Bavachalcone from Cullen corylifolium induces apoptosis and autophagy in HepG2 cells.

BACKGROUND: Cullen corylifolium is a plant widely used in traditional Chinese medicine for its stomachic, anthelmintic, and diuretic properties. Bavachalcone, which is known as a component of C. corylifolium has been reported to inhibit osteoclast differentiation. However, the anticancer efficacy and mechanism of C. corylifolium and bavachalcone have not been studied. HYPOTHESIS/PURPOSE: Our aim is to determine whether C. corylifolium has an anticancer effect and to identify the apoptosis and autophagy mechanism of bavachalcone. STUDY DESIGN/METHOD: The anti-proliferative activity of C. corylifolium and bavachalcone was measured with MTT assay. Apoptosis was analyzed using Annexin V and propidium iodide staining. The expression of apoptosis, cell cycle and autophagy related gene was evaluated by western blot. Cell cycle stage was investigated with TaliⓇ image-based cytometer. Autophagic activity was assessed using monodansylcadaverine (MDC) staining. RESULT: C. corylifolium exhibited potent effect on apoptosis in HepG2 cells. To identify which compound in C. corylifolium is responsible for this effect, we determined the effects of psoralen, psoralidin, bavachalcone, and isobavachalcone on the activity of bid, caspase 3, and PARP. Of all the studied compounds, bavachalcone was the most potent inducer of apoptosis and acted via crosstalk between the intrinsic and extrinsic pathways. In addition, bavachalcone caused cell cycle arrest and decreased the levels of early cell cycle regulatory proteins such as CDK 4 and CDK 2, whereas p21 and p27 levels were increased. We also investigated the extent to which bavachalcone-induced autophagy and apoptosis were related. Phosphorylation and expression of Akt and mTOR were decreased, while the LC3 Ⅱ to LC3Ⅰ ratio was increased in bavachalcone-treated cells. These results suggest that bavachalcone has anticancer activity by promoting both autophagy and apoptosis in HepG2 cells. CONCLUSION: C. corylifolium has an anticancer effect. Especially, bavachalcone has excellent anticancer ability among other components of C. corylifolium by inducing apoptosis and autophagy.

Anticancer effect of Saussurea lappa extract via dual control of apoptosis and autophagy in prostate cancer cells.

To demonstrate the mechanisms of the curative effect of Saussurea lappa ethanol extract (SLE) against prostate cancer, we evaluated the effect of SLE on the induction of apoptosis and autophagy and investigated whether SLE-induced autophagy exerts a pro-survival or pro-apoptotic effect in lymph node carcinoma of the prostate (LNCaP) prostate cancer cells. SLE was prepared using 100% ethanol and added to LNCaP cells for 24 hours. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and cell apoptosis was evaluated by Tali assay. The expression of apoptosis-related mRNA and proteins was analyzed by quantitative real-time RT-PCR and western blotting. SLE treatment decreased the viability of LNCaP cells and increased Bax expression while suppressing the expression of pro-caspases-8/9/3, PARP, Bid, and Bcl-2, thereby inducing apoptosis in LNCaP cells. Cell proliferation related proteins, including p-Akt, androgen receptor, and prostate-specific antigen, were suppressed by SLE treatment. SLE also induced autophagy in LNCaP cells, and inhibition of autophagy enhanced the apoptosis induced by SLE treatment. These results suggest that SLE exerts anticancer effects through the induction of both cellular apoptosis and autophagy, and apoptotic cell death can be facilitated by blocking autophagy in SLE-treated LNCaP cells. Therefore, SLE might be a potential anticancer agent for the treatment of prostate cancer.

Coordination-Driven Self-Assembly and Anticancer Potency Studies of Ruthenium-Cobalt-Based Heterometallic Rectangles.

Three new cobalt-ruthenium heterometallic molecular rectangles, 1-3, were synthesized through the coordination-driven self-assembly of a new cobalt sandwich donor, (η5 -Cp)Co[C4 -trans-Ph2 (4-Py)2 ] (L; Cp: cyclopentyl; Py: pyridine), and one of three dinuclear precursors, [(p-cymene)2 Ru2 (OO∩OO)2 Cl2 ] [OO∩OO: oxalato (A1 ), 5,8-dioxido-1,4-naphthoquinone (A2 ), or 6,11-dioxido-5,12-naphthacenedione (A3 )]. All of the self-assembled architectures were isolated in very good yield (92-94 %) and were fully characterized by spectroscopic analysis; the molecular structures of 2 and 3 were determined by single-crystal X-ray diffraction analysis. The anticancer activities of bimetallic rectangles 1-3 were evaluated with a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, an autophagy assay, and Western blotting. Rectangles 1-3 showed higher cytotoxicity than doxorubicin in AGS human gastric carcinoma cells. In addition, the autophagic activities and apoptotic cell death ratios were increased in AGS cells by treatment with 1-3; the rectangles induced autophagosome formation by promoting LC3-I to LC3-II conversion and apoptotic cell death by increasing caspase-3/7 activity. Our results suggest that rectangles 1-3 induce gastric cancer cell death by modulating autophagy and apoptosis and that they have potential use as agents for the treatment of human gastric cancer.

Combined targeting of high-mobility group box-1 and interleukin-8 to control micrometastasis potential in gastric cancer.

Micrometastasis is the major cause of treatment failure in gastric cancer (GC). Because epithelial-to-mesenchymal transition (EMT) is considered to develop prior to macroscopic metastasis, EMT-promoting factors may affect micrometastasis. This study aimed to evaluate the role of extracellular high-mobility group box-1 (HMGB1) in EMT and the treatment effect of combined targeting of HMGB1 and interleukin-8 (IL-8) at early-stage GC progression through interrupting EMT promotion. Extracellular HMGB1 was induced by human recombinant HMGB1 and pCMV-SPORT6-HMGB1 plasmid transfection. EMT activation was evaluated by immunoblotting, immunofluorescence and immunohistochemistry. Increased migration/invasion activities were evaluated by in vitro transwell migration/invasion assay using all histological types of human GC cell lines (N87, MKN28 SNU-1 and KATOIII), N87-xenograft BALB/c nude mice and human paired serum-tissue GC samples. HMGB1-induced soluble factors were measured by chemiluminescent immunoassay. Inhibition effects of tumor growth and EMT activation by combined targeting of HMGB1 and IL-8 were evaluated in N87-xenograft nude mice. Serum HMGB1 increases along the GC carcinogenesis and reaches maximum before macroscopic metastasis. Overexpressed extracellular HMGB1 promoted EMT activation and increased cell motility/invasiveness through ligation to receptor for advanced glycation end products. HMGB1-induced IL-8 overexpression contributed the HMGB1-induced EMT in GC in vitro and in vivo. Blocking HMGB1 caused significant reduction of tumor growth, and addition of human recombinant IL-8 rescues this antitumor effects. Our results imply the role of HMGB1 in EMT through IL-8 mediation, and a potential mechanism of GC micrometastasis. Our observations suggest combination strategy of HMGB1 and IL-8 as a promising diagnostic and therapeutic target to control GC micrometastasis.

Clinical implications and diagnostic usefulness of correlation between soluble major histocompatibility complex class I chain-related molecule a and protumorigenic cytokines in pancreatic ductal adenocarcinoma.

BACKGROUND: Tumor-derived soluble factors serve as mediators between tumors and surrounding microenvironment to promote tumor growth and metastasis under a complex network. The objective of this study was to evaluate the relationships between soluble major histocompatibility complex class I chain-related molecule A (sMICA) and 4 categories of cytokines (tumor-related proinflammatory, anti-inflammatory, chemotactic/proangiogenic, and growth-stimulatory) in the development and progression of pancreatic ductal adenocarcinoma (PDAC). METHODS: Serum levels of sMICA and 4-categorized cytokines were measured by enzyme-linked immunosorbent assay and chemiluminescent immunoassay, respectively, in 134 individuals (normal, n = 55; chronic pancreatitis, n = 25; PDAC, n = 54). Clinical implications of sMICA and tumor-related cytokines, their correlations, and diagnostic usefulness in PDAC were evaluated. RESULTS: Serum sMICA, which was associated with the development and progression of PDAC, correlated with interferon-γ negatively (P = 0.024), whereas it correlated positively with the anti-inflammatory cytokines interleukin-10 (IL-10) and IL-1 receptor antagonist, and the bifunctional cytokine tumor necrosis factor α, with respect to PDAC development (P < .05). sMICA also correlated positively with the chemotactic/proangiogenic cytokines vascular endothelial growth factor, soluble CD40 ligand, and IL-8, and the tumor growth-stimulatory cytokines epidermal growth factor and transforming growth factor α, with respect to PDAC development and/or progression. Logistic regression analysis validated the diagnostic usefulness of combination use of sMICA and its related cytokines to predict the presence of PDAC and distant metastasis in PDAC, superior to carbohydrate antigen 19-9. CONCLUSIONS: sMICA may be involved in tumor-associated angiogenesis and tumor growth either directly or indirectly by affecting corresponding cytokines as well as causing impairment of natural killer cell cytotoxicity in the development and progression of PDAC. A combination of sMICA and its related cytokines exhibited remarkable diagnostic potential in PDAC.

Serum high mobility group box-1 is a powerful diagnostic and prognostic biomarker for pancreatic ductal adenocarcinoma.

Extracellular high mobility group box-1 (HMGB1) contributes to tumor growth and invasiveness. We evaluated the diagnostic and prognostic ability of serum HMGB1 for pancreatic ductal adenocarcinoma (PDAC). Serum HMGB1 measured by enzyme-linked immunosorbent assay (ELISA) were compared among normal, chronic pancreatitis, PDAC group in both training (n = 25, each group) and independent validation set (n = 45, each group). To determine the usability of serum HMGB1 as a diagnostic predictor of PDAC, receiver operating characteristic (ROC) curves with sensitivity/specificity and logistic regression were evaluated. To assess the HMGB1-associated prognosis of PDAC, Kaplan-Meier survival and Cox proportional-hazards regression were applied. Serum HMGB1 was correlated with presence and advanced-stage of PDAC. Logistic regression exhibited serum HMGB1 was a remarkable biomarker to predict PDAC as a single or multiple-markers; sensitivity/specificity of serum HMGB1 were superior to carbohydrate antigen (CA) 19-9 or carcinoembryonic antigen (CEA) in both training and independent datasets. Kaplan-Meier survival analysis showed PDAC patients with high serum HMGB1 levels (>30 ng/mL; median survival, 192 days) had a worse prognosis than patients with low HMGB1 levels (≤30 ng/mL; 514 days) by log-rank (P = 0.017). Cox proportional-hazards model showed the relative hazard ratios in high-serum HMGB1 group was 3.077 compared with the low-serum HMGB1 group. In conclusion, serum HMGB1 is a desirable diagnostic and prognostic biomarker for PDAC compared with pre-existing PDAC biomarkers, CA19-9 and CEA.

Identification of novel immunogenic human leukocyte antigen-A 2402-binding epitopes of human papillomavirus type 16 E7 for immunotherapy against human cervical cancer.

BACKGROUND: A study was undertaken to identify new immunogenic human leukocyte antigen (HLA)-A 2402-restricted epitopes from human papillomavirus (HPV) type 16 E7 protein for immunotherapy against cervical cancer. METHODS: Synthetic overlapping peptides were screened by measuring the frequency of CD8(+) cytotoxic T lymphocytes (CTLs) producing intracellular interferon-γ (IFN-γ) using flow cytometry and were validated in SiHa cells with a Cr release cytotoxicity assay. In vivo antitumor effects of peptide-sensitized peripheral blood mononuclear cells (PBMCs) and isolated CD8(+) CTLs were evaluated using BALB/c nude mice with SiHa cell xenotransplants. RESULTS: Among 14 overlapping 15-amino acid peptides, E7(61-75) (CDSTLRLCVQSTHVD) and E7(67-81) (LCVQSTHVDIRTLED) induced significantly higher IFN-γ production (P < .05) and showed higher in vitro cytotoxicity against SiHa cells than did cells sensitized with the negative control. To determine the exact HLA-A 2402-restricted epitopes, a total of 25 overlapping 9- or 10-amino acid peptides spanning E7(61-75) and E7(67-81) were synthesized. E7(61-69) (CDSTLRLCV) and E7(67-76) (LCVQSTHVDI) induced significantly greater IFN-γ production as well as increased in vitro cytotoxicity against SiHa cells compared with those of other peptides and the negative control (P < .01), and the antitumor effects of these peptide-sensitized PBMCs were induced by CD8(+) CTLs. E7(61-69) -sensitized and E7(67-76) -sensitized PBMCs and isolated CD8(+) CTLs showed a much greater suppression of tumor growth in vivo compared with that of control groups treated with PBS (P < .01). The authors also confirmed the synergistic antitumor effect of cisplatin followed by E7(67-76) -sensitized PBMCs in vivo. CONCLUSIONS: E7(61-69) and E7(67-76) were identified as novel HPV type 16 E7 epitopes for HLA-A 2402, which could be used for immunotherapy against cervical cancer.

Identification of HLA-A*2402-restricted HCMV immediate early-1 (IE-1) epitopes as targets for CD8+ HCMV-specific cytotoxic T lymphocytes.

BACKGROUND: To identify novel HLA-A*2402-restricted human cytomegalovirus (HCMV) immediate early-1 (IE-1) epitopes for adoptive immunotherapy, we explored 120 overlapping 15-amino acid spanning IE-1. METHODS: These peptides were screened by measuring the frequency of polyclonal CD8+ T cells producing intracellular interferon-gamma (IFN-gamma) using flow cytometry and the epitopes were validated with a HCMV-infected target Cr release cytotoxicity assay. RESULTS: Initial screening was performed with 12 mini-pools of 10 consecutive peptides made from 120 overlapping peptides15-amino acids in length that spanned IE-1. When peripheral blood mononuclear cells (PBMCs) from HLA-A*2402 HCMV-seropositive donors were sensitized with each of the 12 mini-pools, mini-pools 1 and 2 induced the highest frequency of CD8+ cytotoxic T lymphocytes (CTLs) producing IFN-gamma. When PBMCs were stimulated with each of the twenty peptides belonging to mini-pools 1 and 2, peptides IE-11-15MESSAKRKMDPDNPD and IE-15-19AKRKMDPDNPDEGPS induced the greatest quantities of IFN-gamma production and cytotoxicity of HLA-matched HCMV-infected fibroblasts. To determine the exact HLA-A*2402-restricted epitopes within the two IE-1 proteins, we synthesized a total of twenty-one overlapping 9- or 10 amino acid peptides spanning IE-11-15 and IE-15-19. Peptide IE-13-12SSAKRKMDPD induced the greatest quantities of IFN-gamma production and target cell killing by CD8+ CTLs. CONCLUSION: HCMV IE-13-12SSAKRKMDPD is a HLA-A*2402-restricted HCMV IE-1 epitope that can serve as a common target for CD8+ HCMV-specific CTLs.

RU486 suppresses progesterone-induced acrosome reaction in boar spermatozoa.

The effects of progesterone on the acrosome reaction, as well as the effects of RU486 on the progesterone-induced acrosome reaction in capacitated boar spermatozoa, were investigated. Progesterone, a major steroid that is secreted by the cumulus cells of oocyte, clearly induced the acrosome reaction in a dose-dependent manner in capacitated boar spermatozoa, even though it failed to show similar effects in non-capacitated spermatozoa. RU486, a potent antiprogestin, significantly reduced the effects of progesterone on the progesterone-induced acrosome reaction; however, when treated alone, it showed no inhibitory effects on the acrosome reaction. The inhibitory effects of RU486 were also shown to be dose dependent. These results imply that in addition to the wellknown inducer of the acrosome reaction, zona pellucida, progesterone can also induce the acrosome reaction through its specific receptors on spermatozoa after the spermatozoa undergo capacitation.