Rhein exhibits antitumorigenic effects by interfering with the interaction between prolyl isomerase Pin1 and c-Jun

Cho, Jin Hyoung,Chae, Jung-Il,Shim, Jung-Hyun NATIONAL HELLENIC RESEARCH FOUNDATION 2017 ONCOLOGY REPORTS Vol.37 No.3

<P>The Pinl protein (or peptidyl-prolyl cis/trans isomerase) specifically catalyzes the cis/trans isomerization of phosphorylated serine/threonine-proline (Ser/Thr-Pro) bonds and plays an important role in many cellular events through the effects of conformational change in the function of c-Jun, its biological substrate. Pinl expression is involved in essential cellular pathways that mediate cell proliferation, cell cycle progression, tumorigenesis and apoptosis by altering their stability and function, and it is overexpressed in various types of tumors. Pinl phosphorylation has been regarded as a marker of Pinl isomerase activity, and the phosphorylation of Ser/Thr-Pro on protein substrates is prerequisite for its binding activity with Pinl and subsequent isomerization. Since phosphorylation of proteins on Ser/Thr-Pro is a key regulatory mechanism in the control of cell proliferation and transformation, Pinl has become an attractive molecule in cancer research. Many inhibitors of Pinl have been discovered, including several classes of both designed inhibitors and natural products. Anthraquinone compounds possess antitumor properties and have therefore been applied in human and veterinary therapeutics as active substances in medicinal products. Among the anthraquinones, rhein (4,5-dihydroxy9,10-dioxoanthracene-2-carboxylic acid) is a monomeric anthraquinone derivative found mainly in plants in the Polygonaceae family, such as rhubarb and Polygonum cuspidatum. Recent studies have shown that rhein has numerous pharmacological activities, including antitumor effects. Here, we demonstrated the antitumorigenic effect of rhein using cell proliferation assay, anchorage-independent cell transformation, pull-down assay, luciferase promoter activity, fluorescence-activated cell sorting and western blot analysis. The rhein/Pinl association was found to play a regulatory role in cell proliferation and neoplastic cell transformation and the binding of phosphorylated c-Jun (Ser73) with Pinl was markedly decreased and inhibited activator protein 1 or NF-kappa B reporter activity by rhein. Overall, our findings and the accompanying biochemical data demonstrated the antitumorigenic effect of rhein through its interference in Pinl/c-Jun interaction and suggest the possible use of rhein in suppressing the tumor-promoting effects of Pinl. Therefore, rhein may have practical implications for cancer prevention or therapy.</P>

The effect of aberrant maspin expression on the invasive ability of pancreatic ductal adenocarcinoma cells.

Hong, Sung Noh,Lee, Jong Kyun,Choe, Won Hyoek,Ha, Hye Young,Park, Kyoungsook,Sung, In Kyung,Lee, Kyu Taek,Kim, Jae J,Rhee, Jong Chul National Hellenic Research Foundation 2009 ONCOLOGY REPORTS Vol.21 No.2

<P>Maspin is expressed aberrantly in pancreatic ductal adenocarcinoma (PDAC), and its function is still unknown. To explore the role of maspin in PDAC, we constructed wild-type maspin-expressing Panc-1 clones (Panc-1-maspin) by transfecting maspin cDNA into Panc-1, a PDAC-derived cell line that lacks maspin expression. As a control, mock-transfected clones (Panc-1-mock) were constructed using the same method. The invasive ability of the stable transfectants was evaluated with an in vitro invasion assay. The ability of Panc-1-maspin cells to migrate through a Matrigel-coated filter was significantly reduced compared to that of Panc-1-mock cells (p=0.012). In addition, we identified the c.1022A>G variant of maspin in human PDAC cells; however, this polymorphism was not involved in the clinical characteristics of PDAC nor did it alter the invasive ability of PDAC cells. The results of the present study indicate that maspin suppresses the invasive ability of PDAC cells.</P>

Heat-processed neoginseng, KG-135, down-regulates G1 Cyclin-dependent kinase through the proteasome-mediated pathway in HeLa cells.

Lee, Won-Hee,Choi, Joon-Seok,Kim, Hyun Young,Park, Jeong-Hill,Lee, Seung-Ki,Surh, Young-Joon National Hellenic Research Foundation 2009 ONCOLOGY REPORTS Vol.21 No.2

<P>High temperature heat treatment of ginseng (Panax ginseng, C.A. Meyer) generates KG-135 (heat-processed neoginseng) which contains a mixture of three major ginseng saponins, ginsenosides Rk1, Rg3 and Rg5. Ginsenosides, particularly of the diol-type including Rk1, Rg3 and Rg5, have been shown to induce cell growth arrest in various cell types of human cancer. Herein, we report that KG-135 is able to arrest the cell cycle in human cervix adenocarcinoma HeLa cells. KG-135 arrests cells at the G1 phase of the cell cycle with an IC50 value of 69 microg/ml. The G1 phase arrest is associated with down-regulation of Cyclin D1/Cdk4 and Cyclin B1/Cdc2 activities in cells after treatment with KG-135. Furthermore, down-regulation of G1 Cyclin-dependent kinase activities is kinetically well related to the decreased intracellular protein levels of these kinases. In addition, the decrease in the levels of Cyclin D1/Cdk4 and Cyclin B1, but not of Cdc2, is similarly prevented by co-treatment of cells with MG-132, a potent proteasome inhibitor. Thus, the KG-135-induced arrest of the cell cycle at G1 phase in HeLa cells represents a novel mechanism that involves proteasome-mediated degradation of the Cyclins (Cyclin D1 and B1) and Cdk4 proteins.</P>

Enhanced apoptotic effect of combined modality of 9-hydroxypheophorbide alpha-mediated photodynamic therapy and carboplatin on AMC-HN-3 human head and neck cancer cells.

He, Peijie,Ahn, Jin-Chul,Shin, Jang-In,Hwang, Hee-Jun,Kang, Jung-Wook,Lee, Sang-Joon,Chung, Phil-Sang National Hellenic Research Foundation 2009 ONCOLOGY REPORTS Vol.21 No.2

<P>Photodynamic therapy (PDT) has been developed as an effective treatment for malignant disease. Carboplatin (CBDCA), a less nephrotoxic analog of cisdiamminedichloroplatinum (cisplatin), has been widely used for the treatment of multiple malignancies. In this study, we investigated the cytotoxic and apoptotic effect of combined modality of 9-hydroxypheophorbide alpha (9-HPbD)-mediated PDT and CBDCA on AMC-HN-3 human head and neck cancer cell line in vitro. The attached AMC-HN-3 cells were incubated with CBDCA (0.04 mg/ml) for 24 h at 37 degrees C and followed by photosensitization with 9-HPbD for 6 h and laser irradiation with 670 nm diode laser at an intensity of 2.0 J/cm(2) for activating 9-HPbD for 15 min. Then MTT reduction assay and Hoechst 33342 and propidium iodide (PI) double staining were used respectively to measure the cytotoxicity and nuclear morphology at 24 h after PDT. Expression of caspase-3, -9 and poly(ADP-ribose) polymerase (PARP) was detected at 0, 3, 6 and 12 h after irradiation through Western blotting techniques. Compared with PDT and CBDCA alone groups, there was more cytotoxicity and enhanced apoptotic cell death in combination groups. The peaked expression of cleaved form of caspase-3, -9 and PARP occurred approximately 3 h after PDT. There was stronger expression of cleaved caspase-3, -9 and PARP in combination groups than that in PDT or CBDCA alone groups. This study demonstrates that the combined modality resulted in enhanced apoptotic cell death as well as cytotoxic effect on AMC-HN-3 cells in vitro, which suggests the feasibility of combined modality and the possibility of reducing the effective dosage of 9-HPbD and CBDCA and lowering the side effects on normal cells.</P>

(-)-Epigallocatechin-3-gallate inhibits invasion and migration of salivary gland adenocarcinoma cells.

Park, Jong-Hwan,Yoon, Ji-Hye,Kim, Soo-A,Ahn, Sang-Gun,Yoon, Jung-Hoon National Hellenic Research Foundation 2010 ONCOLOGY REPORTS Vol.23 No.2

<P>(-)-Epigallocatechin-3-gallate (EGCG) has inhibitory effect on a variety of cancers by inducing apoptosis and cell cycle arrest or inhibiting angiogenesis and metastasis. EGCG has been found to induce apoptosis in salivary gland carcinoma cells, however, it is not known whether EGCG affects invasion and migration. Thus, this study was performed to clarify whether EGCG affects invasion and migration of salivary gland tumors. Matrigel invasion assay, wound scratch assay and migration assay using commercial kit were performed. beta1 integrin expression and activation of its downstream molecules such as focal adhesion kinase (FAK), AKT and extracellular signal-regulated kinase (ERK) were examined by Western blot. Enzymatic activity of matrix metalloprotease (MMP)-2 and MMP-9 was examined by gelatin zymography. EGCG inhibited effectively invasion and migration of SGT cells in a dose-dependent manner. EGCG also inhibited the activation of beta1 integrin-downstream molecules such as FAK, AKT and ERK as well as the expression of beta1 integrin itself. Moreover, MMP-2 and MMP-9 expression and their enzymatic activity were reduced by EGCG in a dose-dependent manner. These results indicate that EGCG may effectively suppress salivary gland tumors by inhibiting metastasis through beta1 integrin-mediated signaling.</P>

Pyrogallol-induced endothelial cell death is related to GSH depletion rather than ROS level changes.

Han, Yong Hwan,Moon, Hwa Jin,You, Bo Ra,Kim, Sung Zoo,Kim, Suhn Hee,Park, Woo Hyun National Hellenic Research Foundation 2010 ONCOLOGY REPORTS Vol.23 No.1

<P>Pyrogallol (PG) as a polyphenol compound induces apoptosis in several types of cells. Here, we evaluated the effects of PG on endothelial cells (ECs), especially calf pulmonary artery endothelial cells (CPAEC) in relation to the cell growth, ROS and glutathione (GSH) levels. PG dose-dependently inhibited the growth of CPAEC and human umbilical vein endothelial cells (HUVEC) at 24 h. PG also induced apoptosis in CPAEC, which was accompanied by the loss of mitochondrial membrane potential (MMP; DeltaPsim). PG decreased ROS level including O2*- and PG dose-dependently increased GSH depleted cell number in both EC types. N-acetyl-cysteine (NAC; a well-known antioxidant) increased ROS levels in PG-treated CPAEC with the prevention of cell death and GSH depletion. In conclusion, PG inhibited the growth of ECs, especially CPAEC via apoptosis. PG-induced EC death was related to GSH depletion rather than ROS level changes.</P>

Effects and action mechanism of Diospyros kaki on the differentiation of human leukemia HL-60 cells.

Kim, Seung Hyun,Cho, Seung Sik,Simkhada, Jaya Ram,Park, Sung Ju,Lee, Hyo Jeong,Kim, Tae Sung,Yoo, Jin Cheol National Hellenic Research Foundation 2010 ONCOLOGY REPORTS Vol.23 No.1

<P>Diospyros kaki Thunb. (Ebenaceae) is widely distributed in North-East Asian countries. Almost all parts of this plant have been traditionally used as medicine. Human promyelocytic leukemia cells differentiate into monocytes or granulocytes when treated with 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] or all-trans retinoic acid (ATRA). Combination of low doses of ATRA or 1,25-dihydroxyvitamin D3 that do not induce toxicity with another drug is a useful strategy for acute promyelocytic leukemia therapy. Our main aim was to investigate the effect of an acetone extract of D. kaki leaves (KV-1) on HL-60 cell differentiation in combination of ATRA or 1,25-dihydroxyvitamin D3. Treatment of HL-60 cells with zero to 100 microg/ml of KV-1 for 72 h induced a small increase in cell differentiation. Surprisingly, a synergistic induction of differentiation was observed when the HL-60 cells were treated with ATRA or 1,25-(OH)2D3 and the extract. The inhibitors of protein kinase C (PKC) (alpha and betaI) and extracellular signal-regulated kinase (ERK), but not of phosphoinositide 3-kinase (PI3-K) and c-Jun N-terminal kinase (JNK) inhibited the HL-60 differentiation induced by the extract in combination of ATRA or 1,25-(OH)2D3, suggesting that PKC and ERK were involved in the cell differentiation enhancement by the extract. The results indicate that the acetone extract of D. kaki leaves has the ability to enhance HL-60 cell differentiation and suggest that it may be useful in acute promyelocytic leukemia therapy.</P>

Anthocyanins are novel AMPK관1 stimulators that suppress tumor growth by inhibiting mTOR phosphorylation.

Lee, Yun-Kyoung,Lee, Won Sup,Kim, Gon Sup,Park, Ock Jin National Hellenic Research Foundation 2010 ONCOLOGY REPORTS Vol.24 No.6

<P>AMP-activated protein kinase (AMPK) has emerged as a therapeutic target of cancer. AMPK functions as an upstream regulator of proliferative signals such as mammalian target of rapamycin (mTOR), tuberous sclerosis complex (TSC), p70S6 and elongation factor-2, indicating that AMPK can be applied for the inhibition of cancer cell proliferation via modulating the proliferative signaling network. The Akt/mTOR signaling pathway is activated in colon cancer. The well known mTOR inhibitor rapamycin has a disadvantage of feedback stimulation of Akt. Anthocyanins are naturally-occurring mTOR inhibitor possessing Akt inhibitory activities. We have investigated the mTOR inhibitory effect of anthocyanins through the activation of AMPK. In this study, anthocyanins were applied to colon cancer cells and tumor-bearing xenograft models to investigate their anti-proliferative and pro-apoptotic effects, and elucidate the mechanisms that link AMP-activated protein kinase (AMPK) 관1 activation to the survival signal of mTOR. Our results indicated that anthocyanins significantly decreased phospho-mTOR comparable to rapamycin, a synthetic mTOR inhibitor, and this inhibitory effect of anthocyanins on mTOR was completely abrogated by inactivating AMPK관1. Furthermore, suppression of cell growth with anthocyanins was also alleviated in the absence of noticeable AMPK관1 activities. For the first time we have found anthocyanins as novel AMPK관1 activators, and in conditions of AMPK관1 inactivation, anthocyanins lost their ability to inhibit mTOR in HT-29 colon cancer cells. The activation of AMPK관1, and the deactivation of mTOR and Akt were observed in anthocyanins-treated tumor-bearing xenograft models. The results from this study suggest that there is a complex interaction between AMPK관1 and mTOR signaling, and anthocyanins are powerful AMPK관1 activators that inhibit cancer cell growth by inhibiting mTOR phosphorylation.</P>

The effects of N-acetyl cysteine, buthionine sulfoximine, diethyldithiocarbamate or 3-amino-1,2,4-triazole on antimycin A-treated Calu-6 lung cells in relation to cell growth, reactive oxygen species and glutathione.

Han, Yong Hwan,Park, Woo Hyun National Hellenic Research Foundation 2009 ONCOLOGY REPORTS Vol.22 No.2

<P>Antimycin A (AMA) inhibits mitochondrial electron transport between cytochrome b and c. We recently demonstrated that AMA inhibits the growth of lung cancer Calu-6 cells and the changes of reactive oxygen species (ROS) and glutathione (GSH) levels affect apoptosis in Calu-6 cells. Here, we examined the effects of N-acetyl-cysteine (NAC, a well known antioxidant), L-buthionine sulfoximine (BSO, an inhibitor of GSH synthesis), diethyl-dithiocarbamate (DDC, an inhibitor of Cu, Zn-SOD) or 3-amino-1,2,4-triazole (AT, an inhibitor of catalase) on AMA-treated Calu-6 cells in relation to cell death, ROS and GSH levels. Treatment with AMA induced cell growth inhibition, apoptosis and the loss of mitochondrial membrane potential (MMP) (DeltaPsim) in Calu-6 cells. While the intracellular ROS level was decreased in 50 microM AMA-treated Calu-6 cells, O2.- levels among ROS were significantly increased. AMA also induced GSH depletion in Calu-6 cells. Treatment with NAC showed decreasing effect on O2.- levels in AMA-treated cells preventing apoptosis, MMP (DeltaPsim) loss and GSH depletion in these cells. BSO significantly increased GSH depletion and apoptosis in AMA-treated cells. While both DDC and AT increased ROS levels in AMA-treated Calu-6 cells, only DDC intensified GSH depletion and apoptosis. BSO and AT increased the ROS level in Calu-6 control cells, but these agents did not induce apoptosis and GSH depletion. In conclusion, our results suggest that GSH depletion rather than ROS level in AMA-treated Calu-6 cells is more tightly related to apoptosis.</P>

Multiplex genotyping of 1107 SNPs from 232 candidate genes identified an association between IL1A polymorphism and breast cancer risk.

Han, Wonshik,Kang, So Young,Kang, Daehee,Park, Sue K,Lee, Ji-Young,Kim, Ho,Park, Ae Kyung,Noh, Dong-Young National Hellenic Research Foundation 2010 ONCOLOGY REPORTS Vol.23 No.3

<P>We sought to identify genes and polymorphisms associated with breast cancer risk among Korean women using multiplex genotyping. The SNPs (n=1536) of 264 candidate genes were genotyped using the Illumina Golden Gate assay. These genes are involved in the pathways controlling apoptosis/anti-apoptosis, the immune and inflammatory responses, cytokines, DNA repair, cell adhesion, and cell cycle/proliferation. Breast cancer cases (n=209) were recruited from Seoul National University Hospital. Age-matched control subjects (n=209) were selected from a health examinees cohort. Gene-based and SNP-based tests were performed. The final analysis includes 117 cases and 164 controls with 1107 SNPs in 232 genes. Gene-based analyses showed that IL1A, TNFRSF10B, TNFRSF1B, ICAM, and TNFSF9 were significantly associated with breast cancer risk (p<0.01). IL1A was the most significant gene associated with breast cancer risk [p for likelihood ratio test, 1 degree of freedom (df)=6x10(-7); FDR-adjusted p-value, 1df=4x10(-4), 2df=0.0071, respectively]. Individual SNP-based analyses revealed that the rare allele of the IL1A SNP rs2856836, Ex7-592Tright curved arrow C, was strongly associated with breast cancer risk (FDR-adjusted p-value, 1df=0.0027, 2df=0.0162). This SNP was found to increase risk for breast cancer [odds ratio (OR)=2.88, 95% confidence interval (CI)=1.58-5.27 for heterozygote and OR=8.17, 95% CI=2.23-29.99 for rare homozygote]. In summary, we identified a common genetic variant in IL1A strongly associated with breast cancer risk.</P>