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Background and purpose Overexpression or aberrant activation of the T-lymphokine-activated killer cell-originated protein kinase (TOPK) promotes gene expression and growth of solid tumours, implying that TOPK would be a rational target in developing novel anticancer drugs. Acetylshikonin, a diterpenoid compound isolated from Lithospermum erythrorhizon root, exerts a range of biological activities. Here we have investigated whether acetylshikonin, by acting as an inhibitor of TOPK, can attenuate the proliferation of colorectal cancer cells and the growth of patient-derived tumours, in vitro and in vivo. Experimental approach Targets of acetylshikonin, were identified using kinase profiling analysis, kinetic/binding assay, and computational docking analysis and knock-down techniques. Effects of acetylshikonin on colorectal cancer growth and the underlying mechanisms were evaluated in cell proliferation assays, propidium iodide and annexin-V staining analyses and western blots. Patient-derived tumour xenografts in mice (PDX) and immunohistochemistry were used to assess anti-tumour effects of acetylshikonin. Key results Acetylshikonin directly inhibited TOPK activity, interacting with the ATP-binding pocket of TOPK. Acetylshikonin suppressed cell proliferation by inducing cell cycle arrest at the G1 phase, stimulated apoptosis, and increased the expression of apoptotic biomarkers in colorectal cancer cell lines. Mechanistically, acetylshikonin diminished the phosphorylation and activation of TOPK signalling. Furthermore, acetylshikonin decreased the volume of PDX tumours and reduced the expression of TOPK signalling pathway in xenograft tumours. Conclusion and implications Acetylshikonin suppressed growth of colorectal cancer cells by attenuating TOPK signalling. Targeted inhibition of TOPK by acetylshikonin might be a promising new approach to the treatment of colorectal cancer.

BackgroundSkin cancer is one of the most commonly diagnosed cancers worldwide. The 5-year survival rate of the most aggressive late-stage skin cancer ranges between 20 and 30%. Thus, the discovery and investigation of novel target therapeutic agents that can effectively treat skin cancer is of the utmost importance. The T-lymphokine-activated killer cell-originated protein kinase (TOPK), which belongs to the serine-threonine kinase class of the mitogen-activated protein kinase kinase (MAPKK) family, is highly expressed and activated in skin cancer. The present study investigates the role of 3-deoxysappanchalcone (3-DSC), a plant-derived functional TOPK inhibitor, in suppressing skin cancer cell growth.PurposeIn the context of skin cancer prevention and therapy, we clarify the effect and mechanism of 3-DSC on different types of skin cancer and solar-simulated light (SSL)-induced skin hyperplasia.MethodsIn an in vitro study, western blotting and in vitro kinase assays were utilized to determine the protein expression of TOPK and its activity, respectively. Pull-down assay with 3-DSC and TOPK (wild-type and T42A/N172 mutation) was performed to confirm the direct interaction between T42A/N172 amino acid sites of TOPK and 3-DSC. Cell proliferation and anchorage-independent cell growth assays were utilized to determine the effect of 3-DSC on cell growth. In an in vivo study, the thickness of skin and tumor size were measured in the acute SSL-induced inflammation mouse model or SK-MEL-2 cell-derived xenografts mouse model treated with 3-DSC. Immunohistochemistry analysis of tumors isolated from SK-MEL-2 cell-derived xenografts was performed to determine whether cell-based results observed upon 3-DSC treatment could be recapitulated in vivo.Results3-DSC is able to inhibit cell proliferation in skin cancer cells in an anchorage-dependent and anchorage-independent manner by regulation of TOPK and its related signaling pathway in vitro. We also found that application of 3-DSC reduced acute SSL-induced murine skin hyperplasia. Additionally, we observed that 3-DSC decreased SK-MEL-2 cell-derived xenograft tumor growth through attenuating phosphorylation of TOPK and its downstream effectors including ERK, RSK, and c-Jun.ConclusionsOur results suggest that 3-DSC may function in a chemopreventive and chemotherapeutic capacity by protecting against UV-induced skin hyperplasia and inhibiting tumor cell growth by attenuating TOPK signaling, respectively.

The proviral integration site for Moloney murine leukemia virus (PIM) family of serine/threonine-specific kinases consist of three isoforms, that regulate proliferation, apoptosis, metabolism, invasion, and metastasis of cancer cells. Among these, abnormally elevated kinase activity of PIM-1 contributes to the progression of gastric cancer and predicts poor prognosis and a low survival rate in gastric cancer patients. In the present study, we found that resveratrol, one of the representative chemopreventive and anticarcinogenic phytochemicals, directly binds to PIM-1 and thereby inhibits its catalytic activity in human gastric cancer SNU-601 cells. This resulted in suppression of phosphorylation of the proapoptotic Bad, a known substrate of PIM-1. Resveratrol, by inactivating PIM-1, also inhibited anchorage-independent growth and proliferation of SNU-601 cells. To understand the molecular interaction between resveratrol and PIM-1, we conducted docking simulation and found that resveratrol directly binds to the PIM-1 at the ATP-binding pocket. In conclusion, the proapototic and anti-proliferative effects of resveratrol in gastric cancer cells are likely to be mediated through suppression of PIM-1 kinase activity, which may represent a novel mechanism underlying its chemopreventive and anticarcinogenic actions.

// Mee-Hyun Lee 1 , Zigang Dong 1, 6 , Young-Joon Surh 2, 3, 4, 5 and Bu Young Choi 7 1 China-US (Henan) Hormel Cancer Institute, Zhengzhou 450008, China 2 Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, Seoul 08826, South Korea 3 Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 08826, South Korea 4 Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Sciences and Technology, Seoul National University, Seoul 08826, South Korea 5 Cancer Research Institute, Seoul National University, Seoul 110-744, South Korea 6 The Hormel Institute, University of Minnesota, Austin, MN 55912, USA 7 Department of Pharmaceutical Science & Engineering, Seowon University, Cheongju 28674, South Korea Correspondence to: Bu Young Choi, email: bychoi@seowon.ac.kr Young-Joon Surh, email: surh@snu.ac.kr , surhyoungjoon@yahoo.co.kr Keywords: hYSK1, p16 INK4a , MMP-2, tumor migration, hypoxia Received: July 17, 2017 Accepted: August 27, 2017 Published: October 06, 2017 ABSTRACT The alteration of expression of p16 INK4a , a well-known cyclin-dependent kinase inhibitor involved in cell cycle control, in tumors is unclear, especially under hypoxic conditions. To evaluate p16 INK4a regulation, we performed a protein microarray analysis. Among 1,800 proteins in the array, we identified hYSK1 as a novel protein that interacts with the tumor suppressor p16 INK4a . hYSK1, a member of the Ste20 family of serine/threonine protein kinases, promotes cell migration and tumorigenesis and is activated by oxidative stress. However, the molecular mechanisms underlying the oncogenic potential of hYSK1 remain elusive. Here, we report that hYSK1 interacts with p16 INK4a under hypoxic conditions in tumors, where it negatively regulates p16 INK4a , enhancing cancer cell migration. Hypoxic stimulation of hYSK1 reduces p16 INK4a accumulation through p16 promoter regulation to interact with unphosporylated SP-1 and increases matrix metalloproteinase-2 (MMP-2) expression by activating the MMP-2 promoter associated with cell migration and proliferation.Conversely, knocking down hYSK1 expression activated p16 INK4a expression and suppressed MMP-2 expression. Thus, hYSK1 is necessary as a trigger for inactivating p16 INK4a and activating MMP-2 during tumor migration, suggesting that hYSK1 is a specific negative regulator of the tumor suppressor p16 INK4a and may represent a novel molecular target for reactivation of tumor suppressor genes in humans.

Rationale: Signal transducer and activator of transcription-3 (STAT3) plays a pivotal role in cancer biology. Many small-molecule inhibitors that target STAT3 have been developed as potential anticancer drugs. While designing small-molecule inhibitors that target the SH2 domain of STAT3 remains the leading focus for drug discovery, there has been a growing interest in targeting the DNA-binding domain (DBD) of the protein. Methods: We demonstrated the potential antitumor activity of a novel, small-molecule (E)-2-methoxy-4-(3-(4-methoxyphenyl)prop-1-en-1-yl)phenol (MMPP) that directly binds to the DBD of STAT3, in patient-derived non-small cell lung cancer (NSCLC) xenograft model as well as in NCI-H460 cell xenograft model in nude mice. Results: MMPP effectively inhibited the phosphorylation of STAT3 and its DNA binding activity in vitro and in vivo. It induced G1-phase cell cycle arrest and apoptosis through the regulation of cell cycle- and apoptosis-regulating genes by directly binding to the hydroxyl residue of threonine 456 in the DBD of STAT3. Furthermore, MMPP showed a similar or better antitumor activity than that of docetaxel or cisplatin. Conclusion: MMPP is suggested to be a potential candidate for further development as an anticancer drug that targets the DBD of STAT3.

In the present study, the effects of the acetonitrile or water extracts from 400 selected traditional medicinal plants on the growth of PC-3 cells were investigated, and it was demonstrated that an acetonitrile extract of Salvia miltiorrhiza Radix exhibited the most marked cytotoxic effects on PC-3 cells. It was observed that the acetonitrile extract of S. miltiorrhiza Radix induced marked cell cycle arrest and apoptosis in PC-3 cells through the generation of intracellular reactive oxygen species. It was also demonstrated that oral administration of the acetonitrile extract of S. miltiorrhiza Radix decreased the incidence and growth of PC-3 tumor xenografts in nude mice. The results of the present study suggest that the acetonitrile extract of S. miltiorrhiza Radix exhibits marked inhibitory effects on the growth of prostate cancer cells in vitro and in vivo.

5-fluorouracil (5-FU) is a chemotherapeutic agent commonly used for treatment of solid tumors, including colorectal cancer. However, chemoresistance against 5-fluorouracil (5-FU) often limits its success for chemotherapy and, therefore, finding out appropriate adjuvant(s) that might overcome chemoresistance against 5-FU bears a significant importance. In the present study, we have found that α-mangostin can sensitize 5-FU-resistant SNUC5/5-FUR colon cancer cells to apoptosis. Exposure of α-mangostin induced significant DNA damages and increased the intracellular 8-hydroxyguanosine (8-OH-G) and 4-hydroxynonenal (4-HNE) levels in SNUC5 and SNUC5/5-FUR cells. Western blot analysis illustrated that α-mangostin-induced apoptosis was mediated by the activation of the extrinsic and intrinsic pathways in SNUC5/5-FUR cells. In particular, we observed that Fas receptor (FasR) level was lower in SNUC5/5-FUR cells, compared with SNUC5 cells and that silencing FasR attenuated α-mangostin-mediated apoptosis in SNUC5/5-FUR cells. Together, our study illustrates that α-mangostin might be an efficient apoptosis sensitizer that can overcome chemoresistance against 5-FU by activating apoptosis pathway.

// Do Young Lim 1 , Seung Ho Shin 1, 2 , Mee-Hyun Lee 1, 3 , Margarita Malakhova 1 , Igor Kurinov 4 , Qiong Wu 3, 5 , Jinglong Xu 5, 7 , Yanan Jiang 5, 7 , Ziming Dong 5, 7 , Kangdong Liu 3, 5, 6, 7 , Kun Yeong Lee 1 , Ki Beom Bae 1 , Bu Young Choi 8 , Yibin Deng 1 , Ann Bode 1 , Zigang Dong 1, 3, 5, 6, 7 1 The Hormel Institute, University of Minnesota, MN, USA 2 Program in Biomedical Informatics and Computational Biology, University of Minnesota, Minneapolis, MN, USA 3 The China-US (Henan) Cancer Institute, Zhengzhou, Henan, China 4 Cornell University, NE-CAT, Argonne, IL, USA 5 The Pathophysiology Department, The School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, Hunan, China 6 The Affiliated Cancer Hospital, Zhengzhou University, Zhengzhou, Henan, China 7 The Collaborative Innovation Center of Henan Province for Cancer Chemoprevention, Zhengzhou, China 8 Pharmaceutical Science and Engineering, School of Convergence Bioscience and Technology, Seowon University, Cheongju, Chungbuk, South Korea Correspondence to: Zigang Dong, email: zgdong@hi.umn.edu Keywords: catechol, lung cancer, ERK2, c-Myc, natural compound Received: January 06, 2016 Accepted: April 10, 2016 Published: May 07, 2016 ABSTRACT Various carcinogens induce EGFR/RAS/MAPK signaling, which is critical in the development of lung cancer. In particular, constitutive activation of extracellular signal-regulated kinase 2 (ERK2) is observed in many lung cancer patients, and therefore developing compounds capable of targeting ERK2 in lung carcinogenesis could be beneficial. We examined the therapeutic effect of catechol in lung cancer treatment. Catechol suppressed anchorage-independent growth of murine KP2 and human H460 lung cancer cell lines in a dose-dependent manner. Catechol inhibited ERK2 kinase activity in vitro , and its direct binding to the ERK2 active site was confirmed by X-ray crystallography. Phosphorylation of c-Myc, a substrate of ERK2, was decreased in catechol-treated lung cancer cells and resulted in reduced protein stability and subsequent down-regulation of total c-Myc. Treatment with catechol induced G1 phase arrest in lung cancer cells and decreased protein expression related to G1-S progression. In addition, we showed that catechol inhibited the growth of both allograft and xenograft lung cancer tumors in vivo . In summary, catechol exerted inhibitory effects on the ERK2/c-Myc signaling axis to reduce lung cancer tumor growth in vitro and in vivo , including a preclinical patient-derived xenograft (PDX) model. These findings suggest that catechol, a natural small molecule, possesses potential as a novel therapeutic agent against lung carcinogenesis in future clinical approaches.

BACKGROUND: Withania somnifera (known as Ashwagandha) is a medicinal plant used in the ayurvedic medicines in India. Withaferin-A, a withanolide derived from the leaf extract of W. somnifera, has been reported to exhibit anti-tumor activity against various cancer cells, such as leukemia, breast cancer and colon cancer cells. METHODS: We investigated the anti-cancer effects of withaferin-A on the proliferation and migration of human colorectal cancer (HCT116) cells. And we evaluated the effects of withaferin-A on the transcriptional activity of STAT3 and the growth of HCT116 cells in xenograft mouse tumor model. RESULTS: In the present study, we found that withaferin-A inhibited the proliferation and migration of HCT116 cells in a concentration-dependent manner. Treatment of HCT116 cells with withaferin-A attenuated interleukin-6-induced activation of STAT3, which has been implicated in the development and progression of colon cancer. To examine the effect of withaferin-A on HCT116 cells proliferation in vivo, we generated HCT116 cells xenograft tumors in Balb/c nude mice and treated the tumor bearing mice with or without withaferin-A intraperitoneally. Treatment with withaferin-A exhibited significant decrease in the volume and weight of tumors as compared to untreated controls. CONCLUSIONS: The present study suggests that withaferin-A holds the potential to be developed as a small molecule inhibitor of STAT3 for the treatment of HCT116.

Thymoquinone (TQ), a compound isolated from black seed oil (Nigella sativa), has been reported to possess anti-inflammatory and anticancer activities. However, the molecular mechanisms underlying the anticancer effects of TQ remain poorly understood. In the present study, we found that TQ significantly reduced the viability of human colon cancer HCT116 cells in a concentration- and time-dependent manner. Treatment of cells with TQ induced apoptosis, which was associated with the upregulation of Bax and inhibition of Bcl-2 and Bcl-xl expression. TQ also activated caspase-9,-7, and -3, and induced the cleavage of poly-(ADP-ribose) polymerase (PARP). Pretreatment with a pan-caspase inhibitor, z-VAD-fmk, abrogated TQ-induced apoptosis by blocking the cleavage of caspase-3 and PARP. Treatment of cells with TQ also diminished the constitutive phosphorylation, nuclear localization and the reporter gene activity of signal transducer and activator of transcription-3 (STAT3). TQ attenuated the expression of STAT3 target gene products, such as survivin, c-Myc, and cyclin-D1, -D2, and enhanced the expression of cell cycle inhibitory proteins p27 and p21. Treatment with TQ attenuated the phosphorylation of upstream kinases, such as Janus-activated kinase-2 (JAK2), Src kinase and epidermal growth factor receptor (EGFR) tyrosine kinase. Pharmacological inhibition of JAK2 and Src blunted tyrosine phosphorylation of EGFR and STAT3, while treatment with an EGFR tyrosine kinase inhibitor gefitinib inhibited phosphorylation of STAT3 without affecting that of JAK2 and Src in HCT116 cells. Collectively, our study revealed that TQ induced apoptosis in HCT116 cells by blocking STAT3 signaling via inhibition of JAK2- and Src-mediated phosphorylation of EGFR tyrosine kinase.

Background Colorectal cancer is one of the most common causes of cancer death worldwide. Unfortunately, chemotherapies are limited due to many complications and development of resistance and recurrence. The T-lymphokine-activated killer cell-originated protein kinase (TOPK) is highly expressed and activated in colon cancer, and plays an important role in inflammation, proliferation, and survival of cancer cells. Therefore, suppressing TOPK activity and its downstream signaling cascades is considered to be a rational therapeutic/preventive strategy against colon cancers. Purpose 3-Deoxysappanchalcone (3-DSC), a component of Caesalpinia sappan L., is a natural oriental medicine. In this study, we investigated the effects of 3-DSC on colon cancer cell growth and elucidated its underlying molecular mechanism of targeting TOPK. Study design and methods To evaluate the effects of 3-DSC against colon cancer, we performed cell proliferation assays, propidium iodide- and annexin V-staining analyses and Western blotting. Targeting TOPK by 3-DSC was identified by a kinase-binding assay and computational docking models. Results 3-DSC inhibited the kinase activity of TOPK, but not mitogen-activated protein kinase (MEK). The direct binding of 3-DSC with TOPK was explored using a computational docking model and binding assay in vitro and ex vivo. 3-DSC inhibited colon cancer cell proliferation and anchorage-independent cell growth, and induced G2/M cell cycle arrest and apoptosis. Treatment of colon cancer cells with 3-DSC induced expression of protein that are involved in cell cycle (cyclin B1) and apoptosis (cleaved-PARP, cleaved-caspase-3, and cleaved-caspase-7), and suppressed protein expressions of extracellular signal-regulated kinase (ERK)-1/2, ribosomal S6 kinase (RSK), and c-Jun, which are regulated by the upstream kinase, TOPK. Conclusion 3-DSC suppresses colon cancer cell growth by directly targeting the TOPK- mediated signaling pathway.

Background: Colorectal cancer is one of the most common cancer death causes in the worldwide. Still systemic chemotherapies are limited to many complications and relapse. T-lymphokine-activated killer cell-originated protein kinase (TOPK) is highly expressed and activated in colon cancer, and plays important roles in inflammation, proliferation and survival of cancer cells. Therefore, suppression of the TOPK activity and its downstream signaling cascades is considered to be a rational therapeutic/preventive strategy of colon cancers. Purpose: Deoxysappanchalcone (DSC), a component of Caesalpinia sappan L., is a natural oriental medicine. In the present study, we investigated the effects of DSC on colon cancer cell growth and elucidated its underlying molecular mechanism by targeting TOPK. Study Design and Methods: To evaluate the effects of DSC on colon cancers, we performed cell proliferation assay, propidium iodide- or annexin V-staining analysis and western blotting. Targeting TOPK by DSC was identified by kinase/binding assay and computational docking models. Results: DSC inhibited the kinase activity of TOPK but not mitogen-activated protein kinase (MEK). The direct binding of DSC with TOPK was explored using a computational docking model and binding assay in vitro & ex vivo. DSC inhibited colon cancer cell proliferation, anchorage-independent cell growth, and induced G2/M cell cycle arrest and apoptosis. Treatment of colon cancer cells with DSC induced protein expressions which are involved in cell cycle (cyclin B1) and apoptosis (cleaved-PARP, cleaved-caspase-3 and cleaved-caspase-7), and suppressed protein expressions of extracellular signal-regulated kinase (ERK)-1/2, ribosomal S6 kinase (RSK) or cellular jun (c-Jun) which are regulated by the upstream kinases TOPK. Conclusion: DSC suppresses colon cancer cell growth by direct targeting TOPK-mediated signaling pathway. Citation Format: Ran Zhao, Hai Huang, Bu Young Choi, Mengqiu Song, Fanxiang Yin, Xiaorong Fu, Hanyong Chen, Jung Hyun Shim, Mee-Hyun Lee, Zigang Dong. Deoxysappanchalcone inhibits cell growth by regulation of TOPK signaling pathway in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2959.

The alteration expression of p16INK4a, a well-known cyclin-dependent kinase inhibitor involved in cancer cell cycle control, is unclear, especially under hypoxic conditions. To evaluate p16INK4a regulation, we performed a protein microarray analysis. Among 1,800 proteins, we identified hYSK1 as a novel protein that interacts with the tumor suppressor p16INK4a. hYSK1, a member of the Ste20 family of serine/threonine protein kinases, promotes cell migration & tumorigenesis and is activated by oxidative stress. However, the molecular mechanisms underlying the oncogenic potential of hYSK1 remain elusive. Here, we report here that hYSK1 interacts with p16INK4a under hypoxic conditions like tumors, where it negatively regulates p16INK4a, enhancing cancer cell migration. Hypoxic stimulation of hYSK1 reduces p16INK4a accumulation through p16 promoter regulation to interact with unphosporylated SP-1 & increases matrix metalloproteinase-2 (MMP-2) expression by activating the MMP-2 promoter associated with cell migration and proliferation.Conversely, knocking down hYSK1 expression activated p16INK4a expression & suppressed MMP-2 expression.Thus, hYSK1 is necessary as a trigger for inactivating p16INK4a & activating MMP-2 during tumor migration, suggesting that hYSK1 is a specific negative regulator of the tumor suppressor p16INK4a may represent a novel molecular target for reactivation of tumor suppressor genes in humans. Citation Format: Mee-Hyun Lee, Jung-Hyun Shim, Zigang Dong, Young-Joon Surh, Bu Young Choi. Down-regulation of p16INK4a by hYSK1 promotes cancer cell migration under hypoxic conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1718.

Apple is a rich source of bioactive phytochemicals that help improve health by preventing and/or curing many disease processes, including cancer. One of the apple polyphenols is phloretin [2′,4′,6′-Trihydroxy-3-(4-hydroxyphenyl)-propiophenone], which has been widely investigated for its antioxidant, anti-inflammatory and anti-cancer activities in a wide array of preclinical studies. The efficacy of phloretin in suppressing xenograft tumor growth in athymic nude mice implanted with a variety of human cancer cells, and the ability of the compound to interfere with cancer cells signaling, have made it a promising candidate for anti-cancer drug development. Mechanistically, phloretin has been reported to arrest the growth of tumor cells by blocking cyclins and cyclin-dependent kinases and induce apoptosis by activating mitochondria-mediated cell death. The blockade of the glycolytic pathway via downregulation of GLUT2 mRNA and proteins, and the inhibition of tumor cells migration, also corroborates the anti-cancer effects of phloretin. This review sheds light on the molecular targets of phloretin as a potential anti-cancer and anti-inflammatory natural agent.

The c-jun-NH2-kinases (JNK) play a critical role in tumor promoter–induced cell transformation and apoptosis. Here, we showed that the nuclear factor of activated T3 (NFAT3) is phosphorylated by JNK1 or JNK2 at Ser213 and Ser217, which are located in the conserved SP motif. The transactivation domain of NFAT3 is found between amino acids (aa) 113 and 260 and includes the phosphorylation targets of JNK1 and JNK2. NFAT3 transactivation activity was suppressed in JNK1−/− or JNK2−/− mouse embryonic fibroblast (MEF) cells compared with wild-type MEF cells. Moreover, a 3xNFAT-luc reporter gene assay indicated that NFAT3 transcriptional activity was increased in a dose-dependent manner by JNK1 or JNK2. Double mutations at Ser213 and Ser217 suppressed NFAT3 transactivation activity; and SP600125, a JNK inhibitor, suppressed NFAT3-induced 3xNFAT-luciferase activity. Knockdown of JNK1 or JNK2 suppressed foci formation in NIH3T3 cells. Importantly, ectopic expression of NFAT3 inhibited AP-1 activity and suppressed foci formation. Furthermore, knockdown of NFAT3 enhanced Ras-JNK1 or JNK2-induced foci formation in NIH3T3 cells. Taken together, these results provided direct evidence for the anti-oncogenic potential of the NFAT3 transcription factor. [Cancer Res 2007;67(18):8725–35]

In the course of screening for novel anticancer compounds, CR229 (6-Bromo-2,3,4,9-tetrahydro-carbolin-1-one), a novel derivative of beta-carbolin-1-one, was generated as a new scaffold candidate. For the first time, the authors demonstrate that CR229 inhibited the growth of HeLa cells by the induction of cell cycle arrest and apoptosis. Analysis of flow cytometry and western blots of HeLa cells treated with 2.5 microM CR229 revealed an appreciable cell cycle arrest in the G1, G2/M phase and apoptotic induction via the p53-dependent pathway. Furthermore, the release of cytochrome c from mitochondria was detected using confocal microscopy in HeLa cells treated with CR229. Accordingly, these data demonstrate that the anticancer activity of CR229 is associated with: (i) the down-regulation of cyclins and cyclin-dependent kinase; (ii) the induction of p53, p21, and p16; and (iii) the activation of caspase-3.

Acetylshikonin, a natural naphthoquinone derivative compound, has been used for treatment of inflammation and cancer. In the present study, we have investigated whether acetylshikonin could regulate the NF-κB signaling pathway, thereby leading to suppression of tumorigenesis. We observed that acetylshikonin significantly reduced proliferation of several cancer cell lines, including human pancreatic PANC-1 cancer cells. In addition, acetylshikonin inhibited phorbol 12-myristate 13-acetate (PMA) or tumor necrosis-α (TNF-α)-induced NF-κB reporter activity. Proteome cytokine array and real-time RT-PCR results illustrated that acetylshikonin inhibition of PMA-induced production of cytokines was mediated at the transcriptional level and it was associated with suppression of NF-κB activity and matrix metalloprotenases. Finally, we observed that an exposure of acetylshikonin significantly inhibited the anchorage-independent growth of PANC-1 cells. Together, our results indicate that acetylshikonin could serve as a promising therapeutic agent for future treatment of pancreatic cancer.

The oncoprotein c-Jun is a component of the activator protein-1 transcription factor complex, which is involved in cellular proliferation, transformation, and death. The stabilization of c-Jun is critically important for its function. The phosphorylation of c-Jun by c-Jun NH2-terminal kinase 1 and extracellular signal-regulated protein kinases reduces c-Jun ubiquitination resulting in increased stabilization of c-Jun. In this report, we showed that COOH-terminal Src kinase (CSK) binds with and phosphorylates c-Jun at Y26 and Y170. Phosphorylation of c-Jun by CSK, in opposition to c-Jun NH2-terminal kinase 1 and extracellular signal-regulated protein kinases, promoted c-Jun degradation and reduced stability. By promoting c-Jun degradation, CSK helps to maintain a low steady-state level of c-Jun, thereby inhibiting activator protein-1 activity and cell transformation caused by c-Jun. These results indicated that this function of CSK controls cell proliferation under normal growth conditions and may have implications for CSK loss of function in carcinogenesis. (Cancer Res 2006; 66(11): 5729-36)

Background The apoptotic activity of methanol extracts of Impatiens balsamina L. (MEIB) and related mechanisms in human oral squamous cell carcinoma (OSCC) cells have been systematically investigated. Methods The effects of MEIB on human OSCC cell lines were investigated using trypan blue exclusion assay, MTS assay, Western blot, 4′-6-diamidino-2-phenylindole (DAPI) staining, Live/Dead assay, Immunohistochemistry, reverse transcription–polymerase chain reaction, and promoter assay. Results MEIB decreased cell viability and induced apoptosis in HSC-4 cells. Higher levels of p-Akt expression were observed in OSCC than in normal oral mucosa (NOM), and it correlated with poor survival of the patients. MEIB dephosphorylated p-Akt and decreased Akt expression through proteasome-dependent degradation. LY294002 (PI3K inhibitor) decreased p-Akt and Akt, resulting in enhancing MEIB-induced apoptosis. MEIB down-regulated the expression level of survivin protein at the transcriptional level and YM155 (survivin inhibitor) decreased survivin, which facilitated MEIB-induced apoptosis. MEIB and LY294002 significantly increased Bax, thereby inducing the conformational change, mitochondrial translocation, and oligomerization. In addition, MEIB-induced growth inhibition and apoptosis in OSC-20, another human OSCC cells were mediated by regulating Akt and it downstream targets, survivin and Bax. Conclusions These results suggest that MEIB may serve as a potential drug candidate for the treatment of human OSCC.

We have demonstrated that clofilium, a potassium channel blocker, induces apoptosis on human promyelocytic leukemia (HL-60) cells. Cells treated with clofilium led to suppression of viability and proliferation in both time and concentration-dependent manners. Nuclear DAPI staining and electronmicroscopic examination revealed typical nuclear features of apoptosis in cells treated with clofilium that was further verified in DNA fragmentation analysis. Flow cytometry analysis with FITC-annexin V and propidium iodide (PI) revealed that apoptotic cell population with Annexin V+/PI- increased gradually from2% at 0 h, to 20% at 4 h and 29% at 16 h after exposure to 10 microM clofilium in HL-60 cells. Furthermore, fluorometric immunosorbent enzyme assay for activity of caspase-3 showed approximately a 10-fold increase of activity in cells treated with 10 microM of clofilium for 2-3 h compared with the basal level of its activity in untreated control cells. Immunoblotting analysis revealed proteolytic cleavage of caspase-3 and subsequent cleavage of PARP. However, there was no significant change of Bcl-2 and Bax proteins. These results indicate that clofilium exerts antiproliferative action and growth inhibition on HL-60 through induction of apoptosis which is mediated via Bcl-2-insensitive activation of caspase-3, and suggest chemotherapeutic and cytostatic potentials of this compound in human leukemias.

The tumor suppressor protein p16(INK4a) is a member of the INK4 family of cyclin-dependent kinase (Cdk) inhibitors, which are involved in the regulation of the eukaryotic cell cycle. However, the mechanisms underlying the anti-proliferative effects of p16(INK4a) have not been fully elucidated. Using yeast two-hybrid screening, we identified the eukaryotic elongation factor (eEF)1A2 as a novel interacting partner of p16(INK4a). eEF1A2 is thought to function as an oncogene in cancers. The p16(INK4a) protein interacted with all but the D2 (250-327 aa) domain of eEF1A2. Ectopic expression of p16(INK4a) decreased the expression of eEF1A2 and inhibited cancer cell growth. Furthermore, suppression of protein synthesis by expression of p16(INK4a) ex vivo was verified by luciferase reporter activity. Microinjection of p16(INK4a) mRNA into the cytoplasm of Xenopus embryos suppressed the luciferase mRNA translation, whereas the combination of p16(INK4a) and morpholino-eEF1A2 resulted in a further reduction in translational activity. We conclude that the interaction of p16(INK4a) with eEF1A2, and subsequent downregulation of the expression and function of eEF1A2 is a novel mechanism explaining the anti-proliferative effects of p16(INK4a).

Peptidyl prolyl isomerase (PIN1) regulates the functional activity of a subset of phosphoproteins through binding to phosphorylated Ser/Thr-Pro motifs and subsequently isomerization of the phosphorylated bonds. Interestingly, PIN1 is overexpressed in many types of malignancies including breast, prostate, lung and colon cancers. However, its oncogenic functions have not been fully elucidated. Here, we report that PIN1 directly interacts with hypoxia-inducible factor (HIF)-1 alpha in human colon cancer (HCT116) cells. PIN1 binding to HIF-1 alpha occurred in a phosphorylation-dependent manner. We also found that PIN1 interacted with HIF-1 alpha at both exogenous and endogenous levels. Notably, PIN1 binding stabilized the HIF-1 alpha protein, given that their levels were significantly increased under hypoxic conditions. The stabilization of HIF-1 alpha resulted in increased transcriptional activity, consequently upregulating expression of vascular endothelial growth factor, a major contributor to angiogenesis. Silencing of PIN1 or pharmacologic inhibition of its activity abrogated the angiogenesis. By utilizing a bioluminescence imaging technique, we were able to demonstrate that PIN1 inhibition dramatically reduced the tumor volume in a subcutaneous mouse xenograft model and angiogenesis as well as hypoxia-induced transcriptional activity of HIF-1 alpha. These results suggest that PIN1 interacting with HIF-1 alpha is a potential cancer chemopreventive and therapeutic target.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is latent but constitutively activated in many types of cancers. It is well known that STAT3 plays a key role in inflammation-associated tumorigenesis. Curcumin, a natural compound isolated from the traditional Indian spice turmeric (Curcuma longa.,Zingiberaceae), has anti-tumor, anti-proliferative and anti-inflammatory properties. This compound has been extensively used in traditional herbal medicine over the centuries mainly due to its pronounced anti-inflammatory effects. Many studies suggest that curcumin regulates various cancer-related intracellular signaling pathways without damaging normal cells and tissues. Here, we report that curcumin effectively inhibits activation of STAT3 in dose- and time-dependent manners and causes apoptosis of H-Ras transformed breast epithelial cells (H-Ras MCF10A). Specific cysteine residues present in STAT3 appear to be critical for the activity as well as conformation of this transcription factor. We identified the cysteine residue 259 as a putative target of curcumin. We found that the α,β-unsaturated carbonyl moiety of curcumin appears to be essential in its direct binding to STAT3. Tetrahydrocurcumin that lacks the electrophilic α,β-unsaturated carbonyl moiety failed to inhibit STAT3 activation and to induce apoptosis in the same cell line. Taken together, our findings suggest that curcumin can abrogate persistently activated STAT3 signaling through direct interaction, thereby inhibiting STAT3-mediated mammary carcinogenesis. Citation Format: Young-Il Hahn, Su-Jung Kim, Bu-Young Choi, Hye-Kyung Na, Young-Joon Surh. Curcumin induces apoptosis in H-Ras transformed human mammary epithelial cells: Cysteine 259 of STAT3 as a putative target. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1892. doi:10.1158/1538-7445.AM2015-1892

Many beneficial properties have been attributed to (−)−epigallocatechin gallate (EGCG), including chemopreventive, anticarcinogenic, and antioxidant actions. In this study, we investigated the effects of EGCG on the function of glucose-regulated protein 78 (GRP78), which is associated with the multidrug resistance phenotype of many types of cancer cells. Our investigation was directed at elucidating the mechanism of the EGCG and GRP78 interaction and providing evidence about whether EGCG modulates the activity of anticancer drugs through the inhibition of GRP78 function. We found that EGCG directly interacted with GRP78 at the ATP-binding site of protein and regulated its function by competing with ATP binding, resulting in the inhibition of ATPase activity. EGCG binding caused the conversion of GRP78 from its active monomer to the inactive dimer and oligomer forms. Further, we showed that EGCG interfered with the formation of the antiapoptotic GRP78-caspase-7 complex, which resulted in an increased etoposide-induced apoptosis in cancer cells. We also showed that EGCG significantly suppressed the transformed phenotype of breast cancer cells treated with etoposide. Overall, these results strongly suggested that EGCG could prevent the antiapoptotic effect of GRP78, which usually suppresses the caspase-mediated cell death pathways in drug-treated cancer cells, contributing to the development of drug resistance. (Cancer Res 2006; 66(18): 9260-9) (Cancer Res 2006; 66(18): 9260-9)

Very little is known about the role of histone H3 phosphorylation in malignant transformation and cancer development. Here, we examine the function of H3 phosphorylation in cell transformation in vivo. Introduction of small interfering RNA-H3 into JB6 cells resulted in decreased epidermal growth factor (EGF)–induced cell transformation. In contrast, wild-type histone H3 (H3 WT)–overexpressing cells markedly stimulated EGF-induced cell transformation, whereas the H3 mutant S10A cells suppressed transformation. When H3 WT was overexpressed, EGF induction of c-fos and c-jun promoter activity was significantly increased compared with control cells but not in the H3 mutant S10A or S28A cells. In addition, activator protein-1 activity in H3 WT–overexpressing cells was markedly up-regulated by EGF in contrast to the H3 mutant S10A or S28A cells. These results indicate that the phosphorylation of histone H3 at Ser10 is an essential regulatory mechanism for EGF-induced neoplastic cell transformation.

Previously, no member of the mixed-lineage kinase (MLK) protein family was known to function as an oncogene. Here, we demonstrate that MLK-like mitogen-activated protein triple kinase (MLTK)-α, a member of the MLK family, induced neoplastic cell transformation and tumorigenesis in athymic nude mice. Introduction of small interference RNA (siRNA)-MLTK-α into MLTK-α-overexpressing cells dramatically suppressed cell transformation. Nuclear accumulation of the pHisG-MLTK-α fusion protein was observed after epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate treatment. Phosphorylation of downstream mitogen-activated protein kinase-targeted transcription factors including c-Myc, Elk-1, c-Jun, and activating transcription factor (ATF) 2 was also differentially enhanced in MLTK-α-overexpressing cells exposed to epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate stimulation compared with cells expressing mock vector or siRNA-MLTK-α. Very importantly, MLTK-α-overexpressing cells formed fibrosarcomas when injected s.c. into athymic nude mice, whereas almost no tumor formation was observed in mice that received injections of mock or siRNA-MLTK-α stably transfected cells. These results are the first to indicate that MLTK-α plays a key role in neoplastic cell transformation and cancer development.

The tumor suppressor protein p16INK4a is a member of the INK4 family of cyclin-dependent kinase (Cdk) inhibitors, which are involved in the regulation of the eukaryotic cell cycle. However, the mechanisms underlying the anti-proliferative effects of p16INK4a have not been fully elucidated. Using yeast two-hybrid screening, we identified the eukaryotic elongation factor (eEF)1A2 as a novel interacting partner of p16INK4a. eEF1A2 is thought to function as an oncogene in cancers. The p16INK4a protein interacted with all but the D2 (250-327 aa) domain of eEF1A2. Computational docking study predicted that D24/D131 residues of p16INK4a interacted with eEF1A2 and it was confirmed by pull-down assay with mutant p16INK4a (D24A/D131E). Ectopic expression of p16INK4a decreased the expression of eEF1A2 and inhibited cancer cell growth. Furthermore, suppression of protein synthesis by expression of p16INK4a ex vivo was verified by luciferase reporter activity. Microinjection of p16INK4a mRNA into the cytoplasm of Xenopus embryos suppressed the luciferase mRNA translation, whereas the combination of p16INK4a and morpholino-eEF1A2 resulted in a further reduction in translational activity. We conclude that the interaction of p16INK4a with eEF1A2, and subsequent downregulation of the expression and function of eEF1A2 is a novel mechanism explaining the anti-proliferative effects of p16INK4a. Citation Format: Mee-Hyun Lee, Bu Young Choi, Yong-Yeon Cho, Myoung Ok Kim, Sung-Hyun Kim, Cheol-Jung Lee, Ji Hong Song, Ann M. Bode, Zigang Dong, Yong-Joon Surh. Direct down-regulation of eEF1A2 by Tumor suppressor p16INK4a inhibits cancer cell growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2471. doi:10.1158/1538-7445.AM2014-2471

Aberrant gene silencing is highly associated with altered cell cycle regulation during carcinogenesis. In particular, silencing of the CDKN2A tumor suppressor gene, which encodes the p16INK4a protein, has a causal link with several different types of cancers. The p16INK4a protein plays an executional role in cell cycle and senescence through the regulation of the cyclin-dependent kinase (CDK) 4/6 and cyclin D complexes. Several genetic and epigenetic aberrations of CDKN2A lead to enhanced tumorigenesis and metastasis with recurrence of cancer and poor prognosis. In these cases, the restoration of genetic and epigenetic reactivation of CDKN2A is a practical approach for the prevention and therapy of cancer. This review highlights the genetic status of CDKN2A as a prognostic and predictive biomarker in various cancers., Highlights • The status of CDKN2A provides epigenetic/genetic information for the cancer patient. • The correlation of p16INK4a and related biomarkers should be considered for prognosis of cancers. • Epigenetic/genetic modulation of changes in CDKN2A might be a promising cancer preventive/therapeutic strategy.