Apoptosis, also called programmed cell death, is morphologically characterized by cell shrinkage, membrane remodeling, cell blebbing, chromatin condensation and DNA fragmentation with apoptotic bodies.1-3) Apoptosis activation has been considered a good target in cancer therapies. 4,5) In general, apoptosis is regulated by pro-apoptotic and antiapoptotic proteins of the Bcl-2 family and executed through caspases (or cysteine-aspartic proteases), chiefly via two major and inter-related pathways, i.e., the mitochondria-dependent "intrinsic" cytochrome c/caspase-9 pathway and the death receptor-mediated "extrinsic" caspase-8 pathway. [6][7][8] Additionally, apoptosis is controlled by various cell signaling pathways such as the phosphoinositide 3-kinase (PI3K)/AKT survival pathway, regulating apoptosis chiefly by blocking caspase-9 and the mitochondria damaging Bcl-2 family protein, Bad, through phosphorylative inactivation. 9)Tanshinone IIA (Tan IIA,14,6,8,13, Fig. 1A), one of the phytochemical compounds isolated from the Chinese medicinal herb Danshen (root of Salvia miltiorrhiza BUNGE), has been reported to exert diverse biological properties including anti-oxidative, 3,10,11) anti-angiogenic, 12) and anti-inflammatory activities.13) More importantly, anti-cancer activities have been reported including human hepatoma, [14][15][16][17] breast cancer 18) and leukemia. 19) However, there is no report of anticancer activity of Tan IIA on prostate cancer cells. Thus, in the present study, we investigated the cytocidal/apoptosis effect against prostate cancer cell lines of different pathogenetic make-up: LNCaP (p53 null, phosphate and tensin homolog (PTEN) null-high AKT, androgen sensitive) and PC-3 (p53 null, PTEN null-high AKT, androgen receptor null). Our mechanistic investigations suggested an involvement of mitochondria-intrinsic caspase activation cascade and inhibition of PI3K/AKT pathway. MATERIALS AND METHODSTanshinone IIA Isolation The procedure of Tan IIA (Fig. 1A) isolation is as reported by Choi and colleagues. 20)Cell Culture Human prostate cancer cells LNCaP (ATCC CRL 1740, p53 wild type, PTEN mutant-high AKT) and PC3 (ATCC CRL 1435, p53 null, PTEN null-high AKT), and breast cancer cells MDA-MB-231 (ATCC HTB 26, p53 mutant, wild type PTEN-low AKT) were obtained from American Type Culture Collection (ATCC) and maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 2 mmol/l L-glutamine, 10 mmol/l N-(2-hydroxyethyl)piperazine-NЈ-2-ethanesulfonic acid (HEPES), 1 mmol/l sodium pyruvate, and 4.5% D-glucose without antibiotics.Cytotoxicity Assay The cytotoxicity of Tan IIA was assessed by a tetrazolium salt, 2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) assay. LNCaP or PC3 cells were seeded onto 96-well microplates at a density of 1ϫ10 4 cells per well in 100 ml of complete medium. After incubation for 24 h, cells were exposed to var- IIA; 14,16-epoxy-20-nor-5(10),6,8,13,15-abietapentaene-11,12-dione), a phytochemical derived from the roots of Salvia miltio...

The heparan sulfate mimetic PG545 has been shown to exert anti-angiogenic and anti-metastatic activity in vitro and in vivo cancer models. Although much of this activity has been attributed to inhibition of heparanase and heparan sulfate-binding growth factors, it was hypothesized that PG545 may additionally disrupt Wnt signaling, an important pathway underlying the malignancy of pancreatic cancer. We show that PG545, by directly interacting with Wnt3a and Wnt7a, inhibits Wnt/β-catenin signaling leading to inhibition of proliferation in pancreatic tumor cell lines. Additionally, we demonstrate for the first time that the combination of PG545 with gemcitabine has strong synergistic effects on viability, motility and apoptosis induction in several pancreatic cell lines. In an orthotopic xenograft mouse model, combination of PG545 with gemcitabine efficiently inhibited tumor growth and metastasis compared to single treatment alone. Also, PG545 treatment alone decreased the levels of β-catenin and its downstream targets, cyclin D1, MMP-7 and VEGF which is consistent with our in vitro data. Collectively, our findings suggest that PG545 exerts anti-tumor activity by disrupting Wnt/β-catenin signaling and combination with gemcitabine should be considered as a novel therapeutic strategy for pancreatic cancer treatment.

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Defective autophagy and deranged metabolic pathways are common in cancer; pharmacologic targeting of these two pathways could provide a viable therapeutic option. However, how these pathways are regulated by limited availability of growth factors is still unknown. Our study shows that HSulf-1 (endosulfatase), a known tumor suppressor which attenuates heparin sulfate binding growth factor signaling, also regulates interplay between autophagy and lipogenesis. Silencing of HSulf-1 in OV202 and TOV2223 cells (ovarian cancer cell lines) resulted in increased lipid droplets (LDs), reduced autophagic vacuoles (AVs) and less LC3B puncta. In contrast, HSulf-1 proficient cells exhibit more AVs and reduced LDs. Increased LDs in HSulf-1 depleted cells was associated with increased ERK mediated cPLA2S505 phosphorylation. Conversely, HSulf-1 expression in SKOV3 cells reduced the number of LDs and increased the number of AVs compared to vector controls. Furthermore, pharmacological (AACOCF3) and ShRNA mediated downregulation of cPLA2 resulted in reduced LDs, and increased autophagy. Finally, in vivo experiment using OV202 Sh1 derived xenograft show that AACOCF3 treatment effectively attenuated tumor growth and LD biogenesis. Collectively, these results show a reciprocal regulation of autophagy and lipid biogenesis by HSulf-1 in ovarian cancer.

Figure 6: PG545 inhibits β-catenin signaling in AsPC-1 orthotopic xenograft mouse model. (A) Immunohistochemical analysis of β-catenin and β-catenin-regulated proteins, Cyclin D1, MMP-7 and VEGF in pancreatic tumor tissues from mice. Quantitation of staining was performed using 10 fields per analyte. **P < 0.01, ***P < 0.001. (B) Frozen tumor tissues were homogenized on ice and the extracts were subjected to Western blotting. (C) β-catenin and (D) Cyclin D1 levels were quantified by Image J software and plotted relative to the control group in Fig. 6B. **P < 0.01, ***P < 0.001 vs. control.