candidate for pancreatic cancer treatment.

Hydroxyzine Induces Cell Death in Triple-Negative Breast Cancer Cells via Mitochondrial Superoxide and Modulation of Jak2/STAT3 Signaling.

Treatment of triple-negative breast cancer (TNBC) has been limited due to the lack of molecular targets. In this study, we evaluated the cytotoxicity of hydroxyzine, a histamine H1 receptor antagonist in human triple-negative breast cancer BT-20 and HCC-70 cells. Hydroxyzine inhibited the growth of cells in dose- and time-dependent manners. The annexin V/propidium iodide double staining assay showed that hydroxyzine induced apoptosis. The hydroxyzine-induced apoptosis was accompanied down-regulation of cyclins and CDKs, as well as the generation of reactive oxygen species (ROS) without cell cycle arrest. The effect of hydroxyzine on the induction of ROS and apoptosis on TNBC cells was prevented by pre-treatment with ROS scavengers, N-acetyl cysteine or Mito-TEMPO, a mitochondria-targeted antioxidant, indicating that an increase in the generation of ROS mediated the apoptosis induced by hydroxyzine. Western blot analysis showed that hydroxyzine-induced apoptosis was through down-regulation of the phosphorylation of JAK2 and STAT3 by hydroxyzine treatment. In addition, hydroxyzine induced the phosphorylation of JNK and p38 MAPK. Our results indicate that hydroxyzine induced apoptosis via mitochondrial superoxide generation and the suppression of JAK2/STAT3 signaling.

Isolinderalactone sensitizes oxaliplatin-resistance colorectal cancer cells through JNK/p38 MAPK signaling pathways.

BACKGROUND: Isolinderalactone (ILL), a sesquiterpene lactone compound, can be extracted from the root of Lindera aggregate. Physiological activities of ILL, including anti-inflammatory and anti-proliferative effects, have been investigated in multiple diseases. Nevertheless, little is known regarding its anti-cancer activities and the mechanism of action of ILL in targeting human CRC cells. PURPOSE: To determine ILL-mediated anti-proliferative effects on oxaliplatin (Ox)-sensitive and resistant colorectal cancer (CRC) cells and underlying mechanisms involved in its effects focusing on signal transduction. METHODS: Inhibitory effect of ILL on CRC cells was evaluated by analyzing mitochondrial membrane potential (MMP) dysfunction and multi-caspase activity. Apoptosis-regulating proteins and JNK/p38 signaling molecules were monitored by Western blotting. ROS-dependent physiological modifications by ILL were confirmed by pretreatment with N-acetylcysteine (NAC). Moreover, the involvement of JNK/p38 signaling in ROS-mediated apoptosis was verified by treatment with SP600125 (JNK inhibitor) and SB203580 (p38 inhibitor). RESULTS: ILL decreased cell viability and colony formation in both CRC Ox-sensitive (HCT116 and HT29) and Ox-resistant (OxR) (HCT116-OxR and HT29-OxR) cells. ILL induced G2/M phase cell cycle arrest, ROS generation, phosphorylated (p)JNK/p38 MAPK activation, mitochondrial membrane potential (MMP) depolarization, and multi-caspase activation, which eventually triggered apoptotic cell death of CRC cells. In addition, combined treatment with ILL and SP600125, SB203580, or pan-caspase inhibitor (Z-VAD-FMK) prevented decreases in cell viability seen after treatment with ILL alone. Pretreatment with NAC attenuated ILL-mediated apoptosis, ROS production, and p-JNK/p38 expression. CONCLUSION: Taken together, our results suggest that ILL can exert its anticancer effect in CRC Ox-sensitive and OxR cells by inducing ROS-mediated apoptosis through JNK/p38 MAPK signaling pathways. This is the first study demonstrating that ILL has a potential to improve drug efficacy against resistance mechanisms, providing a new insight into therapeutic strategies targeting drug-resistant CRC.

Macrosphelide A Exhibits a Specific Anti-Cancer Effect by Simultaneously Inactivating ENO1, ALDOA, and FH.

Aerobic glycolysis in cancer cells, also known as the Warburg effect, is an indispensable hallmark of cancer. This metabolic adaptation of cancer cells makes them remarkably different from normal cells; thus, inhibiting aerobic glycolysis is an attractive strategy to specifically target tumor cells while sparing normal cells. Macrosphelide A (MSPA), an organic small molecule, is a potential lead compound for the design of anti-cancer drugs. However, its role in modulating cancer metabolism remains poorly understood. MSPA target proteins were screened using mass spectrometry proteomics combined with affinity chromatography. Direct and specific interactions of MSPA with its candidate target proteins were confirmed by in vitro binding assays, competition assays, and simulation modeling. The siRNA-based knockdown of MSPA target proteins indirectly confirmed the cytotoxic effect of MSPA in HepG2 and MCF-7 cancer cells. In addition, we showed that MSPA treatment in the HEPG2 cell line significantly reduced glucose consumption and lactate release. MSPA also inhibited cancer cell proliferation and induced apoptosis by inhibiting critical enzymes involved in the Warburg effect: aldolase A (ALDOA), enolase 1 (ENO1), and fumarate hydratase (FH). Among these enzymes, the purified ENO1 inhibitory potency of MSPA was further confirmed to demonstrate the direct inhibition of enzyme activity to exclude indirect/secondary factors. In summary, MSPA exhibits anti-cancer effects by simultaneously targeting ENO1, ALDOA, and FH.

TM4SF4 and LRRK2 Are Potential Therapeutic Targets in Lung and Breast Cancers through Outlier Analysis.

PURPOSE: To find biomarkers for disease, there have been constant attempts to investigate the genes that differ from those in the disease groups. However, the values that lie outside the overall pattern of a distribution, the outliers, are frequently excluded in traditional analytical methods as they are considered to be 'some sort of problem.' Such outliers may have a biologic role in the disease group. Thus, this study explored new biomarker using outlier analysis, and verified the suitability of therapeutic potential of two genes (TM4SF4 and LRRK2). MATERIALS AND METHODS: Modified Tukey's fences outlier analysis was carried out to identify new biomarkers using the public gene expression datasets. And we verified the presence of the selected biomarkers in other clinical samples via customized gene expression panels and tissue microarrays. Moreover, a siRNA-based knockdown test was performed to evaluate the impact of the biomarkers on oncogenic phenotypes. RESULTS: TM4SF4 in lung cancer and LRRK2 in breast cancer were chosen as candidates among the genes derived from the analysis. TM4SF4 and LRRK2 were overexpressed in the small number of samples with lung cancer (4.20%) and breast cancer (2.42%), respectively. Knockdown of TM4SF4 and LRRK2 suppressed the growth of lung and breast cancer cell lines. The LRRK2 overexpressing cell lines were more sensitive to LRRK2-IN-1 than the LRRK2 under-expressing cell lines. CONCLUSION: Our modified outlier-based analysis method has proved to rescue biomarkers previously missed or unnoticed by traditional analysis showing TM4SF4 and LRRK2 are novel target candidates for lung and breast cancer, respectively.

Isolinderalactone Induces Cell Death via Mitochondrial Superoxide- and STAT3-Mediated Pathways in Human Ovarian Cancer Cells.

The mortality rate of ovarian cancer (OC) worldwide increases with age. OC is an often fatal cancer with a curative rate of only 20-30%, as symptoms often appear after disease progression. Studies have reported that isolinderalactone (ILL), a furanosesquiterpene derivative extracted from the dried root of Lindera aggregata, can inhibit several cancer cell lines' growth. However, the molecular mechanisms underlying ILL activities in human OC cells remain unexplored. This study investigated the antitumor activities of ILL in human OC cells by inducing mitochondrial superoxide (mtSO) and JAK-signal transducer and activator of transcription 3 (STAT3)-dependent cell death. ILL caused cell death in SKOV-3 and OVCAR-3 cells and increased the cell proportion in the subG1 phase. Additionally, ILL significantly induced mtSO production and reduced ROS production. Moreover, ILL downregulated mitochondrial membrane potential and the expression levels of anti-apoptotic Bcl-2 family proteins and superoxide dismutase (SOD)2. Results showed that ILL decreased phosphorylation of serine 727 and tyrosine 705 of STAT3 and expression of survivin, a STAT3-regulated gene. Furthermore, ILL-induced cell death was reversed by pretreatment of Mito-TEMPO, a mitochondria-specific antioxidant. These results suggest that ILL induces cell death by upregulation of mtSO, downregulation of mitochondrial SOD2, and inactivation of the STAT3-mediated pathway.

Deoxypodophyllotoxin, a Lignan from Anthriscus sylvestris, Induces Apoptosis and Cell Cycle Arrest by Inhibiting the EGFR Signaling Pathways in Esophageal Squamous Cell Carcinoma Cells.

Deoxypodophyllotoxin (DPT) derived from Anthriscus sylvestris (L.) Hoffm has attracted considerable interest in recent years because of its anti-inflammatory, antitumor, and antiviral activity. However, the mechanisms underlying DPT mediated antitumor activity have yet to be fully elucidated in esophageal squamous cell carcinoma (ESCC). We show here that DPT inhibited the kinase activity of epidermal growth factor receptor (EGFR) directly, as well as phosphorylation of its downstream signaling kinases, AKT, GSK-3ß, and ERK. We confirmed a direct interaction between DPT and EGFR by pull-down assay using DPT-beads. DPT treatment suppressed ESCC cell viability and colony formation in a time- and dose-dependent manner, as shown by MTT analysis and soft agar assay. DPT also down-regulated cyclin B1 and cdc2 expression to induce G2/M phase arrest of the cell cycle and upregulated p21 and p27 expression. DPT treatment of ESCC cells triggered the release of cytochrome c via loss of mitochondrial membrane potential, thereby inducing apoptosis by upregulation of related proteins. In addition, treatment of KYSE 30 and KYSE 450 cells with DPT increased endoplasmic reticulum stress, reactive oxygen species generation, and multi-caspase activation. Consequently, our results suggest that DPT has the potential to become a new anticancer therapeutic by inhibiting EGFR mediated AKT/ERK signaling pathway in ESCC.

Janus kinase 2 inhibition by Licochalcone B suppresses esophageal squamous cell carcinoma growth.

Esophageal cancer (EC) is one of the leading causes to cancer death in the worldwide and major population of EC is esophageal squamous cell carcinoma (ESCC). Still, ESCC-targeted therapy has not been covered yet. In the present study we have identified that Licochalcone B (Lico B) inhibited the ESCC growth by directly blocking the Janus kinase (JAK) 2 activity and its downstream signaling pathway. Lico B suppressed KYSE450 and KYSE510 ESCC cell growth, arrested cell cycle at G2/M phase and induced apoptosis. Direct target of Lico B was identified by kinase assay and verified with in vitro and ex vivo binding. Computational docking model predicted for Lico B interaction to ATP-binding pocket of JAK2. Furthermore, treatment of JAK2 clinical medicine AZD1480 to ESCC cells showed similar tendency with Lico B. Thus, JAK2 downstream signaling proteins phosphorylation of STAT3 at Y705 and S727 as well as STAT3 target protein Mcl-1 expression was decreased with treatment of Lico B. Our results suggest that Lico B inhibits ESCC cell growth, arrests cell cycle and induces apoptosis, revealing the underlying mechanism involved in JAK2/STAT3 signaling pathways after Lico B treatment. It might provide potential role of Lico B in the treatment of ESCC.

Licochalcone C induces cell cycle G1 arrest and apoptosis in human esophageal squamous carcinoma cells by activation of the ROS/MAPK signaling pathway.

Along with changes in dietary habits and lifestyle, the incidence of esophageal cancer is increasing around the world. Since chemotherapy for esophageal cancer has significant side effects, phytochemicals have attracted attention as an alternative medicine. Licochalcone C (LCC) is a flavonoid compound extracted from Licorice, with a variety of clinical uses including anti-cancer, anti-inflammatory and anti-oxidant effects. Treatment with LCC for 48 h significantly decreased cell viability of esophageal squamous cell carcinoma (ESCC) cells in a dose- and time-dependent manner with IC50 values of 28 µM (KYSE 30), 36 µM (KYSE 70), 19 µM (KYSE 410), 28 µM (KYSE 450) and 26 µM (KYSE 510). LCC induced G1 arrest accompanied by decreased cyclin D1 expression and an increase in the levels of p21 and p27. LCC increased the levels of intracellular ROS, cytochrome C release, and multi-caspase activity, and decreased mitochondrial membrane potential. LCC induced the protein expression of ER stress markers (GRP78 and CHOP) and phosphorylation JNK, c-Jun and p38. We investigated the expression of pro-apoptotic and anti-apoptotic proteins to elucidate the mechanism of apoptosis. Our findings contribute to the understanding of apoptosis mechanism underlying LCC in ESCC cells and provide new insights into the potential clinical opportunities of LCC for ESCC treatment.

Antibody-Based Targeting of Interferon-Beta-1a Mutein in HER2-Positive Cancer Enhances Antitumor Effects Through Immune Responses and Direct Cell Killing.

Type I interferon (IFN) has been approved as an anticancer agent to treat some malignancies. However, IFNs have a short in vivo half-life, systemic toxicity, and poor biophysical properties, which prevent it from being widely used for cancer therapy. This study aimed to construct recombinant IFN-ß-1a mutein immunocytokines that comprise a human epidermal growth factor receptor 2 (HER2)-targeting antibody and IFN-ß muteins with an additional glycosylation, which can overcome the limitation of the cytokine itself. Hence, the molecular design aims to 1) enhance productivity and biophysical properties by adding secondary glycosylation in IFN-ß, 2) increase the therapeutic index of IFN-ß therapy by preferential retention at the tumor by possessing high affinity for HER2-expressing cancer cells, and 3) improve the pharmacokinetics and, thus, the convenience of IFN-ß administration. The yield of trastuzumab-IFN-ß mutein was higher than that of trastuzumab-wild-type IFN-ß in the mammalian cell culture system. Trastuzumab-IFN-ß mutein showed similar IFN activity and HER2-targeting ability equivalent to that of IFN-ß mutein and trastuzumab, respectively. Trastuzumab-IFN-ß mutein directly inhibited the growth of HER2-positive gastric cancer cell lines and was more effective than trastuzumab or IFN-ß mutein alone. Trastuzumab-IFN-ß mutein and IFN-ß mutein displayed enhanced immune cell-mediated cytotoxicity. Collectively, trastuzumab-IFN-ß mutein may have indirect immune cell-mediated antitumor effects and direct cell growth inhibitory effects. Tumor-targeting effect of trastuzumab-IFN-ß mutein was analyzed using in vivo fluorescence imaging. The accumulation of trastuzumab-IFN-ß mutein was observed in HER2-positive tumors rather than other tissues except the liver. To evaluate the both direct tumor growth inhibition effect and indirect immune cell-mediated antitumor effect, we tested the effect of trastuzumab-IFN-ß mutein in HER2-positive cancer xenograft models using nude mice or humanized mice. Trastuzumab-IFN-ß mutein could significantly enhance tumor regression when compared with trastuzumab or IFN-ß mutein. In addition, an increase in tumor-infiltrating lymphocytes was observed in the trastuzumab-IFN-ß mutein-treated group, implying that the tumor-targeting IFN-ß may have an enhanced antitumor effect through increased immune response. Therefore, targeting IFN-ß with an anti-HER2 monoclonal antibody makes the immunocytokine more potent than either agent alone. These novel findings suggest that trastuzumab-IFN-ß mutein merits clinical evaluation as a new candidate of anticancer therapeutics.

Dual inhibition of EGFR and MET by Echinatin retards cell growth and induces apoptosis of lung cancer cells sensitive or resistant to gefitinib.

Patients with non-small-cell lung cancer (NSCLC) containing epidermal growth factor receptor (EGFR) amplification or sensitive mutations initially respond to tyrosine kinase inhibitor gefitinib; however, the treatment is less effective over time. Gefitinib resistance mechanisms include MET gene amplification. A therapeutic strategy targeting MET as well as EGFR can overcome resistance to gefitinib. In the present study we identified Echinatin (Ecn), a characteristic chalcone in licorice, which inhibited both EGFR and MET and strongly altered NSCLC cell growth. The antitumor efficacy of Ecn against gefitinib-sensitive or -resistant NSCLC cells with EGFR mutations and MET amplification was confirmed by suppressing cell proliferation and anchorage-independent colony growth. During the targeting of EGFR and MET, Ecn significantly blocked the kinase activity, which was validated with competitive ATP binding. Inhibition of EGFR and MET by Ecn decreases the phosphorylation of downstream target proteins ERBB3, AKT and ERK compared with total protein expression or control. Ecn induced the G2/M cell cycle arrest, and apoptosis via the intrinsic pathway of caspase-dependent activation. Ecn induced ROS production and GRP78, CHOP, DR5 and DR4 expression as well as depolarized the mitochondria membrane potential. Therefore, our results suggest that Ecn is a promising therapeutic agent in NSCLC therapy.

Retrochalcone Echinatin Triggers Apoptosis of Esophageal Squamous Cell Carcinoma via ROS- and ER Stress-Mediated Signaling Pathways.

Esophageal squamous cell carcinoma (ESCC) is a poor prognostic cancer with a low five-year survival rate. Echinatin (Ech) is a retrochalone from licorice. It has been used as a herbal medicine due to its anti-inflammatory and anti-oxidative effects. However, its anticancer activity or underlying mechanism has not been elucidated yet. Thus, the objective of this study was to investigate the anti-tumor activity of Ech on ESCC by inducing ROS and ER stress dependent apoptosis. Ech inhibited ESCC cell growth in anchorage-dependent and independent analysis. Treatment with Ech induced G2/M phase of cell cycle and apoptosis of ESCC cells. It also regulated their related protein markers including p21, p27, cyclin B1, and cdc2. Ech also led to phosphorylation of JNK and p38. Regarding ROS and ER stress formation associated with apoptosis, we found that Ech increased ROS production, whereas its increase was diminished by NAC treatment. In addition, ER stress proteins were induced by treatment with Ech. Moreover, Ech enhanced MMP dysfunction and caspases activity. Furthermore, it regulated related biomarkers. Taken together, our results suggest that Ech can induce apoptosis in human ESCC cells via ROS/ER stress generation and p38 MAPK/JNK activation.

Thymoquinone induces apoptosis of human epidermoid carcinoma A431 cells through ROS-mediated suppression of STAT3.

Black seed (Nigella sativa) oil has been used in various dermatological applications, and its major constituent, thymoquinone (TQ) has been shown to exhibit antiproliferative activity against various cancer cells. In this study, we tried to provide a mechanistic basis of apoptosis induced by TQ. Skin squamous carcinoma A431 cells were treated with TQ to monitor the apoptosis induced by TQ. Western blot analysis was performed to detect expression of apoptotic or anti-apoptotic proteins. Cell viability and apoptosis were measured by using the MTT test and FACS analysis, respectively. The induction of intracellular reactive oxygen species (ROS) by TQ was evaluated by 2',7'-dichlorofluorescein diacetate staining. In vivo xenograft study was followed to confirm the antiproliferative effect of TQ. Treatment of A431 cells with TQ-induced apoptosis, which was associated with the induction of p53 and Bax, inhibition of Mdm2, Bcl-2, and Bcl-xl expression, and activation of caspase-9, -7, and -3. TQ inhibited the constitutive phosphorylation and DNA binding activity of signal transducer and activator of transcription-3 (STAT3) in A431 cells by blocking the phosphorylation of the upstream kinase, Src. Moreover, the expression of STAT3 target gene products, cyclin D1 and survivin, was attenuated by TQ treatment. The generation of ROS was increased during TQ-induced apoptosis, and the pretreatment of N-acetyl cysteine, a ROS scavenger, reversed the apoptotic effect of TQ. In vivo study with NOD scid gamma (NSG) mice confirmed the inhibitory effect of TQ on the growth of A431 cells. Our results provide the first demonstration that TQ induces the apoptosis of A431 cells through generation of ROS and inhibition of STAT3 signaling.

MED28 Over-Expression Shortens the Cell Cycle and Induces Genomic Instability.

The mammalian mediator complex subunit 28 (MED28) is overexpressed in a variety of cancers and it regulates cell migration/invasion and epithelial-mesenchymal transition. However, transcription factors that increase MED28 expression have not yet been identified. In this study, we performed a luciferase reporter assay to identify and characterize the prospective transcription factors, namely E2F transcription factor 1, nuclear respiratory factor 1, E-26 transforming sequence 1, and CCAAT/enhancer-binding protein ß, which increased MED28 expression. In addition, the release from the arrest at the G1-S or G2-M phase transition after cell cycle synchronization using thymidine or nocodazole, respectively, showed enhanced MED28 expression at the G1-S transition and mitosis. Furthermore, the overexpression of MED28 significantly decreased the duration of interphase and mitosis. Conversely, a knockdown of MED28 using si-RNA increased the duration of interphase and mitosis. Of note, the overexpression of MED28 significantly increased micronucleus and nuclear budding in HeLa cells. In addition, flow cytometry and fluorescence microscopy analyses showed that the overexpression of MED28 significantly increased aneuploid cells. Taken together, these results suggest that MED28 expression is increased by oncogenic transcription factors and its overexpression disturbs the cell cycle, which results in genomic instability and aneuploidy.

UBE2C Overexpression Aggravates Patient Outcome by Promoting Estrogen-Dependent/Independent Cell Proliferation in Early Hormone Receptor-Positive and HER2-Negative Breast Cancer.

We previously showed that UBE2C mRNA expression is significantly associated with poor prognosis only in patients with hormone receptor (HR)+/human epidermal growth factor receptor 2 (HER2)- breast cancer. In this study, we further reanalyzed the correlation between UBE2C mRNA expression and clinical outcomes in patients with HR+/HER2- breast cancer, and we investigated the molecular mechanism underlying the role of UBE2C modulation in disease progression in this subgroup of patients. Univariate and multivariate analyses showed that high UBE2C expression was associated with significantly shorter survival of breast cancer patients with pN0 and pN1 tumors but not pN2/N3 tumors (P < 0.05). In vitro functional experiments in HR+/HER2- breast cancer cells showed that UBE2C expression is a tumorigenic factor, and that estrogen upregulated UBE2C mRNA and protein by directly binding to the UBE2C promoter region. UBE2C knockdown inhibited cell proliferation by affecting cell cycle progression, and UBE2C overexpression was associated with estrogen-independent growth. UBE2C depletion markedly increased the cytotoxicity of tamoxifen by inducing apoptosis. The present findings suggest that UBE2C overexpression is correlated with relapse and promotes estrogen-dependent/independent proliferation in early HR+/HER2- breast cancer.

Development of Human Monoclonal Antibody for Claudin-3 Overexpressing Carcinoma Targeting.

Most malignant tumors originate from epithelial tissues in which tight junctions mediate cell-cell interactions. Tight junction proteins, especially claudin-3 (CLDN3), are overexpressed in various cancers. Claudin-3 is exposed externally during tumorigenesis making it a potential biomarker and therapeutic target. However, the development of antibodies against specific CLDN proteins is difficult, because CLDNs are four-transmembrane domain proteins with high homology among CLDN family members and species. Here, we developed a human IgG1 monoclonal antibody (h4G3) against CLDN3 through scFv phage display using CLDN3-overexpressing stable cells and CLDN3-embedded lipoparticles as antigens. The h4G3 recognized the native conformation of human and mouse CLDN3 without cross-reactivity to other CLDNs. The binding kinetics of h4G3 demonstrated a sub-nanomolar affinity for CLDN3 expressed on the cell surface. The h4G3 showed antibody-dependent cellular cytotoxicity (ADCC) according to CLDN3 expression levels in various cancer cells by the activation of FcγRIIIa (CD16a). The biodistribution of h4G3 was analyzed by intravenous injection of fluorescence-conjugated h4G3 which showed that it localized to the tumor site in xenograft mice bearing CLDN3-expressing tumors. These results indicate that h4G3 recognizes CLDN3 specifically, suggesting its value for cancer diagnosis, antibody-drug conjugates, and potentially as a chimeric antigen receptor (CAR) for CLDN3-expressing pan-carcinoma.