Rhodamine B-based fluorescent probes for molecular mechanism study of the anti-influenza activity of pentacyclic triterpenes.

The antiviral activity of pentacyclic triterpenes has attracted increasing attention. However, the detailed antiviral mechanism remains fully unclear. In the present study, four C28 or C30 modified pentacyclic triterpene probes via conjugating with rhodamine B were designed and synthesized, and their anti-influenza virus activity was evaluated. The results indicated that two compounds 14 and 23 showed significant antiviral activity to influenza A/WSN/33 (H1N1) virus in Madin-Darby canine kidney (MDCK) cells with IC50 values of 8.36 and 8.24 µM, respectively. The mechanism of action studies of representative probe 23 indicated that it could inhibit the membrane fusion by binding with influenza virus hemagglutinin (HA), and the apparent dissociation constant (KD) value for probe 23-HA interaction was successfully evaluated (1.78 × 10-5 M) using surface plasmon resonance spectroscopy. In addition, the subcellular localization of probe 23 in MDCK cells was determined by confocal microscopy and flow cytometry, and the results suggested that fluorescent probe 23 was rapidly taken up in MDCK cells and accumulated in cytoplasm, but no antiviral activity was observed after its entry into cells. The present study further confirmed our previous finding that pentacyclic triterpenes could tightly bind to the viral envelope HA protein, thus blocking the virus entry into host cells.

Proteasome Inhibitor YSY01A Abrogates Constitutive STAT3 Signaling via Down-regulation of Gp130 and JAK2 in Human A549 Lung Cancer Cells.

Proteasome inhibition interfering with many cell signaling pathways has been extensively explored as a therapeutic strategy for cancers. Proteasome inhibitor YSY01A is a novel agent that has shown remarkable anti-tumor effects; however, its mechanisms of action are not fully understood. Here we report that YSY01A is capable of suppressing cancer cell survival by induction of apoptosis. Paradoxically, we find that YSY01A abrogates constitutive activation of STAT3 via proteasome-independent degradation of gp130 and JAK2, but not transcriptional regulation, in human A549 non-small cell lung cancer cells. The reduction in gp130 and JAK2 can be restored by co-treatment with 3-methyladenine, an early-stage autophagy lysosome and type I/III PI3K inhibitor. YSY01A also effectively inhibits cancer cell migration and lung xenograft tumor growth with little adverse effect on animals. Thus, our findings suggest that YSY01A represents a promising candidate for further development of novel anticancer therapeutics targeting the proteasome.

Synthesis and In Vitro Anti-Influenza Virus Evaluation of Novel Sialic Acid (C-5 and C-9)-Pentacyclic Triterpene Derivatives.

The emergence of drug resistant variants of the influenza virus has led to a great need to identify novel and effective antiviral agents. In our previous study, a series of sialic acid (C-2 and C-4)-pentacyclic triterpene conjugates have been synthesized, and a five-fold more potent antiviral activity was observed when sialic acid was conjugated with pentacyclic triterpene via C-4 than C-2. It was here that we further reported the synthesis and anti-influenza activity of novel sialic acid (C-5 and C-9)-pentacyclic triterpene conjugates. Their structures were confirmed by ESI-HRMS, ¹H-NMR, and 13C-NMR spectroscopic analyses. Two conjugates (26 and 42) showed strong cytotoxicity to MDCK cells in the CellTiter-Glo assay at a concentration of 100 µM. However, they showed no significant cytotoxicity to HL-60, Hela, and A549 cell lines in MTT assay under the concentration of 10 µM (except compound 42 showed weak cytotoxicity to HL-60 cell line (10 µM, ~53%)). Compounds 20, 28, 36, and 44 displayed weak potency to influenza A/WSN/33 (H1N1) virus (100 µM, ~20-30%), and no significant anti-influenza activity was found for the other conjugates. The data suggested that both the C-5 acetylamide and C-9 hydroxy of sialic acid were important for its binding with hemagglutinin during viral entry into host cells, while C-4 and C-2 hydroxy were not critical for the binding process and could be replaced with hydrophobic moieties. The research presented herein had significant implications for the design of novel antiviral inhibitors based on a sialic acid scaffold.

Proteasome Inhibitor YSY01A Enhances Cisplatin Cytotoxicity in Cisplatin-Resistant Human Ovarian Cancer Cells.

Cisplatin is one of the most common drugs used for treatment of solid tumors such as ovarian cancer. Unfortunately, the development of resistance against this cytotoxic agent limits its clinical use. Here we report that YSY01A, a novel proteasome inhibitor, is capable of suppressing survival of cisplatin-resistant ovarian cancer cells by inducing apoptosis. And YSY01A treatment enhances the cytotoxicity of cisplatin in drug-resistant ovarian cancer cells. Specifically, YSY01A abrogates regulatory proteins important for cell proliferation and anti-apoptosis including NF-κB p65 and STAT3, resulting in down-regulation of Bcl-2. A dramatic increase in cisplatin uptake was also observed by inductively coupled plasma-mass spectrometry following exposure to YSY01A. Taken together, YSY01A serves as a potential candidate for further development as anticancer therapeutics targeting the proteasome.

Anticancer Effect of a Novel Proteasome Inhibitor, YSY01A, via G2/M Arrest in PC-3M Cells in vitro and in vivo.

YSY01A is a new tripeptideboronic acid and an analog of PS341. However, YSY01A's antitumor effects and mechanism have not yet been elucidated. This study demonstrates that YSY01A inhibited proteasome activity by combining with the chymotrypsin-like (CT-L) site (ß5i/ß5), the post-glutamyl peptide hydrolase (PGPH) site (ß1i/ß1) and the trypsin-like (T-L) site (ß2i/ß2) in special fluorgonic substrates and proteasome probe tests. We explored the anticancer effect using methyl thiazolyltetrazolium (MTT) or sulforhodamine B (SRB), and PC-3M cells were sensitive to YSY01A among the four cancer cell types tested. The YSY01A antiproliferative effect was stronger than that of PS341. In vivo, YSY01A (1.25, 2.25, and 3.25 mg/kg) inhibited PC-3M cell xenograft tumor growth, and the tumor volume inhibition rate was approximately 40% to 60%. YSY01A arrested PC-3M cells in the G2/M phase of the cell cycle by flow cytometry (FCM). Many proteins related to the cell cycle were analyzed using western blot, and YSY01A was shown to increase p21, p27, cyclinB1, P-cdc2 (tyr15) and wee1 protein expression in both cells and tumor tissue in a concentration-dependent manner. YSY01A, a proteasome inhibitor, exerts anticancer effects on PC-3M cells in vitro and in vivo. The mechanism of the YSY01A-mediated antitumor effect is that the cell cycle is arrested at the G2/M stage. This study suggests that YSY01A may be a novel therapeutic agent for prostate cancer.

YSY01A, a Novel Proteasome Inhibitor, Induces Cell Cycle Arrest on G2 Phase in MCF-7 Cells via ERα and PI3K/Akt Pathways.

Given that the proteasome is essential for multiple cellular processes by degrading diverse regulatory proteins, inhibition of the proteasome has emerged as an attractive target for anti-cancer therapy. YSY01A is a novel small molecule compound targeting the proteasome. The compound was found to suppress viability of MCF-7 cells and cause limited cell membrane damage as determined by sulforhodamine B assay (SRB) and CytoTox 96(®) non-radioactive cytotoxicity assay. High-content screening (HCS) further shows that YSY01A treatment induces cell cycle arrest on G2 phase within 24 hrs. Label-free quantitative proteomics (LFQP), which allows extensive comparison of cellular responses following YSY01A treatment, suggests that various regulatory proteins including cell cycle associated proteins and PI3K/Akt pathway may be affected. Furthermore, YSY01A increases p-CDC-2, p-FOXO3a, p53, p21(Cip1) and p27(Kip1) but decreases p-Akt, p-ERα as confirmed by Western blotting. Therefore, YSY01A represents a potential therapeutic for breast cancer MCF-7 by inducing G2 phase arrest via ERα and PI3K/Akt pathways.

Angiogenesis inhibition and cell cycle arrest induced by treatment with Pseudolarix acid B alone or combined with 5-fluorouracil.

Angiogenesis inhibitors combined with chemotherapeutic drugs have significant efficacy in the treatment of a variety of cancers. Pseudolarix acid B (PAB) is a traditional pregnancy-terminating agent, which has previously been shown to reduce tumor growth and angiogenesis. In this study, we used the high content screening assay to examine the effects of PAB on human umbilical vein endothelial cells (HUVECs). Two hepatocarcinoma 22-transplanted mouse models were used to determine PAB efficacy in combination with 5-fluorouracil (5-Fu). Our results suggested that PAB (0.156-1.250 µM) inhibited HUVECs motility in a concentration-dependent manner without obvious cytotoxicity in vitro. In vivo, PAB (25 mg/kg/day) promoted the anti-tumor efficacy of 5-Fu (5 mg/kg/2 days) in combination therapy, resulting in significantly higher tumor inhibition rates, lower microvessel density values, and prolonged survival times. It was also demonstrated that PAB acted by blocking the cell cycle at both the G(1)/S boundary and M phase, down-regulation of vascular endothelial growth factor, hypoxia-inducible factor 1α and cyclin E expression, and up-regulation of cdc2 expression. These observations provide the first evidence that PAB in combination with 5-Fu may be useful in cancer treatment.

Pseudolaric acid B inhibits inducible cyclooxygenase-2 expression via downregulation of the NF-κB pathway in HT-29 cells.

PURPOSE: Pseudolaric acid B (PAB) is a diterpene acid isolated from the root and trunk bark of Pseudolaric kaempferi Gordon. Previous work has found that PAB has anti-inflammatory and anti-tumor effects in xenograft models of human hepatocellular carcinoma. The aim of this study is to evaluate the correlation between anti-cancer and anti-inflammatory effects of PAB and its molecular mechanisms on HT-29 cells. METHODS: Production of prostaglandin E2 (PGE2) in HT-29 cells was evaluated by ELISA. mRNA of cyclooxygenase-2 (COX-2) was analyzed by RT-PCR assay. High-content screening (HCS) method was adopted to detect the cytokine mixture (CM)-induced transcription activity of NF-κB and STAT3. Western blotting was used to evaluate the protein expression levels of inflammatory mediators induced by CM. After treatment with PAB in various concentrations, the inhibition rate of cell proliferation was measured with sulforhodamine B assays. For the in vivo studies, tumor-bearing models xenografted with HT-29 cells were developed in nude mice, and following oral administration with PAB, tumor inhibition rate was calculated. RESULTS: PAB inhibited the PGE2 production in HT-29 cells significantly (P < 0.05) with similar results detected at the COX-2 mRNA level. Furthermore, PAB suppressed the COX-2 protein expression and significant nuclear translocation of NF-κB and STAT3 induced by CM, which correlated with a concomitant degradation of I-κB and a decrease in constitutive STAT3 phosphorylation (P < 0.05). Moreover, various concentrations of PAB inhibited the proliferation of HT-29 cells in a dose- and time-dependent manner. In vivo, after treatment with PAB for 17 days, the tumor weight of the 50 and 100 mg/kg treated groups was 0.62 ± 0.15 and 0.54 ± 0.06 g, respectively. When compared to the control group (0.82 ± 0.16 g), the inhibition rate of tumor weight was 24.2% at 50 mg/kg (P < 0.05) and 34.7% at 100 mg/kg (P < 0.001). CONCLUSIONS: PAB shows potential anti-cancer activity in HT-29 cells, and its molecular mechanisms are related to the anti-inflammatory action.

Effects of CS-1 on A431 cell proliferation, cell cycle, and epidermal growth factor receptor signal transduction.

CS-1, a new alkaloid with a molecular formula of C(21)H(20)O(8)N(2)S, is extracted from traditional Chinese medicine. Previous studies have shown that CS-1 can inhibit the proliferation of several human carcinoma cells in vivo and in vitro. The aims of this study are to investigate the anti-tumor effect and mechanism of CS-1 in epidermal growth factor receptor (EGFR) signaling pathway in human A431 cell line. Through the sulforhodamine B assay, we found that CS-1 inhibited A431 cell proliferation in the concentration- and time-dependent manners. The inhibitory rate ranged from 14.5% to 87.8% after 24 h of incubation. High content screening (HCS) multi-parameters cytotoxicity analysis showed that CS-1 at high concentration had slight cytotoxicity that resulted from the cell permeabilization and slight reduction in total mitochondrial mass, whereas no change in nucleus size/morphology and lysosomal mass-pH was found. The cytotoxicity of CS-1 was not a major reason for its anti-proliferative effect. Cell cycle analysis indicated that CS-1 induced G1-phase arrest in A431 cells in a time-dependent manner at high concentration (2.5 µM), and S-phase arrest at low concentration (0.625 µM). The HCS assay also showed that CS-1 could inhibit the EGFR internalization, extracellular-signal-regulated kinase (Erk)/mitogen-activated protein kinase translocation to nucleus, the accumulation of phosphorylated protein kinase B (Akt), signal transducer and activator of transcription 3 (STAT3), and cyclin D1 in the nucleus. These results were confirmed by the western blot analysis. CS-1 might inhibit the epidermal growth factor binding to its receptor, resulting in the inhibition of the accumulation of phosphorylated Erk and Akt, and STAT3 in the nucleus, and affecting the transcription of cyclin D1 and cell cycle arrest in G1/S phase.

Novel 8-deaza-5,6,7,8-tetrahydroaminopterin derivatives as dihydrofolate inhibitor: design, synthesis and antifolate activity.

We report, for the first time, the synthesis and biological activities of 8-deaza-5,6,7,8-tetrahydroaminopterin 9, and the 5-substituted and 5,10-disubstituted analogues 11, 13, 15, and 17. The analogues were obtained from key compound diethyl 8-deaza-5,6,7,8-tetrahydroaminopterin 8 following the catalytic reduction of the pyridine ring of diethyl 8-deaza aminopterin 5. The five novel 8-deaza-5,6,7,8-tetrahydroaminopterin derivatives were assayed in vitro for their cytotoxicity on BGC-823, HL-60, Bel-7402 and Hela tumor cell lines, and inhibition on recombinant human dihydrofolate reductase (DHFR), among which the most potent molecule (compound 9) was about 4- to 10-fold poorer than MTX on the four kinds of tumor cell lines, and its effect on DHFR was about 17-fold poorer than MTX. The docking studies were followed to explain the biological testing results.

Cytotoxic activity of some Asarum plants.

The cytotoxic activity against some tumor cell lines of 16 commonly used species of Asarum was evaluated in this study. All of these plants were widely used in Asian countries as traditional medicines or folk medicines. Their inhibitory activities against four tumor cell lines (HL-60, BGC-823, KB and Bel-7402) were compared. It was observed that 10 of the tested extracts (eight ethanol extracts and two water extracts) among 32 extracts of these plants showed cytotoxic activity. Those 95% ethanol extractions from A. caudigerellum, A. forbesii, A. inflatum and A. maximum exhibited the highest cytotoxic activity, and 95% ethanol extracts or water extracts of A. sieboldii var. seoulense, A. himalaicum, A. splendens and A. crispulatum showed selective activity against one or two cells among the tested tumor cells. This is the first report of Asarum plants possessing cytotoxic activity against tumor cell lines.

Synthesis and biological activities of 2-oxocycloalkylsulfonamides.

A series of novel 2-oxocycloalkylsulfonamides (4) were synthesized and their structures confirmed by IR, (1)H NMR, and elemental analysis. The bioassay showed that they have fair to excellent fungicidal activities against Botrytis cinerea Pers and Sclerotinia sclerotiorum. Among them, compounds 4A(10), 4A(11), 4A(12), 4B(2), and 4B(3), the EC(50) values of which were 2.12, 3.66, 3.96, 2.38, and 2.43 microg/mL, respectively, displayed excellent fungicidal activity against B. cinerea Pers, and are comparable with commercial fungicide procymidone (the EC(50) value is 2.45 microg/mL). 3D QSAR against B. cinerea Pers was studied, a statistically significant and chemically meaningful CoMFA model was developed and some compounds which have a high predicted activity were forecasted. In addition, the bioassay also showed that the compounds have good inhibitory activities against human tumor cells HL-60, BGC-823, Bel-7402 and KB. It is interesting to point out that the antitumor activities of compounds 4 are in accordance with their fungicidal activity to a great extent: compounds having relatively best antitumor activities (4A(10), 4A(11), 4A(12), and 4B(3)) also displayed excellent fungicidal activity.

Cardenolides from Saussurea stella with cytotoxicity toward cancer cells.

Three new cardenolides, 3-O-beta-D-fucopyranosylstrophanthidin (1), 3-O-beta-D-quinovopyranosylperiplogenin (2), and 3-O-beta-D-glucopyranosyl-(1 --> 4)-alpha- l-rhamnopyranosylcannogenin (3), together with seven known cardenolides (4- 10), were isolated from a cytotoxic ethanol extract of the whole dried plants of Saussurea stella. The structures of these compounds were established by spectroscopic and chemical methods. When the cytotoxicity of compounds 2- 10 toward Bel-7402 human hepatoma cells and BGC-823 human gastric cancer cells was evaluated, all compounds showed IC 50 values of <1 microM for both cell lines. This is the first report of cardenolides occurring in a species of the family Asteraceae.

[Inhibitory effects of 11 coumarin compounds against growth of human bladder carcinoma cell line E-J in vitro].

OBJECTIVE: To screen antitumor active compounds, drug-like or leading compounds from Chinese traditional and herbal drugs. METHODS: Eleven coumarin compounds isolated from the Chinese traditional and herbal drugs were studied for their antitumor activities in vitro by determining the inhibition rates against growth of human bladder carcinoma cell line E-J. RESULTS: It showed that umbelliferone, scoparone, demethylfuropinarine, isopimpinellin, forbesoside, columbianadin, decursin and glycycoumarin inhibited the growth of human bladder carcinoma cell line E-J in vitro and their activities showed a concentration-effect relationship. The inhibitory effects of forbesoside, columbianadin, decursin and umbelliferone, with IC50 values of 7.50x10(-7), 2.30x10(-6), 6.00x10(-6) and 1.30x10(-6) mol/L, respectively, were stronger than those of the other tested compounds. However, xanthotoxin, esculin and sphondin did not inhibit the growth of human bladder carcinoma cell line E-J in this assay condition. CONCLUSION: These findings indicate that forbesoside, columbianadin, esculin, decursin and umbelliferone would be effective or regarded as potent drug-like or leading compounds against human bladder carcinoma.