Vincent Kam Wai Wong

The in Vitro Structure-Related Anti-Cancer Activity of Ginsenosides and Their Derivatives

Panax ginseng has long been used in Asia as a herbal medicine for the prevention and treatment of various diseases, including cancer. The current study evaluated the cytotoxic potency against a variety of cancer cells by using ginseng ethanol extracts (RSE), protopanaxadiol (PPD)-type, protopanaxatriol (PPT)-type ginsenosides fractions, and their hydrolysates, which were prepared by stepwise hydrolysis of the sugar moieties of the ginsenosides. The results showed that the cytotoxic potency of the hydrolysates of RSE and total PPD-type or PPT-type ginsenoside fractions was much stronger than the original RSE and ginsenosides; especially the hydrolysate of PPD-type ginsenoside fractions. Subsequently, two derivatives of protopanaxadiol (1), compounds 2 and 3, were synthesized via hydrogenation and dehydration reactions of compound 1. Using those two derivatives and the original ginsenosides, a comparative study on various cancer cell lines was conducted; the results demonstrated that the cytotoxic potency was generally in the descending order of compound 3 > 20(S)-dihydroprotopanaxadiol (2) > PPD (1) > 20(S)-Rh2 > 20(R)-Rh2 ≈ 20(R)-Rg3 ≈ 20(S)-Rg3. The results clearly indicate the structure-related activities in which the compound with less polar chemical structures possesses higher cytotoxic activity towards cancer cells.

Mechanistic study of saikosaponin‐d (Ssd) on suppression of murine T lymphocyte activation

Saikosaponin‐d (Ssd) is a triterpene saponin derived from the medicinal plant, Bupleurum falcatum L. (Umbelliferae). Previous findings showed that Ssd exhibits a variety of pharmacological and immunomodulatory activities including anti‐inflammatory, anti‐bacterial, anti‐viral and anti‐cancer effects. In the current study we have investigated the effects of Ssd on activated mouse T lymphocytes through the NF‐κB, NF‐AT and AP‐1 signaling pathways, cytokine secretion, and IL‐2 receptor expression. The results demonstrated that Ssd not only suppressed OKT3/CD28‐costimulated human T cell proliferation, it also inhibited PMA, PMA/Ionomycin and Con A‐induced mouse T cell activation in vitro. The inhibitory effect of Ssd on PMA‐induced T cell activation was associated with down‐regulation of NF‐κB signaling through suppression of IKK and Akt activities. In addition, Ssd suppressed both DNA binding activity and the nuclear translocation of NF‐AT and activator protein 1 (AP‐1) of the PMA/Ionomycin‐stimulated T cells. The cell surface markers like IL‐2 receptor (CD25) were also down‐regulated together with decreased production of pro‐inflammatory cytokines of IL‐6, TNF‐α and IFN‐γ. These results indicate that the NF‐κB, NF‐AT and AP‐1 (c‐Fos) signaling pathways are involved in the T cell inhibition evoked by Ssd, so it can be a potential candidate for further study in treating T cell‐mediated autoimmune conditions. J. Cell. Biochem. 107: 303–315, 2009.

Pseudolaric acid B suppresses T lymphocyte activation through inhibition of NF-κB signaling pathway and p38 phosphorylation.

Pseudolaric acid B (PAB) is a major bioactive component of the medicinal plant Pseudolarix kaempferi. Traditional medicine practitioners in Asia have been using the roots of this plant to treat inflammatory and microbial skin diseases for centuries. In the current study, in vitro immunosuppressive effect of PAB and the underlying mechanisms have been investigated. The results showed that PAB dose‐dependently suppressed human T lymphocyte proliferation, IL‐2 production and CD25 expression induced by co‐stimulation of PMA plus ionomycin or of anti‐OKT‐3 plus anti‐CD28. Mechanistic studies showed that PAB significantly inhibited nuclear translocation of NF‐κB p65 and phosphorylation and degradation of IκB‐α evoked by co‐stimulation of PMA plus ionomycin. PAB could also suppress the phosphorylation of p38 in the MAPKs pathway. Based on these evidences, we conclude that PAB suppressed T lymphocyte activation through inhibition of NF‐κB and p38 signaling pathways; this would make PAB a strong candidate for further study as an anti‐inflammatory agent. J. Cell. Biochem. 108: 87–95, 2009.

Total ginsenosides increase coronary perfusion flow in isolated rat hearts through activation of PI3K/Akt-eNOS signaling

BACKGROUND Ginseng is the most popular herb used for treatment of ischemic heart diseases in Chinese community; ginsenosides are considered to be the major active ingredients. However, whether ginsenosides can enhance the coronary artery flow of ischemic heart and, if so, by what mechanisms they do this, remains unclear. METHODS Isolated rat hearts with ischemia/reperfusion injury in Langendorff system were employed for examining the effect of total ginsenosides (TGS) on coronary perfusion flow (CPF). In addition, human aortic endothelial cells (HAECs) were used for mechanistic study. Levels of various vasodilative molecules, intracellular calcium concentration ([Ca²+](i)), and expressions and activation of proteins involving regulation of nitric oxide (NO) signaling pathways in heart tissues and HAECs were determined. RESULTS TGS dose-dependently and significantly increased CPF and improved systolic and diastolic function of the ischemia/reperfused rat heart, while inhibitors of NO synthase (NOS), soluble guanylate cyclase (sGC), heme oxygenase (HO), cyclooxygenase (COX), and potassium channel abolished the vasodilation effect of TGS. Positive control verapamil was effective only in increasing CPF. TGS elevated levels of NO and 6-keto-prostaglandin F₁α, a stable hydrolytic product of prostacyclin I₂ (PGI₂), in both coronary effluents and supernatants of HAECs culturing medium, and augmented [Ca²+](i) in HAECs. TGS significantly up-regulated expression of phosphoinositide 3-kinase (PI3K) and phosphorylations of Akt and endothelial NOS (eNOS) as well. CONCLUSIONS TGS significantly increased CPF of ischemia/reperfused rat hearts through elevation of NO production via activation of PI3K/Akt-eNOS signaling. In addition, PGI₂, EDHF and CO pathways also partially participated in vasodilation induced by TGS.

Neferine Attenuates the Protein Level and Toxicity of Mutant Huntingtin in PC-12 Cells via Induction of Autophagy

Mutant huntingtin aggregation is highly associated with the pathogenesis of Huntington’s disease, an adult-onset autosomal dominant disorder, which leads to a loss of motor control and decline in cognitive function. Recent literature has revealed the protective role of autophagy in neurodegenerative diseases through degradation of mutant toxic proteins, including huntingtin or α-synuclein. Through the GFP-LC3 autophagy detection platform, we have identified neferine, isolated from the lotus seed embryo of Nelumbo nucifera, which is able to induce autophagy through an AMPK-mTOR-dependent pathway. Furthermore, by overexpressing huntingtin with 74 CAG repeats (EGFP-HTT 74) in PC-12 cells, neferine reduces both the protein level and toxicity of mutant huntingtin through an autophagy-related gene 7 (Atg7)-dependent mechanism. With the variety of novel active compounds present in medicinal herbs, our current study suggests the possible protective mechanism of an autophagy inducer isolated from Chinese herbal medicine, which is crucial for its further development into a potential therapeutic agent for neurodegenerative disorders in the future.

New Potential Pharmacological Functions of Chinese Herbal Medicines via Regulation of Autophagy

Autophagy is a universal catabolic cellular process for quality control of cytoplasm and maintenance of cellular homeostasis upon nutrient deprivation and environmental stimulus. It involves the lysosomal degradation of cellular components such as misfolded proteins or damaged organelles. Defects in autophagy are implicated in the pathogenesis of diseases including cancers, myopathy, neurodegenerations, infections and cardiovascular diseases. In the recent decade, traditional drugs with new clinical applications are not only commonly found in Western medicines, but also highlighted in Chinese herbal medicines (CHM). For instance, pharmacological studies have revealed that active components or fractions from Chaihu (Radix bupleuri), Hu Zhang (Rhizoma polygoni cuspidati), Donglingcao (Rabdosia rubesens), Hou po (Cortex magnoliae officinalis) and Chuan xiong (Rhizoma chuanxiong) modulate cancers, neurodegeneration and cardiovascular disease via autophagy. These findings shed light on the potential new applications and formulation of CHM decoctions via regulation of autophagy. This article reviews the roles of autophagy in the pharmacological actions of CHM and discusses their new potential clinical applications in various human diseases.

Hernandezine, a novel AMPK activator induces autophagic cell death in drug-resistant cancers.

Drug resistance hinder most cancer chemotherapies and leads to disease recurrence and poor survival of patients. Resistance of cancer cells towards apoptosis is the major cause of these symptomatic behaviours. Here, we showed that isoquinoline alkaloids, including liensinine, isoliensinine, dauricine, cepharanthine and hernandezine, putatively induce cytotoxicity against a repertoire of cancer cell lines (HeLa, A549, MCF-7, PC3, HepG2, Hep3B and H1299). Proven by the use of apoptosis-resistant cellular models and autophagic assays, such isoquinoline alkaloid-induced cytotoxic effect involves energy- and autophagy-related gene 7 (Atg7)-dependent autophagy that resulted from direct activation of AMP activated protein kinase (AMPK). Hernandezine possess the highest efficacy in provoking such cell death when compared with other examined compounds. We confirmed that isoquinoline alkaloid is structurally varied from the existing direct AMPK activators. In conclusion, isoquinoline alkaloid is a new class of compound that induce autophagic cell death in drug-resistant fibroblasts or cancers by exhibiting its direct activation on AMPK.

Identification of novel autophagic Radix Polygalae fraction by cell membrane chromatography and UHPLC-(Q)TOF-MS for degradation of neurodegenerative disease proteins

With its traditional use in relieving insomnia and anxiety, our previous study has identified onjisaponin B from Radix Polygalae (RP), as a novel autophagic enhancer with potential neuroprotective effects. In current study, we have further identified a novel active fraction from RP, contains 17 major triterpenoid saponins including the onjisaponin B, by the combinational use of cell membrane chromatography (CMC) and ultra-performance liquid chromatography coupled to (quadrupole) time-of-flight mass spectrometry {UHPLC-(Q)TOF-MS}. By exhibiting more potent autophagic effect in cells, the active fraction enhances the clearance of mutant huntingtin, and reduces protein level and aggregation of α-synuclein in a higher extent when compared with onjisaponin B. Here, we have reported for the first time the new application of cell-based CMC and UHPLC-(Q)TOF-MS analysis in identifying new autophagy inducers with neuroprotective effects from Chinese medicinal herb. This result has provided novel insights into the possible pharmacological actions of the active components present in the newly identified active fraction of RP, which may help to improve the efficacy of the traditional way of prescribing RP, and also provide new standard for the quality control of decoction of RP or its medicinal products in the future.

Mutation of cysteine 46 in IKK-beta increases inflammatory responses

Activation of IκB kinase β (IKK-β) and nuclear factor (NF)-κB signaling contributes to cancer pathogenesis and inflammatory disease; therefore, the IKK-β−NF-κB signaling pathway is a potential therapeutic target. Current drug design strategies focus on blocking NF-κB signaling by binding to specific cysteine residues on IKK-β. However, mutations in IKK-β have been found in patients who may eventually develop drug resistance. For these patients, a new generation of IKK-β inhibitors are required to provide novel treatment options. We demonstrate in vitro that cysteine-46 (Cys-46) is an essential residue for IKK-β kinase activity. We then validate the role of Cys-46 in the pathogenesis of inflammation using delayed-type hypersensitivity (DTH) and an IKK-βC46A transgenic mouse model. We show that a novel IKK-β inhibitor, dihydromyricetin (DMY), has anti-inflammatory effects on WT DTH mice but not IKK-βC46A transgenic mice. These findings reveal the role of Cys-46 in the promotion of inflammatory responses, and suggest that Cys-46 is a novel drug-binding site for the inhibition of IKK-β.

Thalidezine, a novel AMPK activator, eliminates apoptosis-resistant cancer cells through energy-mediated autophagic cell death

Cancers illustrating resistance towards apoptosis is one of the main factors causing clinical failure of conventional chemotherapy. Innovative therapeutic methods which can overcome the non-apoptotic phenotype are needed. The AMP-activated protein kinase (AMPK) is the central regulator of cellular energy homeostasis, metabolism, and autophagy. Our previous study showed that the identified natural AMPK activator is able to overcome apoptosis-resistant cancer via autophagic cell death. Therefore, AMPK is an ideal pharmaceutical target for chemoresistant cancers. Here, we unravelled that the bisbenzylisoquinoline alkaloid thalidezine is a novel direct AMPK activator by using biolayer interferometry analysis and AMPK kinase assays. The quantification of autophagic EGFP-LC3 puncta demonstrated that thalidezine increased autophagic flux in HeLa cancer cells. In addition, metabolic stress assay confirmed that thalidezine altered the energy status of our cellular model. Remarkably, thalidezine-induced autophagic cell death in HeLa or apoptosis-resistant DLD-1 BAX-BAK DKO cancer cells was abolished by addition of autophagy inhibitor (3-MA) and AMPK inhibitor (compound C). The mechanistic role of autophagic cell death in resistant cancer cells was further supported through the genetic removal of autophagic gene7 (Atg7). Overall, thalidezine is a novel AMPK activator which has great potential to be further developed into a safe and effective intervention for apoptosis- or multidrug-resistant cancers.